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Sample records for adeno-associated virus mediated

  1. Adeno-associated virus mediated delivery of Tregitope 167 ameliorates experimental colitis

    Institute of Scientific and Technical Information of China (English)

    Sander van der Marel; Anna Majowicz; Karin Kwikkers; Richard van Logtenstein; Anje A te Velde; Anne S De Groot; Sybren L Meijer

    2012-01-01

    AIM:To explore the anti-inflammatory potential of adeno-associated virus-mediated delivery of Tregitope 167 in an experimental colitis model.METHODS:The trinitrobenzene sulfonate (TNBS) model of induced colitis was used in Balb/c mice.Subsequently after intravenous adeno-associated virusmediated regulatory T-cell epitopes (Tregitope) delivery,acute colitis was initiated by intra-rectal administration of 1.5 mg TNBS in 40% ethanol followed by a second treatment with TNBS (0.75 mg in 20% ethanol) 8 d later.Control groups included mice not treated with TNBS (healthy control group) and mice treated by TNBS only (diseased group).At the time of sacrifice colon weight,the disease activity index and histology damage score were determined.Immunohistochemical staining of the colonic tissues was performed to asses the cellular infiltrate and the presence of transcription factor forkhead Box-P3 (Foxp3).Thymus,mesenteric lymph nodes,liver and spleen tissue were collected and the corresponding lymphocyte populations were further assessed by flow cytometry analysis for the expression of CD4+ T cell and regulatory T cell associated markers.RESULTS:The Tregitope 167 treated mice gained an average of 4% over their initial body weight at the time of sacrifice.In contrast,the mice treated with TNBS alone (no Tregitope) developed colitis,and lost 4% of their initial body weight at the time of sacrifice (P < 0.01).The body weight increase that had been observed in the mice pre-treated with Tregitope 167 was substantiated by a lower disease activity index and a decreased colon weight as compared to the diseased control group (P < 0.01 and P < 0.001,respectively).Immunohistochemical staining of the colonic tissues for CD4+ showed that inflammatory cell infiltrates were present in TNBS treated mice with or without administration with tregitope 167 and that these cellular infiltrates consisted mainly of CD4+ cells.For both TNBS treated groups CD4+ T cell infiltrates were

  2. Adeno-associated Virus Mediated LacZ Gene Transfect to Cultured Human Iris Pigment Epithelium Cells

    Institute of Scientific and Technical Information of China (English)

    Chun Zhang; Shibo Tang; Yan Luo; Xiaoling Liang; Jing Ma; Shaofen Lin

    2003-01-01

    Purpose: To study the feasibility of adeno-associated virus mediated gene transfection tocultured human iris pigment epithelium (IPE) cells in vitro.Methods: Recombinant replication deficient adeno-associated viruses (AAV) expressingLacZ gene were produced without helper virus. The LacZ gene was transduced into culturedhuman IPE cells.Results: Cultured human IPE cells stained positively anticytokeratin, The titer ofrAAV-LacZ was 2.1 × 108 virus particles/ml, 42% cultured human IPE cells expressedβ-galactosidase 7 days after transfection and 67% after 14 days.Conclusions: Recombined AAV produced without helper virus can transfer a foreign geneinto human IPE cells with high efficiency in vitro.

  3. Adeno-associated virus mediated gene therapy for retinal degenerative diseases.

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    Stieger, Knut; Cronin, Therese; Bennett, Jean; Rolling, Fabienne

    2011-01-01

    Retinal gene therapy holds great promise for the treatment of inherited and noninherited blinding diseases such as retinitis pigmentosa and age-related macular degeneration. The most widely used vectors for ocular gene delivery are based on adeno-associated virus (AAV) because it mediates long-term transgene expression in a variety of retinal cell types and elicits minimal immune responses. Inherited retinal diseases are nonlethal and have a wide level of genetic heterogeneity. Many of the genes have now been identified and their function elucidated, providing a major step towards the development of gene-based treatments. Extensive preclinical evaluation of gene transfer strategies in small and large animal models is key to the development of successful gene-based therapies for the retina. These preclinical studies have already allowed the field to reach the point where gene therapy to treat inherited blindness has been brought to clinical trial.In this chapter, we focus on AAV-mediated specific gene therapy for inherited retinal degenerative diseases, describing the disease targets, the preclinical studies in animal models and the recent success of the LCA-RPE65 clinical trials.

  4. An adeno-associated virus vector-mediated multiple gene transfer for dopamine synthetic enzymes

    Institute of Scientific and Technical Information of China (English)

    樊东升; 沈扬

    2000-01-01

    Objective: To explore a multiple gene transfer approach with separate adeno-associated virus vectors. Methods: The genes of dopamine synthetic enzymes, tyrosine hydroxylasc (TH), GTP cyclohydrolase I (GCH, an enzyme critical for tetrahydrobioptcrin synthesis), and aromatic L-amino acid decarboxylase (AADC), were cotransduced into 293 cells with separate AAV vectors. Expressions of TH, GCH, and AADC were detected by Western blot analysis. L-dopa and dopamine levels in the ceils were assayed by HPLC. Results: TH, GCH, and AADC proteins were effectively cocxpressed in the transduced cells with three separate AAV vectors, AAV-TH, AAV-GCH, and AAV-AADC. Furthermore, the coexpression of these three proteins resulted in an effectively spontaneous dopainc production in the cotransduced cells. Conclusion: The triple transduction of TH, GCH, and AADC genes with separate AAV vectors is effective, which might be important to gene therapy for Parkinson's disease.

  5. Adeno-Associated Virus 9-Mediated Airway Expression of Antibody Protects Old and Immunodeficient Mice against Influenza Virus

    OpenAIRE

    Adam, Virginie S.; Crosariol, Marco; Kumar, Sachin; Ge, Moyar Q.; Czack, Sarah E.; Roy, Soumitra; Haczku, Angela; Tretiakova, Anna; Wilson, James M.; Limberis, Maria P.

    2014-01-01

    Influenza causes serious and sometimes fatal disease in individuals at risk due to advanced age or immunodeficiencies. Despite progress in the development of seasonal influenza vaccines, vaccine efficacy in elderly and immunocompromised individuals remains low. We recently developed a passive immunization strategy using an adeno-associated virus (AAV) vector to deliver a neutralizing anti-influenza antibody at the site of infection, the nasal airways. Here we show that young, old, and immunod...

  6. Adeno-Associated Virus-Mediated Gene Transfer to Renal Tubule Cells via a Retrograde Ureteral Approach

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    Daniel C. Chung

    2011-11-01

    Full Text Available Background/Aims: Gene therapy involves delivery of exogenous DNA to provide a therapeutic protein. Ideally, a gene therapy vector should be non-toxic, non-immunogenic, easy to produce, and efficient in protecting and delivering DNA into target cells. Methods: Adeno-associated virus (AAV offers these advantages and few, if any, disadvantages, and over 100 isolates exist. We previously showed that AAV-mediated gene therapy can be used to restore vision to patients with Leber’s congenital amaurosis, a disease of childhood blindness. Results: Here we show that novel recombinant AAV2/8 and AAV2/9 transduce kidney tubule cells with high efficiency both in vitroin cell culture and in vivoin mice. In addition, we adapted and modified a retrograde approach to allow for optimal transgene delivery to renal tubular cells that further minimizes the risk of an immunogenic reaction. Conclusions: We believe that recombinant AAV2, especially AAV2/8, gene delivery to renal tubule cells via a retrograde approach represents a viable method for gene therapy for a multitude of renal disorders ranging from autosomal dominant polycystic kidney disease to acute kidney injury.

  7. Adeno-associated virus-mediated doxycycline-regulatable TRAIL expression suppresses growth of human breast carcinoma in nude mice

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    Zheng Liu

    2012-04-01

    Full Text Available Abstract Background Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL functions as a cytokine to selectively kill various cancer cells without toxicity to most normal cells. Numerous studies have demonstrated the potential use of recombinant soluble TRAIL as a cancer therapeutic agent. We have showed previous administration of a recombinant adeno-associated virus (rAAV vector expressing soluble TRAIL results in an efficient suppression of human tumor growth in nude mice. In the present study, we introduced Tet-On gene expression system into the rAAV vector to control the soluble TRAIL expression and evaluate the efficiency of the system in cancer gene therapy. Methods Controllability of the Tet-On system was determined by luciferase activity assay, and Western blotting and enzyme-linked immunoabsorbent assay. Cell viability was determined by MTT assay. The breast cancer xenograft animal model was established and recombinant virus was administrated through tail vein injection to evaluate the tumoricidal activity. Results The expression of soluble TRAIL could be strictly controlled by the Tet-On system in both normal and cancer cells. Transduction of human cancer cell lines with rAAV-TRE-TRAIL&rAAV-Tet-On under the presence of inducer doxycycline resulted in a considerable cell death by apoptosis. Intravenous injection of the recombinant virus efficiently suppressed the growth of human breast carcinoma in nude mice when activated by doxycycline. Conclusion These data suggest that rAAV-mediated soluble TRAIL expression under the control of the Tet-On system is a promising strategy for breast cancer therapy.

  8. Expression of HIV-1 broadly neutralizing antibodies mediated by recombinant adeno-associated virus 8 in vitro and in vivo.

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    Yu, Yongjiao; Fu, Lu; Jiang, Xiaoyu; Guan, Shanshan; Kuai, Ziyu; Kong, Wei; Shi, Yuhua; Shan, Yaming

    2016-12-01

    Despite unremitting efforts since the discovery of human immunodeficiency virus type 1 (HIV-1), an effective vaccine has not been generated. Viral vector-mediated transfer for expression of HIV-1 broadly neutralizing antibodies (BnAbs) is an attractive strategy. In this study, a recombinant adeno-associated virus 8 (rAAV8) vector was used to encode full-length antibodies against HIV-1 in 293T cells and Balb/c mice after gene transfer. The 10E8 or NIH45-46 BnAb was expressed from a single open reading frame by linking the heavy and light chains with a furin cleavage and a 2A self-processing peptide (F2A). The results showed that the BnAbs could be expressed in the 293T cell culture medium. A single intramuscular injection of rAAV8 led to long-term expression of BnAbs in Balb/c mice. The expressed antibodies in the supernatant of 293T cells and in Balb/c mice showed neutralization effects against HIV-1 pseudoviruses. Combined immunization of rAAV8 expressing 10E8 and rAAV8 expressing NIH45-46 in Balb/c mice could increase these neutralization effects on strains of HIV-1 sensitive to 10E8 or NIH45-46 antibody compared with a single injection of rAAV8 expressing either antibody alone. Therefore, the combined immunization may be a potential vaccine approach against HIV-1.

  9. Early, sustained efficacy of adeno-associated virus vector-mediated gene therapy in glycogen storage disease type Ia.

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    Koeberl, D D; Sun, B D; Damodaran, T V; Brown, T; Millington, D S; Benjamin, D K; Bird, A; Schneider, A; Hillman, S; Jackson, M; Beaty, R M; Chen, Y T

    2006-09-01

    The deficiency of glucose-6-phosphatase (G6Pase) underlies life-threatening hypoglycemia and growth retardation in glycogen storage disease type Ia (GSD-Ia). An adeno-associated virus (AAV) vector encoding G6Pase was pseudotyped as AAV8 and administered to 2-week-old GSD-Ia mice (n = 9). Median survival was prolonged to 7 months following vector administration, in contrast to untreated GSD-Ia mice that survived for only 2 weeks. Although GSD-Ia mice were initially growth-retarded, treated mice increased fourfold in weight to normal size. Blood glucose was partially corrected by 2 weeks following treatment, whereas blood cholesterol normalized. Glucose-6-phosphatase activity was partially corrected to 25% of the normal level at 7 months of age in treated mice, and blood glucose during fasting remained lower in treated, affected mice than in normal mice. Glycogen storage was partially corrected in the liver by 2 weeks following treatment, but reaccumulated to pre-treatment levels by 7 months old (m.o.). Vector genome DNA decreased between 3 days and 3 weeks in the liver following vector administration, mainly through the loss of single-stranded genomes; however, double-stranded vector genomes were more stable. Although CD8+ lymphocytic infiltrates were present in the liver, partial biochemical correction was sustained at 7 m.o. The development of efficacious AAV vector-mediated gene therapy could significantly reduce the impact of long-term complications in GSD-Ia, including hypoglycemia, hyperlipidemia and growth failure.

  10. Adeno-associated virus-mediated Bcl-xL prevents aminoglycoside-induced hearing loss in mice

    Institute of Scientific and Technical Information of China (English)

    LIU Yu-he; KE Xiao-mei; QIN Yong; GU Zhi-ping; XIAO Shui-fang

    2007-01-01

    Background Recent studies showed that aminoglycosides destroyed the cochlear cells and induced ototoxicity by producing reactive oxygen species, including free radicals in the mitochondria, damaging the membrane of mitochondria and resulting in apoptotic cell death. Bcl-xL is a well characterized anti-apoptotic member of the Bcl-2 family. The aim of this study was to determine the potential cochlear protective effect of Bcl-xL as a therapeutic agent in the murine model of aminoglycoside ototoxicity.Methods Serotype 2 of adeno-associated virus (AAV2) as a vector encoding the mouse Bcl-xL gene was injected into mice cochleae prior to injection of kanamycin. Bcl-xL expression in vitro and in vivo was examined with Western blotting and immunohistochemistry separately. Cochlear dissection and auditory steady state responses were checked to evaluate the cochlear structure and function.Results The animals in the AAV2-Bcl-xL/kanamycin group displayed better auditory steady state responses hearing thresholds and cochlear structure than those in the artificial perilymph/kanamycin or AAV2-enhanced humanized green fluorescent protein/kanamycin control group at all tested frequencies. The auditory steady state responses hearing thresholds and cochlear structure in the inoculated side were better than that in the contralateral side.Conclusions AAV2-Bcl-xL afforded significant preservation of the cochlear hair cells against ototoxic insults and protected the cochlear function. AAV2-mediated Bcl-xL might be an approach with respect to potential therapeutic application in the cochlear degeneration.

  11. Characterization of Fabry mice treated with recombinant adeno-associated virus 2/8-mediated gene transfer

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    Choi Jin-Ok

    2010-04-01

    Full Text Available Abstract Background Enzyme replacement therapy (ERT with α-galactosidase A (α-Gal A is currently the most effective therapeutic strategy for patients with Fabry disease, a lysosomal storage disease. However, ERT has limitations of a short half-life, requirement for frequent administration, and limited efficacy for patients with renal failure. Therefore, we investigated the efficacy of recombinant adeno-associated virus (rAAV vector-mediated gene therapy for a Fabry disease mouse model and compared it with that of ERT. Methods A pseudotyped rAAV2/8 vector encoding α-Gal A cDNA (rAAV2/8-hAGA was prepared and injected into 18-week-old male Fabry mice through the tail vein. The α-Gal A expression level and globotriaosylceramide (Gb3 levels in the Fabry mice were examined and compared with Fabry mice with ERT. Immunohistochemical and ultrastructural studies were conducted. Results Treatment of Fabry mice with rAAV2/8-hAGA resulted in the clearance of accumulated Gb3 in tissues such as liver, spleen, kidney, heart, and brain with concomitant elevation of α-Gal A enzyme activity. Enzyme activity was elevated for up to 60 weeks. In addition, expression of the α-Gal A protein was identified in the presence of rAAV2/8-hAGA at 6, 12, and 24 weeks after treatment. α-Gal A activity was significantly higher in the mice treated with rAAV2/8-hAGA than in Fabry mice that received ERT. Along with higher α-Gal A activity in the kidney of the Fabry mice treated with gene therapy, immunohistochemical studies showed more α-Gal A expression in the proximal tubules and glomerulus, and less Gb3 deposition in Fabry mice treated with this gene therapy than in mice given ERT. The α-gal A gene transfer significantly reduced the accumulation of Gb3 in the tubules and podocytes of the kidney. Electron microscopic analysis of the kidneys of Fabry mice also showed that gene therapy was more effective than ERT. Conclusions The rAAV2/8-hAGA mediated α-Gal A gene

  12. Adeno-associated virus mediated interferon-gamma inhibits the progression of hepatic fibrosis in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Miao Chen; Guang-Ji Wang; Yong Diao; Rui-An Xu; Hai-Tang Xie; Xin-Yan Li; Jian-Guo Sun

    2005-01-01

    AIM: To investigate the effects of adeno-associated virus (AAV) mediated expression of human interferon-γ for gene therapy in experimental hepatic fibrosisin vitro and in vivo.METHODS: We constructed the recombinant AAV encoding human INF-γ (rAAV- INF-γ) and took the primary rat hepatic stellate cells and carbon tetrachloride induced rats as the experimental hepatic fibrosis model in vitro and in vivo. Immunocytochemistry analysis was used to reveal the expression of α-SMA, the marker protein expressed in hepatic stellate cells. The mRNA expression of TGF-β, TIMP-L, and MMP-13 were analyzed by RT-PCR method. In vivo study, the hydroxyproline content in liver and serum AST, ALT were also detected.RESULTS: In vitro study, AAV vector could mediated efficient expression of human INF-γ,, which inhibit the activation of hepatic stellate cells, decrease the expression of α-SMA and mRNA of TIMP-1, TGF-β, with the MMP-13unchanged. In vivo study, the histological examination revealed that rAAV- INF-γ could inhibit the progression of the hepatic fibrosis. In the rAAV-INF-γ induced group,the hydroxyproline content and serum AST, ALT level were decreased to 177±28 μg/g wet liver, 668.5±140.0,458.4±123.5 U/L, compare with the fibrosis control group 236±31 μg/g wet liver, 1 019.1±276.3, 770.5±154.3 U/L,respectively (P<0.01). mRNA expression of TIMP-1 in the rAAV-INF-γ induced rat liver was decreased while no significant change was observed in TGF-β and MMP-13.CONCLUSION: All these results indicated that rAAV-INF-γhas potential effects for gene therapy of hepatic fibrosis,which could inhibit the progression of hepatic fibrosis.

  13. Recombinant adeno-associated virus-mediated delivery of antisense angiotensin Ⅱ receptor 1 gene attenuates hypertension development

    Institute of Scientific and Technical Information of China (English)

    Xu-guang LI; Jiang-tao YAN; Xi-zheng XU; Jia-ning WANG; Li-ming CHENG; Tao WANG; Ping ZUO; Dao-wen WANG

    2007-01-01

    Aim:The renin-angiotensin system plays a crucial role in the development and establishment of hypertension,and the pharmacological blockade of the system results in a reduction in blood pressure. In the present study,we investigated whether the effects of a novel,double-stranded,recombinant adeno-associated virus vector (rAAV)-mediated antisense angiotensin Ⅱ receptor l (AT1R) gene efficiently prevents the development of hypertension induced by a high-salt diet in adult,male Sprague-Dawley (SD) rats. Methods:A rAAV was prepared with a cassette containing a cytomegalovirus promoter and partial cDNA (660 base pairs) for the AT1R inserted in the antisense direction (rAAV-AT1AS). A single tail vein injection of the rAAV-AT1-AS or rAAV-GFP (green fluorescent protein,a reporter gene) was performed in adult,male SD rats. Two weeks after injection,the animals were fed a diet containing 8% NaCI,and the systolic blood pressure was measured weekly using the tail-cuff method for 12 weeks. Results:The high-salt diet induced a significant rise in systolic blood pressure in the rAAV-GFP-treated animals;however,the rAAV-AT:AS treatment attenuated the rise in blood pressure (142.7±4.5 mmHg vs 117±3.8 mmHg,P<0.01),and the hypotensive effect was maintained until the experiments ended at 12 weeks. In the rAAV-GFP-treated animals AT1 was overexpressed in various tissues,especially in the aorta and kidney at mRNA levels;in contrast,rAAV-AT:AS treatment markedly attenuated AT1 expression. Furthermore,rAAV-AT:AS treatment prevented target organ damages from hypertension,including cardiac dysfunction and renal injury compared to the rAAV-GFP group. Conclusion:These results suggest that rAAVmediated anti-AT1 delivery attenuates the development of hypertension and protects against renal injury and cardiac remodeling.

  14. Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells.

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    Strobel, Benjamin; Klauser, Benedikt; Hartig, Jörg S; Lamla, Thorsten; Gantner, Florian; Kreuz, Sebastian

    2015-10-01

    Cytotoxicity of transgenes carried by adeno-associated virus (AAV) vectors might be desired, for instance, in oncolytic virotherapy or occur unexpectedly in exploratory research when studying sparsely characterized genes. To date, most AAV-based studies use constitutively active promoters (e.g., the CMV promoter) to drive transgene expression, which often hampers efficient AAV production due to cytotoxic, antiproliferative, or unknown transgene effects interfering with producer cell performance. Therefore, we explored artificial riboswitches as novel tools to control transgene expression during AAV production in mammalian cells. Our results demonstrate that the guanine-responsive GuaM8HDV aptazyme efficiently attenuates transgene expression and associated detrimental effects, thereby boosting AAV vector yields up to 23-fold after a single addition of guanine. Importantly, riboswitch-harboring vectors preserved their ability to express functional transgene at high levels in the absence of ligand, as demonstrated in a mouse model of AAV-TGFβ1-induced pulmonary fibrosis. Thus, our study provides the first application-ready biotechnological system-based on aptazymes, which should enable high viral vector yields largely independent of the transgene used. Moreover, the RNA-intrinsic, small-molecule regulatable mode of action of riboswitches provides key advantages over conventional transcription factor-based regulatory systems. Therefore, such riboswitch vectors might be ultimately applied to temporally control therapeutic transgene expression in vivo.

  15. Adeno-associated virus-mediated human IL-10 gene transfer suppresses the development of experimental autoimmune orchitis.

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    Watanabe, M; Kashiwakura, Y; Kusumi, N; Tamayose, K; Nasu, Y; Nagai, A; Shimada, T; Daida, H; Kumon, H

    2005-07-01

    Testicular germ cell-induced autoimmune orchitis is characterized by inflammatory cell infiltration followed by disturbance of spermatogenesis. Experimental autoimmune orchitis (EAO) is an animal model for human immunological male infertility; delayed-type hypersensitivity (DTH) response plays a key role in its induction. Interleukin-10 (IL-10) is a regulatory cytokine that is critical in preventing organ-specific autoimmune inflammation. To determine the effects on EAO of human IL-10 (hIL-10) gene transfer, C3H/He mice immunized by unilateral testicular injury were administered intramuscular (i.m.) injections of adeno-associated viral (AAV) vector-encoding hIL-10 on the day of immunization. Serum hIL-10 was detected beginning at 1 week postinjection, and peaked at 3 weeks. Histological examinations showed a significantly low incidence of orchitis and disturbance of spermatogenesis in AAV hIL-10-treated mice, and the DTH response to autologous testicular cells was significantly suppressed. Immunohistochemical analysis of IFN- and IL-2, T-cell-associated cytokines, in the spleen and testes revealed significantly fewer cytokine-expressing cells after treatment. We conclude that a single i.m. administration of AAV hIL-10 significantly suppresses EAO and hypospermatogenesis by regulating cell-mediated immunity in the testes.

  16. Adeno-associated virus-mediated rescue of the cognitive defects in a mouse model for Angelman syndrome.

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    Jennifer L Daily

    Full Text Available Angelman syndrome (AS, a genetic disorder occurring in approximately one in every 15,000 births, is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes for E6-AP, an ubiquitin ligase. A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. However, we have determined that abnormal calcium/calmodulin-dependent protein kinase II (CaMKII regulation is seen in the maternal UBE3A deletion AS mouse model and is responsible for the major phenotypes. Specifically, there is an increased αCaMKII phosphorylation at the autophosphorylation sites Thr(286 and Thr(305/306, resulting in an overall decrease in CaMKII activity. CaMKII is not produced until after birth, indicating that the deficits associated with AS are not the result of developmental abnormalities. The present studies are focused on exploring the potential to rescue the learning and memory deficits in the adult AS mouse model through the use of an adeno-associated virus (AAV vector to increase neuronal UBE3A expression. These studies show that increasing the levels of E6-AP in the brain using an exogenous vector can improve the cognitive deficits associated with AS. Specifically, the associative learning deficit was ameliorated in the treated AS mice compared to the control AS mice, indicating that therapeutic intervention may be possible in older AS patients.

  17. Recombinant adeno-associated virus-mediated human kallikrein gene therapy protects against hypertensive target organ injuries through inhibiting cell apoptosis

    Institute of Scientific and Technical Information of China (English)

    Jiang-tao YAN; Tao WANG; Dao-wen WANG

    2009-01-01

    Aim: Overexpression of human tissue kallikrein (HK), mediated by recombinant adeno-associated virus (rAAV), decreased blood pres-sure in spontaneous hypertensive rats (SHRs) and reduced injury to the heart, aorta and kidney. In this study, we used both an in vivo animal model and in vitro cell culture system to investigate whether rAAV-rnediated HK gene therapy protects against organ damage by inhibiting cell apoptosis. Methods: rAAV encoding HK(rAAV-HK) or LacZ(rAAV-lacZ) were delivered as a control to spontaneously hypertensive rats (SHRs) and cultured human embryonic kidney (HEK) 293 cells. Results: Treatment with rAAV-HK decreased cell apoptosis in the target organs of SHRs and also inhibited lipopolysaccharide (LPS)-in-duced HEK 293 apoptosis. The rAAV-HK delivery system also increased the levels of apoptosis-inhibiting proteins bcl-2 and bcl-x_L, and decreased the level of Bax and the activity of caspase 3, two promoters of apoptosis. In addition to its role in the inhibition of apopto-sis, rAAV-HK also activated the cell survival and proliferation signaling pathways ERK1/2 and PI3K/AKT. Conclusion: rAAV-mediated HK gene delivery has multiple therapeutic possibilities for treating hypertension, not only by decreasing blood pressure, but also by directly inhibiting end-organ damage.

  18. Cytotoxic-T-Lymphocyte-Mediated Elimination of Target Cells Transduced with Engineered Adeno-Associated Virus Type 2 Vector In Vivo▿

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    Li, Chengwen; Hirsch, Matt; DiPrimio, Nina; Asokan, Aravind; Goudy, Kevin; Tisch, Roland; Samulski, R. Jude

    2009-01-01

    A recent clinical trial in patients with hemophilia B has suggested that adeno-associated virus (AAV) capsid-specific cytotoxic T lymphocytes (CTLs) eliminated AAV-transduced hepatocytes and resulted in therapeutic failure. AAV capsids elicit a CTL response in animal models; however, these capsid-specific CTLs fail to kill AAV-transduced target cells in mice. To better model the human clinical trial data in mice, we introduced an immunodominant epitope derived from ovalbumin (OVA; SIINFEKL) i...

  19. Adeno-associated virus mediated delivery of a non-membrane targeted human soluble CD59 attenuates some aspects of diabetic retinopathy in mice.

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    Mehreen Adhi

    Full Text Available Diabetic retinopathy is the leading cause of visual dysfunction in working adults and is attributed to retinal vascular and neural cell damage. Recent studies have described elevated levels of membrane attack complex (MAC and reduced levels of membrane associated complement regulators including CD55 and CD59 in the retina of diabetic retinopathy patients as well as in animal models of this disease. We have previously described the development of a soluble membrane-independent form of CD59 (sCD59 that when delivered via a gene therapy approach using an adeno-associated virus vector (AAV2/8-sCD59 to the eyes of mice, can block MAC deposition and choroidal neovascularization. Here, we examine AAV2/8-sCD59 mediated attenuation of MAC deposition and ensuing complement mediated damage to the retina of mice following streptozotocin (STZ induced diabetes. We observed a 60% reduction in leakage of retinal blood vessels in diabetic eyes pre-injected with AAV2/8-sCD59 relative to negative control virus injected diabetic eyes. AAV2/8-sCD59 injected eyes also exhibited protection from non-perfusion of retinal blood vessels. In addition, a 200% reduction in retinal ganglion cell apoptosis and a 40% reduction in MAC deposition were documented in diabetic eyes pre-injected with AAV2/8-sCD59 relative to diabetic eyes pre-injected with the control virus. This is the first study characterizing a viral gene therapy intervention that targets MAC in a model of diabetic retinopathy. Use of AAV2/8-sCD59 warrants further exploration as a potential therapy for advanced stages of diabetic retinopathy.

  20. Recombinant adeno-associated virus-mediated microRNA delivery into the postnatal mouse brain reveals a role for miR-134 in dendritogenesis in vivo

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    Mette Christensen

    2010-01-01

    Full Text Available Recent studies using primary neuronal cultures have revealed important roles of the microRNA pathway in the regulation of neuronal development and morphology. For example, miR-134 is involved in dendritogenesis and spine development in hippocampal neurons by regulating local mRNA translation in dendrites. The in vivo roles of microRNAs in these processes are still uninvestigated, partly due to the lack of tools enabling stable in vivo delivery of microRNAs or microRNA inhibitors into neurons of the mammalian brain. Here we describe the construction and validation of a vector-based tool for stable delivery of microRNAs in vivo by use of recombinant adeno-associated virus (rAAV. rAAV-mediated overexpression of miR-134 in neurons of the postnatal mouse brain provided evidence for a negative role of miR-134 in dendritic arborization of cortical layer V pyramidal neurons in vivo, thereby confirming previous findings obtained with cultured neurons. Our system provides researchers with a unique tool to study the role of any candidate microRNA in vivo and can easily be adapted to microRNA loss-of-function studies. This platform should therefore greatly facilitate investigations on the role of microRNAs in synapse development, plasticity and behavior in vivo.

  1. Adeno-associated virus for cystic fibrosis gene therapy

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    S.V. Martini

    2011-11-01

    Full Text Available Gene therapy is an alternative treatment for genetic lung disease, especially monogenic disorders such as cystic fibrosis. Cystic fibrosis is a severe autosomal recessive disease affecting one in 2500 live births in the white population, caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR. The disease is classically characterized by pancreatic enzyme insufficiency, an increased concentration of chloride in sweat, and varying severity of chronic obstructive lung disease. Currently, the greatest challenge for gene therapy is finding an ideal vector to deliver the transgene (CFTR to the affected organ (lung. Adeno-associated virus is the most promising viral vector system for the treatment of respiratory disease because it has natural tropism for airway epithelial cells and does not cause any human disease. This review focuses on the basic properties of adeno-associated virus and its use as a vector for cystic fibrosis gene therapy.

  2. Inhibition of Histone Deacetylation and DNA Methylation Improves Gene Expression Mediated by the Adeno-Associated Virus/Phage in Cancer Cells

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    Amin Hajitou

    2013-10-01

    Full Text Available Bacteriophage (phage, viruses that infect bacteria only, have become promising vectors for targeted systemic delivery of genes to cancer, although, with poor efficiency. We previously designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV. This novel AAV/phage hybrid (AAVP specifically targeted systemic delivery of therapeutic genes into tumors. To advance the AAVP vector, we recently introduced the stress-inducible Grp78 tumor specific promoter and found that this dual tumor-targeted AAVP provides persistent gene expression, over time, in cancer cells compared to silenced gene expression from the CMV promoter in the parental AAVP. Herein, we investigated the effect of histone deacetylation and DNA methylation on AAVP-mediated gene expression in cancer cells and explored the effect of cell confluence state on AAVP gene expression efficacy. Using a combination of AAVP expressing the GFP reporter gene, flow cytometry, inhibitors of histone deacetylation, and DNA methylation, we have demonstrated that histone deacetylation and DNA methylation are associated with silencing of gene expression from the CMV promoter in the parental AAVP. Importantly, inhibitors of histone deacetylases boost gene expression in cancer cells from the Grp78 promoter in the dual tumor-targeted AAVP. However, cell confluence had no effect on AAVP-guided gene expression. Our findings prove that combination of histone deacetylase inhibitor drugs with the Grp78 promoter is an effective approach to improve AAVP-mediated gene expression in cancer cells and should be considered for AAVP-based clinical cancer gene therapy.

  3. Gene therapy strategy for long-term myocardial protection using adeno-associated virus-mediated delivery of heme oxygenase gene.

    Science.gov (United States)

    Melo, Luis G; Agrawal, Reitu; Zhang, Lunan; Rezvani, Mojgan; Mangi, Abeel A; Ehsan, Afshin; Griese, Daniel P; Dell'Acqua, Giorgio; Mann, Michael J; Oyama, Junichi; Yet, Shaw-Fang; Layne, Matthew D; Perrella, Mark A; Dzau, Victor J

    2002-02-05

    Ischemia and oxidative stress are the leading mechanisms for tissue injury. An ideal strategy for preventive/protective therapy would be to develop an approach that could confer long-term transgene expression and, consequently, tissue protection from repeated ischemia/reperfusion injury with a single administration of a therapeutic gene. In the present study, we used recombinant adeno-associated virus (rAAV) as a vector for direct delivery of the cytoprotective gene heme oxygenase-1 (HO-1) into the rat myocardium, with the purpose of evaluating this strategy as a therapeutic approach for long-term protection from ischemia-induced myocardial injury. Human HO-1 gene (hHO-1) was delivered to normal rat hearts by intramyocardial injection. AAV-mediated transfer of the hHO-1 gene 8 weeks before acute coronary artery ligation and release led to a dramatic reduction (>75%) in left ventricular myocardial infarction. The reduction in infarct size was accompanied by decreases in myocardial lipid peroxidation and in proapoptotic Bax and proinflammatory interleukin-1beta protein abundance, concomitant with an increase in antiapoptotic Bcl-2 protein level. This suggested that the transgene exerts its cardioprotective effects in part by reducing oxidative stress and associated inflammation and apoptotic cell death. This study documents the beneficial therapeutic effect of rAAV-mediated transfer, before myocardial injury, of a cytoprotective gene that confers long-term myocardial protection from ischemia/reperfusion injury. Our data suggest that this novel "pre-event" gene transfer approach may provide sustained tissue protection from future repeated episodes of injury and may be beneficial as preventive therapy for patients with or at risk of developing coronary ischemic events.

  4. Cytotoxic-T-lymphocyte-mediated elimination of target cells transduced with engineered adeno-associated virus type 2 vector in vivo.

    Science.gov (United States)

    Li, Chengwen; Hirsch, Matt; DiPrimio, Nina; Asokan, Aravind; Goudy, Kevin; Tisch, Roland; Samulski, R Jude

    2009-07-01

    A recent clinical trial in patients with hemophilia B has suggested that adeno-associated virus (AAV) capsid-specific cytotoxic T lymphocytes (CTLs) eliminated AAV-transduced hepatocytes and resulted in therapeutic failure. AAV capsids elicit a CTL response in animal models; however, these capsid-specific CTLs fail to kill AAV-transduced target cells in mice. To better model the human clinical trial data in mice, we introduced an immunodominant epitope derived from ovalbumin (OVA; SIINFEKL) into the AAV capsid and tested CTL-mediated killing of AAV2-transduced target tissues in vivo. Initially, in vitro experiments demonstrated both classical class I and cross-presentation of the OVA antigen, following endogenous expression or AAV2-OVA vector transduction, respectively. Furthermore, an OVA-specific CTL response was elicited after muscular or systemic injection of the AAV2-OVA vector. Finally, CTL reactivity was enhanced in mice with established SIINFEKL-specific immunity after AAV2-OVA/alpha1 anti-trypsin (AAT) administration. Most importantly, these OVA-specific CTLs decreased AAT expression in mice treated with AAV2-OVA/AAT vector that followed a time course mimicking uncoating kinetics of AAV2 transduction in OVA-immunized mice. These results demonstrate that AAV capsid-derived antigens elicit CD8(+) CTL reactivity, and these CTLs eliminated AAV-transduced target cells in mice. Notably, this model system can be exploited to study the kinetics of capsid presentation from different serotypes of AAV and permit the design of novel strategies to block CTL-mediated killing of AAV-transduced cells.

  5. Recombinant adeno-associated virus-mediated inhibiting of interleukin-4 expression in rat model of asthma

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ Asthma is a chronic disease characterized by reversible airway obstruction, airway hyper- responsiveness, and inflammation of airways. Th2 cells, one sort of CD4+ T lymphocytes, are currently considered to play an important role in the chronic airway inflammation of asthma. Meanwhile, a number of laboratories have clearly established the importance of the Th2-derived cytokine interleukin-4 (IL-4) in mediating the airway inflammatory response. Anti-IL-4 therapy might be beneficial in treatment of chronic asthma.

  6. Adeno-associated virus mediated endostatin gene therapy in combination with topoisomerase inhibitor effectively controls liver tumor in mouse model

    Institute of Scientific and Technical Information of China (English)

    Sung Yi Hong; Myun Hee Lee; Kyung Sup Kim; Hyun Cheol Jung; Jae Kyung Roh; Woo Jin Hyung; Sung Hoon Noh; Seung Ho Choi

    2004-01-01

    AIM: rAAV mediated endostatin gene therapy has been examined as a new method for treating cancer. However,a sustained and high protein delivery is required to achieve the desired therapeutic effects. We evaluated the impact of topoisomerase inhibitors in rAAV delivered endostatin gene therapy in a liver tumor model.METHODS: rAAV containing endostatin expression cassettes were transduced into hepatoma cell lines. To test whether the topoisomerase inhibitor pretreatment increased the expression of endostatin, Western blotting and ELISA were performed. The biologic activity of endostatin was confirmed by endothelial cell proliferation and tube formation assays.The anti-tumor effects of the rAAV-endostatin vector combined with a topoisomerase inhibitor, etoposide, were evaluated in a mouse liver tumor model.RESULTS: Topoisomerase inhibitors, including camptothecin and etoposide, were found to increase the endostatin expression level in vitro. The over-expressed endostatin,as a result of pretreatment with a topoisomerase inhibitor,was also biologically active. In animal experiments, the combined therapy of topoisomerase inhibitor, etoposide with the rAAV-endostatin vector had the best tumorsuppressive effect and tumor foci were barely observed in livers of the treated mice. Pretreatment with an etoposide increased the level of endostatin in the liver and serum of rAAV-endostatin treated mice. Finally, the mice treated with rAAV-endostatin in combination with etoposide showed the longest survival among the experimental models.CONCLUSION: rAAV delivered endostatin gene therapy in combination with a topoisomerase inhibitor pretreatment is an effective modality for anticancer gene therapy.

  7. Adeno-Associated Virus Gene Therapy for Liver Disease

    Science.gov (United States)

    Kattenhorn, Lisa M.; Tipper, Christopher H.; Stoica, Lorelei; Geraghty, Deborah S.; Wright, Teresa L.; Clark, K. Reed; Wadsworth, Samuel C.

    2016-01-01

    The field of adeno-associated virus (AAV) gene therapy has progressed rapidly over the past decade, with the advent of novel capsid serotype and organ-specific promoters, and an increasing understanding of the immune response to AAV administration. In particular, liver-directed therapy has made remarkable strides, with a number of clinical trials currently planned and ongoing in hemophilia A and B, as well as other liver disorders. This review focuses on liver-directed AAV gene therapy, including historic context, current challenges, and future developments. PMID:27897038

  8. Engineering adeno-associated viruses for clinical gene therapy.

    Science.gov (United States)

    Kotterman, Melissa A; Schaffer, David V

    2014-07-01

    Clinical gene therapy has been increasingly successful owing both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among these technologies, delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective and, in one recent case, have led to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers.

  9. Adeno-Associated Virus Vectors (AAV Expressing Phenylalanine Hydroxylase (PAH

    Directory of Open Access Journals (Sweden)

    Ayşegül Akbay Yarpuzlu

    2009-06-01

    Full Text Available Recent articles have appeared in the literature reporting use of adeno-associated virus vectors (AAV expressing phenylalanine hydroxylase in animal trials and suggesting its use in treatment of phenylketonuria (PKU as a form of gene therapy However, agents used in gene therapy to deliver genes are not site-specific and DNA is may be put in the wrong place, causing damage to the organism. The adverse immunogenicity of AAVs also needs to be reconsidered. This letter is written to discuss present unreadiness for Phase 1 clinical trials of gene therapy of PKU. Turk Jem 2009; 13: 18-9

  10. Expression of human nerve growth factor β gene in central nervous system mediated by recombinant adeno-associated viruses type-2 vector

    Institute of Scientific and Technical Information of China (English)

    高凯; 吴勇杰; 吴小兵; 饶春明; 王军志

    2004-01-01

    Background Neurone atrophy and loss are major causes of chronic neurodegenerative disorders such as Alzheimer's disease. Despite many pharmacotherapies for neurodegeneration, there are no accepted treatments. We investigated the feasibility of human nerve growth factor β (hNGFβ) gene expression mediated by recombinant adeno-associated viruses type-2 (rAAV-2) vector in the central nervous system (CNS) after blood brain barrier (BBB) disruption.Methods rAAV-2 containing hNGFβ gene was constructed. The ability of hNGFβ gene mediated by rAAV-2 vector (rAAV-2/hNGFβ) to transfect cells in vitro was confirmed by both ELISA and bioassay of hNGFβ in the culture supernatant of BHK-21 cells infected by rAAV-2/hNGFβ. rAAV-2/hNGFβ and rAAV-2/green fluorescence protein (GFP) were administrated separately to rat brains through internal carotid intubation after BBB disruption with hypertonic mannitol. Brain hNGFβ concentration was measured by ELISA and GFP in brain sections was examined by laser scan confocal microscope.Results After 48 hours, hNGFβ content in supernatant was up to (188.0±28.6) pg/ml when BHK-21 cells were infected by rAAV-2/hNGFβ at multiplicity of infection (MOI)1.0×106 vector genome. Neurone fibre outgrowths were obvious in dorsal root ganglion neurone assays by adding serum free culture medium harvested from BHK-21 cells exposed to rAAV-2/hNGFβ. Whole brain hNGFβ content in rAAV-2/hNGFβ transferred group was up to (636.2±140.6) pg/ml. hNGFβ content of BBB disruption in rAAV-2/hNGFβ infused group increased significantly compared to the control group (P<0.05). GFP expression was clearly observed in brain sections of rAAV-2/GFP transferred group.Conclusion rAAV-2/hNGFβ successfully expresses in the CNS after BBB disruption induced by hypertonic mannitol.

  11. Adeno-associated virus and lentivirus vectors mediate efficient and sustained transduction of cultured mouse and human dorsal root ganglia sensory neurons.

    Science.gov (United States)

    Fleming, J; Ginn, S L; Weinberger, R P; Trahair, T N; Smythe, J A; Alexander, I E

    2001-01-01

    Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of numerous inherited and acquired neurological conditions. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Among contemporary vector systems capable of neuronal transduction, only those based on herpes simplex virus have been extensively evaluated in PNS neurons. We therefore investigated the transduction performance of recombinant adeno-associated virus type 2 (AAV) and VSV-G-pseudotyped lentivirus vectors derived from human immunodeficiency virus (HIV-1) in newborn mouse and fetal human dorsal root ganglia (DRG) sensory neurons. In dissociated mouse DRG cultures both vectors achieved efficient transduction of sensory neurons at low multiplicities of infection (MOIs) and sustained transgene expression within a 28-day culture period. Interestingly, the lentivirus vector selectively transduced neurons in murine cultures, in contrast to human cultures, in which Schwann and fibroblast-like cells were also transduced. Recombinant AAV transduced all three cell types in both mouse and human cultures. After direct microinjection of murine DRG explants, maximal transduction efficiencies of 20 and 200 transducing units per neuronal transductant were achieved with AAV and lentivirus vectors, respectively. Most importantly, both vectors achieved efficient and sustained transduction of human sensory neurons in dissociated cultures, thereby directly demonstrating the exciting potential of these vectors for gene therapy applications in the PNS.

  12. Ocular gene therapy: an evaluation of recombinant adeno-associated virus-mediated gene therapy interventions for the treatment of ocular disease.

    Science.gov (United States)

    Roy, Kamolika; Stein, Linda; Kaushal, Shalesh

    2010-08-01

    Both gene replacement therapy and alteration of host gene expression are playing increasingly important roles in the treatment of ocular diseases. Ocular gene therapy may provide alternatives to current treatments for eye diseases that are either greatly invasive and thus run the risk of complications, that offer only short-term relief from disease symptoms, or that are unable to directly treat vision loss. The success of three separate phase I clinical trials investigating a gene therapy intervention for the treatment of the retinal degenerative disorder Leber's congenital amaurosis (LCA) has unveiled the therapeutic potential of gene therapy. Preliminary results have demonstrated ocular gene transfer, using nonpathogenic recombinant adeno-associated viral (rAAV) vectors specifically, to be a safe, effective, and long-term treatment for LCA, a previously untreatable disorder. Nonpathogenic rAAV vectors offer the potential for long-term treatment. Many of the genes implicated in human ocular diseases have been identified, and animal models for such diseases have been developed, which have greatly facilitated the application of experimental rAAV-mediated gene therapy. This review highlights the key features of rAAV-mediated gene therapy that make it the most suitable gene therapy treatment approach for ocular diseases. Furthermore, it summarizes the current progress of rAAV-mediated gene therapy interventions/applications for a wide variety of ophthalmologic disorders.

  13. Biosafety of recombinant adeno-associated virus vectors.

    Science.gov (United States)

    Dismuke, David J; Tenenbaum, Liliane; Samulski, R Jude

    2013-12-01

    It is hoped that the use of gene transfer technology to treat both monogenetic and acquired diseases may soon become a common therapy option in medicine. For gene therapy to achieve this objective, any gene delivery method will have to meet several criteria, including ease of manufacturing, efficient gene transfer to target tissue, long-term gene expression to alleviate the disease, and most importantly safety in patients. Viral vectors are an attractive choice for use in gene therapy protocols due to their relative efficiency in gene delivery. Since there is inherent risk in using viruses, investigators in the gene therapy community have devoted extensive efforts toward reengineering viral vectors for enhance safety. Here we review the approaches and technologies that are being evaluated for the use of recombinant vectors based upon adeno-associated virus (AAV) in the treatment of a variety of human diseases. AAV is currently the only known human DNA virus that is non-pathogenic and AAV-based vectors are classified as Risk Group 1 agents for all laboratory and animal studies carried out in the US. Although its apparent safety in natural infection and animals appears well documented, we examine the accumulated knowledge on the biology and vectorology of AAV, lessons learned from gene therapy clinical trials, and how this information is impacting current vector design and manufacturing with an overall emphasis on biosafety.

  14. Recombinant adeno-associated virus-mediated human kallikrein gene therapy prevents high-salt diet-induced hypertension without effect on basal blood pressure

    Institute of Scientific and Technical Information of China (English)

    Jiang-tao YAN; Tao WANG; Juan LI; Xiao XIAO; Dao-wen WANG

    2008-01-01

    Aim: To investigate the effects of the expression of human kallikrein (HK) on basal level blood pressure and high-salt diet-induced hypertension. Methods: We delivered the recombinant adeno-associated viral (rAAV)-mediated HK (rAAV-HK) gene and rAAV-LacZ (as the control) to normal, adult Sprague-Dawley rats. The animals were administered a normal diet in the first 4 weeks, followed by a high-salt diet. The expression of HK in the rats was assessed by ELISA and RT-PCR. Blood pressure and Na~ and K~ urinary excretion were monitored. Results: Under the normal diet, no obvious changes in blood pressure and Na+ and K+ urinary excretion were observed. When the high-salt diet was administered, sys-tolic blood pressure in the control animals receiving rAAV-LacZ increased from 122.3±1. 13 mmHg to a stable 142.4±1.77 mmHg 8 weeks after the high-salt diet. In contrast, there was no significant increase in the blood pressure in the rAAV-HK-treated group, in which the blood pressure remained at 121.9±1.73 mmHg. In the rAAV-HK-treated group, Na+ and K+ urinary excretion were higher compared to those of the control group. The morphological analysis showed that HK delivery remarkably protected against renal damage induced by a high-salt intake. Conclusion: Our study indicates that rAAV-mediated human tissue kallikrein gene delivery is a potentially safe method for the long-term treatment of hypertension. More importantly, it could be applied in the salt-sensitive population to prevent the occurrence of hypertension.

  15. Adeno-Associated Virus-Mediated Mini-Agrin Delivery Is Unable to Rescue Disease Phenotype in a Mouse Model of Limb Girdle Muscular Dystrophy Type 2I.

    Science.gov (United States)

    Vannoy, Charles H; Zhou, Haowen; Qiao, Chunping; Xiao, Xiao; Bang, Anne G; Lu, Qi L

    2017-02-01

    Agrin is a basement membrane-specific proteoglycan that can regulate orientation of cytoskeleton proteins and improve function of dystrophic skeletal muscle. In skeletal muscle, agrin binds with high affinity to laminin(s) and α-dystroglycan (α-DG), an integral part of the dystrophin-glycoprotein complex. Miniaturized forms of agrin (mAgrin) have been shown to ameliorate disease pathology in a laminin-α2 knockout mouse model of muscular dystrophy, acting as a link between α-DG and laminin(s). Here, we test whether mAgrin might also improve pathologies associated with FKRP-related dystroglycanopathies, another form of muscular dystrophy characterized by weak interactions between muscle and basement membranes. We demonstrate in vitro that mAgrin enhances laminin binding to primary myoblasts and fibroblasts from an FKRP mutant mouse model and that this enhancement is abrogated when mAgrin is in molar excess relative to laminin. However, in vivo delivery of mAgrin via adeno-associated virus (AAV) into FKRP mutant mice was unable to improve dystrophic phenotypes, both histologically and functionally. These results likely reflect insufficient binding of mAgrin to hypoglycosylated α-DG on muscle fibers and possibly abrogation of binding from molar excess of overexpressed AAV-delivered mAgrin. Further exploration of mAgrin modification is necessary to strengthen its binding to other membrane components, including hypoglycosylated α-DG, for potential therapeutic applications. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  16. The Helper Activities of Different Avian Viruses for Propagation of Recombinant Avian Adeno-Associated Virus

    Institute of Scientific and Technical Information of China (English)

    WANG An-ping; SUN Huai-chang; WANG Jian-ye; WANG Yong-juan; YUAN Wei-feng

    2007-01-01

    To compare the helper activities of different avian viruses for propagation of recombinant avian adeno-associated virus (rAAAV), AAV-293 cells were cotransfected with the AAAV vector pAITR-GFP containing green fluorescent protein (GFP) gene, the AAAV helper vector pcDNA-ARC expressing the rep and cap genes, and the adenovirus helper vector pHelper expressing Ad5 E2A, E4, and VA-RNA genes. Chicken embryonic fibroblast (CEF) or chicken embryonic liver (CEL) cells were cotransfected with the AAAV vector and the AAAV helper vector, followed by infection with Marek's disease virus (MDV), avian adenovirus, chicken embryo lethal orphan (CELO) virus or infectious bursal disease virus (IBDV). Infectious rAAAV particles generated by the two strategies were harvested and titrated on CEF and CEL cells. A significantly higher viral titer was obtained with the helper activity provided by the pHelper vector than by MDV or CELO virus. Further experiments showed that rAAAV-mediated green fluorescent protein (gfp) expression was overtly enhanced by MDV or CELO virus super infection or treatment with sodium butyric acid, but not by IBDV super infection. These data demonstrated that MDV and CELO viruses could provide weak helper activity for propagation of rAAAV, and rAAAV-mediated transgene expression could be enhanced by super infection with the helper viruses.

  17. 腺相关病毒介导的狨猴 P53基因沉默%Adeno-associated virus mediated p53 gene silence in marmosets

    Institute of Scientific and Technical Information of China (English)

    石亮; 张晨; 向志光; 邓仪晨; 苏静芬; 刘云波

    2016-01-01

    目的:在细胞和整体动物水平,利用RNA干扰技术下调绒猴p53基因表达。方法对狨猴p53基因做生物信息学分析,针对靶序列设计shRNA干扰序列,构建在腺相关病毒载体上,转染非洲绿猴肾细胞( cos-7),在细胞水平用荧光定量PCR检测p53mRNA抑制效果,以Western blot 方法检测p53蛋白水平表达变化;优选shRNA干扰序列,包装含shRNA干扰序列的8型腺相关病毒,静脉注射感染狨猴;手术取少量肝脏组织,用Western blot和免疫组化方法检测p53蛋白水平的变化。结果细胞水平研究发现2个有效RNA干扰靶点,mRNA干扰效率分别为(82.7±8.1)%和(80.7±7.5)%(P<0.05);蛋白表达下调(77.3±11.5)%和(73.7±10.7)%(P<0.05);2只绒猴感染病毒后,经活体荧光成像分析可见病毒在肝脏、睾丸、颈部等位置分布,狨猴肝脏P53蛋白经Western blot、免疫组化分析未见明显变化。结论本研究在细胞水平实现绒猴P53基因表达下调,但整体动物水平狨猴肝脏P53蛋白表达未发现明显变化;今后需在感染方式等方面做进一步优化。%Objective To decrease the p53 gene expression at cellular and animal levels in marmoset using RNA interference technique.Methods The shRNA interference sequences were designed and inserted into the adeno-associated virus vector plasmid after bioinformatics analysis.The plasmids were transfected into African green monkey kidney cos-7 cells.The suppression of p53 mRNA was detected by real-time PCR, and the changes of p53 protein expression were detected by Western bolt.The adeno-associated virus-8 was injected through the hind leg vein.The changes of p53 protein expression in the liver tissue was detected by Western blot and immunohistochemistry.Results We screened two RNA interference effective arget sequences.The expression of p53 mRNA was suppressed ( 82.7 ±8.1 )% and ( 80.7 ± 7.5)%, respectively (P<0

  18. Position-independent human beta-globin gene expression mediated by a recombinant adeno-associated virus vector carrying the chicken beta-globin insulator.

    Science.gov (United States)

    Inoue, T; Yamaza, H; Sakai, Y; Mizuno, S; Ohno, M; Hamasaki, N; Fukumaki, Y

    1999-01-01

    The position-independent expression of transgenes in target cells is an essential subject for determining effective gene therapies. The chicken beta-globin insulator blocks the effects of regulatory sequences on transcriptional units at differential domains. We prepared a recombinant adeno-associated virus (rAAV) containing various combinations of the DNase I-hypersensitive site 2 (HS2), 3 (HS3), and 4 (HS4) core elements from the human beta-globin locus control region (LCR), the human beta-globin gene, and the herpes virus thymidine kinase promoter driven neomycin-resistant gene (neoR) (rHS432, rHS43, rHS42, rHS32, and rHS2), and also rAAV containing two copies of the 250-bp core sequence of the chicken beta-globin insulator on both sides of the rHS2 (rIns/HS2/2Ins). After isolating neomycin-resistant mouse erythroleukemia (MEL) cells infected with each rAAV, we analyzed the rAAV genome by Southern blots and polymerase chain reaction (PCR), using primers specific for HS core elements and the human beta-globin gene. All clones contained a single copy of the rAAV genome in the chromosome, however, some of them had a rearranged proviral genome. In five clones with a single unrearranged rAAV genome for each rAAV construct, we assayed the expression of the human b-globin gene relative to the endogenous mouse beta maj-globin gene, using quantitative reverse transcriptase (RT)-PCR. In clones infected with rHS432, the expression level of the human beta-globin gene ranged from 51.6% to 765.6% of that in the mouse beta maj-globin gene. Likewise, in rHS43, the expression level ranged from 36.7% to 259.0%; in rHS42, from 47.8% to 207.0%; in rHS32, from 47.9% to 105.4%; and in rHS2, from 6.1% to 172.1%, indicating a high variability of expression level in clones infected with recombinant virus lacking the insulator. In contrast, in clones infected with rIns/HS2/Ins, the range of expression of the human beta-globin gene ranged from 52.8% to 58.3% of that in the mouse beta maj

  19. Systemic gene delivery to the central nervous system using Adeno-associated virus

    Directory of Open Access Journals (Sweden)

    Mathieu eBOURDENX

    2014-06-01

    Full Text Available Adeno-associated virus (AAV-mediated gene delivery has emerged as an effective and safe tool for both preclinical and clinical studies of neurological disorders. The recent discovery that several serotypes are able to cross the blood-brain-barrier when administered systemically has been a real breakthrough in the field of neurodegenerative diseases. Widespread transgene expression after systemic injection could spark interest as a therapeutic approach. Such strategy will avoid invasive brain surgery and allow non-focal gene therapy promising for CNS diseases affecting large portion of the brain. Here, we will review the recent results achieved through different systemic routes of injection generated in the last decade using systemic AAV-mediated delivery and propose a brief assessment of their values. In particular, we emphasize how the methods used for virus engineering could improve brain transduction after peripheral delivery.

  20. Genome Engineering Using Adeno-associated Virus: Basic and Clinical Research Applications.

    Science.gov (United States)

    Gaj, Thomas; Epstein, Benjamin E; Schaffer, David V

    2016-03-01

    In addition to their broad potential for therapeutic gene delivery, adeno-associated virus (AAV) vectors possess the innate ability to stimulate homologous recombination in mammalian cells at high efficiencies. This process--referred to as AAV-mediated gene targeting--has enabled the introduction of a diverse array of genomic modifications both in vitro and in vivo. With the recent emergence of targeted nucleases, AAV-mediated genome engineering is poised for clinical translation. Here, we review key properties of AAV vectors that underscore its unique utility in genome editing. We highlight the broad range of genome engineering applications facilitated by this technology and discuss the strong potential for unifying AAV with targeted nucleases for next-generation gene therapy.

  1. A novel and highly efficient production system for recombinant adeno-associated virus vector

    Institute of Scientific and Technical Information of China (English)

    WU; Zhijian(伍志坚); WU; Xiaobing(吴小兵); CAO; Hui(曹晖); DONG; Xiaoyan(董小岩); WANG; Hong(王宏); HOU; Yunde(侯云德)

    2002-01-01

    Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.

  2. Capsid modification of adeno-associated virus and tumor targeting gene therapy

    Institute of Scientific and Technical Information of China (English)

    XU ZengHu; ZHOU XiuMei; SHI WenFang; QIAN QiJun

    2008-01-01

    Targeting is critical for successful tumor gene therapy. The adeno-associated virus (AAV) has aroused wide concern due to its excellent advantages over other viral vectors in gene therapy. AAV has a broad infection spectrum, which also results in poor specificity towards tissues or cells and low transduction efficiency. Therefore, it is imperative to improve target and transduction efficiency in AAV-mediated gene therapy. Up to now, researchers have developed many strategies to modify AAV capsids for improving targeting or retargeting only desired cells. These strategies include not only traditional chemical modification, phage display technology, modification of AAV capsid genome, chimeric vectors and so on, but also many novel strategies involved in marker rescue strategy, direct evolution of capsid proteins, direct display random peptides on AAV capsid, AAVP (AAV-Phage), and etc. This review will summarize the advances of researches on the capsid modification of AAV to target malignant cells.

  3. Stable producer cell lines for adeno-associated virus (AAV) assembly.

    Science.gov (United States)

    Chadeuf, Gilliane; Salvetti, Anna

    2010-10-01

    Stable producer cell lines containing both the rep and cap genes and recombinant adeno-associated virus (rAAV) vectors can be infected with a helper virus to provide reliable and efficient production of rAAV stocks. However, the development of these cell lines is time-consuming. The procedure described here is therefore recommended only for studies requiring the production of high amounts of rAAV, such as preclinical studies performed in large animals.

  4. Construction of Adeno-associated Virus System for Human Bone Morphogenetic Protein 7 Gene

    Institute of Scientific and Technical Information of China (English)

    Ke SONG; Nianjing RAO; Meiling CHEN; Yingguang CAO

    2008-01-01

    To construct the recombinant adeno-associated virus (rAAV) vector with human bone morphogenetic protein 7 (BMP7) and observe the BMP7 mRNA expression in vitro, BMP7 CDS se- quence was cloned into expression plasmid pAAV-MCS of AAV Helper Free System. The recombi- nant plasmid was identified with enzyme digestion and sequencing. The recombinant plasmid, pAAV-RC, pHelper were co-transfected into AAV-293 cells according to the calcium phosphate-based protocol. The viral stock was collected by 4 rounds of freeze/thaw. After purified and concentrated,the recombinant virus titer was determined by dot-blot assay. HEK293 cells were transfected with the recombinant virus at different MOI, and the expression of BMP7 mRNA was detected by RT-PCR. The results showed rAAV-BMP7 was constructed and packaged successfully. The physical particle titer was 2.5×1011 vector genomes/mL. There was different expression level of BMP7 mRNA after transfecton. These data suggested that recombinant AAV mediated a stable expression of hBMP7 mRNA in 293 cells. The AAV production method may pave the way of an effective strategy for the jaw bone defection around dental implants.

  5. In vitro and in vivo induction of bone formation based on adeno-associ-ated virus-mediated BMP-7 gene therapy using human adipose-derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Yan KANG; Wei-ming LIAO; Zhen-hua YUAN; Pu-yi SHENG; Long-juan ZHANG; Xiang-wei YUAN; Lei LEI

    2007-01-01

    Aim: To determine whether adeno-associated virus (AAV)-2-mediated, bone mor-phogenetic protein (BMP)-7-expressing human adipose-derived mesenchymal stem cells (ADMS) cells would induce bone formation in vitro and in vivo.Methods:ADMS cells were harvested from patients undergoing selective suction-assisted fipectomy and transduced with AAV carrying the human BMP-7 gene. Non-trans-duced cells and cells transduced with AAV serotype 2 carrying the enhanced green fluorescence protein gene served as controls. ADMS cells were qualita-tively assessed for the production of BMP-7 and osteocalcin, and subjected to alkaline phosphatase (ALP) and Chinalizarin staining. A total of 2.5x 106 cells mixed with type Ⅰ collagen were implanted into the hind limb of severe combined immune-deficient (SCID) mice and subjected to a histological analysis 3 weeks post implantation.Results: Transfection of the ADMS cells achieved an effi-ciency of 99% at d 7. Transduction with AAV2-BMP-7 induced the expression of BMP-7 until d 56, which was markedly increased by d 7. The cells were positively stained for ALP. Osteocalcin production and matrix mineralization further con-firmed that these cells differentiated into osteoblasts and induced bone formation in vitro. A histological examination demonstrated that implantation of BMP-7-expressing ADMS cells could induce new bone formation in vivo.Conclusion: The present in vitro and in vivo study demonstrated that human ADMS cells would be a promising source of autologous mesenchymal stem cells for BMP gene therapy and tissue engineering.

  6. 重组腺相关病毒介导遗传性色盲基因治疗的研究进展%Advance in recombinant adeno-associated virus mediated gene therapy for color blindness

    Institute of Scientific and Technical Information of China (English)

    杨红霞; 邱一果

    2013-01-01

    色盲是缺乏或完全没有辨色能力的一类遗传性疾病,长期被认为是不可治愈性疾病.近年来以腺相关病毒(AAV)为载体介导的基因疗法主要用于对由视蛋白缺乏引起的红绿色盲及由视锥细胞环核苷酸门控离子通道A3(CNGA3)A或B(CNGB3)亚单位基因缺失引起的全色盲的治疗,已在动物实验中获得成功.人类色盲患者与一些实验动物存在着相同的基因缺陷,因此相关的动物实验研究结果用AAV介导的基因疗法为色盲患者进行治疗提供了有用的信息.%Color blindness represents a group of vision disorders characterized by lack of ability to distinguish different colors.The inherited color blindness has been regarded as incurable for a long period of time.Recently,adeno-associated virus(AAV) mediated gene therapy has successfully restored cone system vision in animal models with color blindness caused by different gene mutations.These mutations are presented in human color blindness patients.It is predicted that gene therapy will become a novel treatment for these color blindness victims.In addition,a single gene transfer may achieve long-term correction of color deficiency.

  7. [Innate immune mechanisms against recombinant adeno-associated virus vectors--a review].

    Science.gov (United States)

    Diao, Yong; Xu, Ruian

    2012-05-04

    Recombinant adeno-associated virus (rAAV) is one of the most commonly used vectors for gene therapy. Despite the promising safety profile demonstrated in preclinical trials, the clinic efficacy of using rAAV was hampered by undesired response from the immune system. It is important to understand the mechanisms that lead to the induction of immune response against rAAV. Although a crucial role for innate immunity is shaping adaptive immune responses, the innate immune to rAAV was ignored in the past. Till now, at least three human cell types (dendritic cells, macrophages and endothelial cells) were discovered to be involved in sensing rAAV infection. The engagement of TLR9 by rAAV vector genomes triggers the activation of NF-kappaB signaling cascades, leading to the induction of pro-inflammatory cytokine genes. The viral capsid components are detected by TLR2, and this leads to the production of type I interferon mediated by interferon regulatory factors (IRFs) pathway. Self-complementary rAAV vectors induced higher TLR9 dependent innate immune response than single stranded rAAV. This review highlights the recent findings regarding the innate immune responses to rAAV vectors, the signaling pathways involved, and the impacts of innate immunity on the adaptive immune response to rAAV and its transgene expression.

  8. Effects of adeno-associated virus on adenovirus replication and gene expression during coinfection.

    Science.gov (United States)

    Timpe, Jennifer M; Verrill, Kristin C; Trempe, James P

    2006-08-01

    Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10- to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the E3 gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis.

  9. Adeno associated viral-mediated intraosseous labeling of bone marrow derived cells for CNS tracking.

    Science.gov (United States)

    Selenica, Maj-Linda B; Reid, Patrick; Pena, Gabriela; Alvarez, Jennifer; Hunt, Jerry B; Nash, Kevin R; Morgan, Dave; Gordon, Marcia N; Lee, Daniel C

    2016-05-01

    Inflammation, including microglial activation in the CNS, is an important hallmark in many neurodegenerative diseases. Microglial stimuli not only impact the brain microenvironment by production and release of cytokines and chemokines, but also influence the activity of bone marrow derived cells and blood born macrophage populations. In many diseases including brain disorders and spinal cord injury, researchers have tried to harbor the neuroprotective and repair properties of these subpopulations. Hematopoietic bone marrow derived cells (BMDCs) are of great interest, especially during gene therapy because certain hematopoietic cell subpopulations traffic to the sites of injury and inflammation. The aim of this study was to develop a method of labeling endogenous bone marrow derived cells through intraosseous impregnation of recombinant adeno-associated virus (rAAV) or lentivirus. We utilized rAAV serotype 9 (rAAV-9) or lentivirus for gene delivery of green florescence protein (GFP) to the mouse bone marrow cells. Flow cytometry showed that both viruses were able to efficiently transduce mouse bone marrow cells in vivo. However, the rAAV9-GFP viral construct transduced BMDCs more efficiently than the lentivirus (11.2% vs. 6.8%), as indicated by cellular GFP expression. We also demonstrate that GFP labeled cells correspond to bone marrow cells of myeloid origin using CD11b as a marker. Additionally, we characterized the ability of bone marrow derived, GFP labeled cells to extravasate into the brain parenchyma upon acute and subchronic neuroinflammatory stimuli in the mouse CNS. Viral mediated over expression of chemokine (C-C motif) ligand 2 (CCL2) or intracranial injection of lipopolysaccharide (LPS) recruited GFP labeled BMDCs from the periphery into the brain parenchyma compared to vehicle treated mice. Altogether our findings demonstrate a useful method of labeling endogenous BMDCs via viral transduction and the ability to track subpopulations throughout the body

  10. [Advances in the application of gene therapy for Parkinson's disease with adeno-associated virus].

    Science.gov (United States)

    Chen, Yang; Lü, Ying-Hui; Li, Zhao-Fa

    2014-05-01

    Vectors used to carry foreign genes play an important role in gene therapy, among which, the adeno-associated virus (AAV) has many advantages, such as nonpathogenicity, low immunogenicity, stable and long-term expression and multiple-tissue-type infection, etc. These advantages have made AAV one of the most potential vectors in gene therapy, and widely used in many clinical researches, for example, Parkinson's disease. This paper introduces the biological characteristics of AAV and the latest research progress of AAV carrying neurotrophic factor, dopamine synthesis related enzymes and glutamic acid decarboxylase gene in the gene therapy of Parkinson's disease.

  11. Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis (Florida)

    2012-09-17

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  12. Structural studies of adeno-associated virus serotype 8 capsid transitions associated with endosomal trafficking.

    Science.gov (United States)

    Nam, Hyun-Joo; Gurda, Brittney L; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis

    2011-11-01

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  13. Continuous Collection of Adeno-Associated Virus from Producer Cell Medium Significantly Increases Total Viral Yield.

    Science.gov (United States)

    Benskey, Matthew J; Sandoval, Ivette M; Manfredsson, Fredric P

    2016-02-01

    The ability to efficiently produce large amounts of high-titer recombinant adeno-associated virus (AAV) is a prerequisite to the continued success of AAV as a gene therapy tool targeted toward large-animal preclinical studies or human clinical therapeutics. Current manufacturing procedures necessitate laborious and time-consuming purification procedures to obtain AAV particles of sufficient titer and purity for these demanding biomedical applications. The finding that AAV can be harvested and purified from producer cell medium may represent an efficient alternative to purifying AAV from cellular lysates. Here we sought to determine the maximum duration of time, and frequency within which AAV can be harvested from producer cell medium, in order to maximize the yield obtained from a single transfection preparation. Human embryonic kidney 293T cells were transfected with polyethylenimine to produce AAV2/5 expressing green fluorescent protein (GFP), and cellular medium was harvested every 2 days until a maximum duration of 19 days posttransfection. AAV2/5-GFP was released into producer cell medium at a steady state until 7 days posttransfection, at which time titers dropped dramatically. Harvesting medium every two days resulted in the maximum yield of AAV from a single preparation, and the cumulative yield of AAV harvested from the producer cell medium was 4-fold higher than the yield obtained from a traditional purification of AAV from cellular lysates. The AAV2/5 harvested from medium within the 7-day collection time-course mediated high levels of transduction in vivo, comparable to AAV2/5 harvested from cellular lysates. AAV purified from cell lysates showed increasing amounts of empty particles at 5 and 7 days posttransfection, whereas AAV purified from cell medium did not show an increase in the amount of empty particles throughout the 7-day time course. Finally, we extended these findings to AAV2/9, demonstrating that a comparable ratio of AAV2/9 particles are

  14. Recombinant adeno-associated virus-mediated global anterograde delivery of glial cell line-derived neurotrophic factor to the spinal cord: comparison of rubrospinal and corticospinal tracts in the rat.

    Science.gov (United States)

    Foust, Kevin D; Flotte, Terence R; Reier, Paul J; Mandel, Ronald J

    2008-01-01

    Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of spinal lower motoneurons. Gene delivery is a promising strategy to deliver therapeutic molecules to these vulnerable cells. However, definition of an optimal route of delivery capable of accessing neurons over a considerable extent of the neuraxis represents a significant logistical problem. Intramuscular vector injections are not ideal as this approach would involve hundreds of injections to completely treat an ALS patient and also would be dependent on retrograde transport of the viral platform of choice. Alternatively, upper motoneurons could deliver trophic factors over considerable distances by anterograde transport after a relatively localized intracerebral injection. To test this approach, the present study was designed to compare the corticospinal (CST) and rubrospinal (RST) tracts for their ability to transport recombinant adeno-associated virus serotype 5 (rAAV5)-derived green fluorescent protein (GFP) or glial cell line-derived neurotrophic factor (GDNF) to the spinal cord. Unilateral injections of rAAV5-GFP into the red nucleus (RN) or motor cortex of normal rats produced GFP-positive fibers in the appropriate descending tracts extending to the lumbar spinal cord. For both tracts, GFP-positive axonal projections into the spinal gray matter were consistently observed. GDNF immunohistochemistry demonstrated that confirmed RN injections resulted in GDNF-positive fibers projecting into spinal gray matter as seen in the GFP group. In contrast, confirmed cortical rAAV5-GDNF injections resulted in less evident staining in spinal cord. Spinal cord GDNF levels were elevated at distances up to 72 mm from the injection sites, and confirmed that RST-related GDNF transport to spinal cord surpassed CST-associated delivery.

  15. Adeno-associated virus serotype rh.10 displays strong muscle tropism following intraperitoneal delivery.

    Science.gov (United States)

    Ai, Jianzhong; Li, Jia; Gessler, Dominic J; Su, Qin; Wei, Qiang; Li, Hong; Gao, Guangping

    2017-01-09

    Recombinant adeno-associated virus (rAAV) is an attractive tool for basic science and translational medicine including gene therapy, due to the versatility in its cell and organ transduction. Previous work indicates that rAAV transduction patterns are highly dependent on route of administration. Based on this relationship, we hypothesized that intraperitoneal (IP) administration of rAAV produces unique patterns of tissue tropism. To test this hypothesis, we investigated the transduction efficiency of 12 rAAV serotypes carrying an enhanced green fluorescent protein (EGFP) reporter gene in a panel of 12 organs after IP injection. Our data suggest that IP administration emphasizes transduction patterns that are different from previously reported intravascular delivery methods. Using this approach, rAAV efficiently transduces the liver, pancreas, skeletal muscle, heart and diaphragm without causing significant histopathological changes. Of note, rAAVrh.10 showed excellent muscle transduction following IP administration, highlighting its potential as a new muscle-targeting vector.

  16. Site-specific integration by the adeno-associated virus rep protein.

    Science.gov (United States)

    Recchia, Alessandra; Mavilio, Fulvio

    2011-10-01

    Inserting genetic information at precise locations into the human genome has been the goal of gene transfer technology for almost two decades. The spectacular progress of mammalian genetics has led to the development of technology for genome editing and homologous recombination in human somatic cells that is finally approaching efficiency compatible with clinical application. Site-specific integration, or the insertion of genes at known locations by enzymes with target recognition capacity, has progressed slowly but steadily in recent years, and could very well be the basis of the next generation of gene transfer technology. This review focuses on the use of Rep, the replicase/integrase of the adeno-associated virus (AAV), to insert genes at the natural AAV integration site on human chromosome 19. This region (AAVS1) has characteristics that make it an ideal target for somatic transgenesis.

  17. Chimeric adeno-associated virus and bacteriophage: a potential targeted gene therapy vector for malignant glioma.

    Science.gov (United States)

    Asavarut, Paladd; O'Neill, Kevin; Syed, Nelofer; Hajitou, Amin

    2014-01-01

    The incipient development of gene therapy for cancer has fuelled its progression from bench to bedside in mere decades. Of all malignancies that exist, gliomas are the largest class of brain tumors, and are renowned for their aggressiveness and resistance to therapy. In order for gene therapy to achieve clinical success, a multitude of barriers ranging from glioma tumor physiology to vector biology must be overcome. Many viral gene delivery systems have been subjected to clinical investigation; however, with highly limited success. In this review, the current progress and challenges of gene therapy for malignant glioma are discussed. Moreover, we highlight the hybrid adeno-associated virus and bacteriophage vector as a potential candidate for targeted gene delivery to brain tumors.

  18. Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia.

    Science.gov (United States)

    Su, Wei; Kang, John; Sopher, Bryce; Gillespie, James; Aloi, Macarena S; Odom, Guy L; Hopkins, Stephanie; Case, Amanda; Wang, David B; Chamberlain, Jeffrey S; Garden, Gwenn A

    2016-01-01

    Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. Neonatal microglia are functionally distinct from adult microglia, although the majority of in vitro studies utilize rodent neonatal microglia cultures because of difficulties of culturing adult cells. In addition, cultured microglia are refractory to most methods for modifying gene expression. Here, we developed a novel protocol for culturing adult microglia and evaluated the feasibility and efficiency of utilizing Recombinant Adeno-Associated Virus (rAAV) to modulate gene expression in cultured microglia.

  19. Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor

    Science.gov (United States)

    Pillay, Sirika; Zou, Wei; Cheng, Fang; Puschnik, Andreas S.; Meyer, Nancy L.; Ganaie, Safder S.; Deng, Xuefeng; Wosen, Jonathan E.; Davulcu, Omar; Yan, Ziying; Engelhardt, John F.; Brown, Kevin E.; Chapman, Michael S.

    2017-01-01

    ABSTRACT Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor. IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life

  20. Adeno-associated virus type 2 binding study on model heparan sulfate surface

    Science.gov (United States)

    Negishi, Atsuko; Liu, Jian; McCarty, Douglas; Samulski, Jude; Superfine, Richard

    2003-11-01

    Understanding the mechanisms involved in virus infections is useful in its application in areas such as gene therapy, drug development and delivery, and biosensors. In collaboration with UNC Gene Therapy Center and School of Pharmacy, we are specifically looking at the interaction between human parvovirus adeno-associated virus type 2 (AAV2), a potential viral vector, and heparan sulfate proteoglycan (HSPG), a known cell surface receptor for AAV2. Recent development in glycobiology has shown that some protein-polysaccharide binding is sugar sequence dependent. Heparan sulfate (HS) is a polysaccharide chain of sulfated iduronic/glucuronic and sulfate glucosamine residues and can be differentiated into sequence specific structures by enzymes. These enzymatic modifications, known as heparan sulfate sulfotransferase modified modifications, have been shown to change the biological nature of heparan sulfate such as specific binding to proteins and viruses. For understanding HS-assisted viral infection mechanisms, we are interested in investigating the binding affinity and stability of AAV to different HS structures. We have developed a model heparan sulfate surface in which AAV adsorption studies are done and analyzed using the atomic force microscope (AFM). In addition, a miniArray assay has been created to facilitate to this study. Adsorption studies are done in 4 white LED wells with approximately 3 mm2 reaction areas which minimize sample use and waste.

  1. A simplified purification protocol for recombinant adeno-associated virus vectors

    Directory of Open Access Journals (Sweden)

    Mark Potter

    2014-01-01

    Full Text Available We describe a new rapid, low cost, and scalable method for purification of various recombinant adeno-associated viruses (rAAVs from the lysates of producer cells of either mammalian or insect origin. The method takes advantage of two general biochemical properties of all characterized AAV serotypes: (i low isoelectric point of a capsid and (ii relative biological stability of the viral particle in the acidic environment. A simple and rapid clarification of cell lysate toremove the bulk of proteins and DNA is accomplished by utilizing inexpensive off-the-shelf reagents such as sodium citrate and citric acid. After the low-speed centrifugation step, the supernatant is subjected to cation exchange chromatography via sulfopropyl (SP column. The eluted virus may then be further concentrated by either centrifugal spin devices or tangential flow filtration yielding material of high titer and Good Manufacturing Practice (GMP grade biochemical purity. The protocol is validated for rAAV serotypes 2, 8, and 9. The described method makes rAAV vector technology readily available for the low budget research laboratories and could be easily adapted for a large scale GMP production format.

  2. Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers.

    Science.gov (United States)

    Weitzman, M D; Fisher, K J; Wilson, J M

    1996-03-01

    Replication of a human parvovirus, adeno-associated virus (AAV), is facilitated by coinfection with adeno-virus to provide essential helper functions. We have used the techniques of in situ hybridization and immunocytochemistry to characterize the localization of AAV replication within infected cells, Previous studies have shown that adenovirus establishes foci called replication centers within the nucleus, where adenoviral replication and transcription occur. Our studies indicate that AAV is colocalized with the adenovirus replication centers, where it may utilize adenovirus and cellular proteins for its own replication. Expression of the AAV Rep protein inhibits the normal maturation of the adenovirus centers. Similar experiments were performed with recombinant AAV (rAAV) to establish a relationship between intranuclear localization and rAAV transduction. rAAV efficiently entered the cell, and its genome was faintly detectable in a perinuclear distribution and was mobilized to replication centers when the cell was infected with adenovirus. The recruitment of the replication-defective genome into the intranuclear adenovirus domains resulted in enhanced transduction. These studies illustrate the importance of intracellular compartmentalization for such complex interactions as the relationship between AAV and adenovirus.

  3. Notch1 augments intracellular trafficking of adeno-associated virus type 2.

    Science.gov (United States)

    Ren, Changchun; White, April F; Ponnazhagan, Selvarangan

    2007-02-01

    We report here the significance of the Notch1 receptor in intracellular trafficking of recombinant adeno-associated virus type 2 (rAAV2). RNA profiling of human prostate cancer cell lines with various degrees of AAV transduction indicated a correlation of the amount of Notch1 with rAAV transgene expression. A definitive role of Notch1 in enhancing AAV transduction was confirmed by developing clonal derivatives of DU145 cells overexpressing either full-length or intracellular Notch1. To discern stages of AAV2 transduction influenced by Notch1, competitive binding with soluble heparin and Notch1 antibody, intracellular trafficking using Cy3-labeled rAAV2, and blocking assays for proteasome and dynamin pathways were performed. Results indicated that in the absence or low-level expression of Notch1, only binding of virus was found on the cell surface and internalization was impaired. However, increased Notch1 expression in these cells allowed efficient perinuclear accumulation of labeled capsids. Nuclear transport of the vector was evident by transgene expression and real-time PCR analyses. Dynamin levels were not found to be different among these cell lines, but blocking dynamin function abrogated AAV2 transduction in DU145 clones overexpressing full-length Notch1 but not in clones overexpressing intracellular Notch1. These studies provide evidence for the role of activated Notch1 in intracellular trafficking of AAV2, which may have implications in the optimal use of AAV2 in human gene therapy.

  4. Novel recombinant adeno-associated viruses for Cre activated and inactivated transgene expression in neurons

    Directory of Open Access Journals (Sweden)

    Arpiar eSaunders

    2012-07-01

    Full Text Available Understanding the organization of the nervous system requires methods for dissecting the contributions of each component cell type to circuit function. One widely used approach combines genetic targeting of Cre recombinase to specific cell populations with infection of recombinant adeno-associated viruses (rAAVs whose transgene expression is activated by Cre (Cre-On. Distinguishing how the Cre-expressing neurons differ functionally from neighboring Cre-negative neurons requires rAAVs that are inactivated by Cre (Cre-Off and can be used in tandem with Cre-On viruses. Here we introduce two rAAV vectors that are inactivated by Cre and carry different fluorophore and optogenetic constructs. We demonstrate single and dual rAAV systems to achieve Cre-On and Cre-Off expression in spatially-intermingled cell populations of the striatum. Using these systems, we uncovered cryptic genomic interactions that occur between multiple Cre-sensitive rAAVs or between Cre-sensitive rAAVs and somatic Cre-conditional alleles and devised methods to avoid these interactions. Our data highlight both important experimental caveats associated with Cre-dependent rAAV use as well as opportunities for the development of improved rAAVs for gene delivery.

  5. Effect and Mechanism of Mitomycin C Combined with Recombinant Adeno-Associated Virus Type II against Glioma

    Directory of Open Access Journals (Sweden)

    Hong Ma

    2013-12-01

    Full Text Available The effect of chemotherapy drug Mitomycin C (MMC in combination with recombinant adeno-associated virus II (rAAV2 in cancer therapy was investigated, and the mechanism of MMC affecting rAAV2’s bioactivity was also studied. The combination effect was evaluated by the level of GFP and TNF expression in a human glioma cell line, and the mechanism of MMC effects on rAAV mediated gene expression was investigated by AAV transduction related signal molecules. C57 and BALB/c nude mice were injected with rAAV-EGFP or rAAV-TNF alone, or mixed with MMC, to evaluate the effect of MMC on AAV-mediated gene expression and tumor suppression. MMC was shown to improve the infection activity of rAAV2 both in vitro and in vivo. Enhancement was found to be independent of initial rAAV2 receptor binding stage or subsequent second-strand synthesis of target DNA, but was related to cell cycle retardation followed by blocked genome degradation. In vivo injection of MMC combined with rAAV2 into the tumors of the animals resulted in significant suppression of tumor growth. It was thus demonstrated for the first time that MMC could enhance the expression level of the target gene mediated by rAAV2. The combination of rAAV2 and MMC may be a promising strategy in cancer therapy.

  6. ADENO-ASSOCIATED VIRUS INTRODUCED INTEGRATION AND EXPRESSION OF FOREIGN GENES IN PC12 CELLS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To investigate integration and expression of adeno-associated virus (AAV) vectors in neuronal PC12 cells,the result of which can be applied in further gene therapy of diseases of the central nervous system. Methods Human neurotrophin-3(hNT3)genes were inserted into AAV vectors. Then the recombinat AAV plasmids were encapsidated as recombinant virions. PC12 cells were transfected with the virions and the positive cells were selected by G418. The transfection positive (hNT3 modified)PC12 cells were cultured for several generations and the cellular genomic DNA and total RNA were extracted. We investigated the integration locus of AAV vectors by Southern blot and transcript situation of foreign genes by dot blot. Results The hybridization tests showed that AAV introduced foreign genes were stably integrated, but at random locus, and robustly transcribed in hNT3 modified PC12cells. Conclusion AAV vectors can serve as high efficiency vectors of target genes in neuronal PC12 cells.

  7. Identification of an adeno-associated virus binding epitope for AVB sepharose affinity resin

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    Qiang Wang

    2015-01-01

    Full Text Available Recent successes of adeno-associated virus (AAV–based gene therapy have created a demand for large-scale AAV vector manufacturing and purification techniques for use in clinical trials and beyond. During the development of purification protocols for rh.10, hu.37, AAV8, rh.64R1, AAV3B, and AAV9 vectors, based on a widely used affinity resin, AVB sepharose (GE, we found that, under the same conditions, different serotypes have different affinities to the resin, with AAV3B binding the best and AAV9 the poorest. Further analysis revealed a surface-exposed residue (amino acid number 665 in AAV8 VP1 numbering differs between the high-affinity AAV serotypes (serine in AAV3B, rh.10, and hu.37 and the low-affinity ones (asparagine in AAV8, rh.64R1, and AAV9. The residue locates within a surface-exposed, variable epitope flanked by highly conserved residues. The substitution of the epitope in AAV8, rh.64R1, and AAV9 with the corresponding epitope of AAV3B (SPAKFA resulted in greatly increased affinity to AVB sepharose with no reduction in the vectors’ in vitro potency. The presence of the newly identified AVB-binding epitope will be useful for affinity resin selection for the purification of novel AAV serotypes. It also suggests the possibility of vector engineering to yield a universal affinity chromatography purification method for multiple AAV serotypes.

  8. A Hypoxia-Regulated Adeno-Associated Virus Vector for Cancer-Specific Gene Therapy

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    Hangjun Ruan

    2001-01-01

    Full Text Available The presence of hypoxic cells in human brain tumors is an important factor leading to resistance to radiation therapy. However, this physiological difference between normal tissues and tumors also provides the potential for designing cancer-specific gene therapy. We compared the increase of gene expression under anoxia (<0.01% oxygen produced by 3, 6, and 9 copies of hypoxia-responsive elements (HRE from the erythropoietin gene (Epo, which are activated through the transcriptional complex hypoxia-inducible factor 1 (HIF-1. Under anoxic conditions, nine copies of HIRE (9XHRE yielded 27- to 37-fold of increased gene expression in U-251 MG and U-87 MG human brain tumor cell lines. Under the less hypoxic conditions of 0.3% and 1% oxygen, gene activation by 9XHRE increased expression 11- to 18-fold in these cell lines. To generate a recombinant adeno-associated virus (rAAV in which the transgene can be regulated by hypoxia, we inserted the DNA fragment containing 9XHRE and the LacZ reporter gene into an AAV vector. Under anoxic conditions, this vector produced 79- to 110-fold increase in gene expression. We believe this hypoxia-regulated rAAV vector will provide a useful delivery vehicle for cancer-specific gene therapy.

  9. Ectopic catalase expression in mitochondria by adeno-associated virus enhances exercise performance in mice.

    Science.gov (United States)

    Li, Dejia; Lai, Yi; Yue, Yongping; Rabinovitch, Peter S; Hakim, Chady; Duan, Dongsheng

    2009-08-19

    Oxidative stress is thought to compromise muscle contractility. However, administration of generic antioxidants has failed to convincingly improve performance during exhaustive exercise. One possible explanation may relate to the inability of the supplemented antioxidants to effectively eliminate excessive free radicals at the site of generation. Here, we tested whether delivering catalase to the mitochondria, a site of free radical production in contracting muscle, could improve treadmill performance in C57Bl/6 mice. Recombinant adeno-associated virus serotype-9 (AV.RSV.MCAT) was generated to express a mitochondria-targeted catalase gene. AV.RSV.MCAT was delivered to newborn C57Bl/6 mouse circulation at the dose of 10(12) vector genome particles per mouse. Three months later, we observed a approximately 2 to 10-fold increase of catalase protein and activity in skeletal muscle and the heart. Subcellular fractionation western blot and double immunofluorescence staining confirmed ectopic catalase expression in the mitochondria. Compared with untreated control mice, absolute running distance and body weight normalized running distance were significantly improved in AV.RSV.MCAT infected mice during exhaustive treadmill running. Interestingly, ex vivo contractility of the extensor digitorum longus muscle was not altered. Taken together, we have demonstrated that forced catalase expression in the mitochondria enhances exercise performance. Our result provides a framework for further elucidating the underlying mechanism. It also raises the hope of applying similar strategies to remove excessive, pathogenic free radicals in certain muscle diseases (such as Duchenne muscular dystrophy) and ameliorate muscle disease.

  10. Ectopic catalase expression in mitochondria by adeno-associated virus enhances exercise performance in mice.

    Directory of Open Access Journals (Sweden)

    Dejia Li

    Full Text Available Oxidative stress is thought to compromise muscle contractility. However, administration of generic antioxidants has failed to convincingly improve performance during exhaustive exercise. One possible explanation may relate to the inability of the supplemented antioxidants to effectively eliminate excessive free radicals at the site of generation. Here, we tested whether delivering catalase to the mitochondria, a site of free radical production in contracting muscle, could improve treadmill performance in C57Bl/6 mice. Recombinant adeno-associated virus serotype-9 (AV.RSV.MCAT was generated to express a mitochondria-targeted catalase gene. AV.RSV.MCAT was delivered to newborn C57Bl/6 mouse circulation at the dose of 10(12 vector genome particles per mouse. Three months later, we observed a approximately 2 to 10-fold increase of catalase protein and activity in skeletal muscle and the heart. Subcellular fractionation western blot and double immunofluorescence staining confirmed ectopic catalase expression in the mitochondria. Compared with untreated control mice, absolute running distance and body weight normalized running distance were significantly improved in AV.RSV.MCAT infected mice during exhaustive treadmill running. Interestingly, ex vivo contractility of the extensor digitorum longus muscle was not altered. Taken together, we have demonstrated that forced catalase expression in the mitochondria enhances exercise performance. Our result provides a framework for further elucidating the underlying mechanism. It also raises the hope of applying similar strategies to remove excessive, pathogenic free radicals in certain muscle diseases (such as Duchenne muscular dystrophy and ameliorate muscle disease.

  11. Adeno-associated virus serotype 9 transduction in the central nervous system of nonhuman primates.

    Science.gov (United States)

    Samaranch, Lluis; Salegio, Ernesto A; San Sebastian, Waldy; Kells, Adrian P; Foust, Kevin D; Bringas, John R; Lamarre, Clementine; Forsayeth, John; Kaspar, Brian K; Bankiewicz, Krystof S

    2012-04-01

    Widespread distribution of gene products at clinically relevant levels throughout the CNS has been challenging. Adeno-associated virus type 9 (AAV9) vector has been reported as a good candidate for intravascular gene delivery, but low levels of preexisting antibody titers against AAV in the blood abrogate cellular transduction within the CNS. In the present study we compared the effectiveness of vascular delivery and cerebrospinal fluid (CSF) delivery of AAV9 in transducing CNS tissue in nonhuman primates. Both delivery routes generated similar distribution patterns, although we observed a more robust level of transduction after CSF delivery. Consistent with previous reports administering AAV9, we found greater astrocytic than neuronal tropism via both routes, although we did find a greater magnitude of CNS transduction after CSF delivery compared with intravascular delivery. Last, we have demonstrated that delivery of AAV9 into the CSF does not shield against AAV antibodies. This has obvious implications when developing and/or implementing any clinical trial studies.

  12. Creation of a cardiotropic adeno-associated virus: the story of viral directed evolution

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    Yang Lin

    2013-02-01

    Full Text Available Abstract Adeno-associated virus (AAV is an important vector system for human gene therapy. Although use of AAV serotypes can result in efficient myocardial gene transfer, improvements in the transduction efficiency and specificity are still required. As a method for artificial modification and selection of gene function, directed evolution has been used for diverse applications in genetic engineering of enzymes and proteins. Since 2000, pioneering work has been performed on directed evolution of viral vectors. We further attempted to evolve the AAV using DNA shuffling and in vivo biopanning in a mouse model. An AAVM41 mutant was characterized, which was found to have improved transduction efficiency and specificity in myocardium, an attribute unknown for any natural AAV serotypes. This review focuses on the development of AAV vector for cardiac gene transfer, the history of directed evolution of viral vectors, and our creation of a cardiotropic AAV, which might have implications for the future design and application of viral vectors.

  13. Long-term sex-biased correction of circulating propionic acidemia disease markers by adeno-associated virus vectors.

    Science.gov (United States)

    Guenzel, Adam J; Collard, Renata; Kraus, Jan P; Matern, Dietrich; Barry, Michael A

    2015-03-01

    Propionic academia (PA) occurs because of mutations in the PCCA or PCCB genes encoding the two subunits of propionyl-CoA carboxylase, a pivotal enzyme in the breakdown of certain amino acids and odd-chain fatty acids. There is no cure for PA, but dietary protein restriction and liver transplantation can attenuate its symptoms. We show here that a single intravenous injection of adeno-associated virus 2/8 (AAV8) or AAVrh10 expressing PCCA into PA hypomorphic mice decreased systemic propionylcarnitine and methyl citrate for up to 1.5 years. However, long-term phenotypic correction was always better in male mice. AAV-mediated PCCA expression was similar in most tissues in males and females at early time points and differed only in the liver. Over 1.5 years, luciferase and PCCA expression remained elevated in cardiac tissue for both sexes. In contrast, transgene expression in the liver and skeletal muscles of female, but not male, mice waned—suggesting that these tissues were major sinks for systemic phenotypic correction. These data indicate that single systemic intravenous therapy by AAV vectors can mediate long-term phenotype correction for PA. However, tissue-specific loss of expression in females reduces efficacy when compared with males. Whether similar sex-biased AAV effects occur in human gene therapy remains to be determined.

  14. Supraspinal gene transfer by intrathecal adeno-associated virus serotype 5

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    Daniel J. Schuster

    2014-08-01

    Full Text Available We report the pattern of transgene expression across brain regions after intrathecal delivery of adeno-associated virus serotype 5 (AAV5. Labeling in hindbrain appeared to be primarily neuronal, and was detected in sensory nuclei of medulla, pontine nuclei, and all layers of cerebellar cortex. Expression in midbrain was minimal, and generally limited to isolated neurons and astrocytes in the cerebral peduncles. GFP immunoreactivity (-ir in thalamus was most prominent in medial geniculate nucleus, and otherwise limited to posterior nuclei of the dorsal and lateral margins. Labeling was also observed in neurons and astrocytes of the hippocampal formation and amygdaloid complex. In the hippocampal formation, GFP-ir was found in neuronal cell bodies of the rostral ventral portion, but was largely restricted to fiber-like staining in the molecular layer of dentate gyrus and stratum lacunosum-moleculare of the rostral dorsal region. GFP-ir was seen in neurons and astroglia throughout caudal cortex, whereas in rostral regions of neocortex it was limited to isolated astrocytes and neurons. Labeling was also present in olfactory bulb. These results demonstrate that intrathecal delivery of AAV5 vector leads to transgene expression in discrete CNS regions throughout the rostro-caudal extent of the neuraxis. A caudal-to-rostral gradient of decreasing GFP-ir was present in choroid plexus and Purkinje cells, suggesting that spread of virus through cerebrospinal fluid plays a role in the resulting transduction pattern. Other factors contributing to the observed expression pattern likely include variations in cell-surface receptors and inter-parenchymal space.

  15. Defective-interfering particles of the human parvovirus adeno-associated virus. [uv radiation

    Energy Technology Data Exchange (ETDEWEB)

    Laughlin, C.A.; Myers, M.W.; Risin, D.L.; Carter, B.J.

    1979-04-15

    We have previously shown that adeno-associated virus (AAV) grown in KB cells with a helper adenovirus, produced several classes of particles defined by their buoyant density in CsCl. The predominant density classes were referred to as AAV(1.45), AAV(1.41), AAV (1.35), and AAV(1.32), respectively, where the density of the particle was written in the parentheses. The AAV(1.45) and AAV(1.41) particles which contained standard genomes were the only infectious AAV these infectious AAV particles exhibited autointerference. The ligh-density AAV(1.35) and (1.32) particles contained aberrant (deleted and/or snap-back) genomes. We report here experiments which show that the light-density AAV particles were noninfectious but interfered with the replication of AAV(1.41). The interference was intracellular and resulted in inhibition of synthesis of standard (14.5S) AAV genomes. In some cases there was also a concomitant increase in synthesis of aberrant, shorter AAV DNA. The inhibitory activity of the light-density particles was abolished by uv irradiation. These results show that the population of light AAV particles contained DI particles. The observed autointerference of AAV(1.45) or AAV(1.41) virus is postulated to be due to AAV DI particles. Replication of AAV DI genomes appeared to require the presence of replicating, standard AAV genomes. This is interpreted to mean that progeny strand replication of AAV requires an AAV-specified product, presumably the AAV capsid protein. In contrast to standard, infectious AAV, the AAV DI particles alone do not inhibit replication of the helper adenovirus.

  16. Establishment of a recombinant adeno-associated virus expressing hVEGF165

    Institute of Scientific and Technical Information of China (English)

    Xianghui Huang; Zhibin Shi; Xiaoqian Dang; Chen Zhang; Pengbo Yu; Kunzheng Wang

    2008-01-01

    BACKGROUND: Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration. OBJECTIVE: To construct a non-pathogenic, recombinant adeno-associated virus (AAV) simultaneously expressing human vascular endothelial growth factor 165 (hVEGF165) and green fluorescent protein (GFP). DESIGN, TIME AND SETTING: A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007. MATERIALS: AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. Coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi. METHODS: The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC (the rep/cap-gene containing plasmid), and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP. MAIN OUTCOME MEASURES: Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter (FACS) upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR. RESULTS: The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion

  17. Transient suppression of hepatocellular replication in the mouse liver following transduction with recombinant adeno-associated virus.

    Science.gov (United States)

    Dane, A P; Cunningham, S C; Kok, C Y; Logan, G J; Alexander, I E

    2015-11-01

    Recombinant vectors based on adeno-associated virus (AAV) are proving to be powerful tools for genetic manipulation of the liver, for both discovery and therapeutic purposes. The system can be used to deliver transgene cassettes for expression or, alternatively, DNA templates for genome editing via homologous recombination. The replicative state of target cells is known to influence the efficiency of these processes and knowledge of the host-vector interactions involved is required for optimally effective vector deployment. Here we show, for the first time in vivo, that in addition to the known effects of hepatocellular replication on AAV-mediated gene transfer, the vector itself exerts a potent, albeit transient suppressive effect on cell cycle progression that is relieved on a time course that correlates with the known rate of clearance of input single-stranded vector DNA. This finding requires further mechanistic investigation, delineates an excellent model system for such studies and further deepens our insight into the complexity of interactions between AAV vectors and the cell cycle in a clinically promising target tissue.

  18. Targeted Genome Editing by Recombinant Adeno-Associated Virus (rAAV) Vectors for Generating Genetically Modified Pigs

    Institute of Scientific and Technical Information of China (English)

    Yonglun Luo; Emil Kofod-Olsen; Rikke Christensen; Charlotte Brandt S(φ)rensen; Lars Bolund

    2012-01-01

    Recombinant adeno-associated virus (rAAV) vectors have been extensively used for experimental gene therapy of inherited human diseases.Several advantages,such as simple vector construction,high targeting frequency by homologous recombination,and applicability to many cell types,make rAAV an attractive approach for targeted genome editing.Combined with cloning by somatic cell nuclear transfer (SCNT),this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis,hereditary tyrosinemia type 1,and breast cancer.This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination.We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts,which are subsequently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.

  19. Analysis of particle content of recombinant adeno-associated virus serotype 8 vectors by ion-exchange chromatography.

    Science.gov (United States)

    Lock, Martin; Alvira, Mauricio R; Wilson, James M

    2012-02-01

    Advances in adeno-associated virus (AAV)-mediated gene therapy have brought the possibility of commercial manufacturing of AAV vectors one step closer. To realize this prospect, a parallel effort with the goal of ever-increasing sophistication for AAV vector production technology and supporting assays will be required. Among the important release assays for a clinical gene therapy product, those monitoring potentially hazardous contaminants are most critical for patient safety. A prominent contaminant in many AAV vector preparations is vector particles lacking a genome, which can substantially increase the dose of AAV capsid proteins and lead to possible unwanted immunological consequences. Current methods to determine empty particle content suffer from inconsistency, are adversely affected by contaminants, or are not applicable to all serotypes. Here we describe the development of an ion-exchange chromatography-based assay that permits the rapid separation and relative quantification of AAV8 empty and full vector particles through the application of shallow gradients and a strong anion-exchange monolith chromatography medium.

  20. Recombination and population mosaic of a multifunctional viral gene, adeno-associated virus cap.

    Directory of Open Access Journals (Sweden)

    Yasuhiro Takeuchi

    Full Text Available Homologous recombination is a dominant force in evolution and results in genetic mosaics. To detect evidence of recombination events and assess the biological significance of genetic mosaics, genome sequences for various viral populations of reasonably large size are now available in the GenBank. We studied a multi-functional viral gene, the adeno-associated virus (AAV cap gene, which codes for three capsid proteins, VP1, VP2 and VP3. VP1-3 share a common C-terminal domain corresponding to VP3, which forms the viral core structure, while the VP1 unique N-terminal part contains an enzymatic domain with phospholipase A2 activity. Our recombinant detection program (RecI revealed five novel recombination events, four of which have their cross-over points in the N-terminal, VP1 and VP2 unique region. Comparison of phylogenetic trees for different cap gene regions confirmed discordant phylogenies for the recombinant sequences. Furthermore, differences in the phylogenetic tree structures for the VP1 unique (VP1u region and the rest of cap highlighted the mosaic nature of cap gene in the AAV population: two dominant forms of VP1u sequences were identified and these forms are linked to diverse sequences in the rest of cap gene. This observation together with the finding of frequent recombination in the VP1 and 2 unique regions suggests that this region is a recombination hot spot. Recombination events in this region preserve protein blocks of distinctive functions and contribute to convergence in VP1u and divergence of the rest of cap. Additionally the possible biological significance of two dominant VP1u forms is inferred.

  1. Construction of adeno-associated virus coexpression system for human angiopoietin-1 and VEGF gene

    Institute of Scientific and Technical Information of China (English)

    陈德杰; 谭最; 谢友利; 刘芳

    2004-01-01

    Background Ischemic disease is one of the leading causes of death in the world. In order to further study gene therapy for ischemic disease, we constructed a recombinant plasmid for co-expression of human angiopoietin-1 and vascular endothelial growth factor 165 (VEGF165) gene in adeno-associated virus (AAV) gene delivery system.Methods Human angiopoietin 1 and VEGF165 gene were obtained using PCR. The upstream of angiopoietin 1 contained restriction enzyme site Hind Ⅲ, and the downstream of angiopoietin 1contained restriction enzyme site BamH Ⅰ. The upstream of VEGF165 contained restriction enzyme site Bgl Ⅱ, and the downstream of VEGF165 contained restriction enzyme site BamH Ⅰ . Using the multiple cloning sites (MCS) in plasmid pZero ++ such as BamH Ⅰ , Bgl Ⅱ, Hind Ⅲ, Not Ⅰ , XhoⅠ,Xba Ⅰ , Sal Ⅰ , BspH Ⅰ , Ksp Ⅰ and the corresponding MCS in plasmid pAAV-MCS, angiopoietin 1 and VEGF165 gene were subcloned into pAAV-MCS.Results DNA sequencing revealed that the PCR- amplified angiopoietin 1 and VEGF165 were consistent with NCBI Gene Bank. The recombinant plasmid was identified using PCR and digestion,which proved to be consistent with our hypothesis. In recombinant plasmid, angiopoietin1 and VEGF possessed a CMV promoter and polyA terminator system respectively, thus assuring co-expression of the two genes.Conclusion Successful construction of AAV co-expression system for human angiopoietin 1 and VEGF165 gene will provide the foundation for gene therapy to cure severe ischemic disease.

  2. Effects of Adeno-Associated Virus Serotype and Tissue-Specific Expression on Circulating Biomarkers of Propionic Acidemia

    Science.gov (United States)

    Guenzel, Adam J.; Hillestad, Matthew L.; Matern, Dietrich

    2014-01-01

    Abstract Propionic acidemia (PA) is an autosomal recessive inborn error of metabolism caused by deficiency of propionyl-CoA carboxylase (PCC). This enzyme is composed of six PCCA and six PCCB subunits and mediates a critical step in catabolism of odd chain fatty acids and certain amino acids. Current treatment options for PA are limited to stringent dietary restriction of protein consumption and some patients undergo elective liver transplantation. We previously generated a hypomorphic model of PA, designated Pcca−/−(A138T), with 2% of wild-type enzyme activity that mimics many aspects of the human disease. In this study, we used the differing tissue tropisms of adeno-associated virus (AAV) to probe the ability of liver or muscle-directed gene therapy to treat systemic aspects of this disease that affects many cell types. Systemic therapy with muscle-biased AAV1, liver-biased AAV8, and broadly tropic AAVrh10 mediated significant biochemical corrections in circulating propionylcarnitine (C3) and methyl citrate by all vectors. The innate tissue bias of AAV1 and AAV8 gene expression was made more specific by the use of muscle-specific muscle creatine kinase (specifically MCK6) and hepatocyte-specific transthyretin (TTR) promoters, respectively. Under these targeted conditions, both vectors mediated significant long-term correction of circulating metabolites, demonstrating that correction of muscle and likely other tissue types in addition to liver is necessary to fully correct pathology caused by PA. Liver-specific AAV8-TTR-PCCA mediated better correction than AAV1-MCK-PCCA. These data suggest that targeted gene therapy may be a viable alternative to liver transplantation for PA. They also demonstrate the effects of tissue-specific and broad gene therapy on a cell autonomous systemic genetic disease. PMID:25046265

  3. A novel method for the quantification of adeno-associated virus vectors for RNA interference applications using quantitative polymerase chain reaction and purified genomic adeno-associated virus DNA as a standard.

    Science.gov (United States)

    Wagner, Anke; Röhrs, Viola; Kedzierski, Radoslaw; Fechner, Henry; Kurreck, Jens

    2013-12-01

    Recombinant adeno-associated virus (rAAV) vectors are promising tools in gene therapy, but accurate quantification of the vector dose remains a critical issue for their successful application. We therefore aimed at the precise determination of the titer of self-complementary AAV (scAAV) vectors to improve the reliability of RNA interference (RNAi)-mediated knockdown approaches. Vector titers were initially determined by quantitative polymerase chain reaction (qPCR) using four primer sets targeting different regions within the AAV vector genome (VG) and either coiled or linearized plasmid standards. Despite very low variability between replicates in each assay, these quantification experiments revealed up to 20-fold variation in vector titers. Therefore, we developed a novel approach for the reproducible determination of titers of scAAV vectors based on the use of purified genomic vector DNA as a standard (scAAVStd). Consistent results were obtained in qPCR assays using the four primer sets mentioned above. RNAi-mediated silencing of human cyclophilin B (hCycB) by short hairpin RNA-expressing scAAV vectors was investigated in HeLa cells using two independent vector preparations. We found that the required vector titers for efficient knockdown differed by a factor of 3.5 between both preparations. Hence, we also investigated the number of internalized scAAV vectors, termed transduction units (TUs). TUs were determined by qPCR applying the scAAVStd. Very similar values for 80% hCycB knockdown were obtained for the two AAV vector preparations. Thus, only the determination of TUs, rather than vector concentration, allows for reproducible results in functional analyses using AAV vectors.

  4. Impact of Capsid Conformation and Rep-Capsid Interactions on Adeno-Associated Virus Type 2 Genome Packaging

    OpenAIRE

    Bleker, Svenja; Pawlita, Michael; Kleinschmidt, Jürgen A.

    2006-01-01

    Single-stranded genomes of adeno-associated virus (AAV) are packaged into preformed capsids. It has been proposed that packaging is initiated by interaction of genome-bound Rep proteins to the capsid, thereby targeting the genome to the portal of encapsidation. Here we describe a panel of mutants with amino acid exchanges in the pores at the fivefold axes of symmetry on AAV2 capsids with reduced packaging and reduced Rep-capsid interaction. Mutation of two threonines at the rim of the fivefol...

  5. 介导p73去阻抑肽表达的重组腺伴病毒的构建和鉴定%Construction and identification of recombinant adeno-associated virus vector mediating the expression of the derepressing p73 peptide-p53 (N37)

    Institute of Scientific and Technical Information of China (English)

    白艳霞; 闫利英; 马清涌; 杨广笑; 王全颖

    2012-01-01

    目的 构建编码融合基因NT4-p53(N37)-HA2-TAT的重组腺伴病毒表达载体,为恶性肿瘤基因治疗的实验研究奠定基础.方法 采用互补引物二次PCR法以及T载体克隆法获得p53(N37)基因克隆,酶切后将其连同穿膜肽HA2-TAT片段一起连入pUC19/NT4质粒,再将融合基因NT4-p53(N37)-HA2-TAT亚克隆至腺伴病毒的穿梭质粒pSSHG-CMV中,构建重组质粒pSSHG-CMV/NT4-p53(N37)-HA2-TAT并进行酶切鉴定;应用磷酸钙沉淀法,pSSHCCMV/NT4-p53(N37)-HA2-TAT、辅助质粒pAAV-Ad,腺病毒全基因组质粒pFG140三种质粒共转染HEK293细胞,包装出重组腺伴病毒rAAV/NT4-p53(N37)-HA2-TAT并用斑点杂交法测定重组病毒的滴度;MTT比色法、流式细胞仪观察重组腺伴病毒rAAV/NT4-p53(N37)-HA2-TAT对HepG2细胞的抑制作用.结果 克隆出p53(N37)基因,经酶切及测序证实结果正确;得到高滴度的(2×1013pfu/L)重组腺伴病毒表达载体并对HepG2细胞有明显的抑制作用,且这一作用是通过诱导肿瘤细胞凋亡实现的.结论 通过分子克隆体外重组技术成功制备了rAAV/NT4-p53(N37)-HA2-TAT复制缺陷型重组腺伴病毒,为下一步开展在p53突变或缺失肿瘤中针对p73的靶向性肿瘤基因治疗研究奠定了基础.%Objective To construct a recombinant adeno-associated virus vector mediating the expression of the derepressing p73 peptide-p53(N37) so as to lay a foundation for further research on gene therapy of malignant tumors. Methods The p53(N37) gene was obtained by self-complementary primer PCR and T-vector cloning techniques; then the p53(N37) gene and HA2-TAT segment were cloned into pUC19/NT4 vector after digested with restriction enzyme. The fusion gene of NT4-p53(N37)-HA2-TAT was subcloned into the shuttle plasmid of adeno-associated pSSHG-CMV, and recombinant plasmid pSSHG-CMV/NT4-p53 (N37)-HA2-TAT was constructed and identified by enzyme cutting analysis. The rAAV/NT4-p53 (N37 )-HA2-TAT was produced by using calcium

  6. Attenuation of vesicular stomatitis virus infection of brain using antiviral drugs and an adeno-associated virus-interferon vector.

    Science.gov (United States)

    Wollmann, Guido; Paglino, Justin C; Maloney, Patrick R; Ahmadi, Sebastian A; van den Pol, Anthony N

    2015-01-15

    Vesicular stomatitis virus (VSV) shows promise as a vaccine-vector and oncolytic virus. However, reports of neurotoxicity of VSV remain a concern. We compared 12 antiviral compounds to control infection of VSV-CT9-M51 and VSV-rp30 using murine and human brain cultures, and in vivo mouse models. Inhibition of replication, cytotoxicity and infectivity was strongest with ribavirin and IFN-α and to some extent with mycophenolic acid, chloroquine, and adenine 9-β-d-arabinofuranoside. To generate continuous IFN exposure, we made an adeno-associated virus vector expressing murine IFN; AAV-mIFN-β protected mouse brain cells from VSV, as did a combination of IFN, ribavirin and chloroquine. Intracranial AAV-mIFN-β protected the brain against VSV-CT9-M51. In SCID mice bearing human glioblastoma, AAV-mIFN-β moderately enhanced survival. VSV-CT9-M51 doubled median survival when administered after AAV-mIFN-β; some surviving mice showed complete tumor destruction. Together, these data suggest that AAV-IFN or IFN with ribavirin and chloroquine provide an optimal anti-virus combination against VSV in the brain. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

    Energy Technology Data Exchange (ETDEWEB)

    Lerch, Thomas F.; Chapman, Michael S. (Oregon HSU)

    2012-05-24

    Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites of AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.

  8. Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

    Energy Technology Data Exchange (ETDEWEB)

    Lerch, Thomas F.; Chapman, Michael S., E-mail: chapmami@ohsu.edu

    2012-02-05

    Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites of AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.

  9. Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing.

    Science.gov (United States)

    Adachi, Kei; Enoki, Tatsuji; Kawano, Yasuhiro; Veraz, Michael; Nakai, Hiroyuki

    2014-01-01

    Adeno-associated virus (AAV) capsid engineering is an emerging approach to advance gene therapy. However, a systematic analysis on how each capsid amino acid contributes to multiple functions remains challenging. Here we show proof-of-principle and successful application of a novel approach, termed AAV Barcode-Seq, that allows us to characterize phenotypes of hundreds of different AAV strains in a high-throughput manner and therefore overcomes technical difficulties in the systematic analysis. In this approach, we generate DNA barcode-tagged AAV libraries and determine a spectrum of phenotypes of each AAV strain by Illumina barcode sequencing. By applying this method to AAV capsid mutant libraries tagged with DNA barcodes, we can draw a high-resolution map of AAV capsid amino acids important for the structural integrity and functions including receptor binding, tropism, neutralization and blood clearance. Thus, Barcode-Seq provides a new tool to generate a valuable resource for virus and gene therapy research.

  10. Adeno-associated virus at 50: a golden anniversary of discovery, research, and gene therapy success--a personal perspective.

    Science.gov (United States)

    Hastie, Eric; Samulski, R Jude

    2015-05-01

    Fifty years after the discovery of adeno-associated virus (AAV) and more than 30 years after the first gene transfer experiment was conducted, dozens of gene therapy clinical trials are in progress, one vector is approved for use in Europe, and breakthroughs in virus modification and disease modeling are paving the way for a revolution in the treatment of rare diseases, cancer, as well as HIV. This review will provide a historical perspective on the progression of AAV for gene therapy from discovery to the clinic, focusing on contributions from the Samulski lab regarding basic science and cloning of AAV, optimized large-scale production of vectors, preclinical large animal studies and safety data, vector modifications for improved efficacy, and successful clinical applications.

  11. A novel recombinant adeno-associated virus vector packaging system with HSV-1 amplicon providing helper functions

    Institute of Scientific and Technical Information of China (English)

    舒跃龙; 吴小兵; 杨天忠; 贡惠宇; 侯云德; 颜子颖

    1999-01-01

    A novel packaging system for producing recombinant adeno-associated virus (rAAV) vector was described. Instead of the conventional method for rAAV production by two-plasmid co-transfection followed by superinfection with adenovirus 5, an HSV-1 amplicon system expressing AAV-2 rep and cap genes from their native promoters was used to provide complete helper functions for rAAV replicating and packaging. This HSV-1 ampticon stock consisted of two kinds of infectious HSV-1 virions, a replicating-defective HSV-1 amplicon pseudovirus harboring multi-copies of AAV-2 rep and cap gene and a temperature-sensitive HSV-1 mutant strain ts-KOS. High-titer rAAV was generated with this new packaging system. This packaging system gives a simple and scaleable process for rAAV production.

  12. A phase 1 study to evaluate the safety and immunogenicity of a recombinant HIV type 1 subtype C adeno-associated virus vaccine

    NARCIS (Netherlands)

    Mehendale, Sanjay; van Lunzen, Jan; Clumeck, Nathan; Rockstroh, Jurgen; Vets, Eva; Johnson, Philip R.; Anklesaria, Pervin; Barin, Burc; Boaz, Mark; Kochhar, Sonali; Lehrman, Jennifer; Schmidt, Claudia; Peeters, Mathieu; Schwarze-Zander, Carolynne; Kabamba, Kabeya; Glaunsinger, Tobias; Sahay, Seema; Thakar, Madhuri; Paranjape, Ramesh; Gilmour, Jill; Excler, Jean-Louis; Fast, Patricia; Heald, A1lison E.

    2008-01-01

    A novel prophylactic AIDS vaccine candidate, consisting of single-stranded DNA for HIV-1 subtype C gag, protease, and part of reverse transcriptase genes, enclosed within a recombinant adeno-associated virus serotype-2 protein capsid (tgAAC09) induced T cell responses and antibodies in nonhuman prim

  13. A phase 1 study to evaluate the safety and immunogenicity of a recombinant HIV type 1 subtype C adeno-associated virus vaccine

    NARCIS (Netherlands)

    Mehendale, Sanjay; van Lunzen, Jan; Clumeck, Nathan; Rockstroh, Jurgen; Vets, Eva; Johnson, Philip R.; Anklesaria, Pervin; Barin, Burc; Boaz, Mark; Kochhar, Sonali; Lehrman, Jennifer; Schmidt, Claudia; Peeters, Mathieu; Schwarze-Zander, Carolynne; Kabamba, Kabeya; Glaunsinger, Tobias; Sahay, Seema; Thakar, Madhuri; Paranjape, Ramesh; Gilmour, Jill; Excler, Jean-Louis; Fast, Patricia; Heald, A1lison E.

    A novel prophylactic AIDS vaccine candidate, consisting of single-stranded DNA for HIV-1 subtype C gag, protease, and part of reverse transcriptase genes, enclosed within a recombinant adeno-associated virus serotype-2 protein capsid (tgAAC09) induced T cell responses and antibodies in nonhuman

  14. Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair.

    Science.gov (United States)

    Dupont, Kenneth M; Boerckel, Joel D; Stevens, Hazel Y; Diab, Tamim; Kolambkar, Yash M; Takahata, Masahiko; Schwarz, Edward M; Guldberg, Robert E

    2012-03-01

    Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration.

  15. Immobilization of FLAG-Tagged Recombinant Adeno-Associated Virus 2 onto Tissue Engineering Scaffolds for the Improvement of Transgene Delivery in Cell Transplants.

    Directory of Open Access Journals (Sweden)

    Hua Li

    Full Text Available The technology of virus-based genetic modification in tissue engineering has provided the opportunity to produce more flexible and versatile biomaterials for transplantation. Localizing the transgene expression with increased efficiency is critical for tissue engineering as well as a challenge for virus-based gene delivery. In this study, we tagged the VP2 protein of type 2 adeno-associated virus (AAV with a 3×FLAG plasmid at the N-terminus and packaged a FLAG-tagged recombinant AAV2 chimeric mutant. The mutant AAVs were immobilized onto the tissue engineering scaffolds with crosslinked anti-FLAG antibodies by N-succinimidyl-3-(2-pyridyldithiol propionate (SPDP. Cultured cells were seeded to scaffolds to form 3D transplants, and then tested for viral transduction both in vitro and in vivo. The results showed that our FLAG-tagged AAV2 exerted similar transduction efficiency compared with the wild type AAV2 when infected cultured cells. Following immobilization onto the scaffolds of PLGA or gelatin sponge with anti-FLAG antibodies, the viral mediated transgene expression was significantly improved and more localized. Our data demonstrated that the mutation of AAV capsid targeted for antibody-based immobilization could be a practical approach for more efficient and precise transgene delivery. It was also suggested that the immobilization of AAV might have attractive potentials in applications of tissue engineering involving the targeted gene manipulation in 3D tissue cultures.

  16. Parvovirus B19 promoter at map unit 6 confers autonomous replication competence and erythroid specificity to adeno-associated virus 2 in primary human hematopoietic progenitor cells.

    Science.gov (United States)

    Wang, X S; Yoder, M C; Zhou, S Z; Srivastava, A

    1995-01-01

    The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B19, we constructed a recombinant adeno-associated virus 2 (AAV), in which the authentic AAV promoter at map unit 5 (AAVp5) was replaced by the B19p6 promoter. Although the wild-type (wt) AAV requires a helper virus for its optimal replication, we hypothesized that inserting the B19p6 promoter in a recombinant AAV would permit autonomous viral replication, but only in erythroid progenitor cells. In this report, we provide evidence that the B19p6 promoter is necessary and sufficient to impart autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells. Thus, expression from the B19p6 promoter plays an important role in post-P-antigen receptor erythroid-cell specificity of parvovirus B19. The AAV-B19 hybrid vector system may also prove to be useful in potential gene therapy of human hemoglobinopathies. Images Fig. 2 Fig. 3 Fig. 4 PMID:8618912

  17. Enhanced gene delivery in porcine vasculature tissue following incorporation of adeno-associated virus nanoparticles into porous silicon microparticles.

    Science.gov (United States)

    McConnell, Kellie I; Rhudy, Jessica; Yokoi, Kenji; Gu, Jianhua; Mack, Aaron; Suh, Junghae; La Francesca, Saverio; Sakamoto, Jason; Serda, Rita E

    2014-11-28

    There is an unmet clinical need to increase lung transplant successes, patient satisfaction and to improve mortality rates. We offer the development of a nanovector-based solution that will reduce the incidence of lung ischemic reperfusion injury (IRI) leading to graft organ failure through the successful ex vivo treatment of the lung prior to transplantation. The innovation is in the integrated application of our novel porous silicon (pSi) microparticles carrying adeno-associated virus (AAV) nanoparticles, and the use of our ex vivo lung perfusion/ventilation system for the modulation of pro-inflammatory cytokines initiated by ischemic pulmonary conditions prior to organ transplant that often lead to complications. Gene delivery of anti-inflammatory agents to combat the inflammatory cascade may be a promising approach to prevent IRI following lung transplantation. The rationale for the device is that the microparticle will deliver a large payload of virus to cells and serve to protect the AAV from immune recognition. The microparticle-nanoparticle hybrid device was tested both in vitro on cell monolayers and ex vivo using either porcine venous tissue or a pig lung transplantation model, which recapitulates pulmonary IRI that occurs clinically post-transplantation. Remarkably, loading AAV vectors into pSi microparticles increases gene delivery to otherwise non-permissive endothelial cells.

  18. Prevalence of adeno-associated virus and human papillomavirus DNA in Iranian women with and without cervical cancer.

    Science.gov (United States)

    Shafiei-Jandaghi, Nazanin Zahra; Yavarian, Jila; Faghihloo, Ebrahim; Ghavami, Nastaran; Yousefi Ghalejoogh, Zohreh; Kiani, Seyed Jalal; Shatizadeh Malekshahi, Somayeh; Shahsiah, Reza; Jahanzad, Eisa; Hosseini, Mostafa; Mokhtari Azad, Talat

    2017-02-24

    There is plenty of substantial evidence to support anti-tumor activity of viruses. Adeno-associated virus (AAV) may interact with human papillomavirus (HPV) to modify the risk of cervical neoplasia. The seroprevalence of AAV among women with cervical cancer has been reported to be lower than healthy ones. In spite of this finding, detection of AAV DNA in cervical biopsies does not entirely support the inverse association between AAV seropositivity and cervical cancer. This association is still controversial and requires more thorough evaluation in different countries. The aim of this case-control study was to find the prevalence of AAV and HPV DNA sequences in Iranian women with and without cervical cancer to assess the probable association of AAV infection and cervical cancer. In this study, paraffin-embedded tissue samples of 61 cervical cancer cases and 50 healthy controls (HCs) were investigated for AAV and HPV DNA by semi-nested and nested PCRs respectively. AAV DNA was detected in 7 cases (14%) of HCs and 9 specimens (14.8%) of case group. According to the branching in the phylogenetic tree, AAV2 was the only type detected in this study. Moreover, HPV DNA was detected in 8 cases (16%) of HCs and 44 specimens (72.13%) of case group. In conclusion, a low proportion of cervical biopsies from Iranian women contained AAV-2 genome. No significant difference in correlation between HPV and cervical cancer in presence or absence of AAV genome in cervix was found.

  19. Adeno-associated virus activates an innate immune response in normal human cells but not in osteosarcoma cells.

    Science.gov (United States)

    Laredj, Leila N; Beard, Peter

    2011-12-01

    Adeno-associated virus (AAV) is a small, DNA-containing dependovirus with promising potential as a gene delivery vehicle. Given the variety of applications of AAV-based vectors in the treatment of genetic disorders, numerous studies have focused on the immunogenicity of recombinant AAV. In general, AAV vectors appear not to induce strong inflammatory responses. We have found that AAV2, when it infects the osteosarcoma cells U2OS, can initiate part of its replicative cycle in the absence of helper virus. This does not occur in untransformed cells. We set out to test whether the cellular innate antiviral defenses control this susceptibility and found that, in nonimmune normal human fibroblasts, AAV2 induces type I interferon production and release and the accumulation of nuclear promyelocytic leukemia bodies. AAV fails to mobilize this defense pathway in the U2OS cells. This permissiveness is in large part due to impairment of the viral sensing machinery in these cells. Our investigations point to Toll-like receptor 9 as a potential intracellular sensor that detects AAV2 and triggers the antiviral state in AAV-infected untransformed cells. Efficient sensing of the AAV genome and the ensuing activation of an innate antiviral response are thus crucial cellular events dictating the parvovirus infectivity in host cells.

  20. Immunological inhibition of transplanted liver allografts by adeno-associated virus vector encoding CTLA4Ig in rats

    Institute of Scientific and Technical Information of China (English)

    Sen Lu; Yue Yu; Yun Gao; Guo-Qiang Li; Xue-Hao Wang

    2008-01-01

    BACKGROUND: Blockade interaction between CD28 and B7 with CTLA4Ig has been shown to induce experimental transplantation tolerance. In order to prolong the inhibitory effect of CTLA4Ig, a recombinant adeno-associated virus vector pSNAV expressing CTLA4Ig was constructed, and its effects on transplanted liver allografts were investigated. METHODS:The pSNAV-CTLA4Ig construct was infused into partial liver allografts of rats via the portal vein during transplantation. CTLA4Ig expression in the transplanted livers was detected with reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. Furthermore, real-time quantita-tive PCR was used to measure the expression of IL-2, IFN-γ, IL-4 and IL-10 in the allografts. RESULTS:The expression of CTLA4Ig in the partial allograft was detected successfully and pSNAV-CTLA4Ig improved the survival rate of rats after liver transplantation. Agarose gel analysis of RT-PCR products indicated the presence of CTLA4Ig in the pSNAV-CTLA4Ig treatment group. Cytokines expressed in allografts on day 7 after orthotopic liver transplantation showed that IL-2, IFN-γ, IL-4 and IL-10 mRNA levels decreased in transplant recipients treated with pSNAV-CTLA4Ig compared with those treated with pSNAV-LacZ (1.62±0.09, 1.52±0.11, 1.50± 0.07 and 1.43±0.07 versus 1.29±0.09, 1.32±0.07, 1.34±0.06 and 1.35±0.04, respectively). CONCLUSIONS:pSNAV-CTLA4Ig effectively expressed CTLA4Ig in liver allografts. CTLA4Ig improved the pathological ifndings after liver transplantation. CTLA4Ig induced immune tolerance of liver transplantation, and the mechanism involved induced alteration of Th1 and Th2 cytokine transcripts. The adeno-associated virus vector encoding CTLA4Ig may be useful in the clinical study of transplantation tolerance.

  1. In utero recombinant adeno-associated virus gene transfer in mice, rats, and primates

    Directory of Open Access Journals (Sweden)

    Marrero Luis

    2003-09-01

    Full Text Available Abstract Background Gene transfer into the amniotic fluid using recombinant adenovirus vectors was shown previously to result in high efficiency transfer of transgenes into the lungs and intestines. Adenovirus mediated in utero gene therapy, however, resulted in expression of the transgene for less than 30 days. Recombinant adenovirus associated viruses (rAAV have the advantage of maintaining the viral genome in daughter cells thus providing for long-term expression of transgenes. Methods Recombinant AAV2 carrying green fluorescent protein (GFP was introduced into the amniotic sac of fetal rodents and nonhuman primates. Transgene maintenance and expression was monitor. Results Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates. Expression and secretion of the reporter gene, GFP, was readily demonstrated within 72 hours post-therapy. In long-term studies in rats and nonhuman primates, maintenance of GFP DNA, protein expression, and reporter gene secretion was documented for over one year. Conclusions Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts. Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.

  2. In vivo adeno-associated viral vector-mediated genetic engineering of white and brown adipose tissue in adult mice.

    Science.gov (United States)

    Jimenez, Veronica; Muñoz, Sergio; Casana, Estefania; Mallol, Cristina; Elias, Ivet; Jambrina, Claudia; Ribera, Albert; Ferre, Tura; Franckhauser, Sylvie; Bosch, Fatima

    2013-12-01

    Adipose tissue is pivotal in the regulation of energy homeostasis through the balance of energy storage and expenditure and as an endocrine organ. An inadequate mass and/or alterations in the metabolic and endocrine functions of adipose tissue underlie the development of obesity, insulin resistance, and type 2 diabetes. To fully understand the metabolic and molecular mechanism(s) involved in adipose dysfunction, in vivo genetic modification of adipocytes holds great potential. Here, we demonstrate that adeno-associated viral (AAV) vectors, especially serotypes 8 and 9, mediated efficient transduction of white (WAT) and brown adipose tissue (BAT) in adult lean and obese diabetic mice. The use of short versions of the adipocyte protein 2 or uncoupling protein-1 promoters or micro-RNA target sequences enabled highly specific, long-term AAV-mediated transgene expression in white or brown adipocytes. As proof of concept, delivery of AAV vectors encoding for hexokinase or vascular endothelial growth factor to WAT or BAT resulted in increased glucose uptake or increased vessel density in targeted depots. This method of gene transfer also enabled the secretion of stable high levels of the alkaline phosphatase marker protein into the bloodstream by transduced WAT. Therefore, AAV-mediated genetic engineering of adipose tissue represents a useful tool for the study of adipose pathophysiology and, likely, for the future development of new therapeutic strategies for obesity and diabetes.

  3. In Vivo Adeno-Associated Viral Vector–Mediated Genetic Engineering of White and Brown Adipose Tissue in Adult Mice

    Science.gov (United States)

    Jimenez, Veronica; Muñoz, Sergio; Casana, Estefania; Mallol, Cristina; Elias, Ivet; Jambrina, Claudia; Ribera, Albert; Ferre, Tura; Franckhauser, Sylvie; Bosch, Fatima

    2013-01-01

    Adipose tissue is pivotal in the regulation of energy homeostasis through the balance of energy storage and expenditure and as an endocrine organ. An inadequate mass and/or alterations in the metabolic and endocrine functions of adipose tissue underlie the development of obesity, insulin resistance, and type 2 diabetes. To fully understand the metabolic and molecular mechanism(s) involved in adipose dysfunction, in vivo genetic modification of adipocytes holds great potential. Here, we demonstrate that adeno-associated viral (AAV) vectors, especially serotypes 8 and 9, mediated efficient transduction of white (WAT) and brown adipose tissue (BAT) in adult lean and obese diabetic mice. The use of short versions of the adipocyte protein 2 or uncoupling protein-1 promoters or micro-RNA target sequences enabled highly specific, long-term AAV-mediated transgene expression in white or brown adipocytes. As proof of concept, delivery of AAV vectors encoding for hexokinase or vascular endothelial growth factor to WAT or BAT resulted in increased glucose uptake or increased vessel density in targeted depots. This method of gene transfer also enabled the secretion of stable high levels of the alkaline phosphatase marker protein into the bloodstream by transduced WAT. Therefore, AAV-mediated genetic engineering of adipose tissue represents a useful tool for the study of adipose pathophysiology and, likely, for the future development of new therapeutic strategies for obesity and diabetes. PMID:24043756

  4. 重组8型腺相关病毒介导双荧光素酶基因在小鼠体内的表达%Recombinant adeno-associated virus type 8 mediated dual-luciferase gene expression in mouse

    Institute of Scientific and Technical Information of China (English)

    王刚; 尉迟捷; 董小岩; 田文洪; 吴小兵

    2012-01-01

    目的 利用共表达的分泌型荧光素酶Gluc(gaussia princeps luciferase)和非分泌型荧光素酶Fluc(firefly luciferase)研究重组8型腺相关病毒(recombinant adeno-associated virus type 8,rAAV8)介导的转基因在小鼠体内的表达特点.方法 制备携带双荧光素酶基因的重组8型腺相关病毒rAAV8-Gluc/Fluc,体外感染HEK293细胞并检测上清和胞内Gluc和Fluc活性;将不同剂量的rAAV8-Gluc/Fluc尾静脉注射或肌内注射至BALB/c小鼠,通过尾静脉采血检测Gluc活性,通过活体成像和裂解组织检测Fluc活性.结果 成功制备了rAAV8-Gluc/Fluc,可以有效感染HEK293细胞,同时分泌表达Gluc和胞内表达Fluc;尾静脉注射或肌内注射rAAV8-Gluc/Fluc至小鼠后,外周血Gluc活性均在注射后10 ~20 d达到高峰并稳定持续120 d以上,Gluc活性随注射剂量增加而增高;静脉注射rAAV8-Gluc/Fluc时Fluc主要在肝脏表达,在骨骼肌和心肌有少量表达,而肌内注射时Fluc既在肌内注射局部表达同时也在肝脏中表达.结论 本研究成功制备了携带双荧光素酶基因rAAV8-Gluc/Fluc,研究了其介导的转基因在小鼠体内的表达特点,为rAAV8的临床前应用打下基础.%Objective Recombinant adeno-associated virus type 8 (rAAV8) mediating transgene expression in mice was investigated using co-expressed report gene of secreted Gaussia princeps luciferase (Gluc) and non-secreted firefly luciferase(Fluc).Methods rAAV8-Gluc/Fluc was prepared and infected HEK293 cells to test its performance in vitro.BALB/c mice were received rAAV8-Gluc/Fluc at different doses by intravenous injection (iv) or intramuscular injection (im).Then Gluc activities in blood were measured,the whole-body images for Fluc activities were performed and Fluc activities of tissue lysate were also detected.Results rAAV8-Gluc/Fluc was successfully prepared and could infected HEK293 cells.The Gluc was mainly detected in the culture media while the Fluc was mainly

  5. Adeno-associated virus gene transfer in Morquio A disease - effect of promoters and sulfatase-modifying factor 1.

    Science.gov (United States)

    Alméciga-Díaz, Carlos J; Montaño, Adriana M; Tomatsu, Shunji; Barrera, Luis A

    2010-09-01

    Mucopolysaccharidosis (MPS) IVA is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme N-acetylgalatosamine-6-sulfate sulfatase (GALNS), which leads to the accumulation of keratan sulfate and chondroitin 6-sulfate, mainly in bone. To explore the possibility of gene therapy for Morquio A disease, we transduced the GALNS gene into HEK293 cells, human MPS IVA fibroblasts and murine MPS IVA chondrocytes by using adeno-associated virus (AAV)-based vectors, which carry human GALNS cDNA. The effects of the promoter and the cotransduction with the sulfatase-modifying factor 1 gene (SUMF1) on GALNS activity levels was evaluated. Downregulation of the cytomegalovirus (CMV) immediate early enhancer/promoter was not observed for 10 days post-transduction. The eukaryotic promoters induced equal or higher levels of GALNS activity than those induced by the CMV promoter in HEK293 cells. Transduction of human MPS IVA fibroblasts induced GALNS activity levels that were 15-54% of those of normal human fibroblasts, whereas in transduced murine MPS IVA chondrocytes, the enzyme activities increased up to 70% of normal levels. Cotransduction with SUMF1 vector yielded an additional four-fold increase in enzyme activity, although the level of elevation depended on the transduced cell type. These findings suggest the potential application of AAV vectors for the treatment of Morquio A disease, depending on the combined choice of transduced cell type, selection of promoter, and cotransduction of SUMF1.

  6. Efficacy and safety of myocardial gene transfer of adenovirus, adeno-associated virus and lentivirus vectors in the mouse heart.

    Science.gov (United States)

    Merentie, M; Lottonen-Raikaslehto, L; Parviainen, V; Huusko, J; Pikkarainen, S; Mendel, M; Laham-Karam, N; Kärjä, V; Rissanen, R; Hedman, M; Ylä-Herttuala, S

    2016-03-01

    Gene therapy is a promising new treatment option for cardiac diseases. For finding the most suitable and safe vector for cardiac gene transfer, we delivered adenovirus (AdV), adeno-associated virus (AAV) and lentivirus (LeV) vectors into the mouse heart with sophisticated closed-chest echocardiography-guided intramyocardial injection method for comparing them with regards to transduction efficiency, myocardial damage, effects on the left ventricular function and electrocardiography (ECG). AdV had the highest transduction efficiency in cardiomyocytes followed by AAV2 and AAV9, and the lowest efficiency was seen with LeV. The local myocardial inflammation and fibrosis in the left ventricle (LV) was proportional to transduction efficiency. AdV caused LV dilatation and systolic dysfunction. Neither of the locally injected AAV serotypes impaired the LV systolic function, but AAV9 caused diastolic dysfunction to some extent. LeV did not affect the cardiac function. We also studied systemic delivery of AAV9, which led to transduction of cardiomyocytes throughout the myocardium. However, also diffuse fibrosis was present leading to significantly impaired LV systolic and diastolic function and pathological ECG changes. Compared with widely used AdV vector, AAV2, AAV9 and LeV were less effective in transducing cardiomyocytes but also less harmful. Local administration of AAV9 was safer and more efficient compared with systemic administration.

  7. Vector design Tour de Force: integrating combinatorial and rational approaches to derive novel adeno-associated virus variants.

    Science.gov (United States)

    Marsic, Damien; Govindasamy, Lakshmanan; Currlin, Seth; Markusic, David M; Tseng, Yu-Shan; Herzog, Roland W; Agbandje-McKenna, Mavis; Zolotukhin, Sergei

    2014-11-01

    Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of "virtual family shuffling" to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8 × 10(5) complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8.

  8. Expression of Vascular Endothelial Growth Factor (VEGF) in Human Osteosarcoma Cells Transfected with Adeno-associated Virus-antisense VEGF

    Institute of Scientific and Technical Information of China (English)

    徐卫国; 陈安民; 张衣北; 易成腊

    2004-01-01

    Summary: The expression of protein vascular endothelial growth factor (VEGF) in osteosarcoma cells transfected with adeno-associated virus (rAAV)-antisense VEGF was studied to provide the foundation of osteosarcoma treatment through antivascularization. The rAAV-antisense VEGF at different doses (0, 20, 50, 100, 200, 240 μl) was transfected into osteosarcoma MG-63 cell. The cells and culture supernatants were collected before and after tansfection. The expression of VEGF protein was detected by using immunohistochemical staining (SP) and Western blot. SP and Western-blot tests revealed that the MG-63 Cells transfected with rAAV-antisense VEGF had less staining than those without transfection with rAAV-antisense VEGF, and the staining intensity was negatively correlated with the doses of genes. The corresponding A values of transfected genes with different doses of rAAV-antisense VEGF (0, 20, 50, 100, 200, 240 μA) were 86 614±13 776, 73 245±15 414, 61 078±12 124, 54 657±10 953, 39 802±11 308, 32 014±15 057 respectively,w ith the difference being significant (P<0.05). It was concluded that the expression of VEGF protein in MG-63 cells could be inhibited by rAAV-antisense VEGF.

  9. Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses.

    Science.gov (United States)

    Shigetomi, Eiji; Bushong, Eric A; Haustein, Martin D; Tong, Xiaoping; Jackson-Weaver, Olan; Kracun, Sebastian; Xu, Ji; Sofroniew, Michael V; Ellisman, Mark H; Khakh, Baljit S

    2013-05-01

    Intracellular Ca(2+) transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca(2+) has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca(2+) indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca(2+) signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca(2+) microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca(2+) signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function.

  10. BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector.

    Science.gov (United States)

    Yamamoto, H; Ishimura, M; Ochiai, M; Takada, H; Kusuhara, K; Nakatsu, Y; Tsuzuki, T; Mitani, K; Hara, T

    2016-02-01

    X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells.

  11. Thymosin Beta-4 Recombinant Adeno-associated Virus Enhances Human Nucleus Pulposus Cell Proliferation and Reduces Cell Apoptosis and Senescence

    Institute of Scientific and Technical Information of China (English)

    Yuan-Yi Wang; Qing-San Zhu; Yi-Wei Wang; Ruo-Feng Yin

    2015-01-01

    Background:Thymosin beta-4 (TB-4) is considered key roles in tissue development,maintenance and pathological processes.The study aimed to prove TB-4 positive biological function on nucleus pulposus (NP) cell apoptosis and slowing the process of cell aging while increasing the cell proliferation.Methods:TB-4 recombinant adeno-associated virus (AAV) was constructed and induced to human NP cells.Cell of same group were cultured without gene modification as controlled group.Proliferation capacity and cell apoptosis were observed during 6 passages of the cells.Morphology and expression of the TB-4 gene were documented as parameter of cell activity during cell passage.Results:NP cells with TB-4 transfection has normal TB-4 expression and exocytosis.NP cells with TB-4 transfection performed significantly higher cell activity than that at the control group in each generation.TB-4 recombinant AAV-transfected human NP cells also show slower cell aging,lower cell apoptosis and higher cell proliferation than control group.Conclusions:TB-4 can prevent NP cell apoptosis,slow NP cell aging and promote NP cell proliferation.AAV transfection technique was able to highly and stably express TB-4 in human NP cells,which may provide a new pathway for innovation in the treatment of intervertebral disc degenerative diseases.

  12. Potent anti-melanoma effect by combination of mytomycin C with recombinant adeno-associated virus-mediated tumor-targeting expressed Smac/DIABLO%丝裂霉素C与表达Smac/DIABLO的肿瘤靶向腺相关病毒联用对抗恶性黑色素瘤的效果

    Institute of Scientific and Technical Information of China (English)

    王坚; 林茂; 吴平; 何惠娟; 刘新垣

    2012-01-01

    To investigate whether recombinant adeno-associated virus-mediated tumor-targeting expressed Smac/DIABLO can improve sensitivity of melanoma to mitomycin C both in vitro and in vivo. Methods Tumor-targeting expressed Smac/DIABLO or green fluorescence protein (EGFP) in recombinant adeno-associated virus vectors (rAAV-hTERT/Smac/DIABLO or rAAV-hTERT/EGFP) was constructed. The culture cells were transfected with rAAV-hTERT/Smac/DIABLO or rAAV-hTERT/EGFP. The expression of EGFP in culture cells was observed with fluorescence microscope. Smac/DIABLO expression was detected by RT-PCR method. Proliferation of tumor cells was measured by MTT method. Apoptosis of tumor cells at different drug concentrations was examined by flow cytometry. Synergistic anti-tumor activity of mitomycin C combined with rAAV-hTERT/ Smac/DIABLO was measured by MTT in vitro and animal experiment in vivo. Data was evaluated by SPSS statisticssoftware analysis. Results Green fluorescence could be observed in tumor cells but not in normal cells 48 h after rAAV-hTERT/EGFP transfection. Almost all tumor cells displayed bright yellow-green fluorescence after 96 h. The expression of Smac/DIABLO in rAAV-hTERT/Smac/DIABLO transfected tumor cells showed Smac/DIABLO mRNA band 24 h after transfection and stabilized 48 h after transfection. Tumor cell inhibition rate was increased obviously higher in group of combination of mitomycin C with rAAV-hTERT/Smac/DIABLO than mitomycin C alone (P<0.01). Flow cytometry results indicated that mitomycin C combined with rAAV-hTERT/Smac/DIABLO group had the highest apoptosis-induced effect in groups of negative, mitomycin C, and rAAV-hTERT/Smac/ DIABLO (P<0.01). Animal experiment result indicated that tumor growth was inhibited and survival rate was improved significantly in mitomycin C combined with rAAV-hTERT/Smac/DIABLO group compared to rAAV-hTERT/Smac/DIABLO or mitomycin C aione. Conclusion Tumor-targeting rAAV-hTERT/Smac/DIABLO can improve sensitivity of tumor cells

  13. Mutational Analysis of the Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Construction of AAV2 Vectors with Altered Tropism

    OpenAIRE

    Wu, Pei; Xiao, Wu; Conlon, Thomas; Hughes, Jeffrey; Agbandje-McKenna, Mavis; FERKOL, THOMAS; Flotte, Terence; Muzyczka, Nicholas

    2000-01-01

    Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mut...

  14. Single Amino Acid Changes Can Influence Titer, Heparin Binding, and Tissue Tropism in Different Adeno-Associated Virus Serotypes▿

    Science.gov (United States)

    Wu, Zhijian; Asokan, Aravind; Grieger, Joshua C.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Samulski, R. Jude

    2006-01-01

    Despite the high degree of sequence homology between adeno-associated virus (AAV) serotype 1 and 6 capsids (99.2%), these viruses have different liver transduction profiles when tested as vectors. Examination of the six amino acid residues that differ between AAV1 and AAV6 revealed that a lysine-to-glutamate change (K531E) suppresses the heparin binding ability of AAV6. In addition, the same mutation in AAV6 reduces transgene expression to levels similar to those achieved with AAV1 in HepG2 cells in vitro and in mouse liver following portal vein administration. In corollary, the converse E531K mutation in AAV1 imparts heparin binding ability and increases transduction efficiency. Extraction of vector genomes from liver tissue suggests that the lysine 531 residue assists in preferential transduction of parenchymal cells by AAV6 vectors in comparison with AAV1. Lysine 531 is unique to AAV6 among other known AAV serotypes and is located in a basic cluster near the spikes that surround the icosahedral threefold axes of the AAV capsid. Similar to studies with autonomous parvoviruses, this study describes the first example of single amino acid changes that can explain differential phenotypes such as viral titer, receptor binding, and tissue tropism exhibited by closely related AAV serotypes. In particular, a single lysine residue appears to provide the critical minimum charged surface required for interacting with heparin through electrostatic interaction and simultaneously plays an unrelated yet critical role in the liver tropism of AAV6 vectors. PMID:16943302

  15. An adeno-associated virus-based intracellular sensor of pathological nuclear factor-κB activation for disease-inducible gene transfer.

    Directory of Open Access Journals (Sweden)

    Abdelwahed Chtarto

    Full Text Available Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS. This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

  16. Addressing immune tolerance issues in inflammatory bowel disease and adeno-associated virus based gene transfer

    NARCIS (Netherlands)

    Majowicz, Anna

    2014-01-01

    This thesis is focusing on cell-mediated induction of immune tolerance and consists of two parts. The studies described in Part I report the development of strategies for possible treatment of Inflammatory Bowel Diseases (IBD). Induction of immune tolerance, in IBD mouse model, with the use of regul

  17. Capsid protein expression and adeno-associated virus like particles assembly in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Backovic Ana

    2012-09-01

    Full Text Available Abstract Background The budding yeast Saccharomyces cerevisiae supports replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses and has provided means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus. We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid. In this work, we have investigated the possibility to assemble AAV capsids in yeast. Results To do this, at least two out of three AAV structural proteins, VP1 and VP3, have to be simultaneously expressed in yeast cells and their intracellular stoichiometry has to resemble the one found in the particles derived from mammalian or insect cells. This was achieved by stable co-transformation of yeast cells with two plasmids, one expressing VP3 from its natural p40 promoter and the other one primarily expressing VP1 from a modified AAV2 Cap gene under the control of the inducible yeast promoter Gal1. Among various induction strategies we tested, the best one to yield the appropriate VP1:VP3 ratio was 4.5 hour induction in the medium containing 0.5% glucose and 5% galactose. Following such induction, AAV virus like particles (VLPs were isolated from yeast by two step ultracentrifugation procedure. The transmission electron microscopy analysis revealed that their morphology is similar to the empty capsids produced in human cells. Conclusions Taken together, the results show for the first time that yeast can be used to assemble AAV capsid and, therefore, as a genetic system to identify novel cellular factors involved in AAV biology.

  18. Osteogenic differentiation of recombinant adeno-associated virus 2-transduced murine mesenchymal stem cells and development of an immunocompetent mouse model for ex vivo osteoporosis gene therapy.

    Science.gov (United States)

    Kumar, Sanjay; Mahendra, Gandham; Nagy, Tim R; Ponnazhagan, Selvarangan

    2004-12-01

    Gene therapy for osteopenic conditions including osteoporosis is a potential alternative to pharmacotherapy for cost effectiveness, long-term viability, and the ability to enhance bone mass by anabolic approaches. Increased understanding of mesenchymal stem cell (MSC) lineage differentiation during osteogenesis, and of the molecular pathways involved in bone cell production, provides an opportunity for the advancement of gene therapy approaches for osteopenic conditions. The potential of MSCs in osteoblast differentiation and the relative ease of MSC isolation and culturing offer a promising resource for the development of ex vivo gene therapy for bone defects. In an effort to develop ex vivo gene therapy for osteoporosis, we used gene-modified MSCs in a preclinical mouse model to determine the efficiency of transduction of murine MSCs by recombinant adeno-associated virus 2 (AAV) vectors carrying reporter genes and determined their osteogenic potential after recombinant AAV-mediated expression of bone morphogenic protein 2, known to induce osteoblast differentiation. Although surgical ovariectomy is believed to induce progressive bone loss in mouse models, similar to an osteoporosis-like phenotype in humans, several factors, including hormonal alteration and dietary habits, significantly affect both the onset and progression of the disease. Thus, in the present study, we determined the influence of these factors and developed an immunocompetent mouse model of osteoporosis with degenerative bone loss as in the human pathology.

  19. Hepatorenal correction in murine glycogen storage disease type I with a double-stranded adeno-associated virus vector.

    LENUS (Irish Health Repository)

    Luo, Xiaoyan

    2011-11-01

    Glycogen storage disease type Ia (GSD-Ia) is caused by the deficiency of glucose-6-phosphatase (G6Pase). Long-term complications of GSD-Ia include life-threatening hypoglycemia and proteinuria progressing to renal failure. A double-stranded (ds) adeno-associated virus serotype 2 (AAV2) vector encoding human G6Pase was pseudotyped with four serotypes, AAV2, AAV7, AAV8, and AAV9, and we evaluated efficacy in 12-day-old G6pase (-\\/-) mice. Hypoglycemia during fasting (plasma glucose <100 mg\\/dl) was prevented for >6 months by the dsAAV2\\/7, dsAAV2\\/8, and dsAAV2\\/9 vectors. Prolonged fasting for 8 hours revealed normalization of blood glucose following dsAAV2\\/9 vector administration at the higher dose. The glycogen content of kidney was reduced by >65% with both the dsAAV2\\/7 and dsAAV2\\/9 vectors, and renal glycogen content was stably reduced between 7 and 12 months of age for the dsAAV2\\/9 vector-treated mice. Every vector-treated group had significantly reduced glycogen content in the liver, in comparison with untreated G6pase (-\\/-) mice. G6Pase was expressed in many renal epithelial cells of with the dsAAV2\\/9 vector for up to 12 months. Albuminuria and renal fibrosis were reduced by the dsAAV2\\/9 vector. Hepatorenal correction in G6pase (-\\/-) mice demonstrates the potential of AAV vectors for the correction of inherited diseases of metabolism.

  20. Impact of capsid conformation and Rep-capsid interactions on adeno-associated virus type 2 genome packaging.

    Science.gov (United States)

    Bleker, Svenja; Pawlita, Michael; Kleinschmidt, Jürgen A

    2006-01-01

    Single-stranded genomes of adeno-associated virus (AAV) are packaged into preformed capsids. It has been proposed that packaging is initiated by interaction of genome-bound Rep proteins to the capsid, thereby targeting the genome to the portal of encapsidation. Here we describe a panel of mutants with amino acid exchanges in the pores at the fivefold axes of symmetry on AAV2 capsids with reduced packaging and reduced Rep-capsid interaction. Mutation of two threonines at the rim of the fivefold pore nearly completely abolished Rep-capsid interaction and packaging. This suggests a Rep-binding site at the highly conserved amino acids at or close to the pores formed by the capsid protein pentamers. A different mutant (P. Wu, W. Xiao, T. Conlon, J. Hughes, M. Agbandje-McKenna, T. Ferkol, T. Flotte, and N. Muzyczka, J. Virol. 74:8635-8647, 2000) with an amino acid exchange at the interface of capsid protein pentamers led to a complete block of DNA encapsidation. Analysis of the capsid conformation of this mutant revealed that the pores at the fivefold axes were occupied by VP1/VP2 N termini, thereby preventing DNA introduction into the capsid. Nevertheless, the corresponding capsids had more Rep proteins bound than wild-type AAV, showing that correct Rep interaction with the capsid depends on a defined capsid conformation. Both mutant types together support the conclusion that the pores at the fivefold symmetry axes are involved in genome packaging and that capsid conformation-dependent Rep-capsid interactions play an essential role in the packaging process.

  1. Activation of the cellular unfolded protein response by recombinant adeno-associated virus vectors.

    Directory of Open Access Journals (Sweden)

    Balaji Balakrishnan

    Full Text Available The unfolded protein response (UPR is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER. In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α, activating transcription factor 6 (ATF6 and PKR-like ER kinase (PERK in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold and PERK (up to 8 fold genes 12-48 hours after infection with self-complementary (scAAV2 but less prominent with single-stranded (ssAAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold while AAV6 vectors induced a significant increase on all the three major UPR pathways [6-16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5-2 fold in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively. However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.

  2. The X gene of adeno-associated virus 2 (AAV2) is involved in viral DNA replication.

    Science.gov (United States)

    Cao, Maohua; You, Hong; Hermonat, Paul L

    2014-01-01

    Adeno-associated virus (AAV) (type 2) is a popular human gene therapy vector with a long active transgene expression period and no reported vector-induced adverse reactions. Yet the basic molecular biology of this virus has not been fully addressed. One potential gene at the far 3' end of the AAV2 genome, previously referred to as X (nt 3929 to 4393), overlapping the 3' end of the cap gene, has never been characterized, although we did previously identify a promoter just up-stream (p81). Computer analysis suggested that X was involved in replication and transcription. The X protein was identified during active AAV2 replication using a polyclonal antibody against a peptide starting at amino acid 98. Reagents for the study of X included an AAV2 deletion mutant (dl78-91), a triple nucleotide substitution mutant that destroys all three 5' AUG-initiation products of X, with no effect on the cap coding sequence, and X-positive-293 cell lines. Here, we found that X up-regulated AAV2 DNA replication in differentiating keratinocytes (without helper virus, autonomous replication) and in various forms of 293 cell-based assays with help from wild type adenovirus type 5 (wt Ad5) or Ad5 helper plasmid (pHelper). The strongest contribution by X was seen in increasing wt AAV2 DNA replication in keratinocytes and dl78-91 in Ad5-infected X-positive-293 cell lines (both having multi-fold effects). Mutating the X gene in pAAV-RC (pAAV-RC-3Xneg) yielded approximately a ∼33% reduction in recombinant AAV vector DNA replication and virion production, but a larger effect was seen when using this same X-knockout AAV helper plasmid in X-positive-293 cell lines versus normal 293 cells (again, multi-fold). Taken together these data strongly suggest that AAV2 X encodes a protein involved in the AAV life cycle, particularly in increasing AAV2 DNA replication, and suggests that further studies are warranted.

  3. An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses.

    Science.gov (United States)

    Schreiber, Claire A; Sakuma, Toshie; Izumiya, Yoshihiro; Holditch, Sara J; Hickey, Raymond D; Bressin, Robert K; Basu, Upamanyu; Koide, Kazunori; Asokan, Aravind; Ikeda, Yasuhiro

    2015-08-01

    Adeno-associated viruses (AAV) have evolved to exploit the dynamic reorganization of host cell machinery during co-infection by adenoviruses and other helper viruses. In the absence of helper viruses, host factors such as the proteasome and DNA damage response machinery have been shown to effectively inhibit AAV transduction by restricting processes ranging from nuclear entry to second-strand DNA synthesis. To identify host factors that might affect other key steps in AAV infection, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A), a U2 snRNP-associated protein. Disruption of PHF5A expression selectively enhanced transgene expression from AAV by increasing transcript levels and appears to influence a step after second-strand synthesis in a serotype and cell type-independent manner. Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Notably, adenoviral co-infection and U2 snRNP inhibition appeared to target a common pathway in increasing expression from AAV vectors. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further analysis suggested that U2 snRNP proteins suppress AAV vector transgene expression through direct recognition of intact AAV capsids. In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression. Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors.

  4. An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses.

    Directory of Open Access Journals (Sweden)

    Claire A Schreiber

    2015-08-01

    Full Text Available Adeno-associated viruses (AAV have evolved to exploit the dynamic reorganization of host cell machinery during co-infection by adenoviruses and other helper viruses. In the absence of helper viruses, host factors such as the proteasome and DNA damage response machinery have been shown to effectively inhibit AAV transduction by restricting processes ranging from nuclear entry to second-strand DNA synthesis. To identify host factors that might affect other key steps in AAV infection, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A, a U2 snRNP-associated protein. Disruption of PHF5A expression selectively enhanced transgene expression from AAV by increasing transcript levels and appears to influence a step after second-strand synthesis in a serotype and cell type-independent manner. Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Notably, adenoviral co-infection and U2 snRNP inhibition appeared to target a common pathway in increasing expression from AAV vectors. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further analysis suggested that U2 snRNP proteins suppress AAV vector transgene expression through direct recognition of intact AAV capsids. In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression. Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors.

  5. Insight into the mechanism of inhibition of adeno-associated virus by the Mre11/Rad50/Nbs1 complex.

    Science.gov (United States)

    Lentz, Thomas B; Samulski, R Jude

    2015-01-01

    Adeno-associated virus (AAV) is a dependent virus of the family Parvoviridae. The gene expression and replication of AAV and derived recombinant AAV (rAAV) vectors are severely limited (>10-fold) by the cellular DNA damage-sensing complex made up of Mre11, Rad50, and Nbs1 (MRN). The AAV genome does not encode the means to circumvent this block to productive infection but relies on coinfecting helper virus to do so. Using adenovirus helper proteins E1B55k and E4orf6, which enhance the transduction of AAV via degradation of MRN, we investigated the mechanism through which this DNA damage complex inhibits gene expression from rAAV. We tested the substrate specificity of inhibition and the contribution of different functions of the MRN complex. Our results demonstrate that both single- and double-stranded rAAV vectors are inhibited by MRN, which is in contrast to the predominant model that inhibition is the result of a block to second-strand synthesis. Exploring the contribution of known functions of MRN, we found that inhibition of rAAV does not require downstream DNA damage response factors, including signaling kinases ATM and ATR. The nuclease domain of Mre11 appears to play only a minor role in inhibition, while the DNA binding domain makes a greater contribution. Additionally, mutation of the inverted terminal repeat of the rAAV genome, which has been proposed to be the signal for interaction with MRN, is tolerated by the mechanism of inhibition. These results articulate a model of inhibition of gene expression in which physical interaction is more important than enzymatic activity and several key downstream damage repair factors are dispensable. Many viruses modulate the host DNA damage response (DDR) in order to create a cellular environment permissive for infection. The MRN complex is a primary sensor of damage in the cell but also responds to invading viral genomes, often posing a block to infection. AAV is greatly inhibited by MRN and dependent on coinfecting

  6. CONSTRUCTING ADENO-ASSOCIATED VIRUS-TGFp3 AND COMPARING ITS BIOLOGICAL EFFECT ON PROTEOGLYCAN SYNTHESIS IN DEDIFFERENTIATED NUCLEUS PULPOUS CELLS WITH ADENOVIRUS-TGFβ1

    Institute of Scientific and Technical Information of China (English)

    Jia-ming Sai; You-gu Hu; De-chun Wang

    2007-01-01

    Objective To construct adeno-associated virus (AAV) expression system for transforming growth factor β3 (TGFβ3) and detect its biological effect on proteoglycan synthesis of the earlier and later dedifferentiated rabbit lumbar disc nucleus pulpous (NP) cells, which was compared with that of adenovirus (AV) expression system for TGFβ1.Methods TGFβ3 gene was obtained using PCR. Its upstream contained restriction enzyme site Kpn Ⅰ, and its downstream contained restriction enzyme site Sal Ⅰ. Using the restriction enzyme sites of PCR product of TGFβ3 and the corresponding multiple cloning site (MCS) in plasmid AAV, TGFβ3 was subcloned into AAV. The recombinant plas-mid AAV-TGFβ3 was transfected into H293 cells with Lipofectamine(tm) 2000, and the expression of TGFβ3 gene was detected using immunofluorescent analysis. After AAV-TGFβ3 virus particle with infectious activity was packaged, TGFβ3 expression in NP cells was detected by immunoblotting, and its biological effect on proteoglycan synthesis was detected by antonopulos method and compared with that of AV-TGFβ, in the earlier and later dedifferentiated NP cells.Results For the earlier dedifferentiated NP cells, AAV-TGFβ slowly and stably enhanced proteoglycan synthesis, but AV-TGFβ, rapidly and transiently enhanced its synthesis. For the later dedifferentiated NP cells, AAV-TGFβ3 stably enhanced proteoglycan synthesis, but AV-TGFβ, inhibited its synthesis.Conclusion AAV expression system can mediate TGFβ3 gene to be expressed stably, and AAV-TGFβ3 can enhance proteoglycan synthesis of the earlier and later dedifferentiated NP cells.

  7. Adeno-Associated Vector mediated gene transfer of Transforming Growth Factor-beta1 to normal and osteoarthritic human chondrocytes stimulates cartilage anabolism

    Directory of Open Access Journals (Sweden)

    Ulrich-Vinther M.

    2005-11-01

    Full Text Available The objective of the present study was to investigate whether cartilage anabolism in human primary osteoarthritic chondrocytes could be improved by adeno-associated virus (AAV vector-mediated gene transduction of transforming growth factor TGF-beta1 (TGF-beta1. A bi-cistronic AAV-TGF-beta1-IRES-eGFP (AAV-TGF-beta1 vector was generated and used for transduction of a normal human articular chondrocyte cell line (tsT/AC62 and primary human osteoarthritic articular chondrocytes harvested from 8 patients receiving total knee joint arthroplasty. Transduction efficiency was detected by fluorescent microscopy for gene expression of enhanced green fluorescent protein (eGFP. TGF-beta1 synthesis was determined by ELISA. To assess the influence of TGF-beta1 gene therapy on chondrocyte cartilage metabolism, mRNA expressions of type II collagen, aggrecan, and matrix metalloproteinase 3 (MMP-3 were determined by quantitative real-time PCR. AAV-TGF-beta1 transduction resulted in increased synthesis of TGF-beta1 in both osteoarthritic chondrocytes and the normal articular chondrocyte cell line. The expression levels of the transduced genes were correlated to "multiplicity of infection" (MOI and post-infectious time. In both osteoarthritic chondrocytes and the normal articular chondrocyte cell line, AAV-TGF-beta1 treatment increased mRNA expression of both type II collagen and aggrecan, but decreased MMP-3 mRNA expression. Osteoarthritic chondrocytes and the normal articular chondrocyte cell line could be transduced with equal efficiencies. In conclusion, it was demonstrated that AAV-TGF-beta1 gene transfer stimulates cartilage anabolism and decreases expression of enzymes responsible for cartilage degradation in human osteoarthritic chondrocytes. The results indicate that the AAV vector is an efficient mediator of growth factors to human articular chondrocytes, and that it might be useful in future chondrocyte gene therapy.

  8. 重组腺相关病毒介导的MDA-7基因对人肝癌细胞的选择性凋亡诱导效应%Selective apoptosis-inducing effect of adeno-associated virus mediated gene transfer of MDA-7 on human hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    徐波; 朱光辉; 夏金堂; 翁杰锋; 李书华; 李雯

    2008-01-01

    目的 探讨PEG启动子调控腺相关病人毒介导的黑色素瘤分化相关基因MDA-7对肝癌细胞的选择性凋亡诱导效应.方法 以重组腺相关病人毒rAAV-PEG-MDA-7表达系统感染人肝癌细胞株HepG2细胞和正常人肝细胞株LO2细胞,Westerm Blot检测转染细胞内MDA-7蛋白,MTT法检测细胞增殖抑制率,流式细胞术分析细胞周期、Annexin-Ⅴ、线粒体跨膜电位(△Ψm),RT-PCR检测bcl-2基因mRNA.结果 重组腺相关病毒rAAV-PEG-MDA7可特异性转染HepG2细胞,MDA7蛋白在HepG2细胞中高效表达,并呈时间依赖性;重组腺相关病毒rAAV-PEC-MDA-7可抑制HepG2细胞增殖并诱导其凋亡,细胞周期分析处于G0/G1期细胞百分比明显增多,处于G2/M期的细胞减少,并且可见到较明显的凋亡峰的形成,从24 h开始Annexin-Ⅴ阳性细胞比例增多,△Ψm降低,抗凋亡基因bcl-2 mRNA表达降低.而重组腺相关病毒rAAV-PEG-MDA-7对LO2细胞无类似效应.结论 构建出的重组腺相关病毒rAAV-PEG-MDA-7表达系统可选择性抑制肝癌细胞增殖和诱导其凋亡,其诱导凋亡机制受到bcl-2家族经线粒体途径的调节.%Objective To investigate the selective apoptosis-inducing effect of adeno-associated virus mediated gene transfer of MDA-7 regulated by PEG promoter on human hepatocellular carcinoma cells.Methods We transduced human hepatocellular carcinoma cell line HepG2 and normal human hepatocytes LO2 with rAAV-PEG-MDA-7 and assessed the cell growth inhibition,cell cycle and apoptosis.MDA-7 protein was detected by Western blotting,cell growth-inhibiting rate evaluated by MTT and cell cycle, Annexin-Ⅴ labeling and mitochondrial transmembrane potential were determined by flow eytometry.The expression of bcl-2 mRNA was determined bv RT-PCR.Results rAAV-PEG-MDA-7 was effectively transfected into HepG2 cells.MDA-7 protein was highly expressed in HepG2 cells in a time-dependent manner.rAAV-PEG-MDA-7 suppressed HepG2 cell growth.The cells

  9. Activation of the NF-kappaB pathway by adeno-associated virus (AAV) vectors and its implications in immune response and gene therapy.

    Science.gov (United States)

    Jayandharan, Giridhara R; Aslanidi, George; Martino, Ashley T; Jahn, Stephan C; Perrin, George Q; Herzog, Roland W; Srivastava, Arun

    2011-03-01

    Because our in silico analysis with a human transcription factor database demonstrated the presence of several binding sites for NF-κB, a central regulator of cellular immune and inflammatory responses, in the adeno-associated virus (AAV) genome, we investigated whether AAV uses NF-κB during its life cycle. We used small molecule modulators of NF-κB in HeLa cells transduced with recombinant AAV vectors. VP16, an NF-κB activator, augmented AAV vector-mediated transgene expression up to 25-fold. Of the two NF-κB inhibitors, Bay11, which blocks both the canonical and the alternative NF-κB pathways, totally ablated transgene expression, whereas pyrrolidone dithiocarbamate, which interferes with the classical NF-κB pathway, had no effect. Western blot analyses confirmed the abundance of the nuclear p52 protein component of the alternative NF-κB pathway in the presence of VP16, which was ablated by Bay11, suggesting that AAV transduction activates the alternative NF-κB pathway. In vivo, hepatic AAV gene transfer activated the canonical NF-κB pathway within 2 h, resulting in expression of proinflammatory cytokines and chemokines (likely reflecting the sensing of viral particles by antigen-presenting cells), whereas the alternative pathway was activated by 9 h. Bay11 effectively blocked activation of both pathways without interfering with long-term transgene expression while eliminating proinflammatory cytokine expression. These studies suggest that transient immunosuppression with NF-κB inhibitors before transduction with AAV vectors should lead to a dampened immune response, which has significant implications in the optimal use of AAV vectors in human gene therapy.

  10. Recombinant adeno-associated virus serotype 9 with p65 ribozyme protects H9c2 cells from oxidative stress through inhibiting NF-κB signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Zhan SUN; Yi-Tong MA; Bang-Dang CHEN; Fen LIU

    2014-01-01

    Background Oxidative stress is a major mechanism underlying the pathogenesis of cardiovascular disease. It can trigger inflammatory cascades which are primarily mediated via nuclear factor-κB (NF-κB). The NF-κB transcription factor family includes several subunits (p50, p52, p65, c-Rel, and Rel B) that respond to myocardial ischemia. It has been proved that persistent myocyte NF-κB p65 activation in heart failure exacerbates cardiac remodeling. Mechods A recombinant adeno-associated virus serotype 9 carrying enhanced green fluorescent protein and anti-NF-κB p65 ribozyme (AAV9-R65-CMV-eGFP) was constructed. The cells were assessed by MTT assay, Annexin V–propidium iodide dual staining to study apoptosis. The expression of P65 and P50 were assessed by Western blot to investigate the under-lying molecular mechanisms. Results After stimulation with H2O2 for 6 h, H9c2 cells viability decreased significantly, a large fraction of cells underwent apoptosis. We observed a rescue of H9c2 cells from H2O2-induced apoptosis in pretreatment with AAV9-R65-CMV-eGFP. Moreover, AAV9-R65-CMV-eGFP decreased H2O2-induced P65 expression. Conclusions AAV9-R65-CMV-eGFP protects H9c2 cells from oxidative stress induced apoptosis through down-regulation of P65 expression. These observations indicate that AAV9-R65-CMV-eGFP has the potential to exert cardioprotective effects against oxidative stress, which might be of great importance to clinical efficacy for cardiovascular disease.

  11. 重组腺相关病毒神经肽Y基因转染对癫(癎)大鼠海马病理变化的影响%Effect of recombinant adeno-associated virus-mediated human-derived neuropeptide Y gene transfection on pathological change of the hippocampus in epileptic rat

    Institute of Scientific and Technical Information of China (English)

    董长征; 董秀芳; 李文玲; 岳向勇; 郭韬; 梁传栋; 赵文清

    2012-01-01

    目的 观察重组腺相关病毒介导人源性神经肽Y(rAA V-hNPY-EGFP)基因转染对癫(癎)大鼠海马病理变化的影响.方法 28只Wistar大鼠随机分为点燃组(n=20)和正常对照组(n=8).正常对照组不进行特殊处理,点燃组以大鼠海马内多次注射红藻氨酸(KA)建立慢性癫(癎)模型,造模成功16只,其随机分为模型组和神经肽Y(NPY)治疗组,每组各8只大鼠.NPY治疗组大鼠转染rAA V2/I- hNPY-EGFP基因,模型组未转染.转染4周后,每组取6只大鼠海马行苏木精-伊红染色,2只行电镜观察.结果 苏木精-伊红染色显示:正常对照组大鼠海马CA3区神经元形态正常;模型组海马CA3区神经元丢失,胶质细胞增生;NPY治疗组基因转染后神经元丢失减少.模型组神经元数目为(10.67±7.87)个/视野,正常对照组为(81.42±5.63)个/视野,明显多于模型组(P<0.05);而NPY治疗组神经元数目为(65.73±2.81)个/视野,明显多于模型组(P<0.05).电镜显示:正常对照组神经元结构正常;模型组神经元固缩,线粒体肿胀;NPY治疗组神经元线粒体结构完整.结论 rAA V-hNPY-EGFP基因转染可减轻大鼠癫(癎)发作引起的病理改变,发挥抑制癫(癎)的作用.%Objective To investigate the effect of recombinant adeno-associated virus-mediated human-derived neuropeptide Y gene (rAAV-hNPY-EGFP gene) transfection on pathological change of hippocampus in epileptic rat. Methods A total of 28 Wistar rats were randomly divided into kindling group (n=20) and normal control group (n=8). No special treatment was performed on rats in normal control group. The chronic epileptic models were successfully established in 16 rats by repeated injection of kainic acid into the hippocampi of rats in kindling group which were equally subdivided into two groups: model group and neuropeptide Y (NPY) treatment group. The rats were transfected with rAAV2/1-hNPV-EGFP gene in NPY treatment group and no transfection was made in model

  12. Inhibition of herpes simplex virus type 1 replication by adeno-associated virus rep proteins depends on their combined DNA-binding and ATPase/helicase activities.

    Science.gov (United States)

    Glauser, Daniel L; Seyffert, Michael; Strasser, Regina; Franchini, Marco; Laimbacher, Andrea S; Dresch, Christiane; de Oliveira, Anna Paula; Vogel, Rebecca; Büning, Hildegard; Salvetti, Anna; Ackermann, Mathias; Fraefel, Cornel

    2010-04-01

    Adeno-associated virus (AAV) has previously been shown to inhibit the replication of its helper virus herpes simplex virus type 1 (HSV-1), and the inhibitory activity has been attributed to the expression of the AAV Rep proteins. In the present study, we assessed the Rep activities required for inhibition of HSV-1 replication using a panel of wild-type and mutant Rep proteins lacking defined domains and activities. We found that the inhibition of HSV-1 replication required Rep DNA-binding and ATPase/helicase activities but not endonuclease activity. The Rep activities required for inhibition of HSV-1 replication precisely coincided with the activities that were responsible for induction of cellular DNA damage and apoptosis, suggesting that these three processes are closely linked. Notably, the presence of Rep induced the hyperphosphorylation of a DNA damage marker, replication protein A (RPA), which has been reported not to be normally hyperphosphorylated during HSV-1 infection and to be sequestered away from HSV-1 replication compartments during infection. Finally, we demonstrate that the execution of apoptosis is not required for inhibition of HSV-1 replication and that the hyperphosphorylation of RPA per se is not inhibitory for HSV-1 replication, suggesting that these two processes are not directly responsible for the inhibition of HSV-1 replication by Rep.

  13. Adeno-associated virus type 2 infection activates caspase dependent and independent apoptosis in multiple breast cancer lines but not in normal mammary epithelial cells

    Directory of Open Access Journals (Sweden)

    Tandon Apurva

    2011-08-01

    Full Text Available Abstract Background In normal cells proliferation and apoptosis are tightly regulated, whereas in tumor cells the balance is shifted in favor of increased proliferation and reduced apoptosis. Anticancer agents mediate tumor cell death via targeting multiple pathways of programmed cell death. We have reported that the non-pathogenic, tumor suppressive Adeno-Associated Virus Type 2 (AAV2 induces apoptosis in Human Papillomavirus (HPV positive cervical cancer cells, but not in normal keratinocytes. In the current study, we examined the potential of AAV2 to inhibit proliferation of MCF-7 and MDA-MB-468 (both weakly invasive, as well as MDA-MB-231 (highly invasive human breast cancer derived cell lines. As controls, we used normal human mammary epithelial cells (nHMECs isolated from tissue biopsies of patients undergoing breast reduction surgery. Results AAV2 infected MCF-7 line underwent caspase-independent, and MDA-MB-468 and MDA-MB-231 cell lines underwent caspase-dependent apoptosis. Death of MDA-MB-468 cells was marked by caspase-9 activation, whereas death of MDA-MB-231 cells was marked by activation of both caspase-8 and caspase-9, and resembled a mixture of apoptotic and necrotic cell death. Cellular demise was correlated with the ability of AAV2 to productively infect and differentially express AAV2 non-structural proteins: Rep78, Rep68 and Rep40, dependent on the cell line. Cell death in the MCF-7 and MDA-MB-231 lines coincided with increased S phase entry, whereas the MDA-MB-468 cells increasingly entered into G2. AAV2 infection led to decreased cell viability which correlated with increased expression of proliferation markers c-Myc and Ki-67. In contrast, nHMECs that were infected with AAV2 failed to establish productive infection or undergo apoptosis. Conclusion AAV2 regulated enrichment of cell cycle check-point functions in G1/S, S and G2 phases could create a favorable environment for Rep protein expression. Inherent Rep associated

  14. Intrathecal long-term gene expression by self-complementary adeno-associated virus type 1 suitable for chronic pain studies in rats

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    Janssen William GM

    2006-01-01

    Full Text Available Abstract Background Intrathecal (IT gene transfer is an attractive approach for targeting spinal mechanisms of nociception but the duration of gene expression achieved by reported methods is short (up to two weeks impairing their utility in the chronic pain setting. The overall goal of this study was to develop IT gene transfer yielding true long-term transgene expression defined as ≥ 3 mo following a single vector administration. We defined "IT" administration as atraumatic injection into the lumbar cerebrospinal fluid (CSF modeling a lumbar puncture. Our studies focused on recombinant adeno-associated virus (rAAV, one of the most promising vector types for clinical use. Results Conventional single stranded rAAV2 vectors performed poorly after IT delivery in rats. Pseudotyping of rAAV with capsids of serotypes 1, 3, and 5 was tested alone or in combination with a modification of the inverted terminal repeat. The former alters vector tropism and the latter allows packaging of self-complementary rAAV (sc-rAAV vectors. Combining both types of modification led to the identification of sc-rAAV2/l as a vector that performed superiorly in the IT space. IT delivery of 3 × 10e9 sc-rAAV2/l particles per animal led to stable expression of enhanced green fluorescent protein (EGFP for ≥ 3 mo detectable by Western blotting, quantitative PCR, and in a blinded study by confocal microscopy. Expression was strongest in the cauda equina and the lower sections of the spinal cord and only minimal in the forebrain. Microscopic examination of the SC fixed in situ with intact nerve roots and meninges revealed strong EGFP fluorescence in the nerve roots. Conclusion sc-rAAVl mediates stable IT transgene expression for ≥ 3 mo. Our findings support the underlying hypothesis that IT target cells for gene transfer lack the machinery for efficient conversion of the single-stranded rAAV genome into double-stranded DNA and favor uptake of serotype 1 vectors over 2

  15. Bac-to-Bac杆状病毒昆虫表达系统介导的重组腺相关病毒制备%Production of adeno-associated virus mediated by Bac-to-Bac baculovirus insect expression system

    Institute of Scientific and Technical Information of China (English)

    陈雅宁; 王春荣; 杨宗灿; 姬朝霞; 张延瑞; 韩双印

    2012-01-01

    目的 建立高效的临床级重组腺相关病毒( rAAV)制备方法.方法 将rAAV的反向末端重复序列( ITR)及目的基因表达框(2053 bp)从pAAV-MCS剪切引入杆状病毒载体中构建顺式载体,rAAV血清型2的衣壳和复制蛋白基因在反式载体pFastBacDual中利用真核基因遗漏扫描原理表达,两者分别制备成杆状病毒,共感染昆虫细胞sf9包装rAAV.增强绿色荧光蛋白(EGFP)作为报告基因验证其可行性.结果 顺式和反式杆状病毒载体分别在DH 10BacTM中转座形成杆粒bacmid、昆虫细胞sf9中制备成杆状病毒,病毒滴度可达1×108~3 ×108 v.g./ml.二杆状病毒共感染昆虫细胞sf9,完成rAAV拯救、复制和包装过程,经rAAV-EGFP感染293T细胞测试具有良好的生物学活性.结论 该系统具有高效、安全、简便等特点,为临床级rAAV制备和基因治疗奠定了基础.%Objective To establish an efficient and safe method for production of recombinant adeno-associated viruses (rAAV) of clinical scale.Methods The 2053 bp of rAAV inverted terminal repeat (ITR) and foreign gene expression cassette was cloned into baculovirus vector as cis-acting vector.The capsid and replication proteins of rAAV serum type 2 were expressed in a trans-acting baculovirus vector (pFastBacDual) by leaky scanning mechanism of eukaryotic gene transcription.Both constructs were made into baculovirus which infected insect cells st9 for packaging rAAV.Enhanced green fluorescent protein (EGFP) was used as reporter gene to verify the feasibility of system.Results Transposition of cis-acting and trans-acting vector into bacmid was carried out in DH10BacTM reswctively.Two baculoviruses were prepared in insect cells sf9 and the titers were about 1×108-3×108 v.g./mL.rAAV were rescued,replicated and packaged by co-infection of two baculoviruses into sf9.The biological activity of rAAV-EGFP was proved by infecting 293T cells.Conclusion This system provides an experimental data for making

  16. Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

    Institute of Scientific and Technical Information of China (English)

    LU; Huazhong; (

    2001-01-01

    [1]Chang, J., Jin, J., Lollar, P. et al., Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity, J. Bio. Chem., 1998, 273 (20): 12089-12094.[2]Lai, L., Chen, L., Zhou, H. et al., Clinical phenotype and genetic stability of factor IX gene knock out mice, J. Fudan Uni., 1999, 38 (4): 435-438.[3]Wu, Z. J., Wu, X. B., Hou, Y. D., Generation of a recombinant herps simplex virus which can provide packaging function for recombinant adeno-associated virus, Chinese Sci. Bull., 1999, 44 (8): 715-719.[4]Snyder, R. O., Miao, C. H., Patijn, G. A. et al., Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors, Nat. Genet., 1997, 16 (3): 270-276.[5]Lai, L. H., Chen, L., Wang, J. M. et al., Skeletal muscle-specific expression of human blood coagulation factor IX rescues factor IX deficiency mouse by AAV-mediated gene transfer, Science in China, Ser. C, 1999, 42 (6): 628-634.[6]Snyder, R. O., Miao, C., Meuse, L. et al., Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors, Nat. Med., 1999, 5 (1): 64-70.[7]Kung, S. H., Hagstrom, J. N., Cass, D. et al., Human factor IX corrects the bleeding diathesis of mice with hemophilia B, Blood, 1998, 91(3): 784-790.[8]Hirt, B., Selective extraction of polyoma DNA from infected mouse cell culture, J. Mol. Biol., 1967, 26: 365-369.[9]Sambrook, J., Fritsch, E., Maniatis, T., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, 6, 20-21.[10]Chao, H., Samulski, R. J., Bellinger, D. A. et al., Persistent expression of canine factor IX in hemophilia B canines, Gene Ther., 1999, 6: 1695-1704.[11]Kaufman, R. J., Advances toward gene therapy for hemophilia at the millennium, Hum. Gene Ther., 1999, 10 (13): 2091-2107.[12]Lu, D. R., Zhou, J. M., Zheng, B. et al., Stage I clinical trial of gene

  17. Characterization of Adeno-Associated Viral Vector-Mediated Human Factor VIII Gene Therapy in Hemophilia A Mice.

    Science.gov (United States)

    Greig, Jenny A; Wang, Qiang; Reicherter, Amanda L; Chen, Shu-Jen; Hanlon, Alexandra L; Tipper, Christopher H; Clark, K Reed; Wadsworth, Samuel; Wang, Lili; Wilson, James M

    2017-05-01

    Adeno-associated viral (AAV) vectors are promising vehicles for hemophilia gene therapy, with favorable clinical trial data seen in the treatment of hemophilia B. In an effort to optimize the expression of human coagulation factor VIII (hFVIII) for the treatment of hemophilia A, an extensive study was performed with numerous combinations of liver-specific promoter and enhancer elements with a codon-optimized hFVIII transgene. After generating 42 variants of three reduced-size promoters and three small enhancers, transgene cassettes were packaged within a single AAV capsid, AAVrh10, to eliminate performance differences due to the capsid type. Each hFVIII vector was administered to FVIII knockout (KO) mice at a dose of 10(10) genome copies (GC) per mouse. Criteria for distinguishing the performance of the different enhancer/promoter combinations were established prior to the initiation of the studies. These criteria included prominently the level of hFVIII activity (0.12-2.12 IU/mL) and the pattern of development of anti-hFVIII antibodies. In order to evaluate the impact of capsid on hFVIII expression and antibody formation, one of the enhancer and promoter combinations that exhibited high hFVIII immunogenicity was evaluated using AAV8, AAV9, AAVrh10, AAVhu37, and AAVrh64R1 capsids. The capsids subdivided into two groups: those that generated anti-hFVIII antibodies in ≤20% of mice (AAV8 and AAV9), and those that generated anti-hFVIII antibodies in >20% of mice (AAVrh10, AAVhu37, and AAVrh64R1). The results of this study, which entailed extensive vector optimization and in vivo testing, demonstrate the significant impact that transcriptional control elements and capsid can have on vector performance.

  18. Sodium Chloride Enhances Recombinant Adeno-Associated Virus Production in a Serum-Free Suspension Manufacturing Platform Using the Herpes Simplex Virus System.

    Science.gov (United States)

    Adamson-Small, Laura; Potter, Mark; Byrne, Barry J; Clément, Nathalie

    2017-02-01

    The increase in effective treatments using recombinant adeno-associated viral (rAAV) vectors has underscored the importance of scalable, high-yield manufacturing methods. Previous work from this group reported the use of recombinant herpes simplex virus type 1 (rHSV) vectors to produce rAAV in adherent HEK293 cells, demonstrating the capacity of this system and quality of the product generated. Here we report production and optimization of rAAV using the rHSV system in suspension HEK293 cells (Expi293F) grown in serum and animal component-free medium. Through adjustment of salt concentration in the medium and optimization of infection conditions, titers greater than 1 × 10(14) vector genomes per liter (VG/liter) were observed in purified rAAV stocks produced in Expi293F cells. Furthermore, this system allowed for high-titer production of multiple rAAV serotypes (2, 5, and 9) as well as multiple transgenes (green fluorescent protein and acid α-glucosidase). A proportional increase in vector production was observed as this method was scaled, with a final 3-liter shaker flask production yielding an excess of 1 × 10(15) VG in crude cell harvests and an average of 3.5 × 10(14) total VG of purified rAAV9 material, resulting in greater than 1 × 10(5) VG/cell. These results support the use of this rHSV-based rAAV production method for large-scale preclinical and clinical vector production.

  19. Successful expansion but not complete restriction of tropism of adeno-associated virus by in vivo biopanning of random virus display peptide libraries.

    Directory of Open Access Journals (Sweden)

    Stefan Michelfelder

    Full Text Available Targeting viral vectors to certain tissues in vivo has been a major challenge in gene therapy. Cell type-directed vector capsids can be selected from random peptide libraries displayed on viral capsids in vitro but so far this system could not easily be translated to in vivo applications. Using a novel, PCR-based amplification protocol for peptide libraries displayed on adeno-associated virus (AAV, we selected vectors for optimized transduction of primary tumor cells in vitro. However, these vectors were not suitable for transduction of the same target cells under in vivo conditions. We therefore performed selections of AAV peptide libraries in vivo in living animals after intravenous administration using tumor and lung tissue as prototype targets. Analysis of peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues. The selected clones indeed conferred gene expression in the target tissue while gene expression was undetectable in animals injected with control vectors. However, all of the vectors selected for tumor transduction also transduced heart tissue and the vectors selected for lung transduction also transduced a number of other tissues, particularly and invariably the heart. This suggests that modification of the heparin binding motif by target-binding peptide insertion is necessary but not sufficient to achieve tissue-specific transgene expression. While the approach presented here does not yield vectors whose expression is confined to one target tissue, it is a useful tool for in vivo tissue transduction when expression in tissues other than the primary target is uncritical.

  20. Effects of adeno-associated virus-mediated klotho gene delivery on the expression of runx2 and MMP-13 gene in the bone of the ovariectomy rats%腺相关病毒介导的klotho基因表达对去势大鼠骨Runx2及MMP-13表达的影响

    Institute of Scientific and Technical Information of China (English)

    王艳娇; 马厚勋; 李宝善; 吴平

    2012-01-01

    目的 探讨腺相关病毒介导的klotho(KL)基因表达对去势骨质疏松大鼠的调控作用.方法 SD雌性大鼠随机分为假手术组(S组)和手术组,外科去势术后12周再随机分为模型组(O组)、17β-雌二醇组(E组)、KL基因组(KO组)和空质粒组(GO组),实验12周后处死.取股骨、胫骨测骨密度;冰冻切片及免疫组化法观察肾KL荧光及KL蛋白表达;RT-PCR和免疫组化法检测骨Runx2、MMP-13 mRNA及蛋白表达;HE染色观察骨组织形态学变化.结果 KO组和E组骨密度高于O组和GO组(P<0.05);KO组大鼠肾有小鼠KL基因特异性表达;与O组相比,KO组Runx2 mRNA表达明显上调,MMP-13 mRNA表达显著下调(P<0.05);免疫组化分析KO组Runx2吸光度值为411±96,显著高于O组的353±50(P<0.05);KO组MMP-13吸光度值为397±84,显著低于O组的656±89(P<0.05).KO组、E组和S组大鼠骨小梁排列紧密,连接成网,形态结构较完整,明显优于O组和GO组.结论 KL基因表达上调可减缓去势大鼠骨质疏松症的发展及骨组织微结构的破坏,提示KL基因可能在骨质疏松症的发展中扮演重要角色.%Objective To research the effect of the recombinant adeno-associated virus vector containing klotho gene delivery on the regulating of osteoporosis in ovariectomized rats. Methods Female SD rats were randomly divided into sham operation group (S group) and model group. Model was successfully constructed with ovariectomy after 12 weeks,they were randomly divided into model group (0 group), 17(β-estradiol (E group), klotho gene group (K0 group), empty vector group (GO group), all were sacrificed after 12 weeks. Bone mineral density (BMD) of the femurs and tibia were measured. The fluorescent expression of renal klotho was observed by Cryo-sectioning technique. The Runx2 and MMP-13 mRNA expression of bone tissue were detected by reverse transcription-polymerase chain reaction(RT-PCR). Expression of klotho protein in kidney and Runx

  1. Effect of hydroxyurea and etoposide on transduction of human bone marrow mesenchymal stem and progenitor cell by adeno-associated virus vectors

    Institute of Scientific and Technical Information of China (English)

    Xiao-dong JU; Si-quan LOU; Wei-guo WANG; Jian-qiang PENG; Hua TIAN

    2004-01-01

    AIM: To study the effect of hydroxyurea and etoposide on transduction of human marrow mesenchymal and progenitor stem cells by adeno-associated virus (AAV). METHODS: Isolated human bone marrow mesenchymal stem and progenitor cells (hMSCs) were cultured in DMEM containing 10 % FBS or 5 % FBS and dexamethasone 1 μmol/L respectively. After being treated with hydroxyurea and etoposide, hMSCs were transduced by AAV-LUC.After two days luciferase activity (relative light unites per second or RLU/s) were tested, which indirectly reflected the relative transduction efficiency of different groups, and virus DNA was isolated by Hirt extraction for Southern hybridization. RESULTS: Transduction luciferase activity and transduction efficiency in cultures treated with hydroxyurea and etoposide were significantly higher than that in control cultures. Dividing cells had about 20-fold higher transduction efficiency compared with control cells. Transduction efficiency in stationary cells was about 50 times higher than that in control cells. Southern analysis showed that hydroxyurea and etoposide enhanced second-strand DNA synthesis by rAAV. CONCLUSION: Hydroxyurea and etoposide could increase transduction efficiency of hMSCs by AAV vectors, and stationary cells were more sensitive to these drugs than dividing cells.

  2. A Regulatory Element Near the 3′ End of the Adeno-Associated Virus rep Gene Inhibits Adenovirus Replication in cis by Means of p40 Promoter-Associated Short Transcripts

    Science.gov (United States)

    Hammer, Eva; Gonsior, Melanie; Stutika, Catrin; Heilbronn, Regine

    2016-01-01

    ABSTRACT Adeno-associated virus (AAV) has long been known to inhibit helper adenovirus (Ad) replication independently of AAV Rep protein expression. More recently, replication of Ad serotype 5 (Ad5)/AAV serotype 2 (AAV-2) hybrid vectors was shown to be inhibited in cis by a sequence near the 3′ end of AAV rep, termed the Rep inhibition sequence for adenoviral replication (RIS-Ad). RIS-Ad functions independently of Rep protein expression. Here we demonstrate that inhibition of adenoviral replication by RIS-Ad requires an active AAV p40 promoter and the 5′ half of the intron. In addition, Ad inhibition is critically dependent on the integrity of the p40 transcription start site (TSS) leading to short p40-associated transcripts. These do not give rise to effector molecules capable of inhibiting adenoviral replication in trans, like small polypeptides or microRNAs. Our data point to an inhibitory mechanism in which RNA polymerase II (Pol II) pauses directly downstream of the p40 promoter, leading to interference of the stalled Pol II transcription complex with the adenoviral replication machinery. Whereas inhibition by RIS-Ad is mediated exclusively in cis, it can be overcome by providing a replication-competent adenoviral genome in trans. Moreover, the inhibitory effect of RIS-Ad is not limited to AAV-2 but could also be shown for the corresponding regions of other AAV serotypes, including AAV-5. These findings have important implications for the future generation of Ad5/AAV hybrid vectors. IMPORTANCE Insertion of sequences from the 3′ part of the rep gene of adeno-associated virus (AAV) into the genome of its helper adenovirus strongly reduces adenoviral genome replication. We could show that this inhibition is mediated exclusively in cis without the involvement of trans-acting regulatory RNAs or polypeptides but nevertheless requires an active AAV-2 p40 promoter and p40-associated short transcripts. Our results suggest a novel inhibitory mechanism that has so

  3. Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer.

    Directory of Open Access Journals (Sweden)

    Lina Li

    Full Text Available Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or "CELiD", DNA. CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5 × 10(9 Sf9 cells, and 1-15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.

  4. Prevalence of neutralizing antibodies against liver-tropic adeno-associated virus serotype vectors in 100 healthy Chinese and its potential relation to body constitutions

    Institute of Scientific and Technical Information of China (English)

    Chen Ling; Irene Zolotukhin; Arun Srivastava; Chang-quan Ling; Yuan Wang; Ying-lu Feng; Ya-ni Zhang; Jun Li; Xin-rui Hu; Li-na Wang; Mao-feng Zhong; Xiao-feng Zhai

    2015-01-01

    Recombinant adeno-associated virus (rAAV) serotype 2, 3 and 8 vectors are the most promising liver-tropic AAV serotype vectors. Liver diseases are signiifcant problems in China. However, to date, few studies on AAV neutralizing antibodies (Nabs) were working with the Chinese population or with the rAAV3 vectors. The present study aimed to determine the prevalence of Nabs in Chinese population against wild-type AAV2, AAV3 and AAV8 capsids as wel as additional two AAV3 variants. In addition, we performed a preliminary analysis to investigate the potential inlfuence of traditional Chinese medicine body constitutions on AAV Nabs. Our work demonstrated that the majority of healthy Chinese subjects were positive for AAV Nabs, with the order of AAV2 > AAV3 = AAVLK03 > AAV8. There was no difference between: 1) AAV3 and its variants; 2) male and female subjects; and 3) different age cohorts (≤ 35, 36–50, and ≥ 51 years old). People in the Qi-deifciency constitution had signiifcantly increased AAV8 Nabs than people in the Gentleness constitution. Our studies may have impact on the future clinical design of AAV-based gene therapy in the Chinese population.

  5. Ex vivo intracoronary gene transfer of adeno-associated virus 2 leads to superior transduction over serotypes 8 and 9 in rat heart transplants.

    Science.gov (United States)

    Raissadati, Alireza; Jokinen, Janne J; Syrjälä, Simo O; Keränen, Mikko A I; Krebs, Rainer; Tuuminen, Raimo; Arnaudova, Ralica; Rouvinen, Eeva; Anisimov, Andrey; Soronen, Jarkko; Pajusola, Katri; Alitalo, Kari; Nykänen, Antti I; Lemström, Karl

    2013-11-01

    Heart transplant gene therapy requires vectors with long-lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno-associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar-Furth (WF) recipients. Serial in vivo bioluminescent imaging of syngraft and allograft recipients was performed for 6 months and 4 weeks, respectively. Grafts were removed for PCR-, RT-PCR, and luminometer analysis. In vivo bioluminescent imaging of recipients showed that AAV9 induced a prominent and stable luciferase activity in the abdomen, when compared with AAV2 and AAV8. However, ex vivo analyses revealed that intracoronary perfusion with AAV2 resulted in the highest heart transplant transduction levels in syngrafts and allografts. Ex vivo intracoronary delivery of AAV2 resulted in efficient transgene expression in heart transplants, whereas intracoronary AAV9 escapes into adjacent tissues. In terms of cardiac transduction, these results suggest AAV2 as a potential vector for gene therapy in preclinical heart transplants studies, and highlight the importance of delivery route in gene transfer studies.

  6. Hsp70 gene transfer by adeno-associated virus inhibits MPTP-induced nigrostriatal degeneration in the mouse model of Parkinson disease.

    Science.gov (United States)

    Dong, Zhizhong; Wolfer, David P; Lipp, Hans-Peter; Büeler, Hansruedi

    2005-01-01

    Mitochondrial dysfunction and oxidative stress have been implicated in Parkinson disease (PD). In addition, genetic evidence points to an important role of protein misfolding, aggregation, and failure in the proteasomal degradation of specific neuronal proteins in the pathogenesis of PD. The chaperone heat-shock protein 70 (Hsp70) reduces protein misfolding and aggregation and protects cells against a variety of adverse conditions, including oxidative stress. Moreover, Hsp70 exerts antiapoptotic activity by blocking the function of several key proapoptotic factors. Recently, Hsp70 was shown to inhibit alpha-synuclein toxicity in a Drosophila model of inherited PD. Here we tested the potential of Hsp70 (approved gene symbol HSPA1A) for gene therapy in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of idiopathic PD. We show that Hsp70 gene transfer to dopamine neurons by a recombinant adeno-associated virus significantly protects the mouse dopaminergic system against MPTP-induced dopamine neuron loss and the associated decline in striatal dopamine levels and tyrosine hydroxylase-positive fibers. Hsp70 reduced MPTP-induced apoptosis in the substantia nigra, and unilateral protection of the dopaminergic system by Hsp70 was associated with increased amphetamine-induced turning toward the uninjected side. Collectively, these results suggest that increasing chaperone activity may be beneficial for the treatment of idiopathic PD.

  7. Targeted delivery of self-complementary adeno-associated virus serotype 9 to the brain, using magnetic resonance imaging-guided focused ultrasound.

    Science.gov (United States)

    Thévenot, Emmanuel; Jordão, Jessica F; O'Reilly, Meaghan A; Markham, Kelly; Weng, Ying-Qi; Foust, Kevin D; Kaspar, Brian K; Hynynen, Kullervo; Aubert, Isabelle

    2012-11-01

    Noninvasive drug delivery to the brain remains a major challenge for the treatment of neurological disorders. Transcranial focused ultrasound combined with lipid-coated gas microspheres injected into the bloodstream has been shown to increase the permeability of the blood-brain barrier locally and transiently. Coupled with magnetic resonance imaging, ultrasound can be guided to allow therapeutics administered in the blood to reach brain regions of interest. Using this approach, we perform gene transfer from the blood to specific regions of the mouse brain. Focused ultrasound was targeted to the right hemisphere, at multiple foci, or restricted to one focal point of the hippocampus or the striatum. Doses from 5 × 10(8) to 1.25 × 10(10) vector genomes per gram (VG/g) of self-complementary adeno-associated virus serotype 9 carrying the green fluorescent protein were injected into the tail vein. A dose of 2.5 × 10(9) VG/g was optimal to express the transgene, 12 days later, in neurons, astrocytes, and oligodendrocytes in brain regions targeted with ultrasound, while minimizing the infection of peripheral organs. In the hippocampus and striatum, predominantly neurons and astrocytes were infected, respectively. Transcranial focused ultrasound applications could fulfill a long-term goal of gene therapy: delivering vectors to diseased brain areas directly from the circulation, in a noninvasive manner.

  8. Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer.

    Science.gov (United States)

    Li, Lina; Dimitriadis, Emilios K; Yang, Yu; Li, Juan; Yuan, Zhenhua; Qiao, Chunping; Beley, Cyriaque; Smith, Richard H; Garcia, Luis; Kotin, Robert M

    2013-01-01

    Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or "CELiD", DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5 × 10(9) Sf9 cells, and 1-15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.

  9. Wnt11 gene therapy with adeno-associated virus 9 improves the survival of mice with myocarditis induced by coxsackievirus B3 through the suppression of the inflammatory reaction.

    Science.gov (United States)

    Aoyama, Yutaka; Kobayashi, Koichi; Morishita, Yoshihiro; Maeda, Kengo; Murohara, Toyoaki

    2015-07-01

    The wnt signaling pathway plays important roles in development and in many diseases. Recently several reports suggest that non-canonical Wnt proteins contribute to the inflammatory response in adult animals. However, the effects of Wnt proteins on virus-induced myocarditis have not been explored. Here, we investigated the effect of Wnt11 protein in a model of myocarditis induced by coxsackievirus B3 (CVB3) using recombinant adeno-associated virus 9 (rAAV9). The effect of Wnt11 gene therapy on a CVB3-induced myocarditis model was examined using male BALB/c mice. Mice received a single intravenous injection of either rAAV9-Wnt11 or rAAV9-LacZ 2 weeks before intraperitoneal administration of CVB3. Intravenous injection of the rAAV9 vector resulted in efficient, durable, and relatively cardiac-specific transgene expression. Survival was significantly greater among rAAV9-Wnt11 treated mice than among mice treated with rAAV9-LacZ (87.5% vs. 54.1%, P myocarditis. AAV9-mediated Wnt11 gene therapy produces beneficial effects on cardiac function and increases the survival of mice with CVB3-induced myocarditis through the suppression of both infiltration of inflammatory cells and gene expression of inflammatory cytokines.

  10. NIH oversight of human gene transfer research involving retroviral, lentiviral, and adeno-associated virus vectors and the role of the NIH recombinant DNA advisory committee.

    Science.gov (United States)

    O'Reilly, Marina; Shipp, Allan; Rosenthal, Eugene; Jambou, Robert; Shih, Tom; Montgomery, Maureen; Gargiulo, Linda; Patterson, Amy; Corrigan-Curay, Jacqueline

    2012-01-01

    In response to public and scientific concerns regarding human gene transfer research, the National Institutes of Health (NIH) developed a transparent oversight system that extends to human gene transfer protocols that are either conducted with NIH funding or conducted at institutions that receive NIH funding for recombinant DNA research. The NIH Recombinant DNA Advisory Committee (RAC) has been the primary advisory body to NIH regarding the conduct of this research. Human gene transfer research proposals that are subject to the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH Guidelines) must be submitted to the NIH Office of Biotechnology Activities (OBA), and protocols that raise novel scientific, safety, medical, ethical, or social issues are publicly discussed at the RAC's quarterly public meetings. OBA also convenes gene transfer safety symposia and policy conferences to provide a public forum for scientific experts to discuss emerging issues in the field. This transparent system of review promotes the rapid exchange of important scientific information and dissemination of data. The goal is to optimize the conduct of individual research protocols and to advance gene transfer research generally. This process has fostered the development of retroviral, lentiviral, and adeno-associated viral vector mediated gene delivery.

  11. Cervical cancer isolate PT3, super-permissive for adeno-associated virus replication, over-expresses DNA polymerase δ, PCNA, RFC and RPA

    Directory of Open Access Journals (Sweden)

    Melchert Russell B

    2009-04-01

    Full Text Available Abstract Background Adeno-associated virus (AAV type 2 is an important virus due to its use as a safe and effective human gene therapy vector and its negative association with certain malignancies. AAV, a dependo-parvovirus, autonomously replicates in stratified squamous epithelium. Such tissue occurs in the nasopharynx and anogenitals, from which AAV has been clinically isolated. Related autonomous parvoviruses also demonstrate cell tropism and preferentially replicate in oncogenically transformed cells. Combining these two attributes of parvovirus tropism, squamous and malignant, we assayed if AAV might replicate in squamous cervical carcinoma cell isolates. Results Three primary isolates (PT1-3 and two established cervical cancer cell lines were compared to normal keratinocytes (NK for their ability to replicate AAV. One isolate, PT3, allowed for high levels of AAV DNA replication and virion production compared to others. In research by others, four cellular components are known required for in vitro AAV DNA replication: replication protein A (RPA, replication factor C (RFC, proliferating cell nuclear antigen (PCNA, and DNA polymerase delta (POLD1. Thus, we examined PT3 cells for expression of these components by DNA microarray and real-time quantitative PCR. All four components were over-expressed in PT3 over two representative low-permissive cell isolates (NK and PT1. However, this super-permissiveness did not result in PT3 cell death by AAV infection. Conclusion These data, for the first time, provide evidence that these four cellular components are likely important for AAV in vivo DNA replication as well as in vitro. These data also suggest that PT3 will be a useful reagent for investigating the AAV-permissive transcriptome and AAV anti-cancer effect.

  12. Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation

    Science.gov (United States)

    Stutika, Catrin; Mietzsch, Mario; Gogol-Döring, Andreas; Weger, Stefan; Sohn, Madlen; Chen, Wei; Heilbronn, Regine

    2016-01-01

    Most DNA viruses express small regulatory RNAs, which interfere with viral or cellular gene expression. For adeno-associated virus (AAV), a small ssDNA virus with a complex biphasic life cycle miRNAs or other small regulatory RNAs have not yet been described. This is the first comprehensive Illumina-based RNA-Seq analysis of small RNAs expressed by AAV alone or upon co-infection with helper adenovirus or HSV. Several hotspots of AAV-specific small RNAs were detected mostly close to or within the AAV-ITR and apparently transcribed from the newly identified anti-p5 promoter. An additional small RNA hotspot was located downstream of the p40 promoter, from where transcription of non-coding RNAs associated with the inhibition of adenovirus replication were recently described. Parallel detection of known Ad and HSV miRNAs indirectly validated the newly identified small AAV RNA species. The predominant small RNAs were analyzed on Northern blots and by human argonaute protein-mediated co-immunoprecipitation. None of the small AAV RNAs showed characteristics of bona fide miRNAs, but characteristics of alternative RNA processing indicative of differentially regulated AAV promoter-associated small RNAs. Furthermore, the AAV-induced regulation of cellular miRNA levels was analyzed at different time points post infection. In contrast to other virus groups AAV infection had virtually no effect on the expression of cellular miRNA, which underscores the long-established concept that wild-type AAV infection is apathogenic. PMID:27611072

  13. A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Tratschin, J.D.; West, M.H.P.; Sandbank, T.; Carter, B.J.

    1984-10-01

    The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/ (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

  14. Suppressing tumor growth of nasopharyngeal carcinoma by hTERTC27 polypeptide delivered through adeno-associated virus plus adenovirus vector cocktail

    Institute of Scientific and Technical Information of China (English)

    Xiong Liu; Xiang-Ping Li; Ying Peng; Samuel S.Ng; Hong Yao; Zi-Feng Wang; Xiao-Mei Wang; Hsiang-Fu Kung; Marie C.M.Lin

    2012-01-01

    Nasopharyngeal carcinoma(NPC) is a metastatic carcinoma that is highly prevalent in Southeast Asia.Our laboratory has previously demonstrated that the C-terminal 27-kDa polypeptide of human telomerase reverse transcriptase (hTERTC27) inhibits the growth and tumorigenicity of human glioblastoma and melanoma cells.In this study,we investigated the antitumor effect of hTERTC27 in human C666-1 NPC cells xenografted in a nude mouse model.A cocktail of vectors comprising recombinant adeno-associated virus (rAAV) and recombinant adenovirus (rAdv) that each carry hTERTC27 (rAAV-hTERTC27 and rAdv-hTERTC27; the cocktail was abbreviated to rAAV/rAdv-hTERTC27) was more effective than either rAAV-hTERTC27 or rAdv-hTERTC27 alone in inhibiting the growth of C666-1 NPC xenografts.Furthermore,we established three tumors on each mouse and injected rAAV/rAdv-hTERTC27 into one tumor per mouse.Although hTERTC27 expression could only be detected in the injected tumors,reduced tumor growth was observed in the injected tumor as well as the uninjected tumors,demonstrating that the vector cocktail could provoke an antitumor effect on distant,metastasized tumors.Further studies showed the observed antitumor effects included inducing necrosis and apoptosis and reducing microvessel density.Together,our data suggest that the rAAV/rAdv-hTERTC27 cocktail can potently inhibit NPC tumor growth in both local and metastasized tumors and should be further developed as a novel gene therapy strategy for NPC.

  15. Neonatal intraperitoneal or intravenous injections of recombinant adeno-associated virus type 8 transduce dorsal root ganglia and lower motor neurons.

    Science.gov (United States)

    Foust, Kevin D; Poirier, Amy; Pacak, Christina A; Mandel, Ronald J; Flotte, Terence R

    2008-01-01

    Targeting lower motor neurons (LMNs) for gene delivery could be useful for disorders such as spinal muscular atrophy and amyotrophic lateral sclerosis. LMNs reside in the ventral gray matter of the spinal cord and send axonal projections to innervate skeletal muscle. Studies have used intramuscular injections of adeno-associated virus type 2 (AAV2) to deliver viral vectors to LMNs via retrograde transport. However, treating large areas of the spinal cord in a human would require numerous intramuscular injections, thereby increasing viral titer and risk of immune response. New AAV serotypes, such as AAV8, have a dispersed transduction pattern after intravenous or intraperitoneal injection in neonatal mice, and may transduce LMNs by retrograde transport or through entry into the nervous system. To test LMN transduction after systemic injection, we administered recombinant AAV8 (rAAV8) carrying the green fluorescent protein (GFP) gene by intravenous or intraperitoneal injection to neonatal mice on postnatal day 1. Tissues were harvested 5 and 14 days postinjection and analyzed by real-time polymerase chain reaction and GFP immunohistochemistry to assess the presence of AAV genomes and GFP expression, respectively. Spinal cords were positive for AAV genomes at both time points. GFP immunohistochemistry revealed infrequent labeling of LMNs across all time points and injection routes. Somewhat surprisingly, there was extensive labeling of fibers in the dorsal horns and columns, indicating dorsal root ganglion transduction across all time points and injection routes. Our data suggest that systemic injection of rAAV8 is not an effective delivery route to target lower motor neurons, but could be useful for targeting sensory pathways in chronic pain.

  16. Adeno-associated virus type 2 (AAV2) capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 capsid in vivo.

    Science.gov (United States)

    Li, Chengwen; Hirsch, Matthew; Asokan, Aravind; Zeithaml, Brian; Ma, Hong; Kafri, Tal; Samulski, R Jude

    2007-07-01

    A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kill a cell line stable for capsid expression in vitro and also in a mouse tumor xenograft model in vivo. Parent colon carcinoma (CT26) cells transduced with a large amount of AAV2 vectors in vitro were also destroyed by these CTLs. To determine the effect of CTLs on the elimination of target cells transduced by AAV2 vectors in vivo, we carried out adoptive transfer experiments. CTLs eliminated liver cells with endogenous AAV2 capsid expression but not liver cells transduced by AAV2 vectors, regardless of the reporter genes. Similar results were obtained for rAAV2 transduction in muscle. Our data strongly suggest that AAV vector-transduced cells are rarely eliminated by AAV2 capsid-specific CTLs in vivo, even though the AAV capsid can induce a CTL response. In conclusion, AAV capsid-specific CTLs do not appear to play a role in elimination of rAAV-transduced cells in a mouse model. In addition, our data suggest that the mouse model may not mimic the immune response noted in humans and additional modification to AAV vectors may be required for further study in order to elicit a similar cellular immune response.

  17. Adeno-Associated Virus Type 2 (AAV2) Capsid-Specific Cytotoxic T Lymphocytes Eliminate Only Vector-Transduced Cells Coexpressing the AAV2 Capsid In Vivo▿

    Science.gov (United States)

    Li, Chengwen; Hirsch, Matthew; Asokan, Aravind; Zeithaml, Brian; Ma, Hong; Kafri, Tal; Samulski, R. Jude

    2007-01-01

    A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kill a cell line stable for capsid expression in vitro and also in a mouse tumor xenograft model in vivo. Parent colon carcinoma (CT26) cells transduced with a large amount of AAV2 vectors in vitro were also destroyed by these CTLs. To determine the effect of CTLs on the elimination of target cells transduced by AAV2 vectors in vivo, we carried out adoptive transfer experiments. CTLs eliminated liver cells with endogenous AAV2 capsid expression but not liver cells transduced by AAV2 vectors, regardless of the reporter genes. Similar results were obtained for rAAV2 transduction in muscle. Our data strongly suggest that AAV vector-transduced cells are rarely eliminated by AAV2 capsid-specific CTLs in vivo, even though the AAV capsid can induce a CTL response. In conclusion, AAV capsid-specific CTLs do not appear to play a role in elimination of rAAV-transduced cells in a mouse model. In addition, our data suggest that the mouse model may not mimic the immune response noted in humans and additional modification to AAV vectors may be required for further study in order to elicit a similar cellular immune response. PMID:17475652

  18. Preclinical characterization of a recombinant adeno-associated virus type 1-pseudotyped vector demonstrates dose-dependent injection site inflammation and dissemination of vector genomes to distant sites.

    Science.gov (United States)

    Flotte, Terence R; Conlon, Thomas J; Poirier, Amy; Campbell-Thompson, Martha; Byrne, Barry J

    2007-03-01

    To translate the potential advantages of recombinant adeno-associated virus type 1 (rAAV1) vectors into a clinical application for muscle-directed gene therapy for alpha1 -antitrypsin (AAT) deficiency, we performed safety studies in 170 C57BL/6 mice and 26 New Zealand White rabbits. A mouse toxicology study included 8 cohorts of 10 mice each (5 per sex). Mice were killed either 21 or 90 days after intramuscular injection of doses ranging up to 1x10(13)vector genomes (VG), equivalent to 4 x 10(14)VG/kg. A mouse biodistribution study was performed in 5 cohorts of 10 mice, receiving intramuscular injections at the same doses; as well as in a lower dose cohort (3 x 10(8) VG; equivalent to 1.2 x 10(10)VG/kg); and in 4 other cohorts (excluding the vehicle control) injected with identical doses intravenously. Finally, biodistribution was examined in rabbits, with serial collection of blood and semen, as well as terminal tissue collection. Two significant findings were present, both of which were dose dependent. First, inflammatory cell infiltrates were detected at the site of injection 21, 60, or 90 days after intramuscular injection of 1 x 10(13)VG. This was not associated with loss of transgene expression. Second, vector DNA sequences were detected in most animals, levels being highest with the highest doses and earliest time points. Vector DNA was also present in liver, spleen, kidneys, and a number of other organs, including the gonads of animals receiving the highest dose. Likewise, vector DNA was present in the semen of male rabbits at higher doses. The copy number of vector DNA in the blood and semen declined over time throughout the study. These two dose-dependent findings have served to guide to the design of a phase 1 human trial of rAAV1-AAT.

  19. Attenuation of Dengue Virus Infection by Adeno-Associated Virus-Mediated siRNA Delivery

    Science.gov (United States)

    2004-08-09

    safe and efficacious in Phase I clinical trials for gene therapy of cystic fibrosis and hemophilia B and are regarded as a potential alternative to...retroviral and adenoviral vectors for gene therapy in humans. The AAV vectors have a number of advantages over other vectors. They are not pathogenic and...did not induce significant acute inflammatory responses and, therefore, is useful in gene therapy for DEN infection in humans. In conclusion, we

  20. Package of recombinant adeno-associated virus encoding cell division cycle 2-siRNA%携带cdc2-siRNA重组腺相关病毒的包装

    Institute of Scientific and Technical Information of China (English)

    魏佳军; 王卓然

    2009-01-01

    SETTING: Blank control experiment was performed in the laboratory of Department of Neurology at Wuhan Tongji Hospital between October 2007 and August 2008. MATERIALS: Helper Free adeno-associated virus system (pAAV-MCS-EGFP, p-RC, p-Helper) and AAV-293 cells were purchased from Stratagene. METHODS: The pAAV-MCS-U6-cdc2-siRNA-EGFP expressing plasmid was constructed by molecular biological techniques. The cotransfection of this plasmid, together with p-RC and p-Helper into AAV-293 calls was mediated by calcium acid phosphate. The rAAV encoding cdc2-siRNA (rAAV-U6-cdc2-siRNA-EGFP) was packed and harvested. The silencing effect of this virus on cdc2 gene expression in AAV-293 cells was assessed by Western Blot, and its titer was determined by spot hybridization method. MAIN OUTCOME MEASURES: sequencing of pAAV-MCS-U6-cdc2-siRNA-EGFP plasmid inserting U6-cdc2-siRNA; 3 plasmid pAAV-MCS-U6-cdc2-siRNA-EGFP, p-RC, p-Helper cotransfecting AAV-293 cells; cdc2 expression after rAAV-U6-cdc2-siRNA-EGFP infected AAV-293 cells.RESULTS: ①The successful construction of U6-cdc2-siRNA in pAAV-MCS-EGFP plasmid was proved by DNA sequencing. ②Green fluorescent protein was expressed in AAV-293 cells, and the contransfection was working well. ③The cdc2 gene expression in AAV-293 cells was down-regulated markedly after infection of rAAV-U6-cdc2-siRNA-EGFP. ④The titer of rAAV-U6-cdc2-siRNA-EGFP was 1×10 v.g/mL.CONCLUSION: The package of rAAV encoding cdc2-siRNA is successful. It can silence cdc2 gene effectively.

  1. 腺伴随病毒介导 ADNF-9基因转染对豚鼠卡那霉素致聋的治疗作用%Treatment of adeno-associated virus mediated ADNF-9 gene transfection on guinea pig cochlea deafened by kanamycin

    Institute of Scientific and Technical Information of China (English)

    景阳; 郑国玺; 祝康; 刘晖; 张文

    2015-01-01

    Objective:To observe the effect of rAAV-NT4-ADNF-9 in treating ototoxicity associated with aminoglycoside antibiotics.Methods:The guinea pigs were used,and its auvicle reflex and auditory brain-stem response(ABR)were normal.The ototoxic animal model of guinea pigs were made by the method of hypodemic injection of Kanamycin(400 mg/kg).In animals under general anesthesia,the guinea pigs were cut off the skin of ringt ear sulcus posterior and opened acoustic capsule,then injected the rAAV-NT4-AD-NF-9 10 μL from fossula rotunda.After 14 days,the right acoustic capsule were dislodged.The cochlear were stained with AgNO3 .The cochlear hair cells were observed by contrast phase microscope.Results:Taking the group for rAAV-NT4-ADNF-9 as example,the threshold ABR decreased remarkably after treat-ment(P <0.05).Through the phase contrast microscope the cochlear basilar membrane was complete and the outer hair cells had dot deletion.The thresholds of ABR had no notable differences for the other three groups before and after treatment.Taking comparison between the artificial endolymph control group and empty vector group,through the phase contrast microscope after treatment,hair cells showed a patchy loss in the former group and there was no such phenomenon in the latter group.The average number of hair cells observed in the group of ADNF was significantly higher than that of the artificial endolymph control group and empty vector group(P <0.05).Conclusion:rAAV-NT4-ADNF-9 is able to be transfected into cochle-ar,and to express secretory NT4-ADNF-9 peptide,which preventing hair cells from impairment.%目的:观察包装重组的腺伴随病毒(adeno-associated virus,AAV)rAAV-NT4-ADNF-9对卡拉霉素致聋豚鼠的治疗作用。方法:选用耳廓反射和听性脑干反应(auditory brainstem response,ABR)正常的白色红目豚鼠,在大腿内侧肌肉注射卡那霉素400 mg/(kg·d),连续注射6 d,制备耳聋模型。在全身麻醉的条件

  2. A Single Vector Platform for High-Level Gene Transduction of Central Neurons: Adeno-Associated Virus Vector Equipped with the Tet-Off System.

    Science.gov (United States)

    Sohn, Jaerin; Takahashi, Megumu; Okamoto, Shinichiro; Ishida, Yoko; Furuta, Takahiro; Hioki, Hiroyuki

    2017-01-01

    Visualization of neurons is indispensable for the investigation of neuronal circuits in the central nervous system. Virus vectors have been widely used for labeling particular subsets of neurons, and the adeno-associated virus (AAV) vector has gained popularity as a tool for gene transfer. Here, we developed a single AAV vector Tet-Off platform, AAV-SynTetOff, to improve the gene-transduction efficiency, specifically in neurons. The platform is composed of regulator and response elements in a single AAV genome. After infection of Neuro-2a cells with the AAV-SynTetOff vector, the transduction efficiency of green fluorescent protein (GFP) was increased by approximately 2- and 15-fold relative to the conventional AAV vector with the human cytomegalovirus (CMV) or human synapsin I (SYN) promoter, respectively. We then injected the AAV vectors into the mouse neostriatum. GFP expression in the neostriatal neurons infected with the AAV-SynTetOff vector was approximately 40-times higher than that with the CMV or SYN promoter. By adding a membrane-targeting signal to GFP, the axon fibers of neostriatal neurons were clearly visualized. In contrast, by attaching somatodendritic membrane-targeting signals to GFP, axon fiber labeling was mostly suppressed. Furthermore, we prepared the AAV-SynTetOff vector, which simultaneously expressed somatodendritic membrane-targeted GFP and membrane-targeted red fluorescent protein (RFP). After injection of the vector into the neostriatum, the cell bodies and dendrites of neostriatal neurons were labeled with both GFP and RFP, whereas the axons in the projection sites were labeled only with RFP. Finally, we applied this vector to vasoactive intestinal polypeptide-positive (VIP+) neocortical neurons, one of the subclasses of inhibitory neurons in the neocortex, in layer 2/3 of the mouse primary somatosensory cortex. The results revealed the differential distribution of the somatodendritic and axonal structures at the population level. The

  3. 腺相关病毒介导转化生长因子β1和血管内皮生长因子联合转染促进糖尿病溃疡愈合的生物学效应%Biological effects of co-transfection of transforming growth factor beta 1 and vascular endothelial growth factor mediated by adeno-associated virus on promoting the dermal ulcer healing in diabetes mellitus

    Institute of Scientific and Technical Information of China (English)

    赛佳明; 张慧琴

    2006-01-01

    使溃疡组织中毛细血管密度明显增多,愈合组织中Ⅰ型和Ⅲ型胶原构成比中Ⅰ型胶原的比例明显提高,并有效地促进溃疡愈合.%BACKGROUND: The ulcer wound is hard to heal in diabetic patients,and it is believed to be caused by the microcirculatory disorder of wound and decreased contents of endogenous growth factors in patients with diabetes mellitus.OBJECTIVE: To observe the biological effects of adeno-associated virus (AAV) mediated transforming growth factor beta1 (AAV-TGFβ1) and vascular endothelial growth factor (AAV-VEGF) in promoting the dermal ulcer healing of diabetic rabbits.DESIGN: A randomized controlled animal experiment.SETTINGS: Medical College, Qingdao University; Affiliated Hospital of Medical College, Qingdao University.MATERIALS: The experiments were carried out in the gynecological laboratory, Affiliated Hospital of Medical College, Qingdao University from July 2004 to January 2006. Twenty-four healthy adult New Zealand rabbits were randomly divided into co-transfection group (n=12) and control group (n=12).METHODS: ① The dermal ulcer models of diabetic rabbits was established by injecting alloxan (130 mg/kg) via ear vein, and the ulcer wound was made by operation. ② In the co-transfection group, the wound was locally infiltrated, and injected with AAV-TGFβ1 virus and AAV-VEGF virus (the concentration was 9×106 virus granules/mL respectively). The rabbits in the control group were treated with injection of saline.MAIN OUTCOME MEASURES: ① The levels of TGFβ1 and VEGF gene transcription in the healing tissue were detected with polymerase chain reaction (PCR) at 1 month postoperatively. ② The capillary density in the wound margin was counted with microcirculation microscope at 3 weeks postoperatively. ③ The collagen Ⅰ and Ⅲ were isolated and detected with Western blotting by protein gel electrophoresis and semi-dry electrophoretic transfer. ④ The content of collagen in the ulcer healing issue

  4. α2,3 and α2,6 N-Linked Sialic Acids Facilitate Efficient Binding and Transduction by Adeno-Associated Virus Types 1 and 6

    Science.gov (United States)

    Wu, Zhijian; Miller, Edward; Agbandje-McKenna, Mavis; Samulski, Richard Jude

    2006-01-01

    Recombinant adeno-associated viruses (AAVs) are promising vectors in the field of gene therapy. Different AAV serotypes display distinct tissue tropism, believed to be related to the distribution of their receptors on target cells. Of the 11 well-characterized AAV serotypes, heparan sulfate proteoglycan and sialic acid have been suggested to be the attachment receptors for AAV type 2 and types 4 and 5, respectively. In this report, we identify the receptor for the two closely related serotypes, AAV1 and AAV6. First, we demonstrate using coinfection experiments and luciferase reporter analysis that AAV1 and AAV6 compete for similar receptors. Unlike heparin sulfate, enzymatic or genetic removal of sialic acid markedly reduced AAV1 and AAV6 binding and transduction. Further analysis using lectin staining and lectin competition assays identified that AAV1 and AAV6 use either α2,3-linked or α2,6-linked sialic acid when transducing numerous cell types (HepG2, Pro-5, and Cos-7). Treatment of cells with proteinase K but not glycolipid inhibitor reduced AAV1 and AAV6 infection, supporting the hypothesis that the sialic acid that facilitates infection is associated with glycoproteins rather than glycolipids. In addition, we determined by inhibitor (N-benzyl GalNAc)- and cell line-specific (Lec-1) studies that AAV1 and AAV6 require N-linked and not O-linked sialic acid. Furthermore, a resialylation experiment on a deficient Lec-2 cell line confirmed a 2,3 and 2,6 N-linked sialic acid requirement, while studies of mucin with O-linked sialic acid showed no inhibition effect for AAV1 and AAV6 transduction on Cos-7 cells. Finally, using a glycan array binding assay we determined that AAV1 efficiently binds to NeuAcα2-3GalNAcβ1-4GlcNAc, as well as two glycoproteins with α2,3 and α2,6 N-linked sialic acids. Taken together, competition, genetic, inhibitor, enzymatic reconstitution, and glycan array experiments support α2,3 and α2,6 sialic acids that are present on N

  5. Comparative efficacy and safety of multiple routes of direct CNS administration of adeno-associated virus gene transfer vector serotype rh.10 expressing the human arylsulfatase A cDNA to nonhuman primates.

    Science.gov (United States)

    Rosenberg, Jonathan B; Sondhi, Dolan; Rubin, David G; Monette, Sébastien; Chen, Alvin; Cram, Sara; De, Bishnu P; Kaminsky, Stephen M; Sevin, Caroline; Aubourg, Patrick; Crystal, Ronald G

    2014-09-01

    Metachromatic leukodystrophy (MLD), a fatal disorder caused by deficiency of the lysosomal enzyme arylsulfatase A (ARSA), is associated with an accumulation of sulfatides, causing widespread demyelination in both central and peripheral nervous systems. On the basis of prior studies demonstrating that adeno-associated virus AAVrh.10 can mediate widespread distribution in the CNS of a secreted lysosomal transgene, and as a prelude to human trials, we comparatively assessed the optimal CNS delivery route of an AAVrh.10 vector encoding human ARSA in a large animal model for broadest distribution of ARSA enzyme. Five routes were tested (each total dose, 1.5 × 10(12) genome copies of AAVrh.10hARSA-FLAG): (1) delivery to white matter centrum ovale; (2) deep gray matter delivery (putamen, thalamus, and caudate) plus overlying white matter; (3) convection-enhanced delivery to same deep gray matter locations; (4) lateral cerebral ventricle; and (5) intraarterial delivery with hyperosmotic mannitol to the middle cerebral artery. After 13 weeks, the distribution of ARSA activity subsequent to each of the three direct intraparenchymal administration routes was significantly higher than in phosphate-buffered saline-administered controls, but administration by the intraventricular and intraarterial routes failed to demonstrate measurable levels above controls. Immunohistochemical staining in the cortex, white matter, deep gray matter of the striatum, thalamus, choroid plexus, and spinal cord dorsal root ganglions confirmed these results. Of the five routes studied, administration to the white matter generated the broadest distribution of ARSA, with 80% of the brain displaying more than a therapeutic (10%) increase in ARSA activity above PBS controls. No significant toxicity was observed with any delivery route as measured by safety parameters, although some inflammatory changes were seen by histopathology. We conclude that AAVrh.10-mediated delivery of ARSA via CNS

  6. Trafficking of adeno-associated virus vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells.

    Science.gov (United States)

    Merkel, Steven F; Andrews, Allison M; Lutton, Evan M; Mu, Dakai; Hudry, Eloise; Hyman, Bradley T; Maguire, Casey A; Ramirez, Servio H

    2017-01-01

    Developing therapies for central nervous system (CNS) diseases is exceedingly difficult because of the blood-brain barrier (BBB). Notably, emerging technologies may provide promising new options for the treatment of CNS disorders. Adeno-associated virus serotype 9 (AAV9) has been shown to transduce cells in the CNS following intravascular administration in rodents, cats, pigs, and non-human primates. These results suggest that AAV9 is capable of crossing the BBB. However, mechanisms that govern AAV9 transendothelial trafficking at the BBB remain unknown. Furthermore, possibilities that AAV9 may transduce brain endothelial cells or affect BBB integrity still require investigation. Using primary human brain microvascular endothelial cells as a model of the human BBB, we performed transduction and transendothelial trafficking assays comparing AAV9 to AAV2, a serotype that does not cross the BBB or transduce endothelial cells effectively in vivo. Results of our in vitro studies indicate that AAV9 penetrates brain microvascular endothelial cells barriers more effectively than AAV2, but has reduced transduction efficiency. In addition, our data suggest that (i) AAV9 penetrates endothelial barriers through an active, cell-mediated process, and (ii) AAV9 fails to disrupt indicators of BBB integrity such as transendothelial electrical resistance, tight junction protein expression/localization, and inflammatory activation status. Overall, this report shows how human brain endothelial cells configured in BBB models can be utilized for evaluating transendothelial movement and transduction kinetics of various AAV capsids. Importantly, the use of a human in vitro BBB model can provide import insight into the possible effects that candidate AVV gene therapy vectors may have on the status of BBB integrity. Read the Editorial Highlight for this article on page 192.

  7. Mutational Analysis of the Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Construction of AAV2 Vectors with Altered Tropism

    Science.gov (United States)

    Wu, Pei; Xiao, Wu; Conlon, Thomas; Hughes, Jeffrey; Agbandje-McKenna, Mavis; Ferkol, Thomas; Flotte, Terence; Muzyczka, Nicholas

    2000-01-01

    Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be β-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag

  8. Comparison of murine leukemia virus, human immunodeficiency virus, and adeno-associated virus vectors for gene transfer in multiple myeloma: lentiviral vectors demonstrate a striking capacity to transduce low-proliferating primary tumor cells.

    Science.gov (United States)

    De Vos, John; Bagnis, Claude; Bonnafoux, Lydie; Requirand, Guilhem; Jourdan, Michel; Imbert, Marie-Christine; Jourdan, Eric; Rossi, Jean-François; Mannoni, Patrice; Klein, Bernard

    2003-12-10

    Genetic modification of primary tumor cells by gene transfer is of major interest to study the role of specific genes in the biology of a given malignancy and to modify tumor cells for therapeutic use. Multiple myeloma (MM) is a low-proliferating cancer, with often less than 1% of the cells in the S phase of the cell cycle. As primary myeloma cells are notoriously difficult to transduce, we conducted a comparison of various viral vectors, known to integrate the transgene of interest into the target genome, for their ability to stably promote the expression of an enhanced green fluorescent protein (EGFP) transgene. We compared three murine leukemia virus-based vectors, differing only in their viral envelope, a human immunodeficiency virus (HIV)-based vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), and an adeno-associated virus type 2 vector. Transduction characteristics of these vectors were evaluated in human myeloma cell lines and in primary myeloma cells. Unequivocally, we observed that the VSV-G/HIV vector was the most efficient vector for transducing the cell lines and the only one able to transduce primary myeloma cells reproducibly. The mean percentage of transduced primary myeloma cells was 43.6% (range, 16.3-77.6%), with one round of infection at a low multiplicity of infection, including MM cell samples with less than 1% of cells in the S phase. A quantitative polymerase chain reaction assay demonstrated that this more efficient EGFP expression was associated with a higher GFP copy number in the targeted cell. We propose that lentiviral vectors should be used for transduction of nonproliferating primary tumor cells such as myeloma cells.

  9. A Novel Adeno-Associated Virus-Based Genetic Vaccine Encoding the Hepatitis C Virus NS3/4 Protein Exhibits Immunogenic Properties in Mice Superior to Those of an NS3-Protein-Based Vaccine.

    Directory of Open Access Journals (Sweden)

    Fengqin Zhu

    Full Text Available More than 170 million individuals worldwide are infected with hepatitis C virus (HCV, and up to an estimated 30% of chronically infected individuals will go on to develop progressive liver disease. Despite the recent advances in antiviral treatment of HCV infection, it remains a major public health problem. Thus, development of an effective vaccine is urgently required. In this study, we constructed novel adeno-associated virus (AAV vectors expressing the full-length NS3 or NS3/4 protein of HCV genotype 1b. The expression of the NS3 or NS3/4 protein in HepG2 cells was confirmed by western blotting. C57BL/6 mice were intramuscularly immunised with a single injection of AAV vectors, and the resultant immune response was investigated. The AAV2/rh32.33.NS3/4 vaccine induced stronger humoral and cellular responses than did the AAV2/rh32.33.NS3 vaccine. Our results demonstrate that AAV-based vaccines exhibit considerable potential for the development of an effective anti-HCV vaccine.

  10. Cloning of avian adeno-associated virus genome and rescue of the infectious virus%禽腺联病毒全基因组的克隆及感染性病毒的拯救

    Institute of Scientific and Technical Information of China (English)

    王建业; 孙怀昌; 朱国强

    2007-01-01

    为了克隆禽腺联病毒(Avian adeno-associated virus,AAAV)全基因组用于构建基因转移载体研究,以鸡胚致死孤儿病毒(CELO)作为辅助病毒与AAAV共接种SPF鸡胚进行AAAV的增殖,将AAAV约4.7 kb双链基因组DNA与pCR2.1载体连接,构建了含AAAV全基因组的重组质粒pAAAV并进行了测序.序列分析表明,AAAV YZ-1株的基因组为4 684 bp,两端具有141 bp的末端倒置重复序列和Rep蛋白结合位点特征序列,与GenBank中收录的AAAV DA-1株和VR-865株的核苷酸序列同源性分别为95.0%和92.2%.将pAAAV质粒转染CELO病毒感染的鸡胚肝细胞系,获得了感染性AAAV病毒粒子,结果证明克隆的AAAV基因组中存在与病毒复制和包装相关的正确关键序列,可用于重组AAAV载体的构建.

  11. Neuropathological and behavioral consequences of adeno-associated viral vector-mediated continuous intrastriatal neurotrophin delivery in a focal ischemia model in rats.

    Science.gov (United States)

    Andsberg, Gunnar; Kokaia, Zaal; Klein, Ronald L; Muzyczka, Nicholas; Lindvall, Olle; Mandel, Ronald J

    2002-03-01

    Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were continuously delivered to the striatum at biologically active levels via recombinant adeno-associated viral (rAAV) gene transfer 4-5 weeks prior to 30 min of middle cerebral artery occlusion (MCAO). The magnitude of the deficits in a battery of behavioral tests designed to assess striatal function was highly correlated to the extent of ischemic damage determined by unbiased stereological estimations of striatal neuron numbers. The delivery of neurotrophins lead to mild functional improvements in the ischemia-induced motor impairments assessed 3-5 weeks after the insult, in agreement with a small but significant increase of the survival of dorsolateral striatal neurons. Detailed phenotypic analysis demonstrated that the parvalbumin-containing interneurons were spared to a greater extent by the neurotrophin treatment as compared to the projection neurons, which agreed with the specificity for interneuron transduction by the rAAV vector. These data show the advantage of the never previously performed combination of precise quantification of the ischemia-induced neuropathology along with detailed behavioural analysis for assessing neuroprotection after stroke. We observe that intrastriatal delivery of NGF and BDNF using a viral vector system can mitigate, albeit only moderately, neuronal death following stroke, which leads to detectable functional sparing.

  12. Partial correction of sensitivity to oxidant stress in Friedreich ataxia patient fibroblasts by frataxin-encoding adeno-associated virus and lentivirus vectors.

    Science.gov (United States)

    Fleming, Jane; Spinoulas, Afroditi; Zheng, Maolin; Cunningham, Sharon C; Ginn, Samantha L; McQuilty, Robert C; Rowe, Peter B; Alexander, Ian E

    2005-08-01

    Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of a number of debilitating inherited and acquired neurological conditions. The lack of effective treatments for many such conditions provides a strong rationale for exploring novel therapeutic approaches, including gene therapy. Friedreich ataxia (FRDA), a sensory neuropathy, is a progressive neurodegenerative disease associated with a loss of large sensory neurons from the dorsal root ganglia. Because a mouse model for this well-characterized disease has been generated, we elected to use FRDA as a model disease. In previous studies we achieved efficient and sustained delivery of a reporter gene to PNS sensory neurons, using recombinant adeno-associated viral (AAV) and lentiviral (LV) vectors. In the current study, AAV and LV vectors encoding the human frataxin cDNA were constructed and assessed for frataxin expression and function in primary FRDA patient fibroblast cell lines. FRDA fibroblasts have been shown to exhibit subtle biochemical changes, including increased mitochondrial iron and sensitivity to oxidant stress. Despite the inherent difficulty in working with primary cells, transduction of patient fibroblasts with either vector resulted in the expression of appropriately localized frataxin and partial reversal of phenotype.

  13. Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

    Institute of Scientific and Technical Information of China (English)

    陆华中; 陈立; 王红卫; 伍志坚; 吴小兵; 王学峰; 王鸿利; 卢大儒; 邱信芳; 薛京伦

    2001-01-01

    A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ- ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27% ± 3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors development in the treated mice; and (iv) no local or systemic side-effects and toxicity associated with the gene transfer were found. It demonstrated the potential use of treating hemophilia B by recombinant adeno-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.

  14. Novel Vector Design and Hexosaminidase Variant Enabling Self-Complementary Adeno-Associated Virus for the Treatment of Tay-Sachs Disease.

    Science.gov (United States)

    Karumuthil-Melethil, Subha; Nagabhushan Kalburgi, Sahana; Thompson, Patrick; Tropak, Michael; Kaytor, Michael D; Keimel, John G; Mark, Brian L; Mahuran, Don; Walia, Jagdeep S; Gray, Steven J

    2016-07-01

    GM2 gangliosidosis is a family of three genetic neurodegenerative disorders caused by the accumulation of GM2 ganglioside (GM2) in neuronal tissue. Two of these are due to the deficiency of the heterodimeric (α-β), "A" isoenzyme of lysosomal β-hexosaminidase (HexA). Mutations in the α-subunit (encoded by HEXA) lead to Tay-Sachs disease (TSD), whereas mutations in the β-subunit (encoded by HEXB) lead to Sandhoff disease (SD). The third form results from a deficiency of the GM2 activator protein (GM2AP), a substrate-specific cofactor for HexA. In their infantile, acute forms, these diseases rapidly progress with mental and psychomotor deterioration resulting in death by approximately 4 years of age. After gene transfer that overexpresses one of the deficient subunits, the amount of HexA heterodimer formed would empirically be limited by the availability of the other endogenous Hex subunit. The present study used a new variant of the human HexA α-subunit, μ, incorporating critical sequences from the β-subunit that produce a stable homodimer (HexM) and promote functional interactions with the GM2AP- GM2 complex. We report the design of a compact adeno-associated viral (AAV) genome using a synthetic promoter-intron combination to allow self-complementary (sc) packaging of the HEXM gene. Also, a previously published capsid mutant, AAV9.47, was used to deliver the gene to brain and spinal cord while having restricted biodistribution to the liver. The novel capsid and cassette design combination was characterized in vivo in TSD mice for its ability to efficiently transduce cells in the central nervous system when delivered intravenously in both adult and neonatal mice. This study demonstrates that the modified HexM is capable of degrading long-standing GM2 storage in mice, and it further demonstrates the potential of this novel scAAV vector design to facilitate widespread distribution of the HEXM gene or potentially other similar-sized genes to the nervous system.

  15. Construction of recombinant adeno-associated viral vectors in human neurenergen-3 gene

    Institute of Scientific and Technical Information of China (English)

    Xiangli Wang; Haili Wang; Baojie Mi

    2007-01-01

    BACKGROUND: Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeutic gene.OBJECTIVE: To explore an effective way to construct recombinant adeno-associated viral vectors expression in human neurnnergen-3 gene. DESIGN: Gene directed cloning.SETTING: Central Laboratory of Northern China Coal Medical College.MATERIALS: DH5a competent bacillus coli strain was provided by Capital Medical University; pCDNA3-NT-3 by professor Chen from Bengbu Medical College; pAAV-Laze, pAAV-Helper, pAAV-RC and pAAV-MCS plasmids by Capital Medical University; HEK293 cells by Cell Center of Basic Medical College of Tongji Medical University.METHODS: NT-3 genes which were selected from pCDNA3-NT-3 plasmids were cloned in pAAV-MCS to form a recombinant adeno-associated viral plasmid (pAAV-NT-3). pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were extracted, purified and subjected to enzyme-shearing evaluation. In addition, pAAV-NT-3 and pAAV-LacZ were cotransfected with pHelper and pAAV-RC, respectively into AVV-293 cells with DNA mediated by calcium superphosphate transfection gene; and then, AVV-293 cells were packed into recombinant adeno-associated viral rAAV-NT-3 and rAAV-LacZ. After collection of viral particles, rAAV-LacZ viral stock solution was diluted based on ratio of 10:1 and the mixture was used to infect HT1080 cells. X-gal stain was used to measure virus liter.MAIN OUTCOME MEASURES: Size of targeted gene fragments, validity of vehicle construction and virus liter.RESULTS: Targeted gene NT-3 was successfully inserted into the relative vehicle pAAV and pAAV-NT-3 was correctly recongnized by enzyme-shearing evaluation. Enzyme-shearing electrophoresis demonstrated that pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were successfully extracted and purified.β-galactoside staining in situ indicated that LacZ genes were

  16. Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results of a phase I trial.

    Science.gov (United States)

    Hauswirth, William W; Aleman, Tomas S; Kaushal, Shalesh; Cideciyan, Artur V; Schwartz, Sharon B; Wang, Lili; Conlon, Thomas J; Boye, Sanford L; Flotte, Terence R; Byrne, Barry J; Jacobson, Samuel G

    2008-10-01

    Leber congenital amaurosis (LCA) is a group of autosomal recessive blinding retinal diseases that are incurable. One molecular form is caused by mutations in the RPE65 (retinal pigment epithelium-specific 65-kDa) gene. A recombinant adeno-associated virus serotype 2 (rAAV2) vector, altered to carry the human RPE65 gene (rAAV2-CBSB-hRPE65), restored vision in animal models with RPE65 deficiency. A clinical trial was designed to assess the safety of rAAV2-CBSB-hRPE65 in subjects with RPE65-LCA. Three young adults (ages 21-24 years) with RPE65-LCA received a uniocular subretinal injection of 5.96 x 10(10) vector genomes in 150 microl and were studied with follow-up examinations for 90 days. Ocular safety, the primary outcome, was assessed by clinical eye examination. Visual function was measured by visual acuity and dark-adapted full-field sensitivity testing (FST); central retinal structure was monitored by optical coherence tomography (OCT). Neither vector-related serious adverse events nor systemic toxicities were detected. Visual acuity was not significantly different from baseline; one patient showed retinal thinning at the fovea by OCT. All patients self-reported increased visual sensitivity in the study eye compared with their control eye, especially noticeable under reduced ambient light conditions. The dark-adapted FST results were compared between baseline and 30-90 days after treatment. For study eyes, sensitivity increases from mean baseline were highly significant (p gene therapy for RPE65-LCA that were carried out contemporaneously and reported.

  17. Comparison of efficacy of the disease-specific LOX1- and constitutive cytomegalovirus-promoters in expressing interleukin 10 through adeno-associated virus 2/8 delivery in atherosclerotic mice.

    Directory of Open Access Journals (Sweden)

    Hongqing Zhu

    Full Text Available The development of gene therapy vectors for treating diseases of the cardiovascular system continues at a steady pace. Moreover, in the field of gene therapy the utility of "disease-specific promoters" has strong appeal. Many therapeutic genes, including transforming growth factor beta 1 or interleukin 10, are associated to adverse effects. The use of a disease-specific promoter might minimize toxicity. The lectin-like oxidized low density lipoprotein receptor 1 is a marker of cardiovascular disease and a potential therapeutic target. The lectin-like oxidized low density lipoprotein receptor 1 is known to be up-regulated early during disease onset in a number of cell types at the sites where the disease will be clinically evident. In this study an adeno-associated virus-2 DNA vector (AAV2 using the AAV8 capsid, and containing the full length The lectin-like oxidized low density lipoprotein receptor 1 promoter, was generated and assayed for its ability to express human interleukin 10 in low density lipoprotein receptor knockout mice on high cholesterol diet. The cytomegalovirus early promoter was used for comparison in a similarly structured vector. The two promoters were found to have equal efficacy in reducing atherogenesis as measured by aortic systolic blood velocity, aortic cross sectional area, and aortic wall thickness. This is the first head-to-head comparison of a constitutive with a disease-specific promoter in a therapeutic context. These data strongly suggest that the use of a disease-specific promoter is appropriate for therapeutic gene delivery.

  18. Adeno-associated viral vector serotypes 1 and 5 targeted to the neonatal rat and pig striatum induce widespread transgene expression in the forebrain

    DEFF Research Database (Denmark)

    Kornum, Birgitte R; Stott, Simon R W; Mattsson, Bengt

    2010-01-01

    Viral vector-mediated gene transfer has emerged as a powerful means to target transgene expression in the central nervous system. Here we characterized the efficacy of serotypes 1 and 5 recombinant adeno-associated virus (rAAV) vectors encoding green fluorescent protein (GFP) after stereotaxic....... Our results show that striatal delivery of rAAV5 vectors in the neonatal brain represents a useful tool to express genes of interest both in the basal ganglia and the neocortex. Furthermore, we apply, for the first time, viral vector-mediated gene transfer to the pig brain providing the opportunity...

  19. Partial correction of the CNS lysosomal storage defect in a mouse model of juvenile neuronal ceroid lipofuscinosis by neonatal CNS administration of an adeno-associated virus serotype rh.10 vector expressing the human CLN3 gene.

    Science.gov (United States)

    Sondhi, Dolan; Scott, Emma C; Chen, Alvin; Hackett, Neil R; Wong, Andrew M S; Kubiak, Agnieszka; Nelvagal, Hemanth R; Pearse, Yewande; Cotman, Susan L; Cooper, Jonathan D; Crystal, Ronald G

    2014-03-01

    Juvenile neuronal ceroid lipofuscinosis (JNCL or CLN3 disease) is an autosomal recessive lysosomal storage disease resulting from mutations in the CLN3 gene that encodes a lysosomal membrane protein. The disease primarily affects the brain with widespread intralysosomal accumulation of autofluorescent material and fibrillary gliosis, as well as the loss of specific neuronal populations. As an experimental treatment for the CNS manifestations of JNCL, we have developed a serotype rh.10 adeno-associated virus vector expressing the human CLN3 cDNA (AAVrh.10hCLN3). We hypothesized that administration of AAVrh.10hCLN3 to the Cln3(Δex7/8) knock-in mouse model of JNCL would reverse the lysosomal storage defect, as well as have a therapeutic effect on gliosis and neuron loss. Newborn Cln3(Δex7/8) mice were administered 3 × 10(10) genome copies of AAVrh.10hCLN3 to the brain, with control groups including untreated Cln3(Δex7/8) mice and wild-type littermate mice. After 18 months, CLN3 transgene expression was detected in various locations throughout the brain, particularly in the hippocampus and deep anterior cortical regions. Changes in the CNS neuronal lysosomal accumulation of storage material were assessed by immunodetection of subunit C of ATP synthase, luxol fast blue staining, and periodic acid-Schiff staining. For all parameters, Cln3(Δex7/8) mice exhibited abnormal lysosomal accumulation, but AAVrh.10hCLN3 administration resulted in significant reductions in storage material burden. There was also a significant decrease in gliosis in AAVrh.10hCLN3-treated Cln3(Δex7/8) mice, and a trend toward improved neuron counts, compared with their untreated counterparts. These data demonstrate that AAVrh.10 delivery of a wild-type cDNA to the CNS is not harmful and instead provides a partial correction of the neurological lysosomal storage defect of a disease caused by a lysosomal membrane protein, indicating that this may be an effective therapeutic strategy for JNCL and

  20. Adeno-associated virus-mediated microdystrophin expression protects young mdx muscle from contraction-induced injury

    National Research Council Canada - National Science Library

    Liu, Mingju; Yue, Yongping; Harper, Scott Q; Grange, Robert W; Chamberlain, Jeffrey S; Duan, Dongsheng

    2005-01-01

    ... a CMV.DeltaR4/DeltaC cassette in AAV-5 and evaluated the transduction and muscle contractile profiles in the extensor digitorum longus muscles of young (7-week-old) and adult (9-month-old) mdx mice...

  1. Adeno-associated viral vectors for the treatment of hemophilia

    Science.gov (United States)

    High, Katherine A.; Anguela, Xavier M.

    2016-01-01

    Gene transfer studies for the treatment of hemophilia began more than two decades ago. A large body of pre-clinical work evaluated a variety of vectors and target tissues, but by the start of the new millennium it became evident that adeno-associated viral (AAV)-mediated gene transfer to the liver held great promise as a therapeutic tool. The transition to the clinical arena uncovered a number of unforeseen challenges, mainly in the form of a human-specific immune response against the vector that poses a significant limitation in the application of this technology. While the full nature of this response has not been elucidated, long-term expression of therapeutic levels of factor IX is already a reality for a small number of patients. Extending this success to a greater number of hemophilia B patients remains a major goal of the field, as well as translating this strategy to clinical therapy for hemophilia A. This review summarizes the progress of AAV-mediated gene therapy for the hemophilias, along with its upcoming prospects and challenges. PMID:26614390

  2. 腺相关病毒2-ND4基因转染细胞线粒体的研究%Study on transfection of adeno associated virus 2-ND4 gene into mitochondria

    Institute of Scientific and Technical Information of China (English)

    杨硕; 刘磊; 裴晗; 万幸; 陆朵朵; 胡维琨; 李斌

    2014-01-01

    Background Leber hereditary optic neuropathy (LHON) is mitochondrial DNA (mtDNA) disease and mainly leads to optical nerve degeneration.Its primary mechanism is synthesis disorder of DN4 protein due to variation of mtDNA 11778 locus.So to construct a vector with exogenous normal ND4 and transfect into mitochondria is a key of gene therapy for LHON.Objective This study was to investigate the in vitro transfection of adeno-associated virus (AAV)-ND4 gene into mitochondria.Methods Human renal epithelial cell lines transfected adenovirus E1A (293 cells) were regularly cultured and divided into two groups.Framework plasmids of recombinant AAV-ND4 or simple AAV2 were added to the cell medium respectively.The expression of ND4 in cells were located 12,24,36 and 48 hours after transfected by Y03 dual fluorescent quantum dots staining.The positive response for ND4 showed the green fluorescence.Results Cultured 293 cells grew well with 80% confluence.Abundant green fluorescence particles were seen in cytoplasm in the AAV-ND4 transfected group,but only red fluorescence from mitochondrial protein was seen in the simple AAV transfected group under the fluorescence microscope.Conclusions Exogenous ND4 protein can been successfully transfected into mitochondria using the ND4 gene constructed AAV.This result provides experimental evidence for the further study on gene therapy of LHON.%背景 Leber遗传性视神经病变(LHON)是导致视神经退行性变的线粒体遗传性疾病,主要与线粒体11778位点突变导致ND4蛋白合成异常有关,构建含正常ND4蛋白的载体是基因治疗的关键.由于ND4 DNA存在于线粒体,而转染的外源基因只能进入细胞核,不能作用于突变的线粒体DNA.探讨将ND4基因成功转染到线粒体是LHON的基因治疗的关键. 目的 验证人工合成的ND4基因所构建的重组腺相关病毒(AAV)转染细胞后产生的ND4蛋白能否进入线粒体.方法 常规体外培养转染腺病毒E1A

  3. Perinatal systemic gene delivery using adeno-associated viral vectors

    Directory of Open Access Journals (Sweden)

    Rajvinder eKarda

    2014-11-01

    Full Text Available Neurodegenerative monogenic diseases can also affect a broad range of tissues and organs throughout the body. An effective treatment would require a systemic approach. The intravenous administration of novel therapies is ideal but is hampered by the inability of such drugs to cross the blood-brain barrier and precludes efficacy in the central nervous system. A number of these early lethal intractable diseases also present devastating irreversible pathology at birth or soon after. Therefore, any therapy would ideally be administered during the perinatal period to prevent, stop or ameliorate disease progression. The concept of perinatal gene therapy has moved a step further towards being a feasible approach to treating such disorders. This has primarily been driven by the recent discoveries that particular serotypes of adeno-associated virus (AAV gene delivery vectors have the ability to cross the blood-brain barrier following intravenous administration. Furthermore, this has been safely demonstrated in perinatal mice and non-human primates. This review focuses on the progress made in using AAV to achieve systemic transduction and what this means for developing perinatal gene therapy for early lethal neurodegenerative diseases.

  4. Magnetically enhanced adeno-associated viral vector delivery for human neural stem cell infection.

    Science.gov (United States)

    Kim, Eunmi; Oh, Ji-Seon; Ahn, Ik-Sung; Park, Kook In; Jang, Jae-Hyung

    2011-11-01

    Gene therapy technology is a powerful tool to elucidate the molecular cues that precisely regulate stem cell fates, but developing safe vehicles or mechanisms that are capable of delivering genes to stem cells with high efficiency remains a challenge. In this study, we developed a magnetically guided adeno-associated virus (AAV) delivery system for gene delivery to human neural stem cells (hNSCs). Magnetically guided AAV delivery resulted in rapid accumulation of vectors on target cells followed by forced penetration of the vectors across the plasma membrane, ultimately leading to fast and efficient cellular transduction. To combine AAV vectors with the magnetically guided delivery, AAV was genetically modified to display hexa-histidine (6xHis) on the physically exposed loop of the AAV2 capsid (6xHis AAV), which interacted with nickel ions chelated on NTA-biotin conjugated to streptavidin-coated superparamagnetic iron oxide nanoparticles (NiStNPs). NiStNP-mediated 6xHis AAV delivery under magnetic fields led to significantly enhanced cellular transduction in a non-permissive cell type (i.e., hNSCs). In addition, this delivery method reduced the viral exposure times required to induce a high level of transduction by as much as to 2-10 min of hNSC infection, thus demonstrating the great potential of magnetically guided AAV delivery for numerous gene therapy and stem cell applications.

  5. The potential of adeno-associated viral vectors for gene delivery to muscle tissue.

    Science.gov (United States)

    Wang, Dan; Zhong, Li; Nahid, M Abu; Gao, Guangping

    2014-03-01

    Muscle-directed gene therapy is rapidly gaining attention primarily because muscle is an easily accessible target tissue and is also associated with various severe genetic disorders. Localized and systemic delivery of recombinant adeno-associated virus (rAAV) vectors of several serotypes results in very efficient transduction of skeletal and cardiac muscles, which has been achieved in both small and large animals, as well as in humans. Muscle is the target tissue in gene therapy for many muscular dystrophy diseases, and may also be exploited as a biofactory to produce secretory factors for systemic disorders. Current limitations of using rAAVs for muscle gene transfer include vector size restriction, potential safety concerns such as off-target toxicity and the immunological barrier composing of pre-existing neutralizing antibodies and CD8(+) T-cell response against AAV capsid in humans. In this article, we will discuss basic AAV vector biology and its application in muscle-directed gene delivery, as well as potential strategies to overcome the aforementioned limitations of rAAV for further clinical application. Delivering therapeutic genes to large muscle mass in humans is arguably the most urgent unmet demand in treating diseases affecting muscle tissues throughout the whole body. Muscle-directed, rAAV-mediated gene transfer for expressing antibodies is a promising strategy to combat deadly infectious diseases. Developing strategies to circumvent the immune response following rAAV administration in humans will facilitate clinical application.

  6. Culture of 293 cells for the package of adeno-associated viruses%用于包装腺相关病毒293细胞的培养

    Institute of Scientific and Technical Information of China (English)

    魏佳军; 张苏明; 徐金枝

    2007-01-01

    BACKGROUND: As a main gene engineering vector, adeno-associated virus (AAV) is characterized by its extensive host cells, lasting and stable expression and less immune response to hosts, and is applied widely. But AAV is a kind of defective virus, and need incasing cells to supply E1 protein. As important and special AAV incasing cells, AAV-293 cells can produce E1 in trans. But AAV-293 cells are delicated and cultivated difficultly, and the biological character is easy to be changed. Therefore, it is necessary to establish a culture method of AAV-293 cells to meet the need of gene engineering.OBJECTIVE: To establish a culture method of AAV-293 cells in vitro.DESIGN: An opening study.SETTING: Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: AAV-293 cells line was provided by Stratagene Corporation; high-carbohydrate OMEM (H-DMEM) powder by Gibco Company; there plasmids in AAV Helper-Free by Stratagene Company.METHODS: This experiment was carried out in the neurology laboratory of Tongji Hospital in Wuhan during the period from October 2006 to April 2007. AAV-293 cells were resuscitated and cultivated with H-DMEM growth medium in vitro, and were passaged and stored in liquid nitrogen when the cells monolayer confluence reached 50%. At the same time, their growing state was observed by inverted microscope, and their growth curve was noted. According to whether AAV-293 cells could give out green fluorescence or not (observed by fluorescence inverted microscope) after they were cotransfected with the there AAV system plasmids and infected with AAV supernatant, their biological character of packing AAV was assessed.MAIN OUTCOME MEASURES: ① Morphological observation of AAV-293 cells; ② the growth curve; ③ the package of AAV.RESULTS: ① AAV-293 cells observed by fluorescence inverted microscope were growing adhesively well with irregular polygons, light endochylemas and ambiguous nuclei

  7. Intracranial Injection of Adeno-associated Viral Vectors

    OpenAIRE

    Lowery, Rebecca L.; Ania K Majewska

    2010-01-01

    Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially us...

  8. 禽腺联病毒Rep78和VP3蛋白的原核表达及抗血清制备%Prokaryotic expression of the Rep78 and VP3 proteins of avian adeno-associated virus and preparation of specific antisera

    Institute of Scientific and Technical Information of China (English)

    王建业; 孙怀昌; 朱国强

    2008-01-01

    分别将禽腺联病毒(Avian adeno-associated virus,AAAV)的Rep78基因和VP3基因克隆入pET-47b原核表达载体并转化BL21(DE3)大肠杆菌,在1PTG的诱导下2种目的蛋白均成功得到了表达.SDS-PAGE显示,Rep78蛋白的相对分子质量约为85 000,而VP3蛋白相对分子质量约为60 000.Western-blot分析显示,表达产物均能与抗AAAV的阳性血清反应.将目的蛋白切胶免疫BALB/c小鼠分别制备了针对2种蛋白的多克隆血清.间接免疫荧光试验显示制备的抗血清能够与AAAV抗原特异反应.不与鸡胚致死孤儿病毒(CELO)抗原反应.结果表明,制备的抗Rep78和VP3蛋白的血清可以用于检测重组AAAV载体制备过程中Rep和Cap基因的表达水平.

  9. Identification of adeno-associated viral vectors suitable for intestinal gene delivery and modulation of experimental colitis.

    Science.gov (United States)

    Polyak, Steven; Mach, Annette; Porvasnik, Stacy; Dixon, Lisa; Conlon, Thomas; Erger, Kirsten E; Acosta, Andres; Wright, Amy J; Campbell-Thompson, Martha; Zolotukhin, Irene; Wasserfall, Clive; Mah, Cathryn

    2012-02-01

    Effective gene transfer with sustained gene expression is an important adjunct to the study of intestinal inflammation and future therapy in inflammatory bowel disease. Recombinant adeno-associated virus (AAV) vectors are ideal for gene transfer and long-term transgene expression. The purpose of our study was to identify optimal AAV pseudotypes for transduction of the epithelium in the small intestine and colon, which could be used for studies in experimental colitis. The tropism and transduction efficiencies of AAV pseudotypes 1-10 were examined in murine small intestine and colon 8 wk after administration by real-time PCR and immunohistochemistry. The clinical and histopathological effects of IL-10-mediated intestinal transduction delivered by AAVrh10 were examined in the murine IL-10⁻/⁻ enterocolitis model. Serum IL-10 levels and IL-10 expression were followed by ELISA and real-time PCR, respectively. AAV pseudotypes 4, 7, 8, 9, and 10 demonstrated optimal intestinal transduction. Transgene expression was sustained 8 wk after administration and was frequently observed in enteroendocrine cells. Long-term IL-10 gene expression and serum IL-10 levels were observed following AAV transduction in an IL-10-/- model of enterocolitis. Animals treated with AAVrh10-IL-10 had lower disease activity index scores, higher colon weight-to-length ratios, and lower microscopic inflammation scores. This study identifies novel AAV pseudotypes with small intestine and colon tropism and sustained transgene expression capable of modulating mucosal inflammation in a murine model of enterocolitis.

  10. Surface immobilization of hexa-histidine-tagged adeno-associated viral vectors for localized gene delivery.

    Science.gov (United States)

    Jang, J-H; Koerber, J T; Gujraty, K; Bethi, S R; Kane, R S; Schaffer, D V

    2010-11-01

    Adeno-associated viral (AAV) vectors, which are undergoing broad exploration in clinical trials, have significant promise for therapeutic gene delivery because of their safety and delivery efficiency. Gene delivery technologies capable of mediating localized gene expression may further enhance the potential of AAV in a variety of therapeutic applications by reducing spread outside a target region, which may thereby reduce off-target side effects. We have genetically engineered an AAV variant capable of binding to surfaces with high affinity through a hexa-histidine metal-binding interaction. This immobilized AAV vector system mediates high-efficiency delivery to cells that contact the surface and thus may have promise for localized gene delivery, which may aid numerous applications of AAV delivery to gene therapy.

  11. Recombinant Adeno-Associated Virus-Mediated microRNA Delivery into the Postnatal Mouse Brain Reveals a Role for miR-134 in Dendritogenesis in Vivo

    DEFF Research Database (Denmark)

    Christensen, Mette; Larsen, Lars A; Kauppinen, Sakari;

    2010-01-01

    Recent studies using primary neuronal cultures have revealed important roles of the microRNA pathway in the regulation of neuronal development and morphology. For example, miR-134 is involved in dendritogenesis and spine development in hippocampal neurons by regulating local mRNA translation...

  12. Manufacturing of recombinant adeno-associated viral vectors for clinical trials.

    Science.gov (United States)

    Clément, Nathalie; Grieger, Joshua C

    2016-01-01

    The ability to elicit robust and long-term transgene expression in vivo together with minimal immunogenicity and little to no toxicity are only a few features that make recombinant adeno-associated virus (rAAV) vectors ideally suited for many gene therapy applications. Successful preclinical studies have encouraged the use of rAAV for therapeutic gene transfer to patients in the clinical setting. Nevertheless, the use of rAAV in clinical trials has underscored the need for production and purification systems capable of generating large amounts of highly pure rAAV particles. To date, generating vector quantities sufficient to meet the expanding clinical demand is still a hurdle when using current production systems. In this chapter, we will provide a description of the current methods to produce clinical grade of rAAV under current good manufacturing practice (cGMP) settings.

  13. Adeno-associated viral vector transduction of human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Stender, Stefan; Murphy, Mary; O'Brien, Tim

    2007-01-01

    Mesenchymal stem cells (MSCs) have received considerable attention in the emerging field of regenerative medicine. One aspect of MSC research focuses on genetically modifying the cells with the aim of enhancing their regenerative potential. Adeno-associated virus (AAV) holds promise as a vector...... in human MSCs and to assess whether AAV transduction affects MSC multipotentiality. The results indicated that human MSCs could indeed be transiently transduced in vitro by the AAV2 vector with efficiencies of up to 65%. The percentage of GFP-positive cells peaked at 4 days post-transduction and declined...... rapidly towards 0% after day 8. The level of transgene expression in the GFP-positive population increased 4-fold over a 10,000 fold viral dose increase. This dose-response contrasted with the 200-fold increase observed in similarly transduced 293-cells, indicating a relatively restricted transgene...

  14. Adeno-Associated Viral Vectors Serotype 8 for Cell-Specific Delivery of Therapeutic Genes in the Central Nervous System

    Science.gov (United States)

    Pignataro, Diego; Sucunza, Diego; Vanrell, Lucia; Lopez-Franco, Esperanza; Dopeso-Reyes, Iria G.; Vales, Africa; Hommel, Mirja; Rico, Alberto J.; Lanciego, Jose L.; Gonzalez-Aseguinolaza, Gloria

    2017-01-01

    Adeno-associated viruses (AAVs) have become highly promising tools for research and clinical applications in the central nervous system (CNS). However, specific delivery of genes to the cell type of interest is essential for the success of gene therapy and therefore a correct selection of the promoter plays a very important role. Here, AAV8 vectors carrying enhanced green fluorescent protein (eGFP) as reporter gene under the transcriptional control of different CNS-specific promoters were used and compared with a strong ubiquitous promoter. Since one of the main limitations of AAV-mediated gene delivery lies in its restricted cloning capacity, we focused our work on small-sized promoters. We tested the transduction efficacy and specificity of each vector after stereotactic injection into the mouse striatum. Three glia-specific AAV vectors were generated using two truncated forms of the human promoter for glial fibrillar acidic protein (GFAP) as well as a truncated form of the murine GFAP promoter. All three vectors resulted in predominantly glial expression; however we also observed eGFP expression in other cell-types such as oligodendrocytes, but never in neurons. In addition, robust and neuron-specific eGFP expression was observed using the minimal promoters for the neural protein BM88 and the neuronal nicotinic receptor β2 (CHRNB2). In summary, we developed a set of AAV vectors designed for specific expression in cells of the CNS using minimal promoters to drive gene expression when the size of the therapeutic gene matters. PMID:28239341

  15. Targeted CNS delivery using human MiniPromoters and demonstrated compatibility with adeno-associated viral vectors

    Directory of Open Access Journals (Sweden)

    Charles N de Leeuw

    2014-01-01

    Full Text Available Critical for human gene therapy is the availability of small promoters tools to drive gene expression in a highly specific and reproducible manner. We tackled this challenge by developing human DNA MiniPromoters (MiniPs using computational biology and phylogenetic conservation. MiniPs were tested in mouse as single-copy knock-ins at the Hprt locus on the X chromosome and evaluated for lacZ reporter expression in central nervous system (CNS and non–CNS tissue. Eighteen novel MiniPs driving expression in mouse brain were identified, 2 MiniPs for driving pan-neuronal expression and 17 MiniPs for the mouse eye. Key areas of therapeutic interest were represented in this set: the cerebral cortex, embryonic hypothalamus, spinal cord, bipolar and ganglion cells of the retina, and skeletal muscle. We also demonstrated that three retinal ganglion cell MiniPs exhibit similar cell type specificity when delivered via adeno-associated virus vectors intravitreally. We conclude that our methodology and characterization has resulted in desirable expression characteristics that are intrinsic to the MiniPromoter, not dictated by copy-number effects or genomic location, and results in constructs predisposed to success in adeno-associated virus. These MiniPs are immediately applicable for preclinical studies toward gene therapy in humans and are publicly available to facilitate basic and clinical research, and human gene therapy.

  16. Targeted CNS delivery using human MiniPromoters and demonstrated compatibility with adeno-associated viral vectors

    Science.gov (United States)

    de Leeuw, Charles N; Dyka, Frank M; Boye, Sanford L; Laprise, Stéphanie; Zhou, Michelle; Chou, Alice Y; Borretta, Lisa; McInerny, Simone C; Banks, Kathleen G; Portales-Casamar, Elodie; Swanson, Magdalena I; D’Souza, Cletus A; Boye, Shannon E; Jones, Steven JM; Holt, Robert A; Goldowitz, Daniel; Hauswirth, William W; Wasserman, Wyeth W; Simpson, Elizabeth M

    2014-01-01

    Critical for human gene therapy is the availability of small promoters tools to drive gene expression in a highly specific and reproducible manner. We tackled this challenge by developing human DNA MiniPromoters (MiniPs) using computational biology and phylogenetic conservation. MiniPs were tested in mouse as single-copy knock-ins at the Hprt locus on the X chromosome and evaluated for lacZ reporter expression in central nervous system (CNS) and non–CNS tissue. Eighteen novel MiniPs driving expression in mouse brain were identified, 2 MiniPs for driving pan-neuronal expression and 17 MiniPs for the mouse eye. Key areas of therapeutic interest were represented in this set: the cerebral cortex, embryonic hypothalamus, spinal cord, bipolar and ganglion cells of the retina, and skeletal muscle. We also demonstrated that three retinal ganglion cell MiniPs exhibit similar cell type specificity when delivered via adeno-associated virus vectors intravitreally. We conclude that our methodology and characterization has resulted in desirable expression characteristics that are intrinsic to the MiniPromoter, not dictated by copy-number effects or genomic location, and results in constructs predisposed to success in adeno-associated virus. These MiniPs are immediately applicable for preclinical studies toward gene therapy in humans and are publicly available to facilitate basic and clinical research, and human gene therapy. PMID:24761428

  17. Production and titering of recombinant adeno-associated viral vectors.

    Science.gov (United States)

    McClure, Christina; Cole, Katy L H; Wulff, Peer; Klugmann, Matthias; Murray, Andrew J

    2011-11-27

    In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

  18. Construction and identification of SHH-N gene adeno-associated virus vector and its impact on genes related to proliferation in neural stem cells%SHH-N基因腺相关病毒表达载体的构建及其对神经干细胞增殖相关基因的影响

    Institute of Scientific and Technical Information of China (English)

    刘东升; 崔岩; 申仑; 杜延平; 李桂林; 王任直; 王岩; 张波

    2011-01-01

    Objective To construct and identify SHH-N gene adeno-associated virus vector and to detect its effect on genes controlling to proliferation in neural stem cells(NSCs). Methods The neural stem cells in the subventricular zone of postnatal rat brain were used to collect SHH-N. pSNAV2. 0-CMV-SHH-N-IRES-EGFP was established by enzyme cutting and ligation , and then transfected packaging cell line 293T. Real time PCR was performed after SHH-N transfection of neural stem cells by rAAV-SHH-N-EGFP vector for 48 hours, SHH-N gene expression of infected cells after 2 week examined by fluorescence microscopy. Results ( 1 ) SHH-N gene was coincident with NCBI report. (2) pSNAV2. 0-CMV-SHH-N-IRES-EGFP expression vector and rAAV-SHH-N-EGFP vector was successful established and packaged. (3)Real time PCR was performed after SHH-N infection for 48 hour, induction of Nmyc and Glil in rAAV-SHH-N-EGFP-treated group was enhanced compared to control group. Conclusion rAAV-SHH-N-EGFP vector has been successfully established and it can stably express in neural stem cells. Forced expression of SHH-N in NSCs resulted in enhanced induction of Nmyc and Glil.%目的 构建rAAV-SHH-N-EGFP载体并检测其对神经干细胞增殖相关基因影响.方法 分离并培养大鼠脑室下区神经干细胞,提取RNA、反转录、PCR得到SHH-N的cDNA,克隆入pSNAV2.0-CMV-IRES-EGFP,包装得到腺相关病毒rAAV-SHH-N-EGFP,感染神经干细胞48 h后,采用实时定量PCR检测SHH信号通路下游相关基因的mRNA水平变化,并观察rAAV-SHH-N-EGFP载体在神经干细胞内的表达情况.结果 (1)克隆得到SHH-N基因,测序结果与NCBI报道序列一致.(2)成功构建pSNAV2.0-CMV-SHH-N-EGFP表达载体并鉴定,包装得到rAAV-SHH-N-EGFP.(3)实时定量PCR分析rAAV-SHH-N-EGFP感染神经干细胞48 h后,rAAV-SHH-N-EGFP处理组较感染rAAV-EGFP的对照组,Glil和Nmyc的mRNA水平上调.(4)rAAV-SHH-N-EGFP可有效感染神经干细胞,在感染14 d后稳定表达SHH-N.结论

  19. 人血管内皮生长因子与绿色荧光蛋白标记双基因共表达AAV载体的构建及鉴定%Construction and identification of recombinant adeno- associated virus vector co-expressing human vascular endothelial growth factor and green fluorescent protein

    Institute of Scientific and Technical Information of China (English)

    黄向辉; 时志斌; 王坤正; 党晓谦; 杨佩; 余鹏博

    2008-01-01

    取重组病毒基因组成功扩增出外源目的基因片段hVEGF165,证实重组病毒rAAV-VEGF165-GFP包装成功.结论:成功构建带有绿色荧光蛋白标记并携带hVEGF165基因的无致病性重组腺相关病毒rAAV-VEGF165-GFP.收获的病毒具有较高滴度和感染活性.%BACKGROUND: Vascular endothelial growth factor (VEGF) can specifically promote the division and proliferation of endothelial cells and the revascularization, finally induce angiopoiesis. Recently, VEGF-based gene therapy has been gradually used in clinical trials, but some limits on usually used vectors deserve further studies, including the low transfection efficiency of plasmid vector, the immunogenicity of adnovirus vector to host cells and the potential risk of infection. OBJECTIVE: To construct the non-pathogenic recombinant adeno-associated vires (AAV) co-expressing human vascular endethelial growth factor 165 (hVEGF165) and green fluorescent protein (GFP) label and measure the virus titer and assess its biological activity.DESIGN, TIME AND SETTING: The open experiment was performed at the Vitrus Laboratory of Shanxi Provincial Center for Disease Control and Prevention from March to September 2007.MATERIALS: AAV-293 virus packaging cell line, AAV HT-1080 cells were purchased from Swatagene, USA. E.coli DH5α was a stocked strain from Shanxi Provincial Center for Disease Control and Prevention. AAV Helper Free System (pAAV-IRES-GFP vector containing GFP label) was purchased from Stratagene, USA. Plasmid pUC18-hVEGF165 was constructed previously by Dr.Shi from Department of Orthopaedics of Second Affiliated Hospital of Xi'an Jiaotong University.METHODS: The hVEGF165 gone from plasmid pUC18-hVEGF165 was amplified and inserted into plasmid pAAV-IRES-hrGFP. Then recombinant plasmid pAAV-hVEGF165-IRES-hrGFP, pAAV-RC and pAAV-Helper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of A

  20. Copackaging of multiple adeno-associated viral vectors in a single production step.

    Science.gov (United States)

    Doerfler, Phillip A; Byrne, Barry J; Clément, Nathalie

    2014-10-01

    Limiting factors in large preclinical and clinical studies utilizing adeno-associated virus (AAV) for gene therapy are focused on the restrictive packaging capacity, the overall yields, and the versatility of the production methods for single AAV vector production. Furthermore, applications where multiple vectors are needed to provide long expression cassettes, whether because of long cDNA sequences or the need of different regulatory elements, require that each vector be packaged and characterized separately, directly affecting labor and cost associated with such manufacturing strategies. To overcome these limitations, we propose a novel method of vector production that allows for the packaging of multiple expression cassettes in a single transfection step. Here we combined two expression cassettes in predetermined ratios before transfection and empirically demonstrate that the output vector recapitulates the predicted ratios. Titration by quantitative polymerase chain reaction of AAV vector genome copies using shared or unique genetic elements allowed for delineation of the individual vector contribution to the total preparation that showed the predicted differential packaging outcomes. By copackaging green fluorescent protein (GFP) and mCherry constructs, we demonstrate that both vector genome and infectious titers reiterated the ratios utilized to produce the constructs by transfection. Copackaged therapeutic constructs that only differ in transcriptional elements produced a heterogeneous vector population of both constructs in the predefined ratios. This study shows feasibility and reproducibility of a method that allows for two constructs, differing in either transgene or transcription elements, to be efficiently copackaged and characterized simultaneously, reducing cost of manufacturing and release testing.

  1. The Contrast Tranfecting Efficiency of a Double Stranded Recombinant Adeno -associated Virus Expressing Exendin -4 Compared with Single Stranded AAV%重组双链及单链腺相关病毒介导Exendin -4表达效率的比较

    Institute of Scientific and Technical Information of China (English)

    王俊红; 问姣; 董鹏; 张静; 焦杨; 郭永红

    2015-01-01

    To construct a recombinant double -strands adeno -associated virus(dsAAV)expressing Exendin -4,compared trans-fecting efficiency in cells with a single stranded AAV(ssAAV).The recombinant dsAAV vector pSSHG/Exendin -4 was reconstructed which Exendin -4 by gene engineering method.The recombinant virus was packaged by human embryo kidney 293 cells and the virus titer was measured .The expression GFPs of dsAAV and ssAAV in NIH3T3 cell were detected by fluorescence microscope.Exendin -4 level in the superment of NIH3T3 transferred by dsAAV /pSSHG/Exendin -4or ssAAV /pSSHG/Exendin -4 were determined by ELISA.The expression Exendin -4 of dsAAV and ssAAV in NIH3T3 cell were detected by immunohistochemical.Recombinant dsAAV vector was successfμl constructed and confirmed by restriction enzymes and DNA sequencing.The more higher titer of recombi-nant virus was by homologous recombinationin dsAAV (2.5 ×10 11 pfu)than by ssAAV(2.5 ×109 pfu).There was higher fluorescence intensity in transfected NIH3T3 cells by dsAAV.The Exendin -4 level was higer in supernatant of dsAAV than in ssAAV(181.1 ± 8.75 pmol/mlvs133.81 ±8.09 pmol/ml).The Exendin -4 expression was positive both in dsAAV and ssAAV.The Exendin -4 ex-pression system in dsAAV has relatively high transfecting ability.It should play a significant and fundamental role for further researches about diabetes gene therapy.%比较双链腺相关病毒(Double -stranded adeno -associated virus,dsAAV)及单链 AAV(Single -stranded adeno -associated virus,ssAAV)介导 Exendin -4分泌表达效率。应用基因工程方法构建 dsAAV /pSSHG/Exendin -4,与重组 ssAAV 比较转染 NIH3T3细胞效率及转染后细胞上清 Exendin -4浓度,免疫组化检测 Exendin -4表达。经限制性内切酶及测序证实载体构建成功,测定重组 dsAAV pSSHG/Exendin -4滴度为2.5×1011 pfu,较重组 ssAA 滴度高(2.5×109 pfu);dsAAV /pSSHG/GFP 转染 NIH3T3细

  2. Preparation of a recombinant adeno-associated viral vector with a mutation of human factor IX in large scale and its expression in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A series of adeno-associated viral vectors conraining a mutation of human factor IX (hFIXR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFIXR338Awere obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFIXR338Awas prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFIXR338A viral stocks on a large scale and higher fiter was established,which can be used for industrial purpose. The titer of rAAV-hFIXR338A was more than 1.25x1012 particle/mL, and then, a mammalian cell line, C2C12 and the factor IXknock-out mice were transfected with the rAAV-hFIXR338Ain vitro and in vivo. The results show that the high-level expression of rAAV-hFIXR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng@ (106cells)-1 @ (24 h)-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFIX R338A, which was as high as the expression of rAAV-hFIX -wt (2565.76±64.36) ng@ (106 cells)-1@ (24 h)-1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFIX-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFIXR338A is about 2.46times higher than that of hFIX-wt. It was first reported that a mutation of human factor IX was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFIX R338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFIX.``

  3. A comparative analysis of constitutive promoters located in adeno-associated viral vectors.

    Directory of Open Access Journals (Sweden)

    Lkhagvasuren Damdindorj

    Full Text Available The properties of constitutive promoters within adeno-associated viral (AAV vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV, simian virus 40, and herpes simplex virus thymidine kinase, were constructed and introduced into the HCT116, DLD-1, HT-1080, and MCF-10A cell lines. Quantification of GFP signals in infected cells demonstrated that the CMV promoter produced the highest GFP expression in the six promoters and maintained relatively high GFP expression for up to eight weeks after infection of HCT116, DLD-1, and HT-1080. Exogenous human CDKN2A gene expression was also introduced into DLD-1 and MCF-10A in a similar pattern by using AAV vectors bearing the human β-actin and the CMV promoters. The six constitutive promoters were subsequently placed upstream of the neomycin resistance gene within AAV vectors, and HCT116, DLD-1, and HT-1080 were infected with the resulting vectors. Of the six promoters, the CMV promoter produced the largest number of G418-resistant colonies in all three cell lines. Because AAV vectors have been frequently used as a platform to construct targeting vectors that permit gene editing in human cell lines, we lastly infected the three cell lines with AAV-based targeting vectors against the human PIGA gene in which one of the six promoters regulate the neomycin resistance gene. This assay revealed that the CMV promoter led to the lowest PIGA gene targeting efficiency in the investigated promoters. These results provide a clue to the identification of constitutive promoters suitable to express exogenous genes with AAV vectors, as well as those helpful to conduct efficient gene targeting using AAV-based targeting vectors in human cell lines.

  4. 重组腺相关病毒转导人树突状细胞体外诱导抗肝癌免疫应答%Generation of antitumor response against hepatocellular carcinoma by in vitro transduction of dendritic cells with adeno-associated virus expressing α-fetoprotein

    Institute of Scientific and Technical Information of China (English)

    杜文贞; 于天霞

    2011-01-01

    Objective To investigate the generation of antitumor response against hepatocellular carcinoma by in vitro transduction of dendritic cells (DC)with recombinant adeno-associated virus expressing α-fetoprotein (rAAV-AFP). Methods Peripheral blood mononuclear cells were isolated from healthy volunteers. Adherent peripheral blood mononuclear cells were transduced with AAV-AFP and cultured in the presence of granulocyte macrophage colony stimulating factor and interleukin-4 to generate dendritic cells.MTS assay was used to measure the ability of DC transduced with AAV-AFP ( AAV-AFP + DC) to stimulate the proliferation of T cell. The phenotype and AFP protein expression of DC and the secretion of IFN (interferon)-γ and IL (interleukin)-4 by T cells were detected by flow cytometry. The killing efficacy of cytotoxic T lymphocytes (CTL) activated by AAV-AFP + DC against AFP positive hepatocellular carcinoma cell lines was detected by lactate dehydrogenase (LDH) release assay. Results AAV-AFP + DC expressed HLA Ⅰ (97. 12%), HLAⅡ (97.32%), CD80(38.94%), CD83(60.84%)and CD86(98. 14%). AFP was secreted by 81.2% of AAV-AFP + DC. And it could stimulate effectively the proliferation of T cell.19. 84% of CD4 + T cells and 18.65% of CD8 + T cells activated by AAV-AFP + DC produced IFN-γbut not IL-4 and showed distinct killing activities against AFP positive hepatocellular carcinoma cell lines HepG2 (56. 45% ) and BEL7402 (78. 84% ). Conclusion AAV-AFP + DC can elicit distinct antitumor responses against AFP positive hepatocellular carcinoma cell lines so as to provide a basis for further researches on the clinical application of AAV-AFP + DC in the treatment of hepatocellular carcinoma.%目的 探讨携带甲胎蛋白基因的重组腺相关病毒(rAAV-AFP)转导人树突状细胞(DC)体外诱导抗肝癌免疫应答.方法 分离健康志愿者外周血单核细胞,贴壁细胞转导rAAV-AFP后,在粒细胞巨噬细胞集落刺激因子(GMCSF)和白细胞介素4(IL-4)的联

  5. Good manufacturing practice production of self-complementary serotype 8 adeno-associated viral vector for a hemophilia B clinical trial.

    Science.gov (United States)

    Allay, James A; Sleep, Susan; Long, Scott; Tillman, David M; Clark, Rob; Carney, Gael; Fagone, Paolo; McIntosh, Jenny H; Nienhuis, Arthur W; Davidoff, Andrew M; Nathwani, Amit C; Gray, John T

    2011-05-01

    To generate sufficient clinical-grade vector to support a phase I/II clinical trial of adeno-associated virus serotype 8 (AAV8)-mediated factor IX (FIX) gene transfer for hemophilia B, we have developed a large-scale, good manufacturing practice (GMP)-compatible method for vector production and purification. We used a 293T-based two-plasmid transient transfection system coupled with a three-column chromatography purification process to produce high-quality self-complementary AAV2/8 FIX clinical-grade vector. Two consecutive production campaigns using a total of 432 independent 10-stack culture chambers produced a total of ∼2 × 10(15) vector genomes (VG) by dot-blot hybridization. Benzonase-treated microfluidized lysates generated from pellets of transfected cells were purified by group separation on Sepharose beads followed by anion-exchange chromatography. The virus-containing fractions were further processed by gel filtration and ultrafiltration, using a 100-kDa membrane. The vector was formulated in phosphate-buffered saline plus 0.25% human serum albumin. Spectrophotometric analysis suggested ∼20% full particles, with only low quantities of nonviral proteins were visible on silver-stained sodium dodecyl sulfate-polyacrylamide gels. A sensitive assay for the detection of replication-competent AAV was developed, which did reveal trace quantities of such contaminants in the final product. Additional studies have confirmed the long-term stability of the vector at -80°C for at least 24 months and for at least 24 hr formulated in the clinical diluent and stored at room temperature within intravenous bags. This material has been approved for use in clinical trials in the United States and the United Kingdom.

  6. Successful disabling of the 5' UTR of HCV using adeno-associated viral vectors to deliver modular multimeric primary microRNA mimics.

    Science.gov (United States)

    Bourhill, Tarryn; Arbuthnot, Patrick; Ely, Abdullah

    2016-09-01

    Chronic hepatitis C virus (HCV) infection is a major health concern and is strongly associated with cirrhosis, hepatocellular carcinoma and liver-related mortality. The HCV genome is the template for both protein translation and viral replication and, being RNA, is amenable to direct genetic silencing by RNA interference (RNAi). HCV is a highly mutable virus and is capable of escaping RNAi-mediated silencing. This has highlighted the importance of developing RNAi-based therapy that simultaneously targets multiple regions of the HCV genome. To develop a multi-targeting RNAi activator, a novel approach for the generation of anti-HCV gene therapy was investigated. Five artificial primary miRNA (pri-miR) were each designed to mimic the naturally occurring monomeric pri-miR-31. Potent knockdown of an HCV reporter was seen with four of the five constructs and were processed according to the intended design. The design of the individual pri-miR mimics enabled the modular assembly into multimeric mimics of any possible conformation. Consequently the four potent pri-miR mimics were used to generate polycistronic cassettes, which showed impressive silencing of an HCV target. To further their application as a gene therapy, recombinant adeno-associated viral (rAAV) vectors that express the polycistronic pri-miR mimics were generated. All AAV-delivered anti-HCV pri-miR mimics significantly knocked down the expression of an HCV target and showed inhibition of HCV replicon replication. Here we describe a protocol for the generation of therapeutic rAAVs that express modular polycistronic pri-miR cassettes allowing for rapid alteration and generation of tailored therapeutic constructs against HCV.

  7. Adeno-associated viral vector-induced overexpression of neuropeptide Y Y2 receptors in the hippocampus suppresses seizures

    DEFF Research Database (Denmark)

    Woldbye, David Paul Drucker; Ängehagen, Mikael; Gøtzsche, Casper René;

    2010-01-01

    Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure...... suppression by neuropeptide Y in the hippocampus is predominantly mediated by Y2 receptors, which, together with neuropeptide Y, are upregulated after seizures as a compensatory mechanism. To explore whether such upregulation could prevent seizures, we overexpressed Y2 receptors in the hippocampus using...... and neuropeptide Y had a more pronounced seizure-suppressant effect. These results demonstrate that overexpression of Y2 receptors (alone or in combination with neuropeptide Y) could be an alternative strategy for epilepsy treatment....

  8. Development of next generation adeno-associated viral vectors capable of selective tropism and efficient gene delivery.

    Science.gov (United States)

    Zhang, Chuanling; Yao, Tianzhuo; Zheng, Yongxiang; Li, Zhongjun; Zhang, Qiang; Zhang, Lihe; Zhou, Demin

    2016-02-01

    Virus-based nanoparticles have shown promise as vehicles for delivering therapeutic genes. However, the rational design of viral vectors that enable selective tropism towards particular types of cells and tissues remains challenging. Here, we explored structural-functional relationships of the adeno-associated virus 2 (AAV2) vector by expanding its genetic code during production. As a proof-of-principle, an azide moiety was strategically displayed on the vector capsid as a bioorthogonal chemical reporter. Upon bioorthogonal conjugation of AAV2 with fluorophores and cyclic arginyl-glycyl-aspartic acid ligands at certain modifiable sites, we characterized in vitro and in vivo AAV2 movement and enhanced tropism selectivity towards integrin-expressing tumor cells. Targeting AAV2 vectors resulted in selective killing of U87 glioblastoma cells and derived xenografts via the herpes simplex virus suicide gene thymidine kinase, with the potency of ganciclovir being increased by 25-fold. Our results demonstrated successful rational modification of AAV2 as a targeting delivery vehicle, establishing a facile platform for precision engineering of virus-based nanoparticles in basic research and therapeutic applications.

  9. Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors

    Directory of Open Access Journals (Sweden)

    Sablitzky Fred

    2004-01-01

    Full Text Available Abstract Background Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV and lentiviral (LV vectors into discrete regions of the forebrain. Results Recombinant AAV-Cre, AAV-GFP (green fluorescent protein and LV-Cre-EGFP (enhanced GFP were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection. Conclusion AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.

  10. Adeno-Associated Viral Vectors Transduce Mature Human Adipocytes in Three-Dimensional Slice Cultures.

    Science.gov (United States)

    Kallendrusch, Sonja; Schopow, Nikolas; Stadler, Sonja C; Büning, Hildegard; Hacker, Ulrich T

    2016-10-01

    Adipose tissue plays a pivotal role, both in the regulation of energy homeostasis and as an endocrine organ. Consequently, adipose tissue dysfunction is closely related to insulin resistance, morbid obesity, and metabolic syndrome. To study molecular mechanisms and to develop novel therapeutic strategies, techniques are required to genetically modify mature adipocytes. Here, we report on adeno-associated viral (AAV) vectors as a versatile tool to transduce human mature adipocytes in organotypic three-dimensional tissue cultures.

  11. Novel adeno-associated viral vector delivering the utrophin gene regulator jazz counteracts dystrophic pathology in mdx mice.

    Science.gov (United States)

    Strimpakos, Georgios; Corbi, Nicoletta; Pisani, Cinzia; Di Certo, Maria Grazia; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Gabanella, Francesca; Monaco, Lucia; Mattei, Elisabetta; Passananti, Claudio

    2014-09-01

    Over-expression of the dystrophin-related gene utrophin represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. We developed the artificial zinc finger transcription factor "Jazz" that up-regulates both the human and mouse utrophin promoter. We observed a significant recovery of muscle strength in dystrophic Jazz-transgenic mdx mice. Here we demonstrate the efficacy of an experimental gene therapy based on the systemic delivery of Jazz gene in mdx mice by adeno-associated virus (AAV). AAV serotype 8 was chosen on the basis of its high affinity for skeletal muscle. Muscle-specific expression of the therapeutic Jazz gene was enhanced by adding the muscle α-actin promoter to the AAV vector (mAAV). Injection of mAAV8-Jazz viral preparations into mdx mice resulted in muscle-specific Jazz expression coupled with up-regulation of the utrophin gene. We show a significant recovery from the dystrophic phenotype in mAAV8-Jazz-treated mdx mice. Histological and physiological analysis revealed a reduction of fiber necrosis and inflammatory cell infiltration associated with functional recovery in muscle contractile force. The combination of ZF-ATF technology with the AAV delivery can open a new avenue to obtain a therapeutic strategy for treatment of DMD. © 2014 Wiley Periodicals, Inc.

  12. Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B

    Science.gov (United States)

    Arruda, Valder R.; Stedman, Hansell H.; Haurigot, Virginia; Buchlis, George; Baila, Stefano; Favaro, Patricia; Chen, Yifeng; Franck, Helen G.; Zhou, Shangzhen; Wright, J. Fraser; Couto, Linda B.; Jiang, Haiyan; Pierce, Glenn F.; Bellinger, Dwight A.; Mingozzi, Federico; Nichols, Timothy C.

    2010-01-01

    Muscle represents an important tissue target for adeno-associated viral (AAV) vector-mediated gene transfer of the factor IX (FIX) gene in hemophilia B (HB) subjects with advanced liver disease. Previous studies of direct intramuscular administration of an AAV-FIX vector in humans showed limited efficacy. Here we adapted an intravascular delivery system of AAV vectors encoding the FIX transgene to skeletal muscle of HB dogs. The procedure, performed under transient immunosuppression (IS), resulted in widespread transduction of muscle and sustained, dose-dependent therapeutic levels of canine FIX transgene up to 10-fold higher than those obtained by intramuscular delivery. Correction of bleeding time correlated clinically with a dramatic reduction of spontaneous bleeding episodes. None of the dogs (n = 14) receiving the AAV vector under transient IS developed inhibitory antibodies to canine FIX; transient inhibitor was detected after vector delivery without IS. The use of AAV serotypes with high tropism for muscle and low susceptibility to anti-AAV2 antibodies allowed for efficient vector administration in naive dogs and in the presence of low- but not high-titer anti-AAV2 antibodies. Collectively, these results demonstrate the feasibility of this approach for treatment of HB and highlight the importance of IS to prevent immune responses to the FIX transgene product. PMID:20335222

  13. Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B.

    Science.gov (United States)

    Arruda, Valder R; Stedman, Hansell H; Haurigot, Virginia; Buchlis, George; Baila, Stefano; Favaro, Patricia; Chen, Yifeng; Franck, Helen G; Zhou, Shangzhen; Wright, J Fraser; Couto, Linda B; Jiang, Haiyan; Pierce, Glenn F; Bellinger, Dwight A; Mingozzi, Federico; Nichols, Timothy C; High, Katherine A

    2010-06-10

    Muscle represents an important tissue target for adeno-associated viral (AAV) vector-mediated gene transfer of the factor IX (FIX) gene in hemophilia B (HB) subjects with advanced liver disease. Previous studies of direct intramuscular administration of an AAV-FIX vector in humans showed limited efficacy. Here we adapted an intravascular delivery system of AAV vectors encoding the FIX transgene to skeletal muscle of HB dogs. The procedure, performed under transient immunosuppression (IS), resulted in widespread transduction of muscle and sustained, dose-dependent therapeutic levels of canine FIX transgene up to 10-fold higher than those obtained by intramuscular delivery. Correction of bleeding time correlated clinically with a dramatic reduction of spontaneous bleeding episodes. None of the dogs (n = 14) receiving the AAV vector under transient IS developed inhibitory antibodies to canine FIX; transient inhibitor was detected after vector delivery without IS. The use of AAV serotypes with high tropism for muscle and low susceptibility to anti-AAV2 antibodies allowed for efficient vector administration in naive dogs and in the presence of low- but not high-titer anti-AAV2 antibodies. Collectively, these results demonstrate the feasibility of this approach for treatment of HB and highlight the importance of IS to prevent immune responses to the FIX transgene product.

  14. Phase 2 clinical trial of a recombinant adeno-associated viral vector expressing α1-antitrypsin: interim results.

    LENUS (Irish Health Repository)

    Flotte, Terence R

    2011-10-01

    Recombinant adeno-associated virus (rAAV) vectors offer promise for the gene therapy of α(1)-antitrypsin (AAT) deficiency. In our prior trial, an rAAV vector expressing human AAT (rAAV1-CB-hAAT) provided sustained, vector-derived AAT expression for >1 year. In the current phase 2 clinical trial, this same vector, produced by a herpes simplex virus complementation method, was administered to nine AAT-deficient individuals by intramuscular injection at doses of 6.0×10(11), 1.9×10(12), and 6.0×10(12) vector genomes\\/kg (n=3 subjects\\/dose). Vector-derived expression of normal (M-type) AAT in serum was dose dependent, peaked on day 30, and persisted for at least 90 days. Vector administration was well tolerated, with only mild injection site reactions and no serious adverse events. Serum creatine kinase was transiently elevated on day 30 in five of six subjects in the two higher dose groups and normalized by day 45. As expected, all subjects developed anti-AAV antibodies and interferon-γ enzyme-linked immunospot responses to AAV peptides, and no subjects developed antibodies to AAT. One subject in the mid-dose group developed T cell responses to a single AAT peptide unassociated with any clinical effects. Muscle biopsies obtained on day 90 showed strong immunostaining for AAT and moderate to marked inflammatory cell infiltrates composed primarily of CD3-reactive T lymphocytes that were primarily of the CD8(+) subtype. These results support the feasibility and safety of AAV gene therapy for AAT deficiency, and indicate that serum levels of vector-derived normal human AAT >20 μg\\/ml can be achieved. However, further improvements in the design or delivery of rAAV-AAT vectors will be required to achieve therapeutic target serum AAT concentrations.

  15. Adeno-Associated Viral Vector-Induced Overexpression of Neuropeptide Y Y2 Receptors in the Hippocampus Suppresses Seizures

    Science.gov (United States)

    Woldbye, David P. D.; Angehagen, Mikael; Gotzsche, Casper R.; Elbrond-Bek, Heidi; Sorensen, Andreas T.; Christiansen, Soren H.; Olesen, Mikkel V.; Nikitidou, Litsa; Hansen, Thomas v. O.; Kanter-Schlifke, Irene; Kokaia, Merab

    2010-01-01

    Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure suppression by neuropeptide Y in the hippocampus is…

  16. Noninvasive Imaging Reveals Stable Transgene Expression in Mouse Airways After Delivery of a Nonintegrating Recombinant Adeno-Associated Viral Vector

    NARCIS (Netherlands)

    Vidovic, D; Gijsbers, R; Quiles Jimenez, A; Dooley, J; Van Den Haute, C; Van der Perren, A; Liston, A; Baekelandt, V; Debyser, Z; Carlon, MS

    2015-01-01

    Gene therapy holds promise to cure a wide range of genetic and acquired diseases. Recent successes in recombinant adeno-associated viral vector (rAAV)-based gene therapy in the clinic for hereditary disorders such as Leber's congenital amaurosis and hemophilia B encouraged us to reexplore an rAAV

  17. The use of an adeno-associated viral vector for efficient bicistronic expression of two genes in the central nervous system.

    Science.gov (United States)

    Hutson, Thomas Haynes; Kathe, Claudia; Menezes, Sean Christopher; Rooney, Marie-Claire; Bueler, Hansruedi; Moon, Lawrence David Falcon

    2014-01-01

    Recombinant adeno-associated viral (AAV) vectors are one of the most promising therapeutic delivery systems for gene therapy to the central nervous system (CNS). Preclinical testing of novel gene therapies requires the careful design and production of AAV vectors and their successful application in a model of CNS injury. One major limitation of AAV vectors is their limited packaging capacity (genes (e.g., from two promoters) difficult. An internal ribosomal entry site has been used to express two genes: However, the second transgene is often expressed at lower levels than the first. In addition to this, achieving high levels of transduction in the CNS can be challenging. In this chapter we describe the cloning of a bicistronic AAV vector that uses the foot-and-mouth disease virus 2A sequence to efficiently express two genes from a single promoter. Bicistronic expression of a therapeutic gene and a reporter gene is desirable so that the axons from transduced neurons can be tracked and, after CNS injury, the amount of axonal sprouting or regeneration quantified. We go on to describe how to perform a pyramidotomy model of CNS injury and the injection of AAV vectors into the sensorimotor cortex to provide efficient transduction and bicistronic gene expression in cortical neurons such that transduced axons are detectable in the dorsal columns of the spinal cord.

  18. Successful gene therapy in older Rpe65-deficient dogs following subretinal injection of an adeno-associated vector expressing RPE65.

    Science.gov (United States)

    Annear, Matthew J; Mowat, Freya M; Bartoe, Joshua T; Querubin, Janice; Azam, Selina A; Basche, Mark; Curran, Paul G; Smith, Alexander J; Bainbridge, James W B; Ali, Robin R; Petersen-Jones, Simon M

    2013-10-01

    Young Rpe65-deficient dogs have been used as a model for human RPE65 Leber congenital amaurosis (RPE65-LCA) in proof-of-concept trials of recombinant adeno-associated virus (rAAV) gene therapy. However, there are relatively few reports of the outcome of rAAV gene therapy in Rpe65-deficient dogs older than 2 years of age. The purpose of this study was to investigate the success of this therapy in older Rpe65-deficient dogs. Thirteen eyes were treated in dogs between 2 and 6 years old. An rAAV2 vector expressing the human RPE65 cDNA driven by the human RPE65 promoter was delivered by subretinal injection. Twelve of the 13 eyes had improved retinal function as assessed by electroretinography, and all showed improvement in vision at low lighting intensities. Histologic examination of five of the eyes was performed but found no correlation between electroretinogram (ERG) rescue and numbers of remaining photoreceptors. We conclude that functional rescue is still possible in older dogs and that the use of older Rpe65-deficient dogs, rather than young Rpe65-deficient dogs that have very little loss of photoreceptors, more accurately models the situation when treating human RPE65-LCA patients.

  19. Complete Correction of Hemophilia A with Adeno-Associated Viral Vectors Containing a Full-Size Expression Cassette

    OpenAIRE

    Lu, Hui; Chen, Lingxia; Wang, Jinhui; Huack, Bernd; Sarkar, Rita; Zhou, Shangzhen; Xu, Ray; Ding, Qiulan; Wang, Xuefeng; Wang, HongLi; Xiao, Weidong

    2008-01-01

    Hemophilia A is caused by a deficiency in the factor VIII (FVIII) gene. Constrained by limited packaging capacity, even the 4.3-kb B domain-deleted FVIII remained a challenge for delivery by a single adeno-associated viral (AAV) vector. Studies have shown that up to a 6.6-kb vector sequence may be packaged into AAV virions, which suggested an alternative strategy for hemophilia A gene therapy. To explore the usefulness of AAV vectors carrying an oversized FVIII gene, we constructed the AAV-FV...

  20. Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery

    Directory of Open Access Journals (Sweden)

    Harrison C Brown

    2014-01-01

    Full Text Available Clinical data support the feasibility and safety of adeno-associated viral (AAV vectors in gene therapy applications. Despite several clinical trials of AAV-based gene transfer for hemophilia B, a unique set of obstacles impede the development of a similar approach for hemophilia A. These include (i the size of the factor VIII (fVIII transgene, (ii humoral immune responses to fVIII, (iii inefficient biosynthesis of human fVIII, and (iv AAV vector immunity. Through bioengineering approaches, a novel fVIII molecule, designated ET3, was developed and shown to improve biosynthetic efficiency 10- to 100-fold. In this study, the utility of ET3 was assessed in the context of liver-directed, AAV-mediated gene transfer into hemophilia A mice. Due to the large size of the expression cassette, AAV-ET3 genomes packaged into viral particles as partial genome fragments. Despite this potential limitation, a single peripheral vein administration of AAV-ET3 into immune-competent hemophilia A mice resulted in correction of the fVIII deficiency at lower vector doses than previously reported for similarly oversized AAV-fVIII vectors. Therefore, ET3 appears to improve vector potency and mitigate at least one of the critical barriers to AAV-based clinical gene therapy for hemophilia A.

  1. 转化生长因子-β3与基质金属蛋白酶抑制剂-2联合转染兔骨髓间充质干细胞复合丝素蛋白/壳聚糖生物支架修复兔软骨缺损%Resurfacing rabbit's cartilage defects using mesenchymal stem cells co-transfected with transforming growth factor-β3 and matrix metalloproteinases inhibitors-2 by adeno-associated virus embedded in Silk fibroin/chitosan scaffolds

    Institute of Scientific and Technical Information of China (English)

    孟飞; 吕成昱; 张海宁; 申成凯; 冯尚祥

    2015-01-01

    conditions,we took the third generation of the logarithmic growth phase of rabbit bone marrow mesenchymal stem cells (BMSCs).And we used recombinant adeno-associated virus which carrying each group' s purpose gene to transfected them.After two months we killed the rabbits,as well as visually assessed of cartilage defect repair situation by hematoxylin-eosin staining (HE) staining.And conduct characteristic of cartilage cells that stained with toluidine blue staining and type Ⅱ collagen immunohistochemical staining.Results We observed that:after two months,cartilage-like substance formed in each expermental group' s cariliage defect area,and the co-transfection group induced cartilage were closer to hyaline cartilage.We found the Moran score of difference groups were:concrol group:0.600 ± 0.548,non-transfection group:1.800 ± 0.447,pure TGF-β3 transfected group:3.800 ± 0.837,co-transfection group:5.400 ± 0.548.And the difference of score between co-transfection group and pure TGF-β3 transfected group was statistically significant (P < 0.01).HE staining results suggested that co-transfected cartilage repair group' s result was better than pure TGF-β3 transfected group.Conclusion In animal experiments,pure TGF-β3 transfected rabbit bone marrow mesenchymal stem cells can repair rabbit articular cartilage defects,TGF-β3 and matrix metalloproteinases inhibitors-2 (TIMP-2) co-transfection group was more effective.

  2. Complete correction of hemophilia A with adeno-associated viral vectors containing a full-size expression cassette.

    Science.gov (United States)

    Lu, Hui; Chen, Lingxia; Wang, Jinhui; Huack, Bernd; Sarkar, Rita; Zhou, Shangzhen; Xu, Ray; Ding, Qiulan; Wang, Xuefeng; Wang, Hongli; Xiao, Weidong

    2008-06-01

    Hemophilia A is caused by a deficiency in the factor VIII (FVIII) gene. Constrained by limited packaging capacity, even the 4.3-kb B domain-deleted FVIII remained a challenge for delivery by a single adeno-associated viral (AAV) vector. Studies have shown that up to a 6.6-kb vector sequence may be packaged into AAV virions, which suggested an alternative strategy for hemophilia A gene therapy. To explore the usefulness of AAV vectors carrying an oversized FVIII gene, we constructed the AAV-FVIII vector under the control of a beta-actin promoter with a cytomegalovirus enhancer (CB) and a bovine growth hormone (bGH) poly(A) sequence. The CB promoter plus bGH signal was shown to be 3- to 5-fold more potent than the mini-transthyretin (TTR) promoter with a synthetic poly(A) sequence for directing FVIII expression in the liver. Despite the 5.75-kb genome size of pAAV-CB-FVIII, sufficient AAV vectors were produced for in vivo testing. Approximately 3- to 5-fold more FVIII secretion was observed in animals receiving AAV-CB-FVIII vectors than in those receiving standard-sized AAV-TTR-FVIII vectors. Both the activated partial thromboplastin time assay and the whole blood thromboelastographic analysis confirmed that AAV-FVIII vectors fully corrected the bleeding phenotype of hemophilia mice. These results suggest that AAV vectors with an oversized genome should be useful for not only hemophilia A gene therapy but also other diseases with large cDNA such as muscular dystrophy and cystic fibrosis.

  3. Evaluation of a rapamycin-regulated serotype 2 adeno-associated viral vector in macaque parotid glands

    Science.gov (United States)

    Zheng, Changyu; Voutetakis, Antonis; Metzger, Mark; Wainer, Sandra; Cotrim, Ana P.; Eckhaus, Michael A.; Rivera, Victor M.; Clackson, Tim; Chiorini, John A.; Donahue, Robert E.; Dunbar, Cynthia E.; Baum, Bruce J.

    2009-01-01

    OBJECTIVES Salivary glands are useful target organs for local and systemic gene therapeutics. For such applications, the regulation of transgene expression is important. Previous studies by us in murine submandibular glands showed that a rapamycin transcriptional regulation system in a single serotype 2, adeno-associated viral (AAV2) vector was effective for this purpose. The present study evaluated if such a vector was similarly useful in rhesus macaque parotid glands. METHODS A recombinant AAV2 vector (AAV-TF-RhEpo-2.3w), encoding rhesus erythropoietin (RhEpo) and a rapamycin-inducible promoter, was constructed. The vector was administered to macaques at either of two doses (1.5×1011 [low dose] or 1.5×1012 [high dose] vector genomes) via cannulation of Stensen’s duct. Animals were followed for 12–14 weeks and treated at intervals with rapamycin (0.1 or 0.5 mg/kg) to induce gene expression. Serum chemistry, hematology and RhEpo levels were measured at interval. RESULTS AAV-TF-RhEpo-2.3w administration led to low levels of rapamycin-inducible RhEpo expression in the serum of most macaques. In five animals no significant changes were seen in serum chemistry and hematology values over the study. One macaque, however, developed pneumonia, became anemic and subsequently required euthanasia. After the onset of anemia, a single administration of rapamycin led to significant RhEpo production in this animal. CONCLUSION Administration of AAV-TF-RhEpo-2.3w to macaque parotid glands was generally safe, but led only to low levels of serum RhEpo in healthy animals following rapamycin treatment. PMID:20374510

  4. AAV2/9介导的PD-L1保护移植物的实验研究%EXPERIMENTAL STUDY ON ADENO-ASSOCIATED VIRUS 2/9 MEDIATED PD-L1 GENE TRANSFER TO THE GRAFT PROTECTING ALLO-REJECTION

    Institute of Scientific and Technical Information of China (English)

    罗汕; 蒋小峰; 杨学伟

    2013-01-01

    目的 探讨腺相关病毒介导的PD-L1在移植排斥反应中的作用.方法 用含AAV2/9-PD-L1或LacZ的UW液灌注供体心脏后,移植到受体大鼠体内,观察移植物的转染效率和存活时间.结果 AAV2/9可以高效地介导外源性基因转染移植物,AAV2/9-PD-L1转染移植物后虽能延长移植物的存活时间,但和对照组比较无统计学意义.结论 AAV2/9是一种高效安全的病毒载体,PD-L1局部表达抑制排斥反应的机制还需进一步研究.

  5. Photosensitizers mediated photodynamic inactivation against virus particles.

    Science.gov (United States)

    Sobotta, Lukasz; Skupin-Mrugalska, Paulina; Mielcarek, Jadwiga; Goslinski, Tomasz; Balzarini, Jan

    2015-01-01

    Viruses cause many diseases in humans from the rather innocent common cold to more serious or chronic, life-threatening infections. The long-term side effects, sometimes low effectiveness of standard pharmacotherapy and the emergence of drug resistance require a search for new alternative or complementary antiviral therapeutic approaches. One new approach to inactivate microorganisms is photodynamic antimicrobial chemotherapy (PACT). PACT has evolved as a potential method to inactivate viruses. The great challenge for PACT is to develop a methodology enabling the effective inactivation of viruses while leaving the host cells as untouched as possible. This review aims to provide some main directions of antiviral PACT, taking into account different photosensitizers, which have been widely investigated as potential antiviral agents. In addition, several aspects concerning PACT as a tool to assure viral inactivation in human blood products will be addressed.

  6. Meganuclease-mediated virus self-cleavage facilitates tumor-specific virus replication.

    Science.gov (United States)

    Gürlevik, Engin; Schache, Peter; Goez, Anneliese; Kloos, Arnold; Woller, Norman; Armbrecht, Nina; Manns, Michael P; Kubicka, Stefan; Kühnel, Florian

    2013-09-01

    Meganucleases can specifically cleave long DNA sequence motifs, a feature that makes them an ideal tool for gene engineering in living cells. In a proof-of-concept study, we investigated the use of the meganuclease I-Sce I for targeted virus self-disruption to generate high-specific oncolytic viruses. For this purpose, we provided oncolytic adenoviruses with a molecular circuit that selectively responds to p53 activation by expression of I-Sce I subsequently leading to self-disruption of the viral DNA via heterologous I-Sce I recognition sites within the virus genome. We observed that virus replication and cell lysis was effectively impaired in p53-normal cells, but not in p53-dysfunctional tumor cells. I-Sce I activity led to effective intracellular processing of viral DNA as confirmed by detection of specific cleavage products. Virus disruption did not interfere with E1A levels indicating that reduction of functional virus genomes was the predominant cause for conditional replication. Consequently, tumor-specific replication was further enhanced when E1A expression was additionally inhibited by targeted transcriptional repression. Finally, we demonstrated p53-dependent oncolysis by I-Sce I-expressing viruses in vitro and in vivo, and demonstrated effective inhibition of tumor growth. In summary, meganuclease-mediated virus cleavage represents a promising approach to provide oncolytic viruses with attractive safety profiles.

  7. Transient photoreceptor deconstruction by CNTF enhances rAAV-mediated cone functional rescue in late stage CNGB3-achromatopsia.

    Science.gov (United States)

    Komáromy, András M; Rowlan, Jessica S; Corr, Amanda T Parton; Reinstein, Shelby L; Boye, Sanford L; Cooper, Ann E; Gonzalez, Amaliris; Levy, Britt; Wen, Rong; Hauswirth, William W; Beltran, William A; Aguirre, Gustavo D

    2013-06-01

    Achromatopsia is a genetic disorder of cones, and one of the most common forms is a channelopathy caused by mutations in the β-subunit, CNGB3, of the cone cyclic nucleotide-gated (CNG) channel. Recombinant adeno-associated virus of serotype 5 (rAAV5)-mediated gene transfer of human CNGB3 cDNA to mutant dog cones results in functional and structural rescue in dogs 1 year. We now test a new therapeutic concept by combining gene therapy with the administration of ciliary neurotrophic factor (CNTF). Intravitreal CNTF causes transient dedifferentiation of photoreceptors, a process called deconstruction, whereby visual cells become immature with short outer segments, and decreased retinal function and gene expression that subsequently return to normal. Cone function was successfully rescued in all mutant dogs treated between 14 and 42 months of age with this strategy. CNTF-mediated deconstruction and regeneration of the photoreceptor outer segments prepares the mutant cones optimally for gene augmentation therapy.

  8. Sendai virus, an RNA virus with no risk of genomic integration, delivers CRISPR/Cas9 for efficient gene editing.

    Science.gov (United States)

    Park, Arnold; Hong, Patrick; Won, Sohui T; Thibault, Patricia A; Vigant, Frederic; Oguntuyo, Kasopefoluwa Y; Taft, Justin D; Lee, Benhur

    2016-01-01

    The advent of RNA-guided endonuclease (RGEN)-mediated gene editing, specifically via CRISPR/Cas9, has spurred intensive efforts to improve the efficiency of both RGEN delivery and targeted mutagenesis. The major viral vectors in use for delivery of Cas9 and its associated guide RNA, lentiviral and adeno-associated viral systems, have the potential for undesired random integration into the host genome. Here, we repurpose Sendai virus, an RNA virus with no viral DNA phase and that replicates solely in the cytoplasm, as a delivery system for efficient Cas9-mediated gene editing. The high efficiency of Sendai virus infection resulted in high rates of on-target mutagenesis in cell lines (75-98% at various endogenous and transgenic loci) and primary human monocytes (88% at the ccr5 locus) in the absence of any selection. In conjunction with extensive former work on Sendai virus as a promising gene therapy vector that can infect a wide range of cell types including hematopoietic stem cells, this proof-of-concept study opens the door to using Sendai virus as well as other related paramyxoviruses as versatile and efficient tools for gene editing.

  9. Expression of Lecithin: Cholesterol Acyltransferaseand/or apoA-I Mediated by Recombinant Adeno-as-sociated Virus in Myogenic Cell

    Institute of Scientific and Technical Information of China (English)

    王立峰; 范乐明; 陈丙莺; 刘宝瑞; 王若宁; 魏恩会

    2002-01-01

    Objective Lecithia: cholesterol acyltrmsfer ase (LCAT) is the major enzyme producing most plasma cholesterol esters( CE )and a key partiipant in the process of reverse cholesterol traansfer ( RCT). The aim of the study was to co-express LCAT and its nature activator apoA- I medi ated by recombinant adeno-associated virus vectors in the skeletal muscle cells, and open a new avenue of gene therapy touard the primary or secondary LCAT deficiency. Methods 293T cells were cotrans fected with pDG and rAAVAIL/rAAVL plasmid to produce infectious rAAV, and non-iouic iodixanol gradients centri f ngation followed by heparin affinity chromatography was per formed f or separation . pu rification and concentration of rAAV. The particle numbers of rAAV were assayed by dot-blot, then these vectors transduced C2C12 myoblasts. ELISA and Western Blot asasayed for human apoA- I and 3H-cholesterol labeled radiochemical methods for LCAT activity. Genomic DNA was extracted from transduced C2C12 and analyzed fo the presence of vector sequence by PCR amplifiations. Results The particle mumbers of rAAV were 7× 1014/L (rAAAIL) and 1 × 1014/L (rAAVL). The expres sion of human apoA- I cDNA and/or human LCAT cDNA in transduced C2C12 cells lasted for 3 0 d, even after myoblasts were differentiated into myotubes. PCR products for transgene indiated the long-term persistence of transduced vector sequences. Conclusion The result indicated that the meth ods used for production and purification of rAAV is an effiient and rAAV vector mediate the expres sion and secretion of LCAT and apoA- I gene in C2C12 myoblasts successfully. It suggested that the use of rAAV vectors mediating the high efficiency, long-term expression of human LCAT cDNA and/ or apoA- I cDNA in skeletal muscle in vivo might be a safe and fessible strategy to the gene therapy of LCAT deficiency.

  10. Mutagenesis-mediated virus extinction: Virus-dependent effect of viral load on sensitivity to lethal defection

    OpenAIRE

    Héctor Moreno; Héctor Tejero; Juan Carlos de la Torre; Esteban Domingo; Verónica Martín

    2012-01-01

    Background: Lethal mutagenesis is a transition towards virus extinction mediated by enhanced mutation rates during viral genome replication, and it is currently under investigation as a potential new antiviral strategy. Viral load and virus fitness are known to influence virus extinction. Here we examine the effect or the multiplicity of infection (MOI) on progeny production of several RNA viruses under enhanced mutagenesis. Results: The effect of the mutagenic base analogue 5-fluorouracil (F...

  11. [Mechanisms of Epstein-Barr Virus-Mediated Oncogenesis].

    Science.gov (United States)

    Iwakiri, Dai

    2015-10-01

    Epstein-Barr virus(EBV), a ubiquitous human double-stranded DNA virus, is associated with a variety of malignancies including Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma, and gastric carcinoma. Latent EBV infections have been discovered in cases of EBV -associated cancers, suggesting that EBV latent genes contribute to oncogenesis. Here, I describe mechanisms of oncogenesis associated with EBV, focusing on functions of EBV latent membrane protein(LMP)and EBV-encoded small RNA (EBER). LMP2A, which mimics B cell receptor signaling, and LMP1, which mimics CD40 signaling, collaboratively contribute to malignant lymphoma development. It has been reported that LMP2A-mediated intracellular signaling plays significant roles in epithelial carcinogenesis. However, it has also been demonstrated that EBER, which is expected to have a double-stranded RNA(dsRNA)structure, triggers signal transduction via host viral RNA sensors, RIG-I and TLR3, causing EBV-associated pathogenesis, including carcinogenesis.

  12. The Measles Virus Receptor SLAMF1 Can Mediate Particle Endocytosis.

    Science.gov (United States)

    Gonçalves-Carneiro, Daniel; McKeating, Jane A; Bailey, Dalan

    2017-04-01

    The signaling lymphocyte activation molecule F1 (SLAMF1) is both a microbial sensor and entry receptor for measles virus (MeV). Herein, we describe a new role for SLAMF1 to mediate MeV endocytosis that is in contrast with the alternative, and generally accepted, model that MeV genome enters cells only after fusion at the cell surface. We demonstrated that MeV engagement of SLAMF1 induces dramatic but transient morphological changes, most prominently in the formation of membrane blebs, which were shown to colocalize with incoming viral particles, and rearrangement of the actin cytoskeleton in infected cells. MeV infection was dependent on these dynamic cytoskeletal changes as well as fluid uptake through a macropinocytosis-like pathway as chemical inhibition of these processes inhibited entry. Moreover, we identified a role for the RhoA-ROCK-myosin II signaling axis in this MeV internalization process, highlighting a novel role for this recently characterized pathway in virus entry. Our study shows that MeV can hijack a microbial sensor normally involved in bacterial phagocytosis to drive endocytosis using a complex pathway that shares features with canonical viral macropinocytosis, phagocytosis, and mechanotransduction. This uptake pathway is specific to SLAMF1-positive cells and occurs within 60 min of viral attachment. Measles virus remains a significant cause of mortality in human populations, and this research sheds new light on the very first steps of infection of this important pathogen.IMPORTANCE Measles is a significant disease in humans and is estimated to have killed over 200 million people since records began. According to current World Health Organization statistics, it still kills over 100,000 people a year, mostly children in the developing world. The causative agent, measles virus, is a small enveloped RNA virus that infects a broad range of cells during infection. In particular, immune cells are infected via interactions between glycoproteins found

  13. The Measles Virus Receptor SLAMF1 Can Mediate Particle Endocytosis

    Science.gov (United States)

    Gonçalves-Carneiro, Daniel; McKeating, Jane A.

    2017-01-01

    ABSTRACT The signaling lymphocyte activation molecule F1 (SLAMF1) is both a microbial sensor and entry receptor for measles virus (MeV). Herein, we describe a new role for SLAMF1 to mediate MeV endocytosis that is in contrast with the alternative, and generally accepted, model that MeV genome enters cells only after fusion at the cell surface. We demonstrated that MeV engagement of SLAMF1 induces dramatic but transient morphological changes, most prominently in the formation of membrane blebs, which were shown to colocalize with incoming viral particles, and rearrangement of the actin cytoskeleton in infected cells. MeV infection was dependent on these dynamic cytoskeletal changes as well as fluid uptake through a macropinocytosis-like pathway as chemical inhibition of these processes inhibited entry. Moreover, we identified a role for the RhoA-ROCK-myosin II signaling axis in this MeV internalization process, highlighting a novel role for this recently characterized pathway in virus entry. Our study shows that MeV can hijack a microbial sensor normally involved in bacterial phagocytosis to drive endocytosis using a complex pathway that shares features with canonical viral macropinocytosis, phagocytosis, and mechanotransduction. This uptake pathway is specific to SLAMF1-positive cells and occurs within 60 min of viral attachment. Measles virus remains a significant cause of mortality in human populations, and this research sheds new light on the very first steps of infection of this important pathogen. IMPORTANCE Measles is a significant disease in humans and is estimated to have killed over 200 million people since records began. According to current World Health Organization statistics, it still kills over 100,000 people a year, mostly children in the developing world. The causative agent, measles virus, is a small enveloped RNA virus that infects a broad range of cells during infection. In particular, immune cells are infected via interactions between

  14. Apolipoprotein E Mediates Evasion From Hepatitis C Virus Neutralizing Antibodies.

    Science.gov (United States)

    Fauvelle, Catherine; Felmlee, Daniel J; Crouchet, Emilie; Lee, JiYoung; Heydmann, Laura; Lefèvre, Mathieu; Magri, Andrea; Hiet, Marie-Sophie; Fofana, Isabel; Habersetzer, François; Foung, Steven K H; Milne, Ross; Patel, Arvind H; Vercauteren, Koen; Meuleman, Philip; Zeisel, Mirjam B; Bartenschlager, Ralf; Schuster, Catherine; Baumert, Thomas F

    2016-01-01

    Efforts to develop an effective vaccine against hepatitis C virus (HCV) have been hindered by the propensity of the virus to evade host immune responses. HCV particles in serum and in cell culture associate with lipoproteins, which contribute to viral entry. Lipoprotein association has also been proposed to mediate viral evasion of the humoral immune response, though the mechanisms are poorly defined. We used small interfering RNAs to reduce levels of apolipoprotein E (apoE) in cell culture-derived HCV-producing Huh7.5-derived hepatoma cells and confirmed its depletion by immunoblot analyses of purified viral particles. Before infection of naïve hepatoma cells, we exposed cell culture-derived HCV strains of different genotypes, subtypes, and variants to serum and polyclonal and monoclonal antibodies isolated from patients with chronic HCV infection. We analyzed the interaction of apoE with viral envelope glycoprotein E2 and HCV virions by immunoprecipitation. Through loss-of-function studies on patient-derived HCV variants of several genotypes and subtypes, we found that the HCV particle apoE allows the virus to avoid neutralization by patient-derived antibodies. Functional studies with human monoclonal antiviral antibodies showed that conformational epitopes of envelope glycoprotein E2 domains B and C were exposed after depletion of apoE. The level and conformation of virion-associated apoE affected the ability of the virus to escape neutralization by antibodies. In cell-infection studies, we found that HCV-associated apoE helps the virus avoid neutralization by antibodies against HCV isolated from chronically infected patients. This method of immune evasion poses a challenge for the development of HCV vaccines. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

  15. Multifunctional Virus-Nanoshell Assembly for Targeted Hyperthermia and Viral Gene Therapy for Breast Cancer

    Science.gov (United States)

    2012-06-01

    cancer cells in synergy with gene therapy. We proposed to develop virus- nanoshell assemblies by attaching adeno-associated virus (AAV) to gold... nanoshells (Au NS) through chemical bonds. We have successfully completed majority of tasks 1 and 2 of our Statement of Work. Specifically, we have...therapy, virus, Au nanoshell Multifunctional Virus- Nanoshell Assembly for Targeted Hyperthermia and Viral Gene Therapy for Breast Cancer Dr. Fang Wei

  16. Virus-mediated suppression of host non-self recognition facilitates horizontal transmission of heterologous viruses

    Science.gov (United States)

    Wu, Songsong; Cheng, Jiasen; Fu, Yanping; Chen, Tao; Jiang, Daohong; Ghabrial, Said A.

    2017-01-01

    Non-self recognition is a common phenomenon among organisms; it often leads to innate immunity to prevent the invasion of parasites and maintain the genetic polymorphism of organisms. Fungal vegetative incompatibility is a type of non-self recognition which often induces programmed cell death (PCD) and restricts the spread of molecular parasites. It is not clearly known whether virus infection could attenuate non-self recognition among host individuals to facilitate its spread. Here, we report that a hypovirulence-associated mycoreovirus, named Sclerotinia sclerotiorum mycoreovirus 4 (SsMYRV4), could suppress host non-self recognition and facilitate horizontal transmission of heterologous viruses. We found that cell death in intermingled colony regions between SsMYRV4-infected Sclerotinia sclerotiorum strain and other tested vegetatively incompatible strains was markedly reduced and inhibition barrage lines were not clearly observed. Vegetative incompatibility, which involves Heterotrimeric guanine nucleotide-binding proteins (G proteins) signaling pathway, is controlled by specific loci termed het (heterokaryon incompatibility) loci. Reactive oxygen species (ROS) plays a key role in vegetative incompatibility-mediated PCD. The expression of G protein subunit genes, het genes, and ROS-related genes were significantly down-regulated, and cellular production of ROS was suppressed in the presence of SsMYRV4. Furthermore, SsMYRV4-infected strain could easily accept other viruses through hyphal contact and these viruses could be efficiently transmitted from SsMYRV4-infected strain to other vegetatively incompatible individuals. Thus, we concluded that SsMYRV4 is capable of suppressing host non-self recognition and facilitating heterologous viruses transmission among host individuals. These findings may enhance our understanding of virus ecology, and provide a potential strategy to utilize hypovirulence-associated mycoviruses to control fungal diseases. PMID:28334041

  17. A fragmented adeno-associated viral dual vector strategy for treatment of diseases caused by mutations in large genes leads to expression of hybrid transcripts

    Science.gov (United States)

    McClements, Michelle E.; Charbel Issa, Peter; Blouin, Véronique; MacLaren, Robert E.

    2017-01-01

    Objective Dual vector AAV systems are being utilised to enable gene therapy for disorders in which the disease gene is too large to fit into a single capsid. Fragmented adeno-associated viral (fAAV) vectors containing single inverted terminal repeat truncated transgenes have been considered as one such gene replacement strategy. Here we aim to add to the current understanding of the molecular mechanisms employed by fAAV dual vector systems. Methods Oversized (>8kb) transgene constructs containing ABCA4 coding sequence were packaged as truncated fragments <5kb in size into various AAV serotypes. In vitro transductions with these fAAV vector preparations were conducted with mRNA and protein expression products assessed by way of RT-PCR, qPCR and western blot techniques. Results Transductions with fAAV vector preparations yielded ABCA4 mRNA, but did not generate detectable levels of protein. Sequencing of the transcript population revealed the presence of full length ABCA4 CDS with additional hybrid ABCA4 variants, indicating truncated transgenes without regions of overlap were joining and forming stable hybrid transgenes. In contrast, an ABCA4 overlapping dual vector system (OV) with a defined complementary region generated only full length mRNA transcripts plus detectable ABCA4 protein. Conclusion Despite previous success shown with the fAAV approach, the lack of repeatability and identification of stable hybrid transcripts capable of protein production suggests there is more refinement required before considering this approach in a clinical setting. PMID:28239514

  18. Characterization of cognitive deficits in rats overexpressing human alpha-synuclein in the ventral tegmental area and medial septum using recombinant adeno-associated viral vectors.

    Directory of Open Access Journals (Sweden)

    Hélène Hall

    Full Text Available Intraneuronal inclusions containing alpha-synuclein (a-syn constitute one of the pathological hallmarks of Parkinson's disease (PD and are accompanied by severe neurodegeneration of A9 dopaminergic neurons located in the substantia nigra. Although to a lesser extent, A10 dopaminergic neurons are also affected. Neurodegeneration of other neuronal populations, such as the cholinergic, serotonergic and noradrenergic cell groups, has also been documented in PD patients. Studies in human post-mortem PD brains and in rodent models suggest that deficits in cholinergic and dopaminergic systems may be associated with the cognitive impairment seen in this disease. Here, we investigated the consequences of targeted overexpression of a-syn in the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways. Rats were injected with recombinant adeno-associated viral vectors encoding for either human wild-type a-syn or green fluorescent protein (GFP in the ventral tegmental area and the medial septum/vertical limb of the diagonal band of Broca, two regions rich in dopaminergic and cholinergic neurons, respectively. Histopathological analysis showed widespread insoluble a-syn positive inclusions in all major projections areas of the targeted nuclei, including the hippocampus, neocortex, nucleus accumbens and anteromedial striatum. In addition, the rats overexpressing human a-syn displayed an abnormal locomotor response to apomorphine injection and exhibited spatial learning and memory deficits in the Morris water maze task, in the absence of obvious spontaneous locomotor impairment. As losses in dopaminergic and cholinergic immunoreactivity in both the GFP and a-syn expressing animals were mild-to-moderate and did not differ from each other, the behavioral impairments seen in the a-syn overexpressing animals appear to be determined by the long term persisting neuropathology in the surviving neurons rather than by neurodegeneration.

  19. RNA interference-mediated inhibition of Hepatitis B Virus replication

    Institute of Scientific and Technical Information of China (English)

    TANG Ni; ZHANG Bingqiang; YAN Ge; PU Dan; GAO Xiaolin; Tong-Chuan He; HUANG Ailong

    2004-01-01

    Persistent and recurrent infection of hepatitis B virus (HBV) represents one of the most common and severe viral infections of humans, and has caused a formidable health problem in the affected countries. Currently used antiviral drugs have a very limited success on controlling HBV replication and infection. RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of target mRNA in mammalian and plant cells, has recently been used to knockdown gene expression in various species. In this study, we sought to determine whether RNAi-mediated silencing of HBV viral gene expression could lead to the effective inhibition of HBV replication. We first developed RNAi vectors that expressed small interfering RNA (siRNA) and targeted the HBV core or surface gene sequence. Our results demonstrated that these specific siRNAs efficiently reduced the levels of corresponding viral RNAs and proteins, and thus suppressed viral replication. Treatment with siRNA gave the greatest reduction in the levels of HBsAg (92%) and in HBeAg (85%) respectively in the cultured cell medium. Our findings further demonstrated that the RNAi-mediated antiviral effect was sequence-specific and dose-dependent. Therefore, our findings strongly suggest that RNAi-mediated silencing of HBV viral genes could effectively inhibit the replication of HBV, hence RNAi-based strategy should be further explored as a more efficacious antiviral therapy of HBV infection.

  20. Phylodynamics and human-mediated dispersal of a zoonotic virus.

    Science.gov (United States)

    Talbi, Chiraz; Lemey, Philippe; Suchard, Marc A; Abdelatif, Elbia; Elharrak, Mehdi; Nourlil, Jalal; Jalal, Nourlil; Faouzi, Abdellah; Echevarría, Juan E; Vazquez Morón, Sonia; Rambaut, Andrew; Campiz, Nicholas; Tatem, Andrew J; Holmes, Edward C; Bourhy, Hervé

    2010-10-28

    Understanding the role of humans in the dispersal of predominantly animal pathogens is essential for their control. We used newly developed Bayesian phylogeographic methods to unravel the dynamics and determinants of the spread of dog rabies virus (RABV) in North Africa. Each of the countries studied exhibited largely disconnected spatial dynamics with major geopolitical boundaries acting as barriers to gene flow. Road distances proved to be better predictors of the movement of dog RABV than accessibility or raw geographical distance, with occasional long distance and rapid spread within each of these countries. Using simulations that bridge phylodynamics and spatial epidemiology, we demonstrate that the contemporary viral distribution extends beyond that expected for RABV transmission in African dog populations. These results are strongly supportive of human-mediated dispersal, and demonstrate how an integrated phylogeographic approach will turn viral genetic data into a powerful asset for characterizing, predicting, and potentially controlling the spatial spread of pathogens.

  1. Phylodynamics and human-mediated dispersal of a zoonotic virus.

    Directory of Open Access Journals (Sweden)

    Chiraz Talbi

    Full Text Available Understanding the role of humans in the dispersal of predominantly animal pathogens is essential for their control. We used newly developed Bayesian phylogeographic methods to unravel the dynamics and determinants of the spread of dog rabies virus (RABV in North Africa. Each of the countries studied exhibited largely disconnected spatial dynamics with major geopolitical boundaries acting as barriers to gene flow. Road distances proved to be better predictors of the movement of dog RABV than accessibility or raw geographical distance, with occasional long distance and rapid spread within each of these countries. Using simulations that bridge phylodynamics and spatial epidemiology, we demonstrate that the contemporary viral distribution extends beyond that expected for RABV transmission in African dog populations. These results are strongly supportive of human-mediated dispersal, and demonstrate how an integrated phylogeographic approach will turn viral genetic data into a powerful asset for characterizing, predicting, and potentially controlling the spatial spread of pathogens.

  2. Hendra virus detection using Loop-Mediated Isothermal Amplification.

    Science.gov (United States)

    Foord, Adam J; Middleton, Deborah; Heine, Hans G

    2012-04-01

    Hendra virus (HeV) is a zoonotic paramyxovirus endemic in Australian Pteropus bats (fruit bats or flying foxes). Although bats appear to be unaffected by the virus, HeV can spread from fruit bats to horses, causing severe disease. Human infection results from close contact with the blood, body fluids and tissues of infected horses. HeV is a biosecurity level 4 (BSL-4) pathogen, with a high case-fatality rate in humans and horses. Current assays for HeV detection require complex instrumentation and are generally time consuming. The aim of this study was to develop a Loop-Mediated Isothermal Amplification (LAMP) assay to detect nucleic acid from all known HeV strains in horses without the requirement for complex laboratory equipment. A LAMP assay targeting a conserved region of the HeV P-gene was combined with a Lateral Flow Device (LFD) for detection of amplified product. All HeV isolates, the original HeV isolated in 1994 as well as the most recent isolates from 2011 were detected. Analytical sensitivity and specificity of the HeV-LAMP assay was equal to a TaqMan assay developed previously. Significantly, these assays detected HeV in horses before clinical signs were observed. The combined LAMP-LFD procedure is a sensitive method suitable for HeV diagnosis in a resource-limited situation or where rapid test results are critical.

  3. [Molecular mechanisms of Epstein-Barr virus-mediated carcinogeneis].

    Science.gov (United States)

    Iwakiri, Dai

    2014-01-01

    Epstein-Barr virus (EBV), a ubiquitous human double stranded DNA virus, is associated with a variety of malignancies including Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). These EBV-associated cancers are characterized by the proliferation of monoclonal EBV-infected cells, and viral gene expression in these cells is limited to a subset of latent genes, indicating that EBV latent genes contribute to carcinogenesis. Here I describe the mechanisms of carcinogenesis by EBV, focusing on the function of two EBV latent gens, latent membrane protein 2A (LMP2A) and EBV-encoded small RNA (EBER). LMP2A, which is known to mimic the B cell receptor (BCR) signaling, has been reported to contribute to malignant lymphoma development through the modulation of immune signals. Also, it has been demonstrated that LMP2A-mediated intracellular signaling plays significant roles in epithelial carcinogenesis. On the other hand, it has been demonstrated that EBER, which is expected to form double stranded RNA (dsRNA) structure, triggers a signal transduction from host viral RNA sensors RIG-I and TLR3. Activation of innate immune signals by EBER has been reported to contribute to the pathogenesis of EBV-associated diseases, including cancers.

  4. Virus-mediated FCC iron nanoparticle induced synthesis of uranium dioxide nanocrystals.

    Science.gov (United States)

    Ling, Tao; Yu, Huimin; Shen, Zhongyao; Wang, Hui; Zhu, Jing

    2008-03-19

    A reducing system involving M13 virus-mediated FCC Fe nanoparticles was employed to achieve uranium reduction and synthesize uranium dioxide nanocrystals. Here we show that metastable face-centered cubic (FCC) Fe nanoparticles were fabricated around the surface of the M13 virus during the specific adsorption of the virus towards Fe ions under a reduced environment. The FCC phase of these Fe nanoparticles was confirmed by careful TEM characterization. Moreover, this virus-mediated FCC Fe nanoparticle system successfully reduced contaminable U(VI) into UO(2) crystals with diameters of 2-5 nm by a green and convenient route.

  5. Determination of Anti-Adeno-Associated Viral Vector Neutralizing Antibodies in Patients With Heart Failure in the Cardiovascular Foundation of Colombia (ANVIAS): Study Protocol

    Science.gov (United States)

    Prada, Carlos E; Lopez, Marcos; Castillo, Victor; Echeverria, Luis Eduardo; Serrano, Norma

    2016-01-01

    Background Recent progress in the pathophysiology of heart failure (HF) has led to the development of new therapeutic options such as gene therapy and the use of adeno-associated viral (AAV) vectors. Despite the promising results in early clinical trials of gene therapy for HF, various obstacles have been faced, such as the presence of neutralizing antibodies (NAbs) against the capsid vectors. NAb activity limits vector transduction levels and therefore diminishes the final therapeutic response. Recent studies evaluating the prevalence of NAbs in various populations found considerable geographic variability for each AAV serotype. However, the levels of NAbs in Latin American populations are unknown, becoming a limiting factor to conducting AAV vector therapeutic trials in this population. Objective The goal of this study is to determine for the first time, the prevalence of anti-AAV NAbs for the serotypes 1, 2, and 9 in HF patients from the city of Bucaramanga, Colombia, using the in vitro transduction inhibition assay. Methods We will conduct a cross-sectional study with patients who periodically attend the HF clinic of the Cardiovascular Foundation of Colombia and healthy volunteers matched for age and sex. For all participants, we will evaluate the NAb levels against serotypes AAV1, AAV2, and AAV9. We will determine NAb levels using the in vitro transduction inhibition assay. In addition, participants will answer a survey to evaluate their epidemiological and socioeconomic variables. Participation in the study will be voluntary and all participants will sign an informed consent document before any intervention. Results The project is in the first phase: elaboration of case report forms and the informed consent form, and design of the recruitment strategy. Patient recruitment is expected to begin in the spring of 2016. We expect to have preliminary results, including the titer of the viral vectors, multiplicity of infections that we will use for each serotype

  6. Identification of Multiple Novel Viruses, Including a Parvovirus and a Hepevirus, in Feces of Red Foxes

    Science.gov (United States)

    van der Giessen, Joke; Haagmans, Bart L.; Osterhaus, Albert D. M. E.; Smits, Saskia L.

    2013-01-01

    Red foxes (Vulpes vulpes) are the most widespread members of the order of Carnivora. Since they often live in (peri)urban areas, they are a potential reservoir of viruses that transmit from wildlife to humans or domestic animals. Here we evaluated the fecal viral microbiome of 13 red foxes by random PCR in combination with next-generation sequencing. Various novel viruses, including a parvovirus, bocavirus, adeno-associated virus, hepevirus, astroviruses, and picobirnaviruses, were identified. PMID:23616657

  7. Identification of multiple novel viruses, including a parvovirus and a hepevirus, in feces of red foxes.

    Science.gov (United States)

    Bodewes, Rogier; van der Giessen, Joke; Haagmans, Bart L; Osterhaus, Albert D M E; Smits, Saskia L

    2013-07-01

    Red foxes (Vulpes vulpes) are the most widespread members of the order of Carnivora. Since they often live in (peri)urban areas, they are a potential reservoir of viruses that transmit from wildlife to humans or domestic animals. Here we evaluated the fecal viral microbiome of 13 red foxes by random PCR in combination with next-generation sequencing. Various novel viruses, including a parvovirus, bocavirus, adeno-associated virus, hepevirus, astroviruses, and picobirnaviruses, were identified.

  8. Adeno-Associated Virus Transfer of a Gene Encoding SNAP-25 Resistant to Botulinum Toxin A Attenuates Neuromuscular Paralysis Associated with Botulism

    Science.gov (United States)

    2008-04-02

    refinement of the technology for application to genetic disorders of motor neurons. 15. SUBJECT TERMS Clostridium botulinum, neurotoxiin, serotype...peripherally transported transgenic protein successfully substituted for native cleavage-susceptible S25, as demonstrated by enhanced retention of nerve

  9. Imaging multiple intermediates of single-virus membrane fusion mediated by distinct fusion proteins.

    Science.gov (United States)

    Joo, Kye-Il; Tai, April; Lee, Chi-Lin; Wong, Clement; Wang, Pin

    2010-09-01

    Membrane fusion plays an essential role in the entry of enveloped viruses into target cells. The merging of viral and target cell membranes is catalyzed by viral fusion proteins, which involves multiple sequential steps in the fusion process. However, the fusion mechanisms mediated by different fusion proteins involve multiple transient intermediates that have not been well characterized. Here, we report a synthetic virus platform that allows us to better understand the different fusion mechanisms driven by the diverse types fusion proteins. The platform consists of lentiviral particles coenveloped with a surface antibody, which serves as the binding protein, along with a fusion protein derived from either influenza virus (HAmu) or Sindbis virus (SINmu). By using a single virus tracking technique, we demonstrated that both HAmu- and SINmu-bearing viruses enter cells through clathrin-dependent endocytosis, but they required different endosomal trafficking routes to initiate viral fusion. Direct observation of single viral fusion events clearly showed that hemifusion mediated by SINmu upon exposure to low pH occurs faster than that mediated by HAmu. Monitoring sequential fusion processes by dual labeling the outer and inner leaflets of viral membranes also revealed that the SINmu-mediated hemifusion intermediate is relatively long-lived as compared with that mediated by HAmu. Taken together, we have demonstrated that the combination of this versatile viral platform with the techniques of single virus tracking can be a powerful tool for revealing molecular details of fusion mediated by various fusion proteins.

  10. hTRX-PR39重组腺相关病毒载体的构建%Construction of a hTRX-PR39 recombinant adeno-associated vires vector

    Institute of Scientific and Technical Information of China (English)

    阮喜云; 魏敏; 毕建忠; 杨广笑; 王全颍

    2011-01-01

    Objective To construct a vector of recombinant adeno-associated virus (AAV) containing the fusion gene hTRX-PR39, and explore the role of fusion gene hTRX-PR39 as a target gene in the treatment of cerebral ischemic disease. Methods The constructed fusion gene hTRX-PR39 was inserted into the EcoRI-BamHI site of vector plasmid pSSCMV, and the vector of hTRX-PR39 recombinant AAV was constructed. The recombinant AAV stock was packaged. Renal embryo 293 cells were co-transfected with the recombinant AAV vector of plasmid pSSCMV/hTRX-PR39,packaging plasmid pAAV/Ad and helper adenovirus plasmid pFG140. Recombinant AAV was produced by homologous recombination of the 3 plasmids in renal embryo 293 cells and its titer was measured by quantitative dot blot hybridization. Results The vector of hTRX-PR39 recombinant AAV was successfully constructed. High titer of recombinant AAV was obtained by homologous recombination in renal embryo 293 cells (3.46 x 1012-3.46 × 1013 PFU/mL).Conclusion The recombinant AAV vector encoding the gene hTRX-PR39 was successfully constructed in this study by molecular cloning and in vitro recombination techniques, laying the foundation for further research into genetic therapy of cerebral ischemic disease.%目的 探讨hTRX-PR39融合基因在脑缺血疾病中的治疗作用.方法 将已经构建好的hTRX-PR39融合基因插入到具有相应酶切位点的pSSCMV病毒载体质粒中,得到重组腺相关病毒载体质粒,酶切电泳鉴定.将已构建的重组腺相关病毒载体质粒、腺病毒辅助质粒PFG140和包装质粒pAAV/Ad,三质粒磷酸钙共沉淀法转染293细胞系,通过同源重组获得hTRX-PR39重组腺相关病毒载体,收集病毒,斑点杂交(Dot blot)法测定病毒滴度.结果 成功构建重组腺相关病毒质粒pSSCMV/hTRX-PR39.包装、回收病毒后,Dot b1ot法测定重组病毒滴度为3.46×(1012~1013)PFU/mL.结论 成功构建pSSCMV/hTRX-PR39重组腺相关病毒载体,并包装较高浓度的重组病毒.

  11. Mutagenesis-mediated virus extinction: virus-dependent effect of viral load on sensitivity to lethal defection.

    Directory of Open Access Journals (Sweden)

    Héctor Moreno

    Full Text Available BACKGROUND: Lethal mutagenesis is a transition towards virus extinction mediated by enhanced mutation rates during viral genome replication, and it is currently under investigation as a potential new antiviral strategy. Viral load and virus fitness are known to influence virus extinction. Here we examine the effect or the multiplicity of infection (MOI on progeny production of several RNA viruses under enhanced mutagenesis. RESULTS: The effect of the mutagenic base analogue 5-fluorouracil (FU on the replication of the arenavirus lymphocytic choriomeningitis virus (LCMV can result either in inhibition of progeny production and virus extinction in infections carried out at low multiplicity of infection (MOI, or in a moderate titer decrease without extinction at high MOI. The effect of the MOI is similar for LCMV and vesicular stomatitis virus (VSV, but minimal or absent for the picornaviruses foot-and-mouth disease virus (FMDV and encephalomyocarditis virus (EMCV. The increase in mutation frequency and Shannon entropy (mutant spectrum complexity as a result of virus passage in the presence of FU was more accentuated at low MOI for LCMV and VSV, and at high MOI for FMDV and EMCV. We present an extension of the lethal defection model that agrees with the experimental results. CONCLUSIONS: (i Low infecting load favoured the extinction of negative strand viruses, LCMV or VSV, with an increase of mutant spectrum complexity. (ii This behaviour is not observed in RNA positive strand viruses, FMDV or EMCV. (iii The accumulation of defector genomes may underlie the MOI-dependent behaviour. (iv LCMV coinfections are allowed but superinfection is strongly restricted in BHK-21 cells. (v The dissimilar effects of the MOI on the efficiency of mutagenic-based extinction of different RNA viruses can have implications for the design of antiviral protocols based on lethal mutagenesis, presently under development.

  12. Neuraminidase-Mediated, NKp46-Dependent Immune-Evasion Mechanism of Influenza Viruses

    Directory of Open Access Journals (Sweden)

    Yotam Bar-On

    2013-04-01

    Full Text Available Natural killer (NK cells play an essential role in the defense against influenza virus, one of the deadliest respiratory viruses known today. The NKp46 receptor, expressed by NK cells, is critical for controlling influenza infections, as influenza-virus-infected cells are eliminated through the recognition of the viral hemagglutinin (HA protein by NKp46. Here, we describe an immune-evasion mechanism of influenza viruses that is mediated by the neuraminidase (NA protein. By using various NA blockers, we show that NA removes sialic acid residues from NKp46 and that this leads to reduced recognition of HA. Furthermore, we provide in vivo and in vitro evidence for the existence of this NA-mediated, NKp46-dependent immune-evasion mechanism and demonstrate that NA inhibitors, which are commonly used for the treatment of influenza infections, are useful not only as blockers of virus budding but also as boosters of NKp46 recognition.

  13. p53-Mediated Cellular Response to DNA Damage in Cells with Replicative Hepatitis B Virus

    Science.gov (United States)

    Puisieux, Alain; Ji, Jingwei; Guillot, Celine; Legros, Yann; Soussi, Thierry; Isselbacher, Kurt; Ozturk, Mehmet

    1995-02-01

    Wild-type p53 acts as a tumor suppressor gene by protecting cells from deleterious effects of genotoxic agents through the induction of a G_1/S arrest or apoptosis as a response to DNA damage. Transforming proteins of several oncogenic DNA viruses inactivate tumor suppressor activity of p53 by blocking this cellular response. To test whether hepatitis B virus displays a similar effect, we studied the p53-mediated cellular response to DNA damage in 2215 hepatoma cells with replicative hepatitis B virus. We demonstrate that hepatitis B virus replication does not interfere with known cellular functions of p53 protein.

  14. Influenza virus induces apoptosis via BAD-mediated mitochondrial dysregulation.

    Science.gov (United States)

    Tran, Anh T; Cortens, John P; Du, Qiujiang; Wilkins, John A; Coombs, Kevin M

    2013-01-01

    Influenza virus infection results in host cell death and major tissue damage. Specific components of the apoptotic pathway, a signaling cascade that ultimately leads to cell death, are implicated in promoting influenza virus replication. BAD is a cell death regulator that constitutes a critical control point in the intrinsic apoptosis pathway, which occurs through the dysregulation of mitochondrial outer membrane permeabilization and the subsequent activation of downstream apoptogenic factors. Here we report a novel proviral role for the proapoptotic protein BAD in influenza virus replication. We show that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and that both virus replication and viral protein production are dramatically reduced, which suggests that virus-induced apoptosis is BAD dependent. Our data showed that influenza viruses induced phosphorylation of BAD at residues S112 and S136 in a temporal manner. Viral infection also induced BAD cleavage, late in the viral life cycle, to a truncated form that is reportedly a more potent inducer of apoptosis. We further demonstrate that knockdown of BAD resulted in reduced cytochrome c release and suppression of the intrinsic apoptotic pathway during influenza virus replication, as seen by an inhibition of caspases-3, caspase-7, and procyclic acidic repetitive protein (PARP) cleavage. Our data indicate that influenza viruses carefully modulate the activation of the apoptotic pathway that is dependent on the regulatory function of BAD and that failure of apoptosis activation resulted in unproductive viral replication.

  15. Compromised virus control and augmented perforin-mediated immunopathology in IFN-gamma-deficient mice infected with lymphocytic choriomeningitis virus

    DEFF Research Database (Denmark)

    Nansen, A; Jensen, Teis; Christensen, Jan Pravsgaard

    1999-01-01

    -specific TCR are adoptively transferred before virus challenge, indicating that the disease is the result of an unfortunate balance between virus replication in internal organs, e.g., liver and spleen, and the host response; resetting this balance by increasing host responsiveness will again lead to a rapidly...... in wild-type mice. Our results reveal that IFN-gamma is pivotal to T cell-mediated control of a rapidly invasive stain, whereas it is less important in the acute elimination of a slowly invasive strain. Moreover, the majority of mice infected with the rapidly invasive strain succumb to a wasting syndrome...... mediated by CD8+ effector cells. The primary effector mechanism underlying this disease is perforin-dependent lysis, but other mechanisms are also involved. Wasting disease can be prevented if naive CD8+ cells from mice transgenic for an MHC class I-restricted lymphocytic choriomeningitis virus...

  16. Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of Sugarcane mosaic virus and Sorghum mosaic virus in sugarcane

    Science.gov (United States)

    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) in sugarcane. Six sets of four primers corresponding to the conserved coat protein gene were designed for each virus and their succ...

  17. AAVrh.10-Mediated Expression of an Anti-Cocaine Antibody Mediates Persistent Passive Immunization That Suppresses Cocaine-Induced Behavior

    Science.gov (United States)

    Rosenberg, Jonathan B.; Hicks, Martin J.; De, Bishnu P.; Pagovich, Odelya; Frenk, Esther; Janda, Kim D.; Wee, Sunmee; Koob, George F.; Hackett, Neil R.; Kaminsky, Stephen M.; Worgall, Stefan; Tignor, Nicole; Mezey, Jason G.

    2012-01-01

    Abstract Cocaine addiction is a major problem affecting all societal and economic classes for which there is no effective therapy. We hypothesized an effective anti-cocaine vaccine could be developed by using an adeno-associated virus (AAV) gene transfer vector as the delivery vehicle to persistently express an anti-cocaine monoclonal antibody in vivo, which would sequester cocaine in the blood, preventing access to cognate receptors in the brain. To accomplish this, we constructed AAVrh.10antiCoc.Mab, an AAVrh.10 gene transfer vector expressing the heavy and light chains of the high affinity anti-cocaine monoclonal antibody GNC92H2. Intravenous administration of AAVrh.10antiCoc.Mab to mice mediated high, persistent serum levels of high-affinity, cocaine-specific antibodies that sequestered intravenously administered cocaine in the blood. With repeated intravenous cocaine challenge, naive mice exhibited hyperactivity, while the AAVrh.10antiCoc.Mab-vaccinated mice were completely resistant to the cocaine. These observations demonstrate a novel strategy for cocaine addiction by requiring only a single administration of an AAV vector mediating persistent, systemic anti-cocaine passive immunity. PMID:22486244

  18. Dendritic cells in dengue virus infection: Targets of virus replication and mediators of immunity

    Directory of Open Access Journals (Sweden)

    Michael A. Schmid

    2014-12-01

    Full Text Available Dendritic cells (DCs are sentinels of the immune system and detect pathogens at sites of entry, such as the skin. In addition to the ability of DCs to control infections directly via their innate immune functions, DCs help to prime adaptive B and T cell responses via antigen presentation in lymphoid tissues. Infected Aedes aegypti or Ae. albopictus mosquitoes transmit the four dengue virus (DENV serotypes to humans while probing for small blood vessels in the skin. DENV causes the most prevalent arthropod-borne viral disease in humans, yet no vaccine or specific therapeutic is currently approved. Although primary DENV infection confers life-long protective immunity against re-infection with the same DENV serotype, secondary infection with a different DENV serotype can lead to increased disease severity via cross-reactive T cells or enhancing antibodies. This review summarizes recent findings in humans and animal models about DENV infection of DCs, monocytes and macrophages. We discuss the dual role of DCs as both targets of DENV replication and mediators of innate and adaptive immunity, and summarize immune evasion strategies whereby DENV impairs the function of infected DCs. We suggest that DCs play a key role in priming DENV-specific neutralizing or potentially harmful memory B and T cell responses, and that future DC-directed therapies may help induce protective memory responses and reduce dengue pathogenesis.

  19. Immune-mediated Liver Injury in Hepatitis B Virus Infection

    National Research Council Canada - National Science Library

    In Soo Oh; Su-Hyung Park

    2015-01-01

    Hepatitis B virus (HBV) is responsible for approximately 350 million chronic infections worldwide and is a leading cause of broad-spectrum liver diseases such as hepatitis, cirrhosis and liver cancer...

  20. Insect vector-mediated transmission of plant viruses.

    Science.gov (United States)

    Whitfield, Anna E; Falk, Bryce W; Rotenberg, Dorith

    2015-05-01

    The majority of plant-infecting viruses are transmitted to their host plants by vectors. The interactions between viruses and vector vary in duration and specificity but some common themes in vector transmission have emerged: 1) plant viruses encode structural proteins on the surface of the virion that are essential for transmission, and in some cases additional non-structural helper proteins that act to bridge the virion to the vector binding site; 2) viruses bind to specific sites in or on vectors and are retained there until they are transmitted to their plant hosts; and 3) viral determinants of vector transmission are promising candidates for translational research aimed at disrupting transmission or decreasing vector populations. In this review, we focus on well-characterized insect vector-transmitted viruses in the following genera: Caulimovirus, Crinivirus, Luteovirus, Geminiviridae, Reovirus, Tospovirus, and Tenuivirus. New discoveries regarding these genera have increased our understanding of the basic mechanisms of virus transmission by arthropods, which in turn have enabled the development of innovative strategies for breaking the transmission cycle.

  1. VARICELLA ZOSTER VIRUS-ITS PATHOGENESIS, LATENCY & CELL-MEDIATED IMMUNITY

    Directory of Open Access Journals (Sweden)

    Anis Ahmed

    2013-07-01

    Full Text Available Varicella zoster virus causes primary infection as chickenpox, at which time latencyis established in the neurons of the dorsal root ganglia or ganglia of the cranial nerves.Reactivation produces herpes zoster infection (HZI, commonly called shingles. Anunderstanding of the mechanisms of latency is crucial in developing effective therapies forVZV infections of the nervous system. This article describes the pathogenesis of VZVwhich includes immune response to the virus, immune evasion by the virus, mechanism ofits latency and cell-mediated immunity.

  2. Wolbachia-mediated resistance to dengue virus infection and death at the cellular level.

    Directory of Open Access Journals (Sweden)

    Francesca D Frentiu

    Full Text Available BACKGROUND: Dengue is currently the most important arthropod-borne viral disease of humans. Recent work has shown dengue virus displays limited replication in its primary vector, the mosquito Aedes aegypti, when the insect harbors the endosymbiotic bacterium Wolbachia pipientis. Wolbachia-mediated inhibition of virus replication may lead to novel methods of arboviral control, yet the functional and cellular mechanisms that underpin it are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using paired Wolbachia-infected and uninfected Aedes-derived cell lines and dengue virus, we confirm the phenomenon of viral inhibition at the cellular level. Although Wolbachia imposes a fitness cost to cells via reduced proliferation, it also provides a significant degree of protection from virus-induced mortality. The extent of viral inhibition is related to the density of Wolbachia per cell, with highly infected cell lines showing almost complete protection from dengue infection and dramatically reduced virus titers compared to lines not infected with the bacteria. CONCLUSIONS/SIGNIFICANCE: We have shown that cells infected with Wolbachia display inhibition of dengue virus replication, that the extent of inhibition is related to bacterial density and that Wolbachia infection, although costly, will provide a fitness benefit in some circumstances. Our results parallel findings in mosquitoes and flies, indicating that cell line models will provide useful and experimentally tractable models to study the mechanisms underlying Wolbachia-mediated protection from viruses.

  3. Triptolide T10 enhances AAV-mediated gene transfer in mice striatum.

    Science.gov (United States)

    Ren, Xinmiao; Zhang, Ting; Hu, Jing; Ding, Wei; Wang, Xiaomin

    2010-08-02

    Adeno-associated virus (AAV) mediated gene transfer has been demonstrated to be an effective approach for treating Parkinson's disease (PD). Triptolide T10 is a monomeric compound isolated from tripterygium wilfordii Hook.f. (Thunder God vine), a traditional Chinese herb for anti-inflammatory medications. In the present study, we co-administered T10 with recombinant AAV2 in SH-SY5Y human neuroblastoma cells and in the striatum of C57BL/6 mice, and then evaluated the AAV-mediated gene expression levels. The results have shown that T10 significantly augmented the expression of AAV-mediated gene in a dose-dependent fashion without detectable cytotoxicity. As growing evidence indicated that inflammation contributed to the progression of PD, and the anti-inflammatory effect of T10 was shown in our previous studies, our data of T10 to enhance AAV transduction suggest that T10 might be potentially used as a facilitating reagent for the AAV gene therapy applications in neurodegenerative diseases.

  4. Mechanism of human antibody-mediated neutralization of Marburg virus.

    Science.gov (United States)

    Flyak, Andrew I; Ilinykh, Philipp A; Murin, Charles D; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L; Hashiguchi, Takao; Bornholdt, Zachary A; Slaughter, James C; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G; Ward, Andrew B; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E

    2015-02-26

    The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition.

  5. Virus-mediated archaeal hecatomb in the deep seafloor

    Science.gov (United States)

    Danovaro, Roberto; Dell’Anno, Antonio; Corinaldesi, Cinzia; Rastelli, Eugenio; Cavicchioli, Ricardo; Krupovic, Mart; Noble, Rachel T.; Nunoura, Takuro; Prangishvili, David

    2016-01-01

    Viruses are the most abundant biological entities in the world’s oceans, and they play a crucial role in global biogeochemical cycles. In deep-sea ecosystems, archaea and bacteria drive major nutrient cycles, and viruses are largely responsible for their mortality, thereby exerting important controls on microbial dynamics. However, the relative impact of viruses on archaea compared to bacteria is unknown, limiting our understanding of the factors controlling the functioning of marine systems at a global scale. We evaluate the selectivity of viral infections by using several independent approaches, including an innovative molecular method based on the quantification of archaeal versus bacterial genes released by viral lysis. We provide evidence that, in all oceanic surface sediments (from 1000- to 10,000-m water depth), the impact of viral infection is higher on archaea than on bacteria. We also found that, within deep-sea benthic archaea, the impact of viruses was mainly directed at members of specific clades of Marine Group I Thaumarchaeota. Although archaea represent, on average, ~12% of the total cell abundance in the top 50 cm of sediment, virus-induced lysis of archaea accounts for up to one-third of the total microbial biomass killed, resulting in the release of ~0.3 to 0.5 gigatons of carbon per year globally. Our results indicate that viral infection represents a key mechanism controlling the turnover of archaea in surface deep-sea sediments. We conclude that interactions between archaea and their viruses might play a profound, previously underestimated role in the functioning of deep-sea ecosystems and in global biogeochemical cycles. PMID:27757416

  6. Coat protein-mediated resistance against an Indian isolate of the Cucumber mosaic virus subgroup IB in Nicotiana benthamiana

    Indian Academy of Sciences (India)

    A Srivastava; S K Raj

    2008-06-01

    Coat protein (CP)-mediated resistance against an Indian isolate of the Cucumber mosaic virus (CMV) subgroup IB was demonstrated in transgenic lines of Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transformation. Out of the fourteen independently transformed lines developed, two lines were tested for resistance against CMV by challenge inoculations. The transgenic lines exhibiting complete resistance remained symptomless throughout life and showed reduced or no virus accumulation in their systemic leaves after virus challenge. These lines also showed virus resistance against two closely related strains of CMV. This is the first report of CP-mediated transgenic resistance against a CMV subgroup IB member isolated from India.

  7. Virus infection mediates the effects of elevated CO2 on plants and vectors

    Science.gov (United States)

    Trębicki, Piotr; Vandegeer, Rebecca K.; Bosque-Pérez, Nilsa A.; Powell, Kevin S.; Dader, Beatriz; Freeman, Angela J.; Yen, Alan L.; Fitzgerald, Glenn J.; Luck, Jo E.

    2016-03-01

    Atmospheric carbon dioxide (CO2) concentration has increased significantly and is projected to double by 2100. To increase current food production levels, understanding how pests and diseases respond to future climate driven by increasing CO2 is imperative. We investigated the effects of elevated CO2 (eCO2) on the interactions among wheat (cv. Yitpi), Barley yellow dwarf virus and an important pest and virus vector, the bird cherry-oat aphid (Rhopalosiphum padi), by examining aphid life history, feeding behavior and plant physiology and biochemistry. Our results showed for the first time that virus infection can mediate effects of eCO2 on plants and pathogen vectors. Changes in plant N concentration influenced aphid life history and behavior, and N concentration was affected by virus infection under eCO2. We observed a reduction in aphid population size and increased feeding damage on noninfected plants under eCO2 but no changes to population and feeding on virus-infected plants irrespective of CO2 treatment. We expect potentially lower future aphid populations on noninfected plants but no change or increased aphid populations on virus-infected plants therefore subsequent virus spread. Our findings underscore the complexity of interactions between plants, insects and viruses under future climate with implications for plant disease epidemiology and crop production.

  8. Mathematical Modeling of Cytotoxic Lymphocyte-Mediated Immune Response to Hepatitis B Virus Infection

    Directory of Open Access Journals (Sweden)

    Changjiang Long

    2008-01-01

    Full Text Available Nowak's model of the human immunodeficiency virus (HIV infection has been extensively and successfully used to simulate the interaction between HIV and cytotoxic lymphocyte- (CTL- mediated immune response. However, this model is not available for hepatitis B virus (HBV infection. As the enhanced recruitment of virus-specific CTLs into the liver has been an important novel concept in the pathogenesis of hepatitis B, we develop a specific mathematical model analyzing the relationship between HBV and the CTL-mediated immune response, and the indicator of the liver cell damage, alanine aminotransferase (ALT. The stability condition of the complete recovery equilibrium point at which HBV will be eliminated entirely from the body is discussed. A different set of parameters is used in the simulation, and the results show that the model can interpret the wide variety of clinical manifestations of HBV infection. The model suggests that a rapid and vigorous CTL response is required for resolution of HBV infection.

  9. Impaired cell mediated immunity in haemophilia in the absence of infection with human immunodeficiency virus.

    Science.gov (United States)

    Madhok, R; Gracie, A; Lowe, G D; Burnett, A; Froebel, K; Follett, E; Forbes, C D

    1986-10-18

    The cell mediated immune response was evaluated in vivo in 29 patients with clinically severe haemophilia by means of the dinitrochlorobenzene skin test. All patients had a response below the median normal value, and in 19 the response was on or below the lower limit of the normal range. There was no difference in skin response between patients positive and negative for the human immunodeficiency virus (HIV; formerly known as human T cell lymphotropic virus III or lymphadenopathy associated virus). In the whole group, and in seronegative patients (n = 17), there was an inverse relation between exposure to clotting factor and skin response. In seropositive patients (n = 12) no such association was apparent. This study shows that clotting factor concentrate impairs the cell mediated immune response to a new antigen in the absence of infection with HIV.

  10. BST-2 mediated restriction of simian-human immunodeficiency virus.

    Science.gov (United States)

    Ruiz, Autumn; Lau, David; Mitchell, Richard S; Hill, M Sarah; Schmitt, Kimberly; Guatelli, John C; Stephens, Edward B

    2010-10-25

    Pathogenic simian-human immunodeficiency viruses (SHIV) contain HIV-1 Vpu and SIV Nef, both shown to counteract BST-2 (HM1.24; CD317; tetherin) inhibition of virus release in a species-specific manner. We show that human and pig-tailed BST-2 (ptBST-2) restrict SHIV. We found that sequential "humanization" of the transmembrane domain (TMD) of the pig-tailed BST-2 (ptBST-2) protein resulted in a fluctuation in sensitivity to HIV-1 Vpu. Our results also show that the length of the TMD in human and ptBST-2 proteins is important for BST-2 restriction and susceptibility to Vpu. Taken together, our results emphasize the importance of tertiary structure in BST-2 antagonism and suggests that the HIV-1 Vpu transmembrane domain may have additional functions in vivo unrelated to BST-2 antagonism.

  11. [Mechanisms underlying interferon-mediated host innate immunity during influenza A virus infection].

    Science.gov (United States)

    Chen, Chao; Chi, Xiaojuan; Bai, Qingling; Chen, Jilong

    2015-12-01

    Influenza A virus can create acute respiratory infection in humans and animals throughout the world, and it is still one of the major causes of morbidity and mortality in humans worldwide. Numerous studies have shown that influenza A virus infection induces rapidly host innate immune response. Influenza A virus triggers the activation of signaling pathways that are dependent on host pattern recognition receptors (PRRs) including toll like receptors (TLRs) and RIG-I like receptors (RLRs). Using a variety of regulatory mechanisms, these signaling pathways activate downstream transcript factors that control expression of various interferons and cytokines, such as type I and type III interferons. Thus, these interferons stimulate the transcript of relevant interferon-stimulated genes (ISGs) and expression of the antiviral proteins, which are critical components of host innate immunity. In this review, we will highlight the mechanisms by which influenza A virus infection induces the interferon-mediated host innate immunity.

  12. Cell-specific targeting of lentiviral vectors mediated by fusion proteins derived from Sindbis virus, vesicular stomatitis virus, or avian sarcoma/leukosis virus

    Directory of Open Access Journals (Sweden)

    Marino Michael P

    2010-01-01

    Full Text Available Abstract Background The ability to efficiently and selectively target gene delivery vectors to specific cell types in vitro and in vivo remains one of the formidable challenges in gene therapy. We pursued two different strategies to target lentiviral vector delivery to specific cell types. In one of the strategies, vector particles bearing a membrane-bound stem cell factor sequence plus a separate fusion protein based either on Sindbis virus strain TR339 glycoproteins or the vesicular stomatitis virus G glycoprotein were used to selectively transduce cells expressing the corresponding stem cell factor receptor (c-kit. An alternative approach involved soluble avian sarcoma/leukosis virus receptors fused to cell-specific ligands including stem cell factor and erythropoietin for targeting lentiviral vectors pseudotyped with avian sarcoma/leukosis virus envelope proteins to cells that express the corresponding receptors. Results The titers of unconcentrated vector particles bearing Sindbis virus strain TR339 or vesicular stomatitis virus G fusion proteins plus stem cell factor in the context of c-kit expressing cells were up to 3.2 × 105 transducing units per ml while vector particles lacking the stem cell factor ligand displayed titers that were approximately 80 fold lower. On cells that lacked the c-kit receptor, the titers of stem cell factor-containing vectors were approximately 40 times lower compared to c-kit-expressing cells. Lentiviral vectors pseudotyped with avian sarcoma/leukosis virus subgroup A or B envelope proteins and bearing bi-functional bridge proteins encoding erythropoietin or stem cell factor fused to the soluble extracellular domains of the avian sarcoma/leukosis virus subgroup A or B receptors resulted in efficient transduction of erythropoietin receptor or c-kit-expressing cells. Transduction of erythropoietin receptor-expressing cells mediated by bi-functional bridge proteins was found to be dependent on the dose, the

  13. Cell type mediated resistance of vesicular stomatitis virus and Sendai virus to ribavirin.

    Science.gov (United States)

    Shah, Nirav R; Sunderland, Amanda; Grdzelishvili, Valery Z

    2010-06-22

    Ribavirin (RBV) is a synthetic nucleoside analog with broad spectrum antiviral activity. Although RBV is approved for the treatment of hepatitis C virus, respiratory syncytial virus, and Lassa fever virus infections, its mechanism of action and therapeutic efficacy remains highly controversial. Recent reports show that the development of cell-based resistance after continuous RBV treatment via decreased RBV uptake can greatly limit its efficacy. Here, we examined whether certain cell types are naturally resistant to RBV even without prior drug exposure. Seven different cell lines from various host species were compared for RBV antiviral activity against two nonsegmented negative-strand RNA viruses, vesicular stomatitis virus (VSV, a rhabdovirus) and Sendai virus (SeV, a paramyxovirus). Our results show striking differences between cell types in their response to RBV, ranging from virtually no antiviral effect to very effective inhibition of viral replication. Despite differences in viral replication kinetics for VSV and SeV in the seven cell lines, the observed pattern of RBV resistance was very similar for both viruses, suggesting that cellular rather than viral determinants play a major role in this resistance. While none of the tested cell lines was defective in RBV uptake, dramatic variations were observed in the long-term accumulation of RBV in different cell types, and it correlated with the antiviral efficacy of RBV. While addition of guanosine neutralized RBV only in cells already highly resistant to RBV, actinomycin D almost completely reversed the RBV effect (but not uptake) in all cell lines. Together, our data suggest that RBV may inhibit the same virus via different mechanisms in different cell types depending on the intracellular RBV metabolism. Our results strongly point out the importance of using multiple cell lines of different origin when antiviral efficacy and potency are examined for new as well as established drugs in vitro.

  14. Cell type mediated resistance of vesicular stomatitis virus and Sendai virus to ribavirin.

    Directory of Open Access Journals (Sweden)

    Nirav R Shah

    Full Text Available Ribavirin (RBV is a synthetic nucleoside analog with broad spectrum antiviral activity. Although RBV is approved for the treatment of hepatitis C virus, respiratory syncytial virus, and Lassa fever virus infections, its mechanism of action and therapeutic efficacy remains highly controversial. Recent reports show that the development of cell-based resistance after continuous RBV treatment via decreased RBV uptake can greatly limit its efficacy. Here, we examined whether certain cell types are naturally resistant to RBV even without prior drug exposure. Seven different cell lines from various host species were compared for RBV antiviral activity against two nonsegmented negative-strand RNA viruses, vesicular stomatitis virus (VSV, a rhabdovirus and Sendai virus (SeV, a paramyxovirus. Our results show striking differences between cell types in their response to RBV, ranging from virtually no antiviral effect to very effective inhibition of viral replication. Despite differences in viral replication kinetics for VSV and SeV in the seven cell lines, the observed pattern of RBV resistance was very similar for both viruses, suggesting that cellular rather than viral determinants play a major role in this resistance. While none of the tested cell lines was defective in RBV uptake, dramatic variations were observed in the long-term accumulation of RBV in different cell types, and it correlated with the antiviral efficacy of RBV. While addition of guanosine neutralized RBV only in cells already highly resistant to RBV, actinomycin D almost completely reversed the RBV effect (but not uptake in all cell lines. Together, our data suggest that RBV may inhibit the same virus via different mechanisms in different cell types depending on the intracellular RBV metabolism. Our results strongly point out the importance of using multiple cell lines of different origin when antiviral efficacy and potency are examined for new as well as established drugs in vitro.

  15. Viral glycoprotein-mediated cell fusion assays using vaccinia virus vectors.

    Science.gov (United States)

    Bossart, Katharine N; Broder, Christopher C

    2004-01-01

    The vaccinia virus-based expression of viral envelope glycoprotein genes-derived from enveloped viruses that infect their respective host cells through a pH-independent mechanism of membrane fusion-has been a powerful tool in helping to characterize these important attachment and fusion proteins. The cellular expression of these viral envelope glycoproteins has allowed for the measurement of membrane fusion events using cell-cell fusion or syncytia formation. This method has been enhanced by the addition of a reporter-gene system to the vaccinia virus-based cell-cell fusion assay. This improvement has provided a high-throughput and quantitative aspect to this assay, which can serve as a surrogate for virus entry and is therefore ideally suited in the characterization of numerous enveloped viruses, including biological safety level-4 (BSL-4) agents. This chapter will detail the methods of the vaccinia virus-based reporter-gene fusion assay and how it may be used to characterize the fusion mediated by the BSL-4-classified Hendra and Nipah viruses.

  16. Robust RNA silencing-mediated resistance to Plum pox virus under variable abiotic and biotic conditions.

    Science.gov (United States)

    Di Nicola, Elisa; Tavazza, Mario; Lucioli, Alessandra; Salandri, Laura; Ilardi, Vincenza

    2014-10-01

    Some abiotic and biotic conditions are known to have a negative impact on post-transcriptional gene silencing (PTGS), thus representing a potential concern for the production of stable engineered virus resistance traits. However, depending on the strategy followed to achieve PTGS of the transgene, different responses to external conditions can be expected. In the present study, we utilized the Nicotiana benthamiana–Plum pox virus (PPV) pathosystem to evaluate in detail the stability of intron-hairpin(ihp)-mediated virus resistance under conditions known to adversely affect PTGS. The ihp plants grown at low or high temperatures were fully resistant to multiple PPV challenges, different PPV inoculum concentrations and even to a PPV isolate differing from the ihp construct by more than 28% at the nucleotide level. In addition, infections of ihp plants with viruses belonging to Cucumovirus, Potyvirus or Tombusvirus, all known to affect PTGS at different steps, were not able to defeat PPV resistance. Low temperatures did not affect the accumulation of transgenic small interfering RNAs (siRNAs), whereas a clear increase in the amount of siRNAs was observed during infections sustained by Cucumber mosaic virus and Potato virus Y. Our results show that the above stress factors do not represent an important concern for the production,through ihp-PTGS technology, of transgenic plants having robust virus resistance traits.

  17. Biosafety considerations of RNAi-mediated virus resistance in fruit-tree cultivars and in rootstock.

    Science.gov (United States)

    Lemgo, Godwin Nana Yaw; Sabbadini, Silvia; Pandolfini, Tiziana; Mezzetti, Bruno

    2013-12-01

    A major application of RNA interference (RNAi) is envisaged for the production of virus-resistant transgenic plants. For fruit trees, this remains the most, if not the only, viable option for the control of plant viral disease outbreaks in cultivated orchards, due to the difficulties associated with the use of traditional and conventional disease-control measures. The use of RNAi might provide an additional benefit for woody crops if silenced rootstock can efficiently transmit the silencing signal to non-transformed scions, as has already been demonstrated in herbaceous plants. This would provide a great opportunity to produce non-transgenic fruit from transgenic rootstock. In this review, we scrutinise some of the concerns that might arise with the use of RNAi for engineering virus-resistant plants, and we speculate that this virus resistance has fewer biosafety concerns. This is mainly because RNAi-eliciting constructs only express small RNA molecules rather than proteins, and because this technology can be applied using plant rootstock that can confer virus resistance to the scion, leaving the scion untransformed. We discuss the main biosafety concerns related to the release of new types of virus-resistant plants and the risk assessment approaches in the application of existing regulatory systems (in particular, those of the European Union, the USA, and Canada) for the evaluation and approval of RNAi-mediated virus-resistant plants, either as transgenic varieties or as plant virus resistance induced by transgenic rootstock.

  18. CD55 is a key complement regulatory protein that counteracts complement-mediated inactivation of Newcastle Disease Virus.

    Science.gov (United States)

    Rangaswamy, Udaya S; Cotter, Christopher R; Cheng, Xing; Jin, Hong; Chen, Zhongying

    2016-08-01

    Newcastle disease virus (NDV) is being developed as an oncolytic virus for virotherapy. In this study we analysed the regulation of complement-mediated inactivation of a recombinant NDV in different host cells. NDV grown in human cells was less sensitive to complement-mediated virus inactivation than NDV grown in embryonated chicken eggs. Additionally, NDV produced from HeLa-S3 cells is more resistant to complement than NDV from 293F cells, which correlated with higher expression and incorporation of complement regulatory proteins (CD46, CD55 and CD59) into virions from HeLa-S3 cells. Further analysis of the recombinant NDVs individually expressing the three CD molecules showed that CD55 is the most potent in counteracting complement-mediated virus inactivation. The results provide important information on selecting NDV manufacture substrate to mitigate complement-mediated virus inactivation.

  19. CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Xianglian Ge

    2016-01-01

    Full Text Available Clustered interspaced short palindromic repeats (CRISPR/CRISPR-associated protein 9 (Cas9-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low. Gene targeting with adeno-associated virus (AAV vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases. However, whether CRISPR/Cas9-mediated double strand breaks and AAV based donor DNA mediated homologous recombination approaches could be combined to create a novel CRISPR/Cas9-AAV genetic tool for highly specific gene editing is not clear. Here we demonstrate that using CRISPR/Cas9-AAV, we could successfully knock-in a DsRed reporter gene at the basic motifleucine zipper transcription factor (NRL locus in human embryonic stem cells. For the first time, this study provides the proof of principle that these two technologies can be used together. CRISPR/Cas9-AAV, a new genome editing tool, offers a platform for the manipulation of human genome.

  20. CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells.

    Science.gov (United States)

    Ge, Xianglian; Xi, Haitao; Yang, Fayu; Zhi, Xiao; Fu, Yanghua; Chen, Ding; Xu, Ren-He; Lin, Ge; Qu, Jia; Zhao, Junzhao; Gu, Feng

    2016-11-29

    Clustered interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low. Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases. However, whether CRISPR/Cas9-mediated double strand breaks and AAV based donor DNA mediated homologous recombination approaches could be combined to create a novel CRISPR/Cas9-AAV genetic tool for highly specific gene editing is not clear. Here we demonstrate that using CRISPR/Cas9-AAV, we could successfully knock-in a DsRed reporter gene at the basic motifleucine zipper transcription factor (NRL) locus in human embryonic stem cells. For the first time, this study provides the proof of principle that these two technologies can be used together. CRISPR/Cas9-AAV, a new genome editing tool, offers a platform for the manipulation of human genome.

  1. Preparation of rAAV/hFⅨ by HSV/AAV hybrid helper virus and evaluation of its safety

    Institute of Scientific and Technical Information of China (English)

    CHEN Li; CHEN Haoming; ZOU Beiyan; WU Zhijian; WU Xiaobing; LU Daru; XUE Jinglun

    2003-01-01

    The recombinant adeno-associated viral vector with human coagulation Factor Ⅸ minigene which was regulated by CMV promoter was constructed. Large quantity of recombinant adeno-associated viral particles (rAAV/ hFⅨ) was prepared by the HSV/AAV hybrid helper virus method. Southern dot blot assay and QC-PCR indicated that the titer of the virus was 3.6×1012 v.g./mL. It demonstrated that this method can effectively overcome the hurdles of mass production of AAV vector. Followed by an intramuscular injection of viral vectors (7.5×1011 v.g./mouse) in the quadriceps femoris, an elevation of human Factor Ⅸ expression in the plasma of hemophilia B mice was detected (387 ng/mL) and persisted more than 12 weeks. The level of anti-virus antibody in plasma aligned with the Factor Ⅸ expression curve. The QC-PCR method is easier and more accurate than traditional dothybridization for determination of the titer of recombinant adeno-associated virus. Moreover, there are no HSV particles existing in produced AAV assayed by RT-PCR. AAV is the only virus that has been amplified from AAV-injected muscle by PCR.

  2. Adeno-associated viral vector serotypes 1 and 5 targeted to the neonatal rat and pig striatum induce widespread transgene expression in the forebrain

    DEFF Research Database (Denmark)

    Kornum, Birgitte R; Stott, Simon R W; Mattsson, Bengt;

    2010-01-01

    . Our results show that striatal delivery of rAAV5 vectors in the neonatal brain represents a useful tool to express genes of interest both in the basal ganglia and the neocortex. Furthermore, we apply, for the first time, viral vector-mediated gene transfer to the pig brain providing the opportunity...

  3. Tn7-mediated Introduction of DNA into Bacmid-cloned Pseudorabies Virus Genome for Rapid Construction of Recombinant Viruses

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    lacZα-mini-attTn7 was inserted into the intergenic region between the gG and gD genes in a PRV bacterial artificial chromosome (BAC) by homologous recombination in E. coli. The resulting recombinant BAC (pBeckerZF1) was confirmed by PCR and sequencing. Green fluorescent protein (GFP) gene was then transposed into pBeckerZF1 by transposon Tn7 to generate pBeckerZF2. Recombinant viruses vBeckerZF1 and vBeckerZF2 were generated by transfection with the corresponding BAC pBeckerZF1 or pBeckerZF2. The titers and cytopathic effect (CPE) observed for by vBeckerZF1 and vBeckerZF2 was comparable to that of the parental virus vBecker3. vBeckerZF2 was serial passaged for five rounds in cell culture, and the mini-Tn7 insertion was stably maintained in viral genome. These results show that recombinant viruses can be rapidly and reliably created by Tn7-mediated transposition. This technology should accelerate greatly the pace at which recombinant PRV can be generated and, thus, facilitate the use of recombinant viruses for detailed mutagenic studies.

  4. Platelets promote liver immunopathology contributing to hepatitis B virus-mediated hepatocarcinogenesis.

    Science.gov (United States)

    Sitia, Giovanni

    2014-06-01

    Chronic hepatitis B virus (HBV) infection is a major risk factor for the development of hepatocellular carcinoma (HCC). Among the pathogenetic factors triggered by HBV, virus-specific CD8(+) T cells play and important role in disease pathogenesis by promoting necroinflammatory liver damage. Accordingly, amelioration of immune-mediated chronic liver injury may prevent HCC. Platelets facilitate this process by sustaining the hepatic accumulation of virus-specific CD8(+) T cells and subsequently other virus nonspecific inflammatory cells that contribute to liver disease. Importantly, a recent study shows that the long-term use of clinically relevant doses of the anti-platelet drugs aspirin and clopidogrel, administered after the onset of liver disease, in an HBV transgenic mouse model of immune-mediated chronic hepatitis and HCC, can prevent hepatocarcinogenesis improving overall survival. Platelets therefore, act as key players in the pathogenesis of HBV-associated liver cancer supporting the notion that immune-mediated necroinflammatory liver disease is sufficient to trigger HCC and that interference with platelet activation may have clinical implications for HCC prevention.

  5. CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells.

    Science.gov (United States)

    Yuen, Kit-San; Chan, Chi-Ping; Wong, Nok-Hei Mickey; Ho, Chau-Ha; Ho, Ting-Hin; Lei, Ting; Deng, Wen; Tsao, Sai Wah; Chen, Honglin; Kok, Kin-Hang; Jin, Dong-Yan

    2015-03-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.

  6. Role of CD137 signaling in dengue virus-mediated apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Nagila, Amar [Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Netsawang, Janjuree [Faculty of Medical Technology, Rangsit University, Bangkok (Thailand); Srisawat, Chatchawan [Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Noisakran, Sansanee [Dengue Hemorrhagic Fever Research Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok (Thailand); Morchang, Atthapan; Yasamut, Umpa [Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Puttikhunt, Chunya [Dengue Hemorrhagic Fever Research Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok (Thailand); Kasinrerk, Watchara [Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai (Thailand); Biomedical Technology Research Center, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency at Chiang Mai University, Chiang Mai (Thailand); and others

    2011-07-08

    Highlights: {yields} For the first time the role of CD137 in dengue virus (DENV) infection. {yields} Induction of DENV-mediated apoptosis by CD137 signaling. {yields} Sensitization to CD137-mediated apoptosis by dengue virus capsid protein (DENV C). {yields} Nuclear localization of DENV C is required for CD137-mediated apoptosis. -- Abstract: Hepatic dysfunction is a well recognized feature of dengue virus (DENV) infection. However, molecular mechanisms of hepatic injury are still poorly understood. A complex interaction between DENV and the host immune response contributes to DENV-mediated tissue injury. DENV capsid protein (DENV C) physically interacts with the human death domain-associated protein Daxx. A double substitution mutation in DENV C (R85A/K86A) abrogates Daxx interaction, nuclear localization and apoptosis. Therefore we compared the expression of cell death genes between HepG2 cells expressing DENV C and DENV C (R85A/K86A) using a real-time PCR array. Expression of CD137, which is a member of the tumor necrosis factor receptor family, increased significantly in HepG2 cells expressing DENV C compared to HepG2 cells expressing DENV C (R85A/K86A). In addition, CD137-mediated apoptotic activity in HepG2 cells expressing DENV C was significantly increased by anti-CD137 antibody compared to that of HepG2 cells expressing DENV C (R85A/K86A). In DENV-infected HepG2 cells, CD137 mRNA and CD137 positive cells significantly increased and CD137-mediated apoptotic activity was increased by anti-CD137 antibody. This work is the first to demonstrate the contribution of CD137 signaling to DENV-mediated apoptosis.

  7. Tissue-protective effects of NKG2A in immune-mediated clearance of virus infection.

    Science.gov (United States)

    Ely, Kenneth H; Matsuoka, Mitsuo; DeBerge, Matthew P; Ruby, Jessica A; Liu, Jun; Schneider, Mark J; Wang, Yan; Hahn, Young S; Enelow, Richard I

    2014-01-01

    Virus infection triggers a CD8(+) T cell response that aids in virus clearance, but also expresses effector functions that may result in tissue injury. CD8(+) T cells express a variety of activating and inhibiting ligands, though regulation of the expression of inhibitory receptors is not well understood. The ligand for the inhibitory receptor, NKG2A, is the non-classical MHC-I molecule Qa1(b), which may also serve as a putative restricting element for the T cell receptors of purported regulatory CD8(+) T cells. We have previously shown that Qa1(b)-null mice suffer considerably enhanced immunopathologic lung injury in the context of CD8(+) T cell-mediated clearance of influenza infection, as well as evidence in a non-viral system that failure to ligate NKG2A on CD8(+) effector T cells may represent an important component of this process. In this report, we examine the requirements for induction of NKG2A expression, and show that NKG2A expression by CD8(+) T cells occurs as a result of migration from the MLN to the inflammatory lung environment, irrespective of peripheral antigen recognition. Further, we confirmed that NKG2A is a mediator in limiting immunopathology in virus infection using mice with a targeted deletion of NKG2A, and infecting the mutants with two different viruses, influenza and adenovirus. In neither infection is virus clearance altered. In influenza infection, the enhanced lung injury was associated with increased chemoattractant production, increased infiltration of inflammatory cells, and significantly enhanced alveolar hemorrhage. The primary mechanism of enhanced injury was the loss of negative regulation of CD8(+) T cell effector function. A similar effect was observed in the livers of mutant mice infected intravenously with adenovirus. These results demonstrate the immunoregulatory role of CD8(+) NKG2A expression in virus infection, which negatively regulates T cell effector functions and contributes to protection of tissue integrity during

  8. A mediator embedded micro-immunosensing unit for electrochemical detection on viruses within physiological saline media

    Science.gov (United States)

    Pires, Nuno M. M.; Dong, Tao; Yang, Zhaochu; Høivik, Nils; Zhao, Xinyan

    2011-11-01

    To provide a time- and cost-saving alternative to the conventional methods for virus detection in biological media, this work presents an electrochemical micro-immunosensor based on the nickel hexacyanoferrate (NiHCF) redox mediator film coating the interdigitated microelectrodes (IDMEs). By chelation binding with no additional cross-linker, the 6xHis-tagged antibodies were immobilized on a NiHCF film. Secondly, an immunoassay response was enhanced by employing microbeads coated with 6xHis antibody. The electrochemical properties and the stability of the NiHCF film modified IDMEs were evaluated by cyclic voltammetry. The bead-induced impedance variations at the electrode film/electrolyte interface were characterized by electrochemical impedance spectroscopy and verified using FEM simulation. Experiments of virus detection were conducted through targeting the antigens of the vital infectious salmon viruses, such as infectious salmon anaemia virus, infectious pancreatic necrosis virus and salmonid alphavirus subtype 3. The micro-immunosensor exhibited detection limits as low as 10 pg ml-1 and detection sensitivities as high as 57.5 kΩ µM-1 within a physiological saline solution. Tests for multiple antigen-antibody interactions showed good detection specificity, as confirmed by ELISA. By incorporating the microfluidic network, electrochemical impedance micro-immunosensing units can be realized in a fully integrated platform for multiplex virus detection in tissue samples.

  9. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice.

    Science.gov (United States)

    Yang, Yang; Wang, Lili; Bell, Peter; McMenamin, Deirdre; He, Zhenning; White, John; Yu, Hongwei; Xu, Chenyu; Morizono, Hiroki; Musunuru, Kiran; Batshaw, Mark L; Wilson, James M

    2016-03-01

    Many genetic liver diseases in newborns cause repeated, often lethal, metabolic crises. Gene therapy using nonintegrating viruses such as adeno-associated virus (AAV) is not optimal in this setting because the nonintegrating genome is lost as developing hepatocytes proliferate. We reasoned that newborn liver may be an ideal setting for AAV-mediated gene correction using CRISPR-Cas9. Here we intravenously infuse two AAVs, one expressing Cas9 and the other expressing a guide RNA and the donor DNA, into newborn mice with a partial deficiency in the urea cycle disorder enzyme, ornithine transcarbamylase (OTC). This resulted in reversion of the mutation in 10% (6.7-20.1%) of hepatocytes and increased survival in mice challenged with a high-protein diet, which exacerbates disease. Gene correction in adult OTC-deficient mice was lower and accompanied by larger deletions that ablated residual expression from the endogenous OTC gene, leading to diminished protein tolerance and lethal hyperammonemia on a chow diet.

  10. Anti-gp120 minibody gene transfer to female genital epithelial cells protects against HIV-1 virus challenge in vitro.

    Directory of Open Access Journals (Sweden)

    Ussama M Abdel-Motal

    Full Text Available BACKGROUND: Although cervico-vaginal epithelial cells of the female lower genital tract provide the initial defense system against HIV-1 infection, the protection is sometimes incomplete. Thus, enhancing anti-HIV-1 humoral immunity at the mucosal cell surface by local expression of anti-HIV-1 broadly neutralizing antibodies (BnAb that block HIV-1 entry would provide an important new intervention that could slow the spread of HIV/AIDS. METHODS AND FINDINGS: This study tested the hypothesis that adeno-associated virus (AAV-BnAb gene transfer to cervico-vaginal epithelial cells will lead to protection against HIV-1. Accordingly, a recombinant AAV vector that encodes human b12 anti-HIV gp120 BnAb as a single-chain variable fragment Fc fusion (scFvFc, or "minibody" was constructed. The secreted b12 minibody was shown to be biologically functional in binding to virus envelope protein, neutralizing HIV-1 and importantly, blocking transfer and infectivity of HIV-1(bal in an organotypic human vaginal epithelial cell (VEC model. Furthermore, cervico-vaginal epithelial stem cells were found to be efficiently transduced by the optimal AAV serotype mediated expression of GFP. CONCLUSION: This study provides the foundation for a novel microbicide strategy to protect against sexual transmission of HIV-1 by AAV transfer of broadly neutralizing antibody genes to cervico-vaginal epithelial stem cells that could replenish b12 BnAb secreting cells through multiple menstrual cycles.

  11. ONCOLYTIC VIRUS-MEDIATED REVERSAL OF IMPAIRED TUMOR ANTIGEN PRESENTATION

    Directory of Open Access Journals (Sweden)

    Shashi Ashok Gujar

    2014-04-01

    Full Text Available Anti-tumor immunity can eliminate existing cancer cells and also maintain a constant surveillance against possible relapse. Such an antigen-specific adaptive response begins when tumor-specific T cells become activated. T cell activation requires two signals on antigen presenting cells (APCs: antigen presentation through MHC molecules and co-stimulation. In the absence of one or both of these signals, T cells remain inactivated or can even become tolerized. Cancer cells and their associated microenvironment strategically hinder the processing and presentation of tumor antigens and consequently prevent the development of anti-tumor immunity. Many studies, however, demonstrate that interventions that overturn tumor-associated immune evasion mechanisms can establish anti-tumor immune responses of therapeutic potential. One such intervention is oncolytic virus (OV-based anti-cancer therapy. Here we discuss how OV-induced immunological events override tumor-associated antigen presentation impairment and promote appropriate T cell:APC interaction. Detailed understanding of this phenomenon is pivotal for devising the strategies that will enhance the efficacy of OV-based anti-cancer therapy by complementing its inherent oncolytic

  12. Pseudorabies virus can escape from CRISPR-Cas9-mediated inhibition.

    Science.gov (United States)

    Peng, Zhiyuan; Ouyang, Ting; Pang, Daxin; Ma, Teng; Chen, Xinrong; Guo, Ning; Chen, Fuwang; Yuan, Lin; Ouyang, Hongsheng; Ren, Linzhu

    2016-09-02

    The CRISPR-Cas9 system is a newly developed genome-engineering tool used to inhibit virus infection by targeting the conserved regions of the viral genomic DNA. In the present study, we constructed a cell line stably expressing Cas9 endonuclease and sgRNA targeting the conserved UL30 gene of pseudorabies virus (PRV). During the PRV infection, the CRISPR-Cas9 system was efficient in cleaving the UL30 gene in each passage. However, deletions and insertions occurred at low passages, while substitutions were frequently observed at high passages. Furthermore, copy numbers and virus titers of PRV were significantly increased in a passage-dependent manner, indicating that viral genomic replication and assembly were more effective at the high passages than at low passages. These results demonstrated that PRV could escape from CRISPR-Cas9-mediated inhibition. Therefore, whether the CRISPR-Cas9 system is suitable for antiviral application should be considered and carefully verified.

  13. Molecular determinants of dengue virus 2 envelope protein important for virus entry in FcγRIIA-mediated antibody-dependent enhancement of infection

    Energy Technology Data Exchange (ETDEWEB)

    Chotiwan, Nunya; Roehrig, John T. [Arboviral Diseases Branch, Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Schlesinger, Jacob J. [Department of Medicine, University of Rochester, Rochester, NY 14642 (United States); Blair, Carol D. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Huang, Claire Y.-H., E-mail: yxh0@cdc.gov [Arboviral Diseases Branch, Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States)

    2014-05-15

    Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fcγ receptors (FcγR) on FcγR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcγRIIA, and binding of virus-Ab complexes with FcγRIIA alone is not sufficient for ADE of infection. The FcγRIIA mainly plays an auxiliary role in concentrating the virus–Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE. - Highlights: • KKK305/307/310 in DENV2 E-DIII is critical for virus attachment in ADE and non-ADE infection. • Binding of DENV2–Ab complex with FcγRII alone is not sufficient for virus entry in ADE infection. • Other primary receptors were required for DENV2 internalization during FcγRII–mediated ADE. • G104 and L135 of DENV2 E are critical for virus-mediated membrane fusion. • DENV2 virus-mediated membrane fusion is required for both ADE and non-ADE infection.

  14. Sustained Small Interfering RNA-Mediated Human Immunodeficiency Virus Type 1 Inhibition in Primary Macrophages

    OpenAIRE

    2003-01-01

    Small interfering RNAs (siRNAs) can induce potent gene silencing by degradation of cognate mRNA. However, in dividing cells, the silencing lasts only 3 to 7 days, presumably because of siRNA dilution with cell division. Here, we investigated if sustained siRNA-mediated silencing of human immunodeficiency virus type 1 (HIV-1) is possible in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor...

  15. Mannosyl Glycodendritic Structure Inhibits DC-SIGN-Mediated Ebola Virus Infection in cis and in trans

    Science.gov (United States)

    Lasala, Fátima; Arce, Eva; Otero, Joaquín R.; Rojo, Javier; Delgado, Rafael

    2003-01-01

    We have designed a glycodendritic structure, BH30sucMan, that blocks the interaction between dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and Ebola virus (EBOV) envelope. BH30sucMan inhibits DC-SIGN-mediated EBOV infection at nanomolar concentrations. BH30sucMan may counteract important steps of the infective process of EBOV and, potentially, of microorganisms shown to exploit DC-SIGN for cell entry and infection. PMID:14638512

  16. AAV-mediated expression of BAG1 and ROCK2-shRNA promote neuronal survival and axonal sprouting in a rat model of rubrospinal tract injury.

    Science.gov (United States)

    Challagundla, Malleswari; Koch, Jan Christoph; Ribas, Vinicius Toledo; Michel, Uwe; Kügler, Sebastian; Ostendorf, Thomas; Bradke, Frank; Müller, Hans Werner; Bähr, Mathias; Lingor, Paul

    2015-07-01

    A lesion to the rat rubrospinal tract is a model for traumatic spinal cord lesions and results in atrophy of the red nucleus neurons, axonal dieback, and locomotor deficits. In this study, we used adeno-associated virus (AAV)-mediated over-expression of BAG1 and ROCK2-shRNA in the red nucleus to trace [by co-expression of enhanced green fluorescent protein (EGFP)] and treat the rubrospinal tract after unilateral dorsal hemisection. We investigated the effects of targeted gene therapy on neuronal survival, axonal sprouting of the rubrospinal tract, and motor recovery 12 weeks after unilateral dorsal hemisection at Th8 in rats. In addition to the evaluation of BAG1 and ROCK2 as therapeutic targets in spinal cord injury, we aimed to demonstrate the feasibility and the limits of an AAV-mediated protein over-expression versus AAV.shRNA-mediated down-regulation in this traumatic CNS lesion model. Our results demonstrate that BAG1 and ROCK2-shRNA both promote neuronal survival of red nucleus neurons and enhance axonal sprouting proximal to the lesion.

  17. Non-classical phase diagram for virus bacterial coevolution mediated by clustered regularly interspaced short palindromic repeats.

    Science.gov (United States)

    Han, Pu; Deem, Michael W

    2017-02-01

    CRISPR is a newly discovered prokaryotic immune system. Bacteria and archaea with this system incorporate genetic material from invading viruses into their genomes, providing protection against future infection by similar viruses. The condition for coexistence of prokaryots and viruses is an interesting problem in evolutionary biology. In this work, we show an intriguing phase diagram of the virus extinction probability, which is more complex than that of the classical predator-prey model. As the CRISPR incorporates genetic material, viruses are under pressure to evolve to escape recognition by CRISPR. When bacteria have a small rate of deleting spacers, a new parameter region in which bacteria and viruses can coexist arises, and it leads to a more complex coexistence patten for bacteria and viruses. For example, when the virus mutation rate is low, the virus extinction probability changes non-montonically with the bacterial exposure rate. The virus and bacteria coevolution not only alters the virus extinction probability, but also changes the bacterial population structure. Additionally, we show that recombination is a successful strategy for viruses to escape from CRISPR recognition when viruses have multiple proto-spacers, providing support for a recombination-mediated escape mechanism suggested experimentally. Finally, we suggest that the re-entrant phase diagram, in which phages can progress through three phases of extinction and two phases of abundance at low spacer deletion rates as a function of exposure rate to bacteria, is an experimentally testable phenomenon. © 2017 The Author(s).

  18. Citrus tristeza virus p23: a unique protein mediating key virus-host interactions

    Directory of Open Access Journals (Sweden)

    Ricardo eFlores

    2013-05-01

    Full Text Available The large RNA genome of CTV (ca. 20 kb contains 12 open reading frames (ORFs, with the 3’-terminal one corresponding to a protein of 209 amino acids (p23 that is expressed from an abundant subgenomic RNA. p23, an RNA-binding protein with a putative Zn-finger domain and some basic motifs, is unique to CTV because no homologues have been found in other closteroviruses, including the type species of the genus Beet yellows virus (despite both viruses having many homologous genes. Consequently, p23 might have evolved for the specific interaction of CTV with its citrus hosts. From a functional perspective p23 has been involved in many roles: i regulation of the asymmetrical accumulation of CTV RNA strands, ii induction of the seedling yellows syndrome in sour orange and grapefruit, iii intracellular suppression of RNA silencing, iv elicitation of CTV-like symptoms when expressed ectopically as a transgene in several Citrus spp., and v enhancement of systemic infection (and virus accumulation in sour orange and CTV release from the phloem in p23-expressing transgenic sweet and sour orange. Moreover, transformation of Mexican lime with intron-hairpin constructs designed for the co-inactivation of p23 and the two other CTV silencing suppressors results in complete resistance against the homologous virus. From a cellular point of view, recent data indicate that p23 accumulates preferentially in the nucleolus, being the first closterovirus protein with such a subcellular localization, as well as in plasmodesmata. These major accumulation sites most likely determine some of the functional roles of p23.

  19. Rescue of cardiomyopathy through U7snRNA-mediated exon skipping in Mybpc3-targeted knock-in mice.

    Science.gov (United States)

    Gedicke-Hornung, Christina; Behrens-Gawlik, Verena; Reischmann, Silke; Geertz, Birgit; Stimpel, Doreen; Weinberger, Florian; Schlossarek, Saskia; Précigout, Guillaume; Braren, Ingke; Eschenhagen, Thomas; Mearini, Giulia; Lorain, Stéphanie; Voit, Thomas; Dreyfus, Patrick A; Garcia, Luis; Carrier, Lucie

    2013-07-01

    Exon skipping mediated by antisense oligoribonucleotides (AON) is a promising therapeutic approach for genetic disorders, but has not yet been evaluated for cardiac diseases. We investigated the feasibility and efficacy of viral-mediated AON transfer in a Mybpc3-targeted knock-in (KI) mouse model of hypertrophic cardiomyopathy (HCM). KI mice carry a homozygous G>A transition in exon 6, which results in three different aberrant mRNAs. We identified an alternative variant (Var-4) deleted of exons 5-6 in wild-type and KI mice. To enhance its expression and suppress aberrant mRNAs we designed AON-5 and AON-6 that mask splicing enhancer motifs in exons 5 and 6. AONs were inserted into modified U7 small nuclear RNA and packaged in adeno-associated virus (AAV-U7-AON-5+6). Transduction of cardiac myocytes or systemic administration of AAV-U7-AON-5+6 increased Var-4 mRNA/protein levels and reduced aberrant mRNAs. Injection of newborn KI mice abolished cardiac dysfunction and prevented left ventricular hypertrophy. Although the therapeutic effect was transient and therefore requires optimization to be maintained over an extended period, this proof-of-concept study paves the way towards a causal therapy of HCM.

  20. Human CD8+ T cells mediate protective immunity induced by a human malaria vaccine in human immune system mice.

    Science.gov (United States)

    Li, Xiangming; Huang, Jing; Zhang, Min; Funakoshi, Ryota; Sheetij, Dutta; Spaccapelo, Roberta; Crisanti, Andrea; Nussenzweig, Victor; Nussenzweig, Ruth S; Tsuji, Moriya

    2016-08-31

    A number of studies have shown that CD8+ T cells mediate protective anti-malaria immunity in a mouse model. However, whether human CD8+ T cells play a role in protection against malaria remains unknown. We recently established human immune system (HIS) mice harboring functional human CD8+ T cells (HIS-CD8 mice) by transduction with HLA-A∗0201 and certain human cytokines using recombinant adeno-associated virus-based gene transfer technologies. These HIS-CD8 mice mount a potent, antigen-specific HLA-A∗0201-restricted human CD8+ T-cell response upon immunization with a recombinant adenovirus expressing a human malaria antigen, the Plasmodium falciparum circumsporozoite protein (PfCSP), termed AdPfCSP. In the present study, we challenged AdPfCSP-immunized HIS-CD8 mice with transgenic Plasmodium berghei sporozoites expressing full-length PfCSP and found that AdPfCSP-immunized (but not naïve) mice were protected against subsequent malaria challenge. The level of the HLA-A∗0201-restricted, PfCSP-specific human CD8+ T-cell response was closely correlated with the level of malaria protection. Furthermore, depletion of human CD8+ T cells from AdPfCSP-immunized HIS-CD8 mice almost completely abolished the anti-malaria immune response. Taken together, our data show that human CD8+ T cells mediate protective anti-malaria immunity in vivo.

  1. Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus.

    Directory of Open Access Journals (Sweden)

    I-Chueh Huang

    Full Text Available Interferon-inducible transmembrane proteins 1, 2, and 3 (IFITM1, 2, and 3 are recently identified viral restriction factors that inhibit infection mediated by the influenza A virus (IAV hemagglutinin (HA protein. Here we show that IFITM proteins restricted infection mediated by the entry glycoproteins (GP(1,2 of Marburg and Ebola filoviruses (MARV, EBOV. Consistent with these observations, interferon-β specifically restricted filovirus and IAV entry processes. IFITM proteins also inhibited replication of infectious MARV and EBOV. We observed distinct patterns of IFITM-mediated restriction: compared with IAV, the entry processes of MARV and EBOV were less restricted by IFITM3, but more restricted by IFITM1. Moreover, murine Ifitm5 and 6 did not restrict IAV, but efficiently inhibited filovirus entry. We further demonstrate that replication of infectious SARS coronavirus (SARS-CoV and entry mediated by the SARS-CoV spike (S protein are restricted by IFITM proteins. The profile of IFITM-mediated restriction of SARS-CoV was more similar to that of filoviruses than to IAV. Trypsin treatment of receptor-associated SARS-CoV pseudovirions, which bypasses their dependence on lysosomal cathepsin L, also bypassed IFITM-mediated restriction. However, IFITM proteins did not reduce cellular cathepsin activity or limit access of virions to acidic intracellular compartments. Our data indicate that IFITM-mediated restriction is localized to a late stage in the endocytic pathway. They further show that IFITM proteins differentially restrict the entry of a broad range of enveloped viruses, and modulate cellular tropism independently of viral receptor expression.

  2. Nigrostriatal rAAV-mediated GDNF Overexpression Induces Robust Weight Loss in a Rat Model of Age-related Obesity

    OpenAIRE

    Manfredsson, Fredric P; Tumer, Nihal; Erdos, Benedek; Landa, Tessa; Broxson, Christopher S; Sullivan, Layla F; Rising, Aaron C; Foust, Kevin D.; Zhang, Yi; Muzyczka, Nicholas; Gorbatyuk, Oleg S.; Scarpace, Philip J.; Mandel, Ronald J.

    2009-01-01

    Intraventricular administration of glial cell line–derived neurotrophic factor (GDNF) in primate and humans to study Parkinson's disease (PD) has revealed the potential for GDNF to induce weight loss. Our previous data indicate that bilateral continuous hypothalamic GDNF overexpression via recombinant adeno-associated virus (rAAV) results in significant failure to gain weight in young rats and weight loss in aged rats. Based on these previous results, we hypothesized that because the nigrostr...

  3. Ultrasound-mediated oncolytic virus delivery and uptake for increased therapeutic efficacy: state of art

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    Nande R

    2015-11-01

    Full Text Available Rounak Nande,1 Candace M Howard,2 Pier Paolo Claudio,3,4 1Department of Biochemistry and Microbiology, Marshall University School of Medicine, Huntington, WV, 2Department of Radiology, University of Mississippi Medical Center, Jackson, MS, 3Department of BioMolecular Sciences and National Center for Natural Products Research, School of Pharmacy, University of Mississippi, MS, 4Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS, USA Abstract: The field of ultrasound (US has changed significantly from medical imaging and diagnosis to treatment strategies. US contrast agents or microbubbles (MB are currently being used as potential carriers for chemodrugs, small molecules, nucleic acids, small interfering ribonucleic acid, proteins, adenoviruses, and oncolytic viruses. Oncolytic viruses can selectively replicate within and destroy a cancer cell, thus making them a powerful therapeutic in treating late-stage or metastatic cancer. These viruses have been shown to have robust activity in clinical trials when injected directly into tumor nodules. However limitations in oncolytic virus’ effectiveness and its delivery approach have warranted exploration of ultrasound-mediated delivery. Gene therapy bearing adenoviruses or oncolytic viruses can be coupled with MBs and injected intravenously. Following application of US energy to the target region, the MBs cavitate, and the resulting shock wave enhances drug, gene, or adenovirus uptake. Though the underlying mechanism is yet to be fully understood, there is evidence to suggest that mechanical pore formation of cellular membranes allows for the temporary uptake of drugs. This delivery method circumvents the limitations due to stimulation of the immune system that prevented intravenous administration of viruses. This review provides insight into this intriguing new frontier on the delivery of oncolytic viruses to tumor sites.Keywords: microbubbles, ultrasound

  4. Novel mutations in Marburg virus glycoprotein associated with viral evasion from antibody mediated immune pressure.

    Science.gov (United States)

    Kajihara, Masahiro; Nakayama, Eri; Marzi, Andrea; Igarashi, Manabu; Feldmann, Heinz; Takada, Ayato

    2013-04-01

    Marburg virus (MARV) and Ebola virus, members of the family Filoviridae, cause lethal haemorrhagic fever in humans and non-human primates. Although the outbreaks are concentrated mainly in Central Africa, these viruses are potential agents of imported infectious diseases and bioterrorism in non-African countries. Recent studies demonstrated that non-human primates passively immunized with virus-specific antibodies were successfully protected against fatal filovirus infection, highlighting the important role of antibodies in protective immunity for this disease. However, the mechanisms underlying potential evasion from antibody mediated immune pressure are not well understood. To analyse possible mutations involved in immune evasion in the MARV envelope glycoprotein (GP) which is the major target of protective antibodies, we selected escape mutants of recombinant vesicular stomatitis virus (rVSV) expressing MARV GP (rVSVΔG/MARVGP) by using two GP-specific mAbs, AGP127-8 and MGP72-17, which have been previously shown to inhibit MARV budding. Interestingly, several rVSVΔG/MARVGP variants escaping from the mAb pressure-acquired amino acid substitutions in the furin-cleavage site rather than in the mAb-specific epitopes, suggesting that these epitopes are recessed, not exposed on the uncleaved GP molecule, and therefore inaccessible to the mAbs. More surprisingly, some variants escaping mAb MGP72-17 lacked a large proportion of the mucin-like region of GP, indicating that these mutants efficiently escaped the selective pressure by deleting the mucin-like region including the mAb-specific epitope. Our data demonstrate that MARV GP possesses the potential to evade antibody mediated immune pressure due to extraordinary structural flexibility and variability.

  5. rAAV vector-mediated gene therapy for experimental ischemic stroke

    Directory of Open Access Journals (Sweden)

    Li Zhao-Jian

    2008-01-01

    Full Text Available The safest viral vector system for gene therapy is based on recombinant adeno-associated virus (rAAV up to date in Phase I clinical trials, which has been developed rapidly and applied for ischemic stroke gene therapy in animal experiments since the past seven years. rAAV vector has made great progress in improving gene delivery by modification of the capsid and increasing transgene expression by encapsidation of double-stranded rAAV genome. And in all, nine therapeutic genes in 12 animal studies were successfully delivered using rAAV vector to ischemic brain via different approaches in rat or mice stroke models for gene therapy and the results suggested that rAAV could mediate genes′ expression efficiently; most of them displayed evidently therapeutic efficacy with satisfactory biological safety. Gene therapy involving rAAV vector seems effective in attenuation of ischemic damage in stroke and has greatly promising potential use for patients in the future. In this review, we will focus on the basic biology and development of rAAV vector itself as well as the recent progress in the use of this vector for ischemic stroke gene therapy in animal experiments.

  6. Concurrent Reactivation of Herpes Simplex and Varicella Zoster Viruses Confirmed by the Loop-Mediated Isothermal Amplification Assay

    Directory of Open Access Journals (Sweden)

    Tsukane Kobayashi

    2014-01-01

    Full Text Available Concurrent reactivation of herpes simplex and varicella zoster viruses is rare. Here, we describe the case of an elderly patient with herpes labialis and herpes zoster manifesting as a right-side facial eruption with vesicles and crusting. The loop-mediated isothermal amplification (LAMP assay demonstrated the presence of both herpes simplex virus type 1 and varicella zoster virus in swab samples taken from the face, which was confirmed by real-time PCR, suggesting concurrent reactivation of both viruses. The use of the LAMP assay in the present case indicates its usefulness in the diagnosis of atypical herpes infections.

  7. Avian leukosis virus subgroup J triggers caspase-1-mediated inflammatory response in chick livers.

    Science.gov (United States)

    Liu, Xue-lan; Shan, Wen-jie; Jia, Li-juan; Yang, Xu; Zhang, Jin-jing; Wu, Ya-rong; Xu, Fa-zhi; Li, Jin-nian

    2016-04-02

    Many pathogens trigger caspase-1-mediated innate immune responses. Avian leukosis virus subgroup J (ALV-J) causes serious immunosuppression and diverse tumors in chicks. The caspase-1 inflammasome mechanism of response to ALV-J invading remains unclear. Here we investigated the expression of caspase-1, the inflammasome adaptor NLRP3, IL-1β and IL-18 in response to ALV-J infection in the liver of chick. We found caspase-1 mRNA expression was elevated at 5 dpi and peaked at 7 dpi in ALV-J infected animals. Corresponding to this, the expressions of NLRP3 and proinflammatory cytokines IL-1β and IL-18 were significantly increased at 5 or 7 dpi. In addition, caspase-1 protein expression and inflammatory cell infiltration were induced after virus infection. These results indicated that ALV-J infection could trigger the caspase-1- mediated inflammatory response in chicks. Thus, an understanding of the inflammatory responses can provide a better insight into the pathogenicity of ALV-J and a possible anti-virus target for ALV-J infection.

  8. Kinetics of cellular uptake of viruses and nanoparticles via clathrin-mediated endocytosis

    Science.gov (United States)

    Banerjee, Anand; Berezhkovskii, Alexander; Nossal, Ralph

    2016-02-01

    Several viruses exploit clathrin-mediated endocytosis to gain entry into host cells. This process is also used extensively in biomedical applications to deliver nanoparticles (NPs) to diseased cells. The internalization of these nano-objects is controlled by the assembly of a clathrin-containing protein coat on the cytoplasmic side of the plasma membrane, which drives the invagination of the membrane and the formation of a cargo-containing endocytic vesicle. Current theoretical models of receptor-mediated endocytosis of viruses and NPs do not explicitly take coat assembly into consideration. In this paper we study cellular uptake of viruses and NPs with a focus on coat assembly. We characterize the internalization process by the mean time between the binding of a particle to the membrane and its entry into the cell. Using a coarse-grained model which maps the stochastic dynamics of coat formation onto a one-dimensional random walk, we derive an analytical formula for this quantity. A study of the dependence of the mean internalization time on NP size shows that there is an upper bound above which this time becomes extremely large, and an optimal size at which it attains a minimum. Our estimates of these sizes compare well with experimental data. We also study the sensitivity of the obtained results on coat parameters to identify factors which significantly affect the internalization kinetics.

  9. Tissue-protective effects of NKG2A in immune-mediated clearance of virus infection.

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    Kenneth H Ely

    Full Text Available Virus infection triggers a CD8(+ T cell response that aids in virus clearance, but also expresses effector functions that may result in tissue injury. CD8(+ T cells express a variety of activating and inhibiting ligands, though regulation of the expression of inhibitory receptors is not well understood. The ligand for the inhibitory receptor, NKG2A, is the non-classical MHC-I molecule Qa1(b, which may also serve as a putative restricting element for the T cell receptors of purported regulatory CD8(+ T cells. We have previously shown that Qa1(b-null mice suffer considerably enhanced immunopathologic lung injury in the context of CD8(+ T cell-mediated clearance of influenza infection, as well as evidence in a non-viral system that failure to ligate NKG2A on CD8(+ effector T cells may represent an important component of this process. In this report, we examine the requirements for induction of NKG2A expression, and show that NKG2A expression by CD8(+ T cells occurs as a result of migration from the MLN to the inflammatory lung environment, irrespective of peripheral antigen recognition. Further, we confirmed that NKG2A is a mediator in limiting immunopathology in virus infection using mice with a targeted deletion of NKG2A, and infecting the mutants with two different viruses, influenza and adenovirus. In neither infection is virus clearance altered. In influenza infection, the enhanced lung injury was associated with increased chemoattractant production, increased infiltration of inflammatory cells, and significantly enhanced alveolar hemorrhage. The primary mechanism of enhanced injury was the loss of negative regulation of CD8(+ T cell effector function. A similar effect was observed in the livers of mutant mice infected intravenously with adenovirus. These results demonstrate the immunoregulatory role of CD8(+ NKG2A expression in virus infection, which negatively regulates T cell effector functions and contributes to protection of tissue

  10. Virus-induced gene silencing reveals signal transduction components required for the Pvr9-mediated hypersensitive response in Nicotiana benthamiana.

    Science.gov (United States)

    Tran, Phu-Tri; Choi, Hoseong; Choi, Doil; Kim, Kook-Hyung

    2016-08-01

    Resistance to pathogens mediated by plant resistance (R) proteins requires different signaling transduction components and pathways. Our previous studies revealed that a potyvirus resistance gene in pepper, Pvr9, confers a hypersensitive response (HR) to pepper mottle virus in Nicotiana benthamiana. Our results show that the Pvr9-mediated HR against pepper mottle virus infection requires HSP90, SGT1, NDR1, but not EDS1. These results suggest that the Pvr9-mediated HR is possibly related to the SA pathway but not the ET, JA, ROS or NO pathways.

  11. Porcine aminopeptidase N mediated polarized infection by porcine epidemic diarrhea virus in target cells

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    Cong, Yingying; Li, Xiaoxue; Bai, Yunyun [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China); Lv, Xiaonan [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China); CAS Key Lab for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience & Technology of China, Beijing 100090 (China); Herrler, Georg [Institute for Virology, University of Veterinary Medicine, Hannover D-30559 (Germany); Enjuanes, Luis [Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid (Spain); Zhou, Xingdong [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China); Qu, Bo [Faculty of Life Sciences, Northeast Agricultural University, Harbin 150030 (China); Meng, Fandan [Institute for Virology, University of Veterinary Medicine, Hannover D-30559 (Germany); Cong, Chengcheng [College Animal Husbandry and Veterinary Medicine, Shenyang Agricultural University, Shenyang 110161 (China); Ren, Xiaofeng; Li, Guangxing [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China)

    2015-04-15

    Infection of polarized intestinal epithelial cells by porcine epidemic diarrhea virus (PEDV) was characterized. Indirect immunofluorescence assay, real-time PCR, and transmission electron microscopy confirmed PEDV can be successfully propagated in immortalized swine small intestine epithelial cells (IECs). Infection involved porcine aminpeptidase N (pAPN), a reported cellular receptor for PEDV, transient expression of pAPN and siRNA targeted pAPN increased and decreased the infectivity of PEDV in IECs, respectively. Subsequently, polarized entry into and release from both Vero E6 and IECs was analyzed. PEDV entry into polarized cells and pAPN grown on membrane inserts occurs via apical membrane. The progeny virus released into the medium was also quantified which demonstrated that PEDV is preferentially released from the apical membrane. Collectively, our data demonstrate that pAPN, the cellular receptor for PEDV, mediates polarized PEDV infection. These results imply the possibility that PEDV infection may proceed by lateral spread of virus in intestinal epithelial cells. - Highlights: • PEDV infection of polarized intestinal epithelial cells (IECs) was characterized. • Porcine aminpeptidase N (pAPN) facilitated PEDV infection in IECs. • PEDV entry into and release from polarized cell via its apical membrane. • PEDV infection may proceed by lateral spread of virus in IECs.

  12. Influenza C virus NS1 protein counteracts RIG-I-mediated IFN signalling

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    Vlasak Reinhard

    2011-02-01

    Full Text Available Abstract The nonstructural proteins 1 (NS1 from influenza A and B viruses are known as the main viral factors antagonising the cellular interferon (IFN response, inter alia by inhibiting the retinoic acid-inducible gene I (RIG-I signalling. The cytosolic pattern-recognition receptor RIG-I senses double-stranded RNA and 5'-triphosphate RNA produced during RNA virus infections. Binding to these ligands activates RIG-I and in turn the IFN signalling. We now report that the influenza C virus NS1 protein also inhibits the RIG-I-mediated IFN signalling. Employing luciferase-reporter assays, we show that expression of NS1-C proteins of virus strains C/JJ/50 and C/JHB/1/66 considerably reduced the IFN-β promoter activity. Mapping of the regions from NS1-C of both strains involved in IFN-β promoter inhibition showed that the N-terminal 49 amino acids are dispensable, while the C-terminus is required for proper modulation of the IFN response. When a mutant RIG-I, which is constitutively active without ligand binding, was employed, NS1-C still inhibited the downstream signalling, indicating that IFN inhibitory properties of NS1-C are not necessarily linked to an RNA binding mechanism.

  13. Egg drop syndrome virus enters duck embryonic fibroblast cells via clathrin-mediated endocytosis.

    Science.gov (United States)

    Huang, Jingjing; Tan, Dan; Wang, Yang; Liu, Caihong; Xu, Jiamin; Wang, Jingyu

    2015-12-02

    Previous studies of egg drop syndrome virus (EDSV) is restricted to serological surveys, disease diagnostics, and complete viral genome analysis. Consequently, the infection characteristics and entry routes of EDSV are poorly understood. Therefore, we aimed to explore the entry pathway of EDSV into duck embryonic fibroblast (DEF) cells as well as the infection characteristics and proliferation of EDSV in primary DEF and primary chicken embryo liver (CEL) cells. Transmission electron microscopy revealed that the virus triggered DEF cell membrane invagination as early as 10 min post-infection and that integrated endocytic vesicles formed at 20 min post-infection. The virus yield in EDSV-infected DEF cells treated with chlorpromazine (CPZ), sucrose, methyl-β-cyclodextrin (MβCD), or NH4Cl was measured by quantitative real-time PCR. Compared with the mock treatment, CPZ and sucrose greatly inhibited the production of viral progeny in a dose-dependent manner, while MβCD treatment did not result in a significant difference. Furthermore, NH4Cl had a strong inhibitory effect on the production of EDSV progeny. In addition, indirect immunofluorescence demonstrated that virus particles clustered on the surface of DEF cells treated with CPZ or sucrose. These results indicate that EDSV enters DEF cells through clathrin-mediated endocytosis followed by a pH-dependent step, which is similar to the mechanism of entry of human adenovirus types 2 and 5. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Infection of hepatitis B virus in extrahepatic endothelial tissues mediated by endothelial progenitor cells

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    Zhang Lili

    2007-04-01

    Full Text Available Abstract Background Hepatitis B virus (HBV replication has been reported to be involved in many extrahepatic viral disorders; however, the mechanism by which HBV is trans-infected into extrahepatic tissues such as HBV associated myocarditis remains largely unknown. Results In this study, we showed that human cord blood endothelial progenitor cells (EPCs, but not human umbilical vein endothelial cells (HUVECs could be effectively infected by uptake of HBV in vitro. Exposure of EPCs with HBV resulted in HBV DNA and viral particles were detected in EPCs at day 3 after HBV challenge, which were peaked around day 7 and declined in 3 weeks. Consistently, HBV envelope surface and core antigens were first detected in EPCs at day 3 after virus challenge and were retained to be detectable for 3 weeks. In contrast, HBV covalently closed circular DNA was not detected in EPCs at any time after virus challenge. Intravenous transplantation of HBV-treated EPCs into myocardial infarction and acute renal ischemia mouse model resulted in incorporation of HBV into injured heart, lung, and renal capillary endothelial tissues. Conclusion These results strongly support that EPCs serve as virus carrier mediating HBV trans-infection into the injured endothelial tissues. The findings might provide a novel mechanism for HBV-associated myocarditis and other HBV-related extrahepatic diseases as well.

  15. Loop-mediated Isothermal Amplification Assay to Rapidly Detect Wheat Streak Mosaic Virus in Quarantined Plants

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    Siwon Lee

    2015-12-01

    Full Text Available We developed a loop-mediated isothermal amplification (LAMP method to rapidly diagnose Wheat streak mosaic virus (WSMV during quarantine inspections of imported wheat, corn, oats, and millet. The LAMP method was developed as a plant quarantine inspection method for the first time, and its simplicity, quickness, specificity and sensitivity were verified compared to current reverse transcription-polymerase chain reaction (RT-PCR and nested PCR quarantine methods. We were able to quickly screen for WSMV at quarantine sites with many test samples; thus, this method is expected to contribute to plant quarantine inspections.

  16. Amphotericin B Increases Influenza A Virus Infection by Preventing IFITM3-Mediated Restriction

    OpenAIRE

    Tsai-Yu Lin; Christopher R. Chin; Aaron R. Everitt; Simon Clare; Jill M. Perreira; George Savidis; Aaron M. Aker; Sinu P. John; David Sarlah; Erick M. Carreira; Stephen J. Elledge; Paul Kellam; Abraham L. Brass

    2013-01-01

    Summary The IFITMs inhibit influenza A virus (IAV) replication in vitro and in vivo. Here, we establish that the antimycotic heptaen, amphotericin B (AmphoB), prevents IFITM3-mediated restriction of IAV, thereby increasing viral replication. Consistent with its neutralization of IFITM3, a clinical preparation of AmphoB, AmBisome, reduces the majority of interferon’s protective effect against IAV in vitro. Mechanistic studies reveal that IFITM1 decreases host-membrane fluidity, suggesting both...

  17. Endothelial progenitor cell mediates transport of hepatitis B virus into myocardial tissue

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Background Hepatitis B virus (HBV) replication has been reported to be involved in many extrahepatic viral disorders;however,the mechanism by which HBV is transinfected into extrahepatic tissues such as myocardium and causes HBV associated myocarditis remains largely unknown.Methods In this study,endothelial progenitor cells (EPCs) were infected by HBV and then transfused into ischemic model of mice.HBV surface and core antigen as well as mutation of HBV particles were detected by immunonistochemistry,fluorescent activated cell sorter and transmission electron microscopy in vitro and in vivo.Results Human cord blood EPCs, but not human umbilical vein endothelial cells (HUVECs) could be effectively infected by taking up HBV in vitro.HBV envelope surface and core antigen expressions were first detectable in EPCs at day 3 after virus challenge,sustained for up to 11 days,and decreased thereafter.Similarly,the virus particles were the most abundant in EPCs ln the first week observed by a transmission electron microscope,and declined in 3 weeks after HBV infection.HBV DNA but not HBV cccDNA in EPCs were detectable even 3 weeks after virus challenge,as shown by PCR analysis.Furthermore,intravenous transplantation of HBV-treated EPCs into myocardial infarction Sprague & Dawley rats model resulted in incorporation of both EPCs and HBV into injured endothelial tissues of capillaries in the ischemic border zone.Conclusions These results strongly support that EPCs serve as virus carrier mediating HBV trans-infection into the injured myocardial tissues.The findings might suggest a novel mechanism for HBV-associated myocarditis.

  18. Clinical development of reovirus for cancer therapy: An oncolytic virus with immune-mediated antitumor activity

    Science.gov (United States)

    Gong, Jun; Sachdev, Esha; Mita, Alain C; Mita, Monica M

    2016-01-01

    Reovirus is a double-stranded RNA virus with demonstrated oncolysis or preferential replication in cancer cells. The oncolytic properties of reovirus appear to be dependent, in part, on activated Ras signaling. In addition, Ras-transformation promotes reovirus oncolysis by affecting several steps of the viral life cycle. Reovirus-mediated immune responses can present barriers to tumor targeting, serve protective functions against reovirus systemic toxicity, and contribute to therapeutic efficacy through antitumor immune-mediated effects via innate and adaptive responses. Preclinical studies have demonstrated the broad anticancer activity of wild-type, unmodified type 3 Dearing strain reovirus (Reolysin®) across a spectrum of malignancies. The development of reovirus as an anticancer agent and available clinical data reported from 22 clinical trials will be reviewed. PMID:27019795

  19. Clinical development of reovirus for cancer therapy: An oncolytic virus with immune-mediated antitumor activity.

    Science.gov (United States)

    Gong, Jun; Sachdev, Esha; Mita, Alain C; Mita, Monica M

    2016-03-26

    Reovirus is a double-stranded RNA virus with demonstrated oncolysis or preferential replication in cancer cells. The oncolytic properties of reovirus appear to be dependent, in part, on activated Ras signaling. In addition, Ras-transformation promotes reovirus oncolysis by affecting several steps of the viral life cycle. Reovirus-mediated immune responses can present barriers to tumor targeting, serve protective functions against reovirus systemic toxicity, and contribute to therapeutic efficacy through antitumor immune-mediated effects via innate and adaptive responses. Preclinical studies have demonstrated the broad anticancer activity of wild-type, unmodified type 3 Dearing strain reovirus (Reolysin(®)) across a spectrum of malignancies. The development of reovirus as an anticancer agent and available clinical data reported from 22 clinical trials will be reviewed.

  20. Antagonizing Interferon-Mediated Immune Response by Porcine Reproductive and Respiratory Syndrome Virus

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    Rong Wang

    2014-01-01

    Full Text Available Interferons (IFNs are important components in innate immunity involved in the first line of defense to protect host against viral infection. Porcine reproductive and respiratory syndrome virus (PRRSV leads to severe economic losses for swine industry since being first identified in early 1990s. PRRSV interplays with host IFN production and IFN-activated signaling, which may contribute to the delayed onset and low level of neutralizing antibodies, as well as weak cell-mediated immune response in infected pigs. PRRSV encodes several proteins that act as antagonists for the IFN signaling. In this review, we summarized the various strategies used by PRRSV to antagonize IFN production and thwart IFN-activated antiviral signaling, as well as the variable interference with IFN-mediated immune response by different PRRSV strains. Thorough understanding of the interaction between PRRSV and host innate immune response will facilitate elucidation of PRRSV pathogenesis and development of a better strategy to control PRRS.

  1. Antagonizing interferon-mediated immune response by porcine reproductive and respiratory syndrome virus.

    Science.gov (United States)

    Wang, Rong; Zhang, Yan-Jin

    2014-01-01

    Interferons (IFNs) are important components in innate immunity involved in the first line of defense to protect host against viral infection. Porcine reproductive and respiratory syndrome virus (PRRSV) leads to severe economic losses for swine industry since being first identified in early 1990s. PRRSV interplays with host IFN production and IFN-activated signaling, which may contribute to the delayed onset and low level of neutralizing antibodies, as well as weak cell-mediated immune response in infected pigs. PRRSV encodes several proteins that act as antagonists for the IFN signaling. In this review, we summarized the various strategies used by PRRSV to antagonize IFN production and thwart IFN-activated antiviral signaling, as well as the variable interference with IFN-mediated immune response by different PRRSV strains. Thorough understanding of the interaction between PRRSV and host innate immune response will facilitate elucidation of PRRSV pathogenesis and development of a better strategy to control PRRS.

  2. Immunity and AAV-mediated gene therapy for muscular dystrophies in large animal models and human trials

    OpenAIRE

    2011-01-01

    Adeno-associated viral (AAV) vector-mediated gene replacement for the treatment of muscular dystrophy represents a promising therapeutic strategy in modern medicine. One major obstacle in using AAV vectors for in vivo gene delivery is the development of host immune responses to the viral capsid protein and transgene products as evidenced in animal models and human trials for a range of genetic diseases. Here, we review immunity against AAV vector and transgene in the context of gene delivery ...

  3. AAV-Mediated Gene Delivery in a Feline Model of Sandhoff Disease Corrects Lysosomal Storage in the Central Nervous System

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    Hannah E. Rockwell

    2015-04-01

    Full Text Available Sandhoff disease (SD is an autosomal recessive neurodegenerative disease caused by a mutation in the gene for the β-subunit of β-N-acetylhexosaminidase (Hex, resulting in the inability to catabolize ganglioside GM2 within the lysosomes. SD presents with an accumulation of GM2 and its asialo derivative GA2, primarily in the central nervous system. Myelin-enriched glycolipids, cerebrosides and sulfatides, are also decreased in SD corresponding with dysmyelination. At present, no treatment exists for SD. Previous studies have shown the therapeutic benefit of adeno-associated virus (AAV vector-mediated gene therapy in the treatment of SD in murine and feline models. In this study, we treated presymptomatic SD cats with AAVrh8 vectors expressing feline Hex in the thalamus combined with intracerebroventricular (Thal/ICV injections. Treated animals showed clearly improved neurologic function and quality of life, manifested in part by prevention or attenuation of whole-body tremors characteristic of untreated animals. Hex activity was significantly elevated, whereas storage of GM2 and GA2 was significantly decreased in tissue samples taken from the cortex, cerebellum, thalamus, and cervical spinal cord. Treatment also increased levels of myelin-enriched cerebrosides and sulfatides in the cortex and thalamus. This study demonstrates the therapeutic potential of AAV for feline SD and suggests a similar potential for human SD patients.

  4. AAV8-mediated in vivo overexpression of miR-155 enhances the protective capacity of genetically attenuated malarial parasites.

    Science.gov (United States)

    Hentzschel, Franziska; Hammerschmidt-Kamper, Christiane; Börner, Kathleen; Heiss, Kirsten; Knapp, Bettina; Sattler, Julia M; Kaderali, Lars; Castoldi, Mirco; Bindman, Julia G; Malato, Yann; Willenbring, Holger; Mueller, Ann-Kristin; Grimm, Dirk

    2014-12-01

    Malaria, caused by protozoan Plasmodium parasites, remains a prevalent infectious human disease due to the lack of an efficient and safe vaccine. This is directly related to the persisting gaps in our understanding of the parasite's interactions with the infected host, especially during the clinically silent yet essential liver stage of Plasmodium development. Previously, we and others showed that genetically attenuated parasites (GAP) that arrest in the liver induce sterile immunity, but only upon multiple administrations. Here, we comprehensively studied hepatic gene and miRNA expression in GAP-injected mice, and found both a broad activation of IFNγ-associated pathways and a significant increase of murine microRNA-155 (miR-155), that was especially pronounced in non-parenchymal cells including liver-resident macrophages (Kupffer cells). Remarkably, ectopic upregulation of this miRNA in the liver of mice using robust hepatotropic adeno-associated virus 8 (AAV8) vectors enhanced GAP's protective capacity substantially. In turn, this AAV8-mediated miR-155 expression permitted a reduction of GAP injections needed to achieve complete protection against infectious parasite challenge from previously three to only one. Our study highlights a crucial role of mammalian miRNAs in Plasmodium liver infection in vivo and concurrently implies their great potential as future immune-augmenting agents in improved vaccination regimes against malaria and other diseases.

  5. Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends survival in models of inherited ALS.

    Science.gov (United States)

    Foust, Kevin D; Salazar, Desirée L; Likhite, Shibi; Ferraiuolo, Laura; Ditsworth, Dara; Ilieva, Hristelina; Meyer, Kathrin; Schmelzer, Leah; Braun, Lyndsey; Cleveland, Don W; Kaspar, Brian K

    2013-12-01

    Mutations in superoxide dismutase 1 (SOD1) are linked to familial amyotrophic lateral sclerosis (ALS) resulting in progressive motor neuron death through one or more acquired toxicities. Involvement of wild-type SOD1 has been linked to sporadic ALS, as misfolded SOD1 has been reported in affected tissues of sporadic patients and toxicity of astrocytes derived from sporadic ALS patients to motor neurons has been reported to be reduced by lowering the synthesis of SOD1. We now report slowed disease onset and progression in two mouse models following therapeutic delivery using a single peripheral injection of an adeno-associated virus serotype 9 (AAV9) encoding an shRNA to reduce the synthesis of ALS-causing human SOD1 mutants. Delivery to young mice that develop aggressive, fatal paralysis extended survival by delaying both disease onset and slowing progression. In a later-onset model, AAV9 delivery after onset markedly slowed disease progression and significantly extended survival. Moreover, AAV9 delivered intrathecally to nonhuman primates is demonstrated to yield robust SOD1 suppression in motor neurons and glia throughout the spinal cord and therefore, setting the stage for AAV9-mediated therapy in human clinical trials.

  6. Exosome-associated AAV2 vector mediates robust gene delivery into the murine retina upon intravitreal injection

    Science.gov (United States)

    Wassmer, Sarah J.; Carvalho, Livia S.; György, Bence; Vandenberghe, Luk H.; Maguire, Casey A.

    2017-01-01

    Widespread gene transfer to the retina is challenging as it requires vector systems to overcome physical and biochemical barriers to enter and diffuse throughout retinal tissue. We investigated whether exosome-associated adeno-associated virus, (exo-AAV) enabled broad retinal targeting following intravitreal (IVT) injection, as exosomes have been shown to traverse biological barriers and mediate widespread distribution upon systemic injection. We packaged an AAV genome encoding green fluorescent protein (GFP) into conventional AAV2 and exo-AAV2 vectors. Vectors were IVT injected into the eyes of adult mice. GFP expression was noninvasively monitored by fundus imaging and retinal expression was analyzed 4 weeks post-injection by qRT-PCR and histology. Exo-AAV2 outperformed conventional AAV2 in GFP expression based on fundus image analysis and qRT-PCR. Exo-AAV2 demonstrated deeper penetration in the retina, efficiently reaching the inner nuclear and outer plexiform, and to a lesser extent the outer nuclear layer. Cell targets were ganglion cells, bipolar cells, Müller cells, and photoreceptors. Exo-AAV2 serves as a robust gene delivery tool for murine retina, and the simplicity of production and isolation should make it widely applicable to basic research of the eye. PMID:28361998

  7. Chemical Genomics Identifies the PERK-Mediated Unfolded Protein Stress Response as a Cellular Target for Influenza Virus Inhibition

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    Sara Landeras-Bueno

    2016-04-01

    Full Text Available Influenza A viruses generate annual epidemics and occasional pandemics of respiratory disease with important consequences for human health and the economy. Therefore, a large effort has been devoted to the development of new anti-influenza virus drugs directed to viral targets, as well as to the identification of cellular targets amenable to anti-influenza virus therapy. Here we have addressed the identification of such potential cellular targets by screening collections of drugs approved for human use. We reasoned that screening with a green fluorescent protein-based recombinant replicon system would identify cellular targets involved in virus transcription/replication and/or gene expression and hence address an early stage of virus infection. By using such a strategy, we identified Montelukast (MK as an inhibitor of virus multiplication. MK inhibited virus gene expression but did not alter viral RNA synthesis in vitro or viral RNA accumulation in vivo. The low selectivity index of MK prevented its use as an antiviral, but it was sufficient to identify a new cellular pathway suitable for anti-influenza virus intervention. By deep sequencing of RNA isolated from mock- and virus-infected human cells, treated with MK or left untreated, we showed that it stimulates the PERK-mediated unfolded protein stress response. The phosphorylation of PERK was partly inhibited in virus-infected cells but stimulated in MK-treated cells. Accordingly, pharmacological inhibition of PERK phosphorylation led to increased viral gene expression, while inhibition of PERK phosphatase reduced viral protein synthesis. These results suggest the PERK-mediated unfolded protein response as a potential cellular target to modulate influenza virus infection.

  8. Baculovirus-mediated promoter assay and transcriptional analysis of white spot syndrome virus orf427 gene

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    Yu Li

    2005-08-01

    Full Text Available Abstract Background White spot syndrome virus (WSSV is an important pathogen of the penaeid shrimp with high mortalities. In previous reports, Orf427 of WSSV is characterized as one of the three major latency-associated genes of WSSV. Here, we were interested to analyze the promoter of orf427 and its expression during viral pathogenesis. Results in situ hybridization revealed that orf427 was transcribed in all the infected tissues during viral lytic infection and the translational product can be detected from the infected shrimp. A time-course RT-PCR analysis indicated that transcriptional products of orf427 could only be detected after 6 h post virus inoculation. Furthermore, a baculovirus-mediated promoter analysis indicated that the promoter of orf427 failed to express the EGFP reporter gene in both insect SF9 cells and primary shrimp cells. Conclusion Our data suggested that latency-related orf427 might not play an important role in activating virus replication from latent phase due to its late transcription during the lytic infection.

  9. Immune-mediated thrombocytopenia in horses infected with equine infectious anemia virus.

    Science.gov (United States)

    Clabough, D L; Gebhard, D; Flaherty, M T; Whetter, L E; Perry, S T; Coggins, L; Fuller, F J

    1991-11-01

    An adult horse infected with a virulent, cell culture-adapted strain of equine infectious anemia virus (EIAV) developed cyclical thrombocytopenia in which the nadir of platelet counts coincided with peak febrile responses. In order to investigate the mechanism of thrombocytopenia during acute febrile episodes, four adult horses were experimentally infected with the wild-type Wyoming strain of EIAV. Platelet counts decreased from baseline as rectal temperature increased. Serum reverse transcriptase activity increased above background levels in all horses, coincident with increase in rectal temperature. All horses developed an EIAV-specific immune response detectable by Western immunoblot by postinfection day 10. Increases in platelet-associated immunoglobulins G and M were detectable by direct fluorescent-antibody test and flow cytometric assay. Viral replication in bone marrow megakaryocytes was not detectable by in situ hybridization. Results suggest an immune-mediated mechanism of thrombocytopenia in horses infected with EIAV. Despite an inability to identify virion particles in association with platelet-bound antibody, the cyclical nature of the thrombocytopenia and the occurrence of a marked cell-free viremia concomitant with fever and thrombocytopenia suggest immune complex deposition on platelets. We propose that clearance of virus and antibody-coated platelets from the peripheral circulation by hepatic Kupffer cells and splenic macrophages may target infectious virus particles, in the form of immune complexes, to host cells most permissive for in vivo viral replication.

  10. Fluorogenic Detection of Duck Tembusu Virus( DTMUV ) by Loop-mediated Isothermal Amplification(LAMP)

    Institute of Scientific and Technical Information of China (English)

    Zhang; Lin; Wang; Bin; Zhang; Wei; Zhang; Xiumei

    2014-01-01

    This study was to develop an efficient and simple method for the detection of duck Tembusu virus( DTMUV) by loop-mediated isothermal amplification( LAMP). Six pairs of LAMP primers were designed according to the conserved region of the DTMUV E gene sequence in Gen Bank,which were then used for the optimization of various reaction components and reaction system of specific LAMP for DTMUV. Further the fluorescent reagent SYBR Green I and a certain proportion of calcium and manganese ion were used to determin the color development of products for visible analysis instead of agarose gel electrophoresis. The results showed that the sensitivity SYBR Green I as the fluorescent reagent was 10 copies viruses per μL,which is 100 times higher than normal PCR method,while the detection limit of combined use of calcium and manganese ion was 1 000 copies viruses per μL. Although the sensitivity of mixture of calcium and manganese ion is lower than SYBR Green I,it can avoid the aerosol contamination. The fluorogenic analysis-based LAMP system established in our study has a high sensitivity and avoid the cross contamination,which is of huge potential in research institutions,grass-roots laboratories and field testing and can provide effective means to completely curb the occurrence and spreading of DTMUV.

  11. Development of loop-mediated isothermal amplification for rapid detection of avian leukosis virus subgroup A.

    Science.gov (United States)

    Wang, Yongqiang; Kang, Zhonghui; Gao, Yulong; Qin, Liting; Chen, Lei; Wang, Qi; Li, Jiukuan; Gao, Honglei; Qi, Xiaole; Lin, Huan; Wang, Xiaomei

    2011-04-01

    This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for distinguishing avian leukosis virus (ALV) subgroup A from other subgroups of the virus. On the basis of the results of sequence comparison and the sequence characteristics of ALV subgroups, a LAMP method was designed to target the gp85 segment for detection of ALV-A. Under optimal reaction conditions, ALV-A LAMP produced neither cross-reactions with other major subgroups (including subgroups J, B, C, and E) nor nonspecific reactions with other common avian infectious diseases. A sensitivity test showed that this method can detect 20 copies of proviral nucleic acid sequence within 45 min, which is 100 times more sensitive than the conventional polymerase chain reaction (PCR). This method can detect subgroup A virus rapidly and the results can be assessed based on color changes. The whole reaction process can be performed without opening the lid of the reaction tube, which reduces the possibility of contamination greatly and simplifies the detection process, indicating the considerable potential of this method for in situ application in the future.

  12. Detection of Papaya leaf distortion mosaic virus by reverse-transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Shen, Wentao; Tuo, Decai; Yan, Pu; Li, Xiaoying; Zhou, Peng

    2014-01-01

    Papaya leaf distortion mosaic virus (PLDMV) can infect transgenic papaya resistant to a related pathogen, Papaya ringspot virus (PRSV), posing a substantial threat to papaya production in China. Current detection methods, however, are unable to be used for rapid detection in the field. Here, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PLDMV, using a set of four RT-LAMP primers designed based on the conserved sequence of PLDMV CP. The RT-LAMP method detected specifically PLDMV and was highly sensitive, with a detection limit of 1.32×10(-6) μg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR, while also requiring significantly less time and equipment. The effectiveness of RT-LAMP and one-step RT-PCR in detecting the virus were compared using 90 field samples of non-transgenic papaya and 90 field samples of commercialized PRSV-resistant transgenic papaya from Hainan Island. None of the non-transgenic papaya tested positive for PLDMV using either method. In contrast, 19 of the commercialized PRSV-resistant transgenic papaya samples tested positive by RT-LAMP assay, and 6 of those tested negative by RT-PCR. Therefore, the PLDMV-specific RT-LAMP is a simple, rapid, sensitive, and cost-effective tool in the field diagnosis and control of PLDMV.

  13. Reverse transcription loop-mediated isothermal amplification assay for rapid detection of Papaya ringspot virus.

    Science.gov (United States)

    Shen, Wentao; Tuo, Decai; Yan, Pu; Yang, Yong; Li, Xiaoying; Zhou, Peng

    2014-08-01

    Papaya ringspot virus (PRSV) and Papaya leaf distortion mosaic virus (PLDMV), which causes disease symptoms similar to PRSV, threaten commercial production of both non-transgenic-papaya and PRSV-resistant transgenic papaya in China. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to detect PLDMV was developed previously. In this study, the development of another RT-LAMP assay to distinguish among transgenic, PRSV-infected and PLDMV-infected papaya by detection of PRSV is reported. A set of four RT-LAMP primers was designed based on the highly conserved region of the P3 gene of PRSV. The RT-LAMP method was specific and sensitive in detecting PRSV, with a detection limit of 1.15×10(-6)μg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR. Field application of the RT-LAMP assay demonstrated that samples positive for PRSV were detected only in non-transgenic papaya, whereas samples positive for PLDMV were detected only in commercialized PRSV-resistant transgenic papaya. This suggests that PRSV remains the major limiting factor for non-transgenic-papaya production, and the emergence of PLDMV threatens the commercial transgenic cultivar in China. However, this study, combined with the earlier development of an RT-LAMP assay for PLDMV, will provide a rapid, sensitive and cost-effective diagnostic power to distinguish virus infections in papaya.

  14. Hepatitis C virus genomic RNA dimerization is mediated via a kissing complex intermediate

    Science.gov (United States)

    Shetty, Sumangala; Kim, Seungtaek; Shimakami, Tetsuro; Lemon, Stanley M.; Mihailescu, Mihaela-Rita

    2010-01-01

    With over 200 million people infected with hepatitis C virus (HCV) worldwide, there is a need for more effective and better-tolerated therapeutic strategies. The HCV genome is a positive-sense; single-stranded RNA encoding a large polyprotein cleaved at multiple sites to produce at least ten proteins, among them an error-prone RNA polymerase that confers a high mutation rate. Despite considerable overall sequence diversity, in the 3′-untranslated region of the HCV genomic RNA there is a 98-nucleotide (nt) sequence named X RNA, the first 55 nt of which (X55 RNA) are 100% conserved among all HCV strains. The X55 region has been suggested to be responsible for in vitro dimerization of the genomic RNA in the presence of the viral core protein, although the mechanism by which this occurs is unknown. In this study, we analyzed the X55 region and characterized the mechanism by which it mediates HCV genomic RNA dimerization. Similar to a mechanism proposed previously for the human immunodeficiency 1 virus (HIV-1) genome, we show that dimerization of the HCV genome involves formation of a kissing complex intermediate, which is converted to a more stable extended duplex conformation in the presence of the core protein. Mutations in the dimer linkage sequence loop sequence that prevent RNA dimerization in vitro significantly reduced but did not completely ablate the ability of HCV RNA to replicate or produce infectious virus in transfected cells. PMID:20360391

  15. (Pro)renin receptor mediates both angiotensin II-dependent and -independent oxidative stress in neuronal cells.

    Science.gov (United States)

    Peng, Hua; Li, Wencheng; Seth, Dale M; Nair, Anand R; Francis, Joseph; Feng, Yumei

    2013-01-01

    The binding of renin or prorenin to the (pro)renin receptor (PRR) promotes angiotensin (Ang) II formation and mediates Ang II-independent signaling pathways. In the central nervous system (CNS), Ang II regulates blood pressure via inducing oxidative stress; however, the role of PRR-mediated Ang II-independent signaling pathways in oxidative stress in the CNS remains undefined. To address this question, Neuro-2A cells were infected with control virus or an adeno-associated virus encoding the human PRR. Human PRR over-expression alone increased ROS levels, NADPH oxidase activity, as well as NADPH oxidase (NOX) isoforms 2 and 4 mRNA expression levels and these effects were not blocked by losartan. Moreover, the increase in NOX 2 and NOX 4 mRNA levels, NADPH oxidase activity, and ROS levels induced by PRR over-expression was prevented by mitogen activated protein kinase/extracellular signal-regulated kinase 1 and 2 (MAPK/ERK1/2) inhibition, and phosphoinositide 3 kinase/Akt (IP3/Akt) inhibition, indicating that PRR regulates NOX activity and ROS formation in neuro-2A cells through Ang II-independent ERK1/2 and IP3/Akt activation. Interestingly, at a concentration of 2 nM or higher, prorenin promoted Ang II formation, and thus further increased the ROS levels in cultured Neuro-2A cells via PRR. In conclusion, human PRR over-expression induced ROS production through both angiotensin II-dependent and -independent mechanisms. We showed that PRR-mediated angiotensin II-independent ROS formation is associated with activation of the MAPK/ERK1/2 and PI3/Akt signaling pathways and up-regulation of mRNA level of NOX 2 and NOX4 isoforms in neuronal cells.

  16. Distinct Patterns of IFITM-Mediated Restriction of Filoviruses, SARS Coronavirus, and Influenza A Virus

    Science.gov (United States)

    2011-01-06

    West Nile viruses . In contrast, they do not inhibit replication of murine leukemia virus (MLV), or the entry processes of amphotropic MLV, Machupo virus ...MACV), Lassa virus (LASV), or lympho- cytic choriomeningitis virus (LCMV). Although IFITM proteins are induced by type I and II interferons, most...processes of several highly pathogenic viruses – Marburg virus , Ebola virus , and SARS coronavirus – are similarly disrupted by IFITM proteins. We

  17. Cell-mediated infection of cervix derived epithelial cells with primary isolates of human immunodeficiency virus.

    Science.gov (United States)

    Tan, X; Phillips, D M

    1996-01-01

    We have previously demonstrated that HIV-infected transformed T-cells or monocytes adhere to monolayers of CD4-negative epithelial cells. Adhesion is soon followed by budding of HIV from infected mononuclear cells onto the surface of epithelial cells. Epithelial cells subsequently take up virus and become productively infected. Based on these findings, we proposed that sexual transmission of HIV may involve cell-mediated infection of intact mucosal epithelia of the urogenital tract. However, it has become increasingly clear that primary cells and HIV strains isolated from patients are more appropriate models for HIV infection than established cell lines and lab strains of virus. In the studies described here, we infected cervix-derived epithelial monolayers with primary monocytes infected with patient isolates of non-syncytial inducing (NSI) macrophage-tropic strains of HIV. Under the culture conditions employed, HIV-infected primary monocytes do not remain adherent to the apical surface of the epithelium, as did HIV-infected transformed cells. Instead, following adherence, the primary cells migrate between epithelial cells. Virus is secreted from a pseudopod as HIV-infected primary monocytes pass between cells of the epithelium. Productive infection of the epithelium was detected by p24 ELISA and PCR Southern blot analysis. Infection can be blocked by sera from HIV-seropositive individuals or by certain sulfated polysaccharides. These findings support the supposition that transmission of HIV may occur via cell-mediated infection of intact epithelia. The observations also hint at the possibility that-HIV-infected monocyte/macrophages in semen or cervical-vaginal secretions could cross intact epithelia by passing between epithelial cells. Blocking studies suggest that it may be possible to inhibit sexual transmission of HIV either by antibodies in genital tract secretions or by a topical formulation containing certain sulfated polysaccharides.

  18. Axl Mediates ZIKA Virus Entry in Human Glial Cells and Modulates Innate Immune Responses

    Directory of Open Access Journals (Sweden)

    Laurent Meertens

    2017-01-01

    Full Text Available ZIKA virus (ZIKV is an emerging pathogen responsible for neurological disorders and congenital microcephaly. However, the molecular basis for ZIKV neurotropism remains poorly understood. Here, we show that Axl is expressed in human microglia and astrocytes in the developing brain and that it mediates ZIKV infection of glial cells. Axl-mediated ZIKV entry requires the Axl ligand Gas6, which bridges ZIKV particles to glial cells. Following binding, ZIKV is internalized through clathrin-mediated endocytosis and traffics to Rab5+ endosomes to establish productive infection. During entry, the ZIKV/Gas6 complex activates Axl kinase activity, which downmodulates interferon signaling and facilitates infection. ZIKV infection of human glial cells is inhibited by MYD1, an engineered Axl decoy receptor, and by the Axl kinase inhibitor R428. Our results highlight the dual role of Axl during ZIKV infection of glial cells: promoting viral entry and modulating innate immune responses. Therefore, inhibiting Axl function may represent a potential target for future antiviral therapies.

  19. Interleukin 10 Mediated By Herpes Simplex Virus Vectors Suppresses Neuropathic Pain Induced by Human Immunodeficiency Virus gp120 in Rats

    Science.gov (United States)

    Zheng, Wenwen; Huang, Wan; Liu, Shue; Levitt, Roy C.; Candiotti, Keith A.; Lubarsky, David A.; Hao, Shuanglin

    2014-01-01

    Background Human Immunodeficiency Virus (HIV)-associated sensory neuropathy is a common neurological complication of HIV infection affecting up to 30% of HIV-positive individuals. However, the exact neuropathological mechanisms remain unknown, which hinders our ability to develop effective treatments for HIV-neuropathic pain (NP). In this study, we tested the hypothesis that inhibition of proinflammatory factors with overexpression of interleukin (IL)-10 reduces HIV-related NP in a rat model. Methods NP was induced by the application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve. The hindpaws of rats were inoculated with nonreplicating herpes simplex virus (HSV) vectors expressing antiinflammatory cytokine IL-10 or control vector. Mechanical threshold was tested using von Frey filaments before and after treatments with the vectors. The mechanical threshold response was assessed over time using the area under curves (AUC). The expression of phosphorylated p38 mitogen-activated kinase, as tumor necrosis factor alpha, stromal cell-derived factor-1α (SDF-1α), and C-X-C chemokine receptor type 4 in both the lumbar spinal cord and the L4/5 dorsal root ganglia (DRG) was examined at 14 and 28 days after vector inoculation using Western blots. Results We found that in the gp120-induced NP model, IL-10 overexpression mediated by the HSV vector resulted in a significant elevation of the mechanical threshold that was apparent on day 3 after vector inoculation compared with the control vector (PIL-10 lasted more than 28 days. The AUC in the HSV vector expressing IL-10 was increased compared with that in the control vector (PIL-10 reversed the upregulation of phosphorylated p38 mitogen-activated kinase, as tumor necrosis factor alpha, stromal cell-derived factor-1α (SDF-1α), and C-X-C chemokine receptor type 4 expression at 14 and/or 28 days in the DRG and/or the spinal dorsal horn. Conclusions Our studies demonstrate that blocking the signaling of these

  20. The cricket paralysis virus suppressor inhibits microRNA silencing mediated by the Drosophila Argonaute-2 protein.

    Directory of Open Access Journals (Sweden)

    Corinne Besnard-Guérin

    Full Text Available Small RNAs are potent regulators of gene expression. They also act in defense pathways against invading nucleic acids such as transposable elements or viruses. To counteract these defenses, viruses have evolved viral suppressors of RNA silencing (VSRs. Plant viruses encoded VSRs interfere with siRNAs or miRNAs by targeting common mediators of these two pathways. In contrast, VSRs identified in insect viruses to date only interfere with the siRNA pathway whose effector Argonaute protein is Argonaute-2 (Ago-2. Although a majority of Drosophila miRNAs exerts their silencing activity through their loading into the Argonaute-1 protein, recent studies highlighted that a fraction of miRNAs can be loaded into Ago-2, thus acting as siRNAs. In light of these recent findings, we re-examined the role of insect VSRs on Ago-2-mediated miRNA silencing in Drosophila melanogaster. Using specific reporter systems in cultured Schneider-2 cells and transgenic flies, we showed here that the Cricket Paralysis virus VSR CrPV1-A but not the Flock House virus B2 VSR abolishes silencing by miRNAs loaded into the Ago-2 protein. Thus, our results provide the first evidence that insect VSR have the potential to directly interfere with the miRNA silencing pathway.

  1. Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus.

    Science.gov (United States)

    He, Wenqian; Tan, Gene S; Mullarkey, Caitlin E; Lee, Amanda J; Lam, Mannie Man Wai; Krammer, Florian; Henry, Carole; Wilson, Patrick C; Ashkar, Ali A; Palese, Peter; Miller, Matthew S

    2016-10-18

    The generation of strain-specific neutralizing antibodies against influenza A virus is known to confer potent protection against homologous infections. The majority of these antibodies bind to the hemagglutinin (HA) head domain and function by blocking the receptor binding site, preventing infection of host cells. Recently, elicitation of broadly neutralizing antibodies which target the conserved HA stalk domain has become a promising "universal" influenza virus vaccine strategy. The ability of these antibodies to elicit Fc-dependent effector functions has emerged as an important mechanism through which protection is achieved in vivo. However, the way in which Fc-dependent effector functions are regulated by polyclonal influenza virus-binding antibody mixtures in vivo has never been defined. Here, we demonstrate that interactions among viral glycoprotein-binding antibodies of varying specificities regulate the magnitude of antibody-dependent cell-mediated cytotoxicity induction. We show that the mechanism responsible for this phenotype relies upon competition for binding to HA on the surface of infected cells and virus particles. Nonneutralizing antibodies were poor inducers and did not inhibit antibody-dependent cell-mediated cytotoxicity. Interestingly, anti-neuraminidase antibodies weakly induced antibody-dependent cell-mediated cytotoxicity and enhanced induction in the presence of HA stalk-binding antibodies in an additive manner. Our data demonstrate that antibody specificity plays an important role in the regulation of ADCC, and that cross-talk among antibodies of varying specificities determines the magnitude of Fc receptor-mediated effector functions.

  2. Efficient gene silencing mediated by tobacco rattle virus in an emerging model plant physalis.

    Directory of Open Access Journals (Sweden)

    Ji-Si Zhang

    Full Text Available The fruit of Physalis has a berry and a novelty called inflated calyx syndrome (ICS, also named the 'Chinese lantern'. Elucidation of the underlying developmental mechanisms of fruit diversity demands an efficient gene functional inference platform. Here, we tested the application of the tobacco rattle virus (TRV-mediated gene-silencing system in Physalis floridana. First, we characterized the putative gene of a phytoene desaturase in P. floridana (PfPDS. Infecting the leaves of the Physalis seedlings with the PfPDS-TRV vector resulted in a bleached plant, including the developing leaves, floral organs, ICS, berry, and seed. These results indicated that a local VIGS treatment can efficiently induce a systemic mutated phenotype. qRT-PCR analyses revealed that the bleaching extent correlated to the mRNA reduction of the endogenous PfPDS. Detailed comparisons of multiple infiltration and growth protocols allowed us to determine the optimal methodologies for VIGS manipulation in Physalis. We subsequently utilized this optimized VIGS methodology to downregulate the expression of two MADS-box genes, MPF2 and MPF3, and compared the resulting effects with gene-downregulation mediated by RNA interference (RNAi methods. The VIGS-mediated gene knockdown plants were found to resemble the mutated phenotypes of floral calyx, fruiting calyx and pollen maturation of the RNAi transgenic plants for both MPF2 and MPF3. Moreover, the two MADS-box genes were appeared to have a novel role in the pedicel development in P. floridana. The major advantage of VIGS-based gene knockdown lies in practical aspects of saving time and easy manipulation as compared to the RNAi. Despite the lack of heritability and mosaic mutation phenotypes observed in some organs, the TRV-mediated gene silencing system provides an alternative efficient way to infer gene function in various developmental processes in Physalis, thus facilitating understanding of the genetic basis of the evolution

  3. Efficient gene silencing mediated by tobacco rattle virus in an emerging model plant physalis.

    Science.gov (United States)

    Zhang, Ji-Si; Zhao, Jing; Zhang, Shaohua; He, Chaoying

    2014-01-01

    The fruit of Physalis has a berry and a novelty called inflated calyx syndrome (ICS, also named the 'Chinese lantern'). Elucidation of the underlying developmental mechanisms of fruit diversity demands an efficient gene functional inference platform. Here, we tested the application of the tobacco rattle virus (TRV)-mediated gene-silencing system in Physalis floridana. First, we characterized the putative gene of a phytoene desaturase in P. floridana (PfPDS). Infecting the leaves of the Physalis seedlings with the PfPDS-TRV vector resulted in a bleached plant, including the developing leaves, floral organs, ICS, berry, and seed. These results indicated that a local VIGS treatment can efficiently induce a systemic mutated phenotype. qRT-PCR analyses revealed that the bleaching extent correlated to the mRNA reduction of the endogenous PfPDS. Detailed comparisons of multiple infiltration and growth protocols allowed us to determine the optimal methodologies for VIGS manipulation in Physalis. We subsequently utilized this optimized VIGS methodology to downregulate the expression of two MADS-box genes, MPF2 and MPF3, and compared the resulting effects with gene-downregulation mediated by RNA interference (RNAi) methods. The VIGS-mediated gene knockdown plants were found to resemble the mutated phenotypes of floral calyx, fruiting calyx and pollen maturation of the RNAi transgenic plants for both MPF2 and MPF3. Moreover, the two MADS-box genes were appeared to have a novel role in the pedicel development in P. floridana. The major advantage of VIGS-based gene knockdown lies in practical aspects of saving time and easy manipulation as compared to the RNAi. Despite the lack of heritability and mosaic mutation phenotypes observed in some organs, the TRV-mediated gene silencing system provides an alternative efficient way to infer gene function in various developmental processes in Physalis, thus facilitating understanding of the genetic basis of the evolution and development

  4. The development of a viral mediated CRISPR/Cas9 system with doxycycline dependent gRNA expression for inducible in vitro and in vivo genome editing.

    Directory of Open Access Journals (Sweden)

    Christopher A. de Solis

    2016-08-01

    Full Text Available The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV. Specifically, we developed an inducible gRNA (gRNAi AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as one day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e. Cas9 mouse, CRISPRi, etc., and therefore it likely can be used to render these systems inducible as well.

  5. AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating

    Energy Technology Data Exchange (ETDEWEB)

    Tenney, Rebeca M.; Bell, Christie L.; Wilson, James M., E-mail: wilsonjm@mail.med.upenn.edu

    2014-04-15

    Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8's robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII and IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype. - Highlights: • We construct chimeric vectors to identify determinants of AAV8 liver transduction. • An AAV2-based vector with 17 AAV8 residues exhibited high liver transduction in mice. • This vector also surpassed AAV2 in cell entry, nuclear entry and onset of expression. • Most chimeric vector particles were uncoated at 6 weeks, like AAV8 and unlike AAV2. • Chimera retained heparin binding and was antigenically distinct from AAV2 and AAV8.

  6. Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome.

    Science.gov (United States)

    Chang, Qing; Wang, Jianjun; Li, Qi; Kim, Yeunjung; Zhou, Binfei; Wang, Yunfeng; Li, Huawei; Lin, Xi

    2015-06-17

    Mutations in the potassium channel subunit KCNQ1 cause the human severe congenital deafness Jervell and Lange-Nielsen (JLN) syndrome. We applied a gene therapy approach in a mouse model of JLN syndrome (Kcnq1(-/-) mice) to prevent the development of deafness in the adult stage. A modified adeno-associated virus construct carrying a Kcnq1 expression cassette was injected postnatally (P0-P2) into the endolymph, which resulted in Kcnq1 expression in most cochlear marginal cells where native Kcnq1 is exclusively expressed. We also found that extensive ectopic virally mediated Kcnq1 transgene expression did not affect normal cochlear functions. Examination of cochlear morphology showed that the collapse of the Reissner's membrane and degeneration of hair cells (HCs) and cells in the spiral ganglia were corrected in Kcnq1(-/-) mice. Electrophysiological tests showed normal endocochlear potential in treated ears. In addition, auditory brainstem responses showed significant hearing preservation in the injected ears, ranging from 20 dB improvement to complete correction of the deafness phenotype. Our results demonstrate the first successful gene therapy treatment for gene defects specifically affecting the function of the stria vascularis, which is a major site affected by genetic mutations in inherited hearing loss.

  7. Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome

    Science.gov (United States)

    Chang, Qing; Wang, Jianjun; Li, Qi; Kim, Yeunjung; Zhou, Binfei; Wang, Yunfeng; Li, Huawei; Lin, Xi

    2015-01-01

    Mutations in the potassium channel subunit KCNQ1 cause the human severe congenital deafness Jervell and Lange-Nielsen (JLN) syndrome. We applied a gene therapy approach in a mouse model of JLN syndrome (Kcnq1−/− mice) to prevent the development of deafness in the adult stage. A modified adeno-associated virus construct carrying a Kcnq1 expression cassette was injected postnatally (P0–P2) into the endolymph, which resulted in Kcnq1 expression in most cochlear marginal cells where native Kcnq1 is exclusively expressed. We also found that extensive ectopic virally mediated Kcnq1 transgene expression did not affect normal cochlear functions. Examination of cochlear morphology showed that the collapse of the Reissner’s membrane and degeneration of hair cells (HCs) and cells in the spiral ganglia were corrected in Kcnq1−/− mice. Electrophysiological tests showed normal endocochlear potential in treated ears. In addition, auditory brainstem responses showed significant hearing preservation in the injected ears, ranging from 20 dB improvement to complete correction of the deafness phenotype. Our results demonstrate the first successful gene therapy treatment for gene defects specifically affecting the function of the stria vascularis, which is a major site affected by genetic mutations in inherited hearing loss. PMID:26084842

  8. rAAV-mediated delivery of brain-derived neurotrophic factor promotes neurite outgrowth and protects neurodegeneration in focal ischemic model.

    Science.gov (United States)

    Zhang, Jingyu; Yu, Zhigang; Yu, Zhiqiang; Yang, Zichao; Zhao, Hong; Liu, Luran; Zhao, Jiexu

    2011-06-20

    Stroke is one of the neurological diseases which lead to permanently neuronal damage after temporary or long-term occlusion of vessels or after heart attack. However, there are few efficient strategies to prevent or treat this kind of insult in clinical because the consequence is irreversible and could be long-lasting after the onset of stroke. Gene therapy especially using viral system has long been addressed to be of great potential to reduce the damage. Here, we generated recombinant adeno-associated virus (rAAV) carrying brain-derived neurotrophic factor (BDNF) gene. Cells infected with rAAV-BDNF could be able to produce functional BDNF which promoted neurite outgrowth and protected neurons from apoptosis induced by serum deprivation. Further more, single injection of rAAV showed neuroprotection against cell death in focal ischemic model. These results showed that rAAV-mediated gene delivery is functional, which shed light to the future application of viral system-based gene therapy in clinical.

  9. The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread

    Energy Technology Data Exchange (ETDEWEB)

    Delpeut, Sebastien; Noyce, Ryan S. [The Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5 (Canada); IWK Health Centre, Canadian Center for Vaccinology, Goldbloom Pavilion, Halifax, Nova Scotia, Canada B3H 1X5 (Canada); Richardson, Christopher D., E-mail: chris.richardson@dal.ca [The Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5 (Canada); IWK Health Centre, Canadian Center for Vaccinology, Goldbloom Pavilion, Halifax, Nova Scotia, Canada B3H 1X5 (Canada); The Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia (Canada)

    2014-04-15

    The entry of canine distemper virus (CDV) is a multistep process that involves the attachment of CDV hemagglutinin (H) to its cellular receptor, followed by fusion between virus and cell membranes. Our laboratory recently identified PVRL4 (nectin-4) to be the epithelial receptor for measles and canine distemper viruses. In this study, we demonstrate that the V domain of PVRL4 is critical for CDV entry and virus cell-to-cell spread. Furthermore, four key amino acid residues within the V domain of dog PVRL4 and two within the CDV hemagglutinin were shown to be essential for receptor-mediated virus entry. - Highlights: • PVRL4 (nectin-4) is the epithelial cell receptor for measles and canine distemper viruses. • V domain of PVRL4 is critical for CDV entry, cell-to-cell spread, and syncytia formation. • Chimeric PVRL1 backbone substituted with the V domain of PVRL4 can function as a receptor. • Amino acids (F132/P133/A134/G135) within the V domain are essential for PVRL4 receptor activity. • Amino acids (P493/Y539) within CDV H protein are essential for PVRL4 receptor interaction.

  10. Development of primers for loop-mediated isothermal amplification of Philippine white spot syndrome virus isolates

    Directory of Open Access Journals (Sweden)

    Benedict A. Maralit

    2012-09-01

    Full Text Available Because there are currently no effective chemicals or drugs to completely treat bacterial andviral diseases, effective means of prevention such as early detection methods are continuously beingexplored and fully exhausted. Over the past few years, we have seen the development of cutting edgetechnologies to specifically and efficiently detect White Spot Syndrome Virus (WSSV, one of the majorviruses that devastated global shrimp aquaculture. The recent discovery of loop mediated isothermalamplification (LAMP brought about opportunities for fast diagnosis of a lot of animal diseases includinghumans. Here, the development of alternative LAMP primers specifically for Philippine WSSV isolates wasdiscussed. The sensitivity of the established detection protocol was found to be at 0.3954 pg of shrimpDNA template while exhibiting high specificity to the viral target. In addition, alternative visualizationtechniques and comparison with other detection protocols are also discussed.

  11. Real-time fluorescence loop-mediated isothermal amplification for the diagnosis of hemorrhagic enteritis virus.

    Science.gov (United States)

    Liu, Xuemei; Li, Yuhao; Xu, Chenggang; Qin, Jianru; Hao, Jianyong; Feng, Min; Tan, Liqiang; Jia, Weixin; Liao, Ming; Cao, Weisheng

    2014-04-01

    Suspected cases of hemorrhagic enteritis associated with hemorrhagic enteritis virus (HEV) are becoming more frequent among yellow chickens in the Guangdong Province of China. In this study, we have developed a one-step, ecumenical, real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay for the rapid diagnosis of HEV. The RealAmp assay was performed at 63°C and reduced the assay time to 15min, using a simple and portable device, the ESE-Quant Tube Scanner. The detection limit of DNA was 1fg/μl, and the detection was specific only to HEV. We also used nested PCR to evaluate the application of the RealAmp assay. The coincidence rate of the two methods was 100%. Our data indicated that the RealAmp assay provides a sensitive, specific, and user-friendly diagnostic tool for the identification and quantification of HEV for field diagnosis and in laboratory research.

  12. Cell transformation mediated by chromosomal deoxyribonucleic acid of polyoma virus-transformed cells

    Energy Technology Data Exchange (ETDEWEB)

    Della Valle, G.; Fenton, R.G.; Basilico, C.

    1981-05-01

    To study the mechanism of deoxyribonucleic acid (DNA)-mediated gene transfer, normal rat cells were transfected with total cellular DNA extracted from polyoma virus-transformed cells. This resulted in the appearance of the transformed phenotype in 1 x 10/sup -6/ to 3 x 10/sup -6/ of the transfected cells. Transformation was invariably associated with the acquisition of integrated viral DNA sequences characteristic of the donor DNA. This was caused not by the integration of free DNA molecules, but by the transfer of large DNA fragments (10 to 20 kilobases) containing linked cellular and viral sequences. Although Southern blot analysis showed that integration did not appear to occur in a homologus region of the recipient chromosome, the frequency of transformation was rather high when compared with that of purified polyoma DNA, perhaps due to ''position'' effects or to the high efficiency of recombination of large DNA fragments.

  13. White spot syndrome virus enters crayfish hematopoietic tissue cells via clathrin-mediated endocytosis.

    Science.gov (United States)

    Huang, Jiajun; Li, Fang; Wu, Junjun; Yang, Feng

    2015-12-01

    White spot syndrome virus (WSSV) is a major pathogen of aquacultured shrimp. However, the mechanism of its entry remains poorly understood. In this study, by analyzing the internalization of WSSV using crayfish hematopoietic tissue (HPT) cells, we showed that WSSV virions were engulfed by cell membrane invaginations sharing the features of clathrin-coated pits and then internalized into coated cytoplasmic vesicles. Further investigation indicated that WSSV internalization was significantly inhibited by chlorpromazine (CPZ) but not genistein. The internalized virions were colocalized with endogenous clathrin as well as transferrin which undergoes clathrin-dependent uptake. Preventing endosome acidification by ammonium chloride (NH4Cl) or chloroquine (CQ) dramatically reduced WSSV entry as well. Moreover, disturbance of dynamin activity or depletion of membrane cholesterol also blocked WSSV uptake. These data indicate that WSSV enters crayfish HPT cells via clathrin-mediated endocytosis in a pH-dependent manner, and membrane cholesterol as well as dynamin is critical for efficient viral entry.

  14. Vaccinia virus-mediated melanin production allows MR and optoacoustic deep tissue imaging and laser-induced thermotherapy of cancer.

    Science.gov (United States)

    Stritzker, Jochen; Kirscher, Lorenz; Scadeng, Miriam; Deliolanis, Nikolaos C; Morscher, Stefan; Symvoulidis, Panagiotis; Schaefer, Karin; Zhang, Qian; Buckel, Lisa; Hess, Michael; Donat, Ulrike; Bradley, William G; Ntziachristos, Vasilis; Szalay, Aladar A

    2013-02-26

    We reported earlier the delivery of antiangiogenic single chain antibodies by using oncolytic vaccinia virus strains to enhance their therapeutic efficacy. Here, we provide evidence that gene-evoked production of melanin can be used as a therapeutic and diagnostic mediator, as exemplified by insertion of only one or two genes into the genome of an oncolytic vaccinia virus strain. We found that produced melanin is an excellent reporter for optical imaging without addition of substrate. Melanin production also facilitated deep tissue optoacoustic imaging as well as MRI. In addition, melanin was shown to be a suitable target for laser-induced thermotherapy and enhanced oncolytic viral therapy. In conclusion, melanin as a mediator for thermotherapy and reporter for different imaging modalities may soon become a versatile alternative to replace fluorescent proteins also in other biological systems. After ongoing extensive preclinical studies, melanin overproducing oncolytic virus strains might be used in clinical trials in patients with cancer.

  15. Mutations That Alter Use of Hepatitis C Virus Cell Entry Factors Mediate Escape From Neutralizing Antibodies

    Science.gov (United States)

    FAUVELLE, CATHERINE; ZAHID, MUHAMMAD NAUMAN; TUREK, MARINE; HEYDMANN, LAURA; CURY, KARINE; HAYER, JULIETTE; COMBET, CHRISTOPHE; COSSET, FRANÇOIS–LOÏC; PIETSCHMANN, THOMAS; HIET, MARIE–SOPHIE; BARTENSCHLAGER, RALF; HABERSETZER, FRANÇOIS; DOFFOËL, MICHEL; KECK, ZHEN–YONG; FOUNG, STEVEN K. H.; ZEISEL, MIRJAM B.; STOLL–KELLER, FRANÇOISE; BAUMERT, THOMAS F.

    2017-01-01

    BACKGROUND & AIMS The development of vaccines and other strategies to prevent hepatitis C virus (HCV) infection is limited by rapid viral evasion. HCV entry is the first step of infection; this process involves several viral and host factors and is targeted by host-neutralizing responses. Although the roles of host factors in HCV entry have been well characterized, their involvement in evasion of immune responses is poorly understood. We used acute infection of liver graft as a model to investigate the molecular mechanisms of viral evasion. METHODS We studied factors that contribute to evasion of host immune responses using patient-derived antibodies, HCV pseudoparticles, and cell culture–derived HCV that express viral envelopes from patients who have undergone liver transplantation. These viruses were used to infect hepatoma cell lines that express different levels of HCV entry factors. RESULTS By using reverse genetic analyses, we identified altered use of host-cell entry factors as a mechanism by which HCV evades host immune responses. Mutations that alter use of the CD81 receptor also allowed the virus to escape neutralizing antibodies. Kinetic studies showed that these mutations affect virus–antibody interactions during postbinding steps of the HCV entry process. Functional studies with a large panel of patient-derived antibodies showed that this mechanism mediates viral escape, leading to persistent infection in general. CONCLUSIONS We identified a mechanism by which HCV evades host immune responses, in which use of cell entry factors evolves with escape from neutralizing antibodies. These findings advance our understanding of the pathogenesis of HCV infection and might be used to develop antiviral strategies and vaccines. PMID:22503792

  16. Satellite RNA-mediated Reduction of Cucumber Mosaic Virus Genomic RNAs Accumulation in Nicotiana tabacum

    Institute of Scientific and Technical Information of China (English)

    Qiansheng LIAO; Liping ZHU; Zhiyou DU; Rong ZENG; Junli FENG; Jishuang CHEN

    2007-01-01

    Satellite RNAs (satRNAs) are molecular parasites that interfere with the pathogenesis of the helper viruses.In this study,the relative accumulation of cucumber mosaic virus (CMV)-Fny genomic RNAs with or without satRNAs were quantitatively analyzed by real-time RT-PCR.The results showed that satRs apparently attenuated the symptoms of CMV-Fny on Nicotiana tabacum by depressing the accumulation of CMV-Fny genomic RNAs,tested as open reading frames.The accumulation of CMV-Fny la,2a,2b,3a,and CP genes was much higher than that of CMV-Fny with satRs added(CMV-Fsat),at different inoculation times.CMV-Fny△2b,in which the complete 2b gene and 41 amino acids at the C-terminal of the 2a gene were deleted,caused only a slight mosaic effect on N.tabacum seedlings,similar to that of CMVFsat,but the addition of satRs to CMV-Fny△2b showed further decrease in the accumulation of CMVFny△2b genomic RNAs.Our results indicated that the attenuation of CMV,by adding satRs or deleting the 2b gene,was due to the low accumulation of CMV genomic RNAs,and that satRNA-mediated reduction of CMV genomic RNAs accumulation in N.tabacum was possibly related to the 2b gene.

  17. RNAi-mediated resistance to Cassava brown streak Uganda virus in transgenic cassava.

    Science.gov (United States)

    Yadav, Jitender S; Ogwok, Emmanuel; Wagaba, Henry; Patil, Basavaprabhu L; Bagewadi, Basavaraj; Alicai, Titus; Gaitan-Solis, Eliana; Taylor, Nigel J; Fauquet, Claude M

    2011-09-01

    Cassava brown streak disease (CBSD), caused by Cassava brown streak Uganda virus (CBSUV) and Cassava brown streak virus (CBSV), is of new epidemic importance to cassava (Manihot esculenta Crantz) production in East Africa, and an emerging threat to the crop in Central and West Africa. This study demonstrates that at least one of these two ipomoviruses, CBSUV, can be efficiently controlled using RNA interference (RNAi) technology in cassava. An RNAi construct targeting the near full-length coat protein (FL-CP) of CBSUV was expressed constitutively as a hairpin construct in cassava. Transgenic cassava lines expressing small interfering RNAs (siRNAs) against this sequence showed 100% resistance to CBSUV across replicated graft inoculation experiments. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed the presence of CBSUV in leaves and some tuberous roots from challenged controls, but not in the same tissues from transgenic plants. This is the first demonstration of RNAi-mediated resistance to the ipomovirus CBSUV in cassava.

  18. Disruption of Rpp1-mediated soybean rust immunity by virus-induced gene silencing.

    Science.gov (United States)

    Cooper, Bret; Campbell, Kimberly B; McMahon, Michael B; Luster, Douglas G

    2013-01-01

    Phakopsora pachyrhizi, a fungus that causes rust disease on soybean, has potential to impart significant yield loss and disrupt food security and animal feed production. Rpp1 is a soybean gene that confers immunity to soybean rust, and it is important to understand how it regulates the soybean defense system and to use this knowledge to protect commercial crops. It was previously discovered that some soybean proteins resembling transcription factors accumulate in the nucleus of Rpp1 soybeans. To determine if they contribute to immunity, Bean pod mottle virus was used to attenuate or silence the expression of their genes. Rpp1 plants subjected to virus-induced gene silencing exhibited reduced amounts of RNA for 5 of the tested genes, and the plants developed rust-like symptoms after subsequent inoculation with fungal spores. Symptoms were associated with the accumulation of rust fungal RNA and protein. Silenced plants also had reduced amounts of RNA for the soybean Myb84 transcription factor and soybean isoflavone O-methyltransferase, both of which are important to phenylpropanoid biosynthesis and lignin formation, crucial components of rust resistance. These results help resolve some of the genes that contribute to Rpp1-mediated immunity and improve upon the knowledge of the soybean defense system. It is possible that these genes could be manipulated to enhance rust resistance in otherwise susceptible soybean cultivars.

  19. Epstein-Barr virus reactivation associated with diminished cell-mediated immunity in antarctic expeditioners

    Science.gov (United States)

    Mehta, S. K.; Pierson, D. L.; Cooley, H.; Dubow, R.; Lugg, D.

    2000-01-01

    Epstein-Barr virus (EBV) reactivation and cell-mediated immune (CMI) responses were followed in 16 Antarctic expeditioners during winter-over isolation at 2 Australian National Antarctic Research Expedition stations. Delayed-type hypersensitivity (DTH) skin testing was used as an indicator of the CMI response, that was evaluated 2 times before winter isolation and 3 times during isolation. At all 5 evaluation times, 8 or more of the 16 subjects had a diminished CMI response. Diminished DTH was observed on every test occasion in 4/16 subjects; only 2/16 subjects exhibited normal DTH responses for all 5 tests. A polymerase chain reaction (PCR) assay was used to detect EBV DNA in saliva specimens collected before, during, and after the winter isolation. EBV DNA was present in 17% (111/642) of the saliva specimens; all 16 subjects shed EBV in their saliva on at least 1 occasion. The probability of EBV shedding increased (P = 0.013) from 6% before or after winter isolation to 13% during the winter period. EBV appeared in saliva during the winter isolation more frequently (P viruses.

  20. An EDS1 orthologue is required for N-mediated resistance against tobacco mosaic virus.

    Science.gov (United States)

    Peart, Jack R; Cook, Graeme; Feys, Bart J; Parker, Jane E; Baulcombe, David C

    2002-03-01

    In Arabidopsis, EDS1 is essential for disease resistance conferred by a structural subset of resistance (R) proteins containing a nucleotide-binding site, leucine-rich-repeats and amino-terminal similarity to animal Toll and Interleukin-1 (so-called TIR-NBS-LRR proteins). EDS1 is not required by NBS-LRR proteins that possess an amino-terminal coiled-coil motif (CC-NBS-LRR proteins). Using virus-induced gene silencing (VIGS) of a Nicotiana benthaminana EDS1 orthologue, we investigated the role of EDS1 in resistance specified by structurally distinct R genes in transgenic N. benthamiana. Resistance against tobacco mosaic virus mediated by tobacco N, a TIR-NBS-LRR protein, was EDS1-dependent. Two other R proteins, Pto (a protein kinase), and Rx (a CC-NBS-LRR protein) recognizing, respectively, a bacterial and viral pathogen did not require EDS1. These data, together with the finding that expression of N. benthamiana and Arabidopsis EDS1 mRNAs are similarly regulated, lead us to conclude that recruitment of EDS1 by TIR-NBS-LRR proteins is evolutionarily conserved between dicotyledenous plant species in resistance against bacterial, oomycete and viral pathogens. We further demonstrate that VIGS is a useful approach to dissect resistance signaling pathways in a genetically intractable plant species.

  1. Rapid detection of squash leaf curl virus by loop-mediated isothermal amplification.

    Science.gov (United States)

    Kuan, Cheng-Ping; Wu, Min-Tze; Lu, Yi-Lin; Huang, Hung-Chang

    2010-10-01

    A loop-mediated isothermal amplification (LAMP) assay was employed to develop a simple and efficient system for the detection of squash leaf curl virus (SLCV) in diseased plants of squash (Cucurbita pepo) and melon (Cucumis melo). Completion of LAMP assay required 30-60 min under isothermal conditions at 65 degrees C by employing a set of four primers targeting SLCV. Although the sensitivity of the LAMP assay and the polymerase chain reaction (PCR) assay was comparable at high virus concentrations, the LAMP assay was by a 10-fold dilution factor more sensitive than the PCR assay for the detection of SLCV in diseased plants. No reaction was detected in the tissues of healthy plants by either the LAMP or the PCR. The LAMP products can be visualized by staining directly in the tube with SYBR Safe DNA gel stain dye. The sensitivity of the SYBR Safe DNA gel stain is similar to analysis by gel electrophoresis. Although both the LAMP and the PCR methods were capable of detecting SLCV in infected tissues of squash and melon, the LAMP method would be more useful than the PCR method for detection of SLCV infection in cucurbitaceous plants because it is more rapid, simple, accurate and sensitive.

  2. RNAi-mediated silencing of Atp6i and Atp6i haploinsufficiency prevents both bone loss and inflammation in a mouse model of periodontal disease.

    Directory of Open Access Journals (Sweden)

    Hongbing Jiang

    Full Text Available Periodontal disease affects about 80% of adults in America, and is characterized by oral bacterial infection-induced gingival inflammation, oral bone resorption, and tooth loss. Periodontitis is also associated with other diseases such as rheumatoid arthritis, diabetes, and heart disease. Although many efforts have been made to develop effective therapies for this disease, none have been very effective and there is still an urgent need for better treatments and preventative strategies. Herein we explored for the first time the possibility that adeno-associated virus (AAV-mediated RNAi knockdown could be used to treat periodontal disease with improved efficacy. For this purpose, we used AAV-mediated RNAi knockdown of Atp6i/TIRC7 gene expression to target bone resorption and gingival inflammation simultaneously. Mice were infected with the oral pathogen Porphyromonas gingivalis W50 (P. gingivalis in the maxillary periodontium to induce periodontitis. We found that Atp6i depletion impaired extracellular acidification and osteoclast-mediated bone resorption. Furthermore, local injection of AAV-shRNA-Atp6i/TIRC7 into the periodontal tissues in vivo protected mice from P. gingivalis infection-stimulated bone resorption by >85% and decreased the T-cell number in periodontal tissues. Notably, AAV-mediated Atp6i/TIRC7 knockdown also reduced the expression of osteoclast marker genes and inflammation-induced cytokine genes. Atp6i(+/- mice with haploinsufficiency were similarly protected from P. gingivalis infection-stimulated bone loss and gingival inflammation. This suggests that AAV-shRNA-Atp6i/TIRC7 therapeutic treatment may significantly improve the health of millions who suffer from P. gingivalis-mediated periodontal disease.

  3. The requirement of multiple defense genes in soybean Rsv1-mediated extreme resistance to soybean mosaic virus.

    Science.gov (United States)

    Zhang, Chunquan; Grosic, Sehiza; Whitham, Steven A; Hill, John H

    2012-10-01

    Soybean mosaic virus (SMV) is a major viral pathogen of soybean. Among the three SMV resistance genes, Rsv1 mediates extreme resistance (ER) against most SMV strains, including the β-glucuronidase-tagged G2 isolate that was previously used in studies of Rsv1. Using virus-induced gene silencing (VIGS), we screened 82 VIGS constructs to identify genes that play a role in Rsv1-mediated ER to SMV infection. The target genes included putative Rsv1 candidate genes, soybean orthologs to known defense-signaling genes, and 62 WRKY transcription factors. We identified eight VIGS constructs that compromised Rsv1-mediated resistance when the target genes were silenced, including GmEDR1, GmEDS1, GmHSP90, GmJAR1, GmPAD4, and two WRKY transcription factors. Together, our results provide new insight into the soybean signaling network required for ER against SMV.

  4. Amphotericin B Increases Influenza A Virus Infection by Preventing IFITM3-Mediated Restriction

    Directory of Open Access Journals (Sweden)

    Tsai-Yu Lin

    2013-11-01

    Full Text Available The IFITMs inhibit influenza A virus (IAV replication in vitro and in vivo. Here, we establish that the antimycotic heptaen, amphotericin B (AmphoB, prevents IFITM3-mediated restriction of IAV, thereby increasing viral replication. Consistent with its neutralization of IFITM3, a clinical preparation of AmphoB, AmBisome, reduces the majority of interferon’s protective effect against IAV in vitro. Mechanistic studies reveal that IFITM1 decreases host-membrane fluidity, suggesting both a possible mechanism for IFITM-mediated restriction and its negation by AmphoB. Notably, we reveal that mice treated with AmBisome succumbed to a normally mild IAV infection, similar to animals deficient in Ifitm3. Therefore, patients receiving antifungal therapy with clinical preparations of AmphoB may be functionally immunocompromised and thus more vulnerable to influenza, as well as other IFITM3-restricted viral infections.

  5. 重组腺相关病毒质量控制的qPCR技术研究进展%Advances in Real-Time Quantitative PCR Technology for Quality Control of Recombinant Adeno-Associated Virus

    Institute of Scientific and Technical Information of China (English)

    肖桂清; 杨会勇; 刁勇

    2014-01-01

    综述实时荧光定量PCR(qPCR)技术在重组腺相关病毒(rAAV)的基因组滴度测定中的应用成果,以及qPCR技术在rAAV质量控制过程中的应用,如感染滴度及rcAAV污染率的测定等.分析qPCR技术存在的适用范围窄、易产生系统性误差和易受rAAV杂质干扰等问题的原因,并探讨相关的解决策略.

  6. Effects of adeno-associated virus (AAV) of transforming growth factors β1 and β3 (TGFβ1,3) on promoting synthesis of glycosaminoglycan and collagen type Ⅱ of dedifferentiated nucleus pulposus (NP) cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type Ⅱ of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type Ⅱ were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type Ⅱ, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.

  7. Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages.

    Science.gov (United States)

    Song, Erwei; Lee, Sang-Kyung; Dykxhoorn, Derek M; Novina, Carl; Zhang, Dong; Crawford, Keith; Cerny, Jan; Sharp, Phillip A; Lieberman, Judy; Manjunath, N; Shankar, Premlata

    2003-07-01

    Small interfering RNAs (siRNAs) can induce potent gene silencing by degradation of cognate mRNA. However, in dividing cells, the silencing lasts only 3 to 7 days, presumably because of siRNA dilution with cell division. Here, we investigated if sustained siRNA-mediated silencing of human immunodeficiency virus type 1 (HIV-1) is possible in terminally differentiated macrophages, which constitute an important reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor in macrophages, and the viral structural gene for p24 were targeted either singly or in combination. When transfected 2 days prior to infection, both CCR5 and p24 siRNAs effectively reduced HIV-1 infection for the entire 15-day period of observation, and combined targeting of both genes abolished infection. To investigate whether exogenously introduced siRNA is maintained stably in macrophages, we tested the kinetics of siRNA-mediated viral inhibition by initiating infections at various times (2 to 15 days) after transfection with CCR5 and p24 siRNAs. HIV suppression mediated by viral p24 siRNA progressively decreased and was lost by day 7 posttransfection. In contrast, viral inhibition by cellular CCR5 knockdown was sustained even when transfection preceded infection by 15 days, suggesting that the continued presence of target RNA may be needed for persistence of siRNA. The longer sustenance of CCR5 relative to p24 siRNA in uninfected macrophages was also confirmed by detection of internalized siRNA by modified Northern blot analysis. We also tested the potential of p24 siRNA to stably silence HIV in the setting of an established infection where the viral target gene is actively transcribed. Under these circumstances, long-term suppression of HIV replication could be achieved with p24 siRNA. Thus, siRNAs can induce potent and long-lasting HIV inhibition in nondividing cells such as macrophages.

  8. Gene expression profiles of human liver cells mediated by hepatitis B virus X protein

    Institute of Scientific and Technical Information of China (English)

    Wei-ying ZHANG; Fu-qing XU; Chang-liang SHAN; Rong XIANG; Li-hong YE; Xiao-dong ZHANG

    2009-01-01

    Aim: To demonstrate the gene expression profiles mediated by hepatitis B virus X protein (HBx), we characterized the molecular features of pathogenesis associated with HBx in a human liver cell model.Methods: We examined gene expression profiles in L-O2-X cells, an engineered L-O2 cell line that constitutively expresses HBx, relative to L-O2 cells using an Agilent 22 K human 70-mer oligonucleotide microarray representing more than 21,329 unique, well-characterized Homo sapiens genes, Western blot analysis and RNA interference (RNAi) targeting HBx mRNA validated the overexpression of proliferating cell nuclear antigen (PCNA) and Bcl-2 in L-O2-X cells. Meanwhile, the BrdU incorporation assay was used to test cell proliferation mediated by upregulated cyclooxygenase-2 (COX-2).Results: The microarray showed that the expression levels of 152 genes were remarkably altered; 82 of the genes were upregulated and 70 genes were downregulated in L-O2-X cells. The altered genes were associated with signal transduction pathways, cell cycle, metastasis, transcriptional regulation, immune response, metabolism, and other processes. PCNA and Bcl-2 were upregulated in L-O2-X cells. Furthermore, we found that COX-2 upregulation in L-O2-X cells enhanced proliferation using the BrdU incorporation assay, whereas indomethacin (an inhibitor of COX-2) abolished the promotion.Conclusion: Our findings provide new evidence that HBx is able to regulate many genes that may be involved in the car-cinogenesis. These regulated genes mediated by HBx may serve as molecular targets for the prevention and treatment of hepatocellular carcinoma.

  9. Increasing the Efficiency of CRISPR/Cas9-mediated Precise Genome Editing of HSV-1 Virus in Human Cells

    Science.gov (United States)

    Lin, Chaolong; Li, Huanhuan; Hao, Mengru; Xiong, Dan; Luo, Yong; Huang, Chenghao; Yuan, Quan; Zhang, Jun; Xia, Ningshao

    2016-01-01

    Genetically modified HSV-1 viruses serve as promising vectors for tumour therapy and vaccine development. The CRISPR/Cas9 system is one of the most powerful tools for precise gene editing of the genomes of organisms. However, whether the CRISPR/Cas9 system can precisely and efficiently make gene replacements in the genome of HSV-1 remains essentially unknown. Here, we reported CRISPR/Cas9-mediated editing of the HSV-1 genome in human cells, including the knockout and replacement of large genes. In established cells stably expressing CRISPR/Cas9, gRNA in coordination with Cas9 could direct a precise cleavage within a pre-defined target region, and foreign genes were successfully used to replace the target gene seamlessly by HDR-mediated gene replacement. Introducing the NHEJ inhibitor SCR7 to the CRISPR/Cas9 system greatly facilitated HDR-mediated gene replacement in the HSV-1 genome. We provided the first genetic evidence that two copies of the ICP0 gene in different locations on the same HSV-1 genome could be simultaneously modified with high efficiency and with no off-target modifications. We also developed a revolutionized isolation platform for desired recombinant viruses using single-cell sorting. Together, our work provides a significantly improved method for targeted editing of DNA viruses, which will facilitate the development of anti-cancer oncolytic viruses and vaccines. PMID:27713537

  10. Fc receptors do not mediate African swine fever virus replication in macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Alcami, A.; Vinuela, E. (Centro de Biologia Molecular, Universidad Autonoma, Madrid (Spain))

    1991-04-01

    Titration experiments in swine macrophages have shown that African swine fever virus infectivity was not enhanced in the presence of antiviral antibodies. The early viral protein synthesis and the viral DNA replication in swine macrophages infected with virus-antibody complexes were inhibited in the presence of high doses of uv-inactivated virus, which saturated specific virus receptors, but not when Fc receptors were saturated with antibodies. These results indicate that African swine fever virus does not infect swine macrophages through Fc receptors and that the normal entry pathway through virus receptors is not bypassed by the virus-antibody complexes.

  11. Protection from Ebola Virus Mediated by Cytotoxic T Lymphocytes Specific for the Viral Nucleoprotein

    Science.gov (United States)

    2001-03-01

    be required for optimal protection from Ebola virus. Ebola viruses are associated with outbreaks of highly lethal hemorrhagic fever in humans and...nonhuman primates. The Ebola Zaire viruses responsible for outbreaks of human dis- ease in 1976 and 1995 had case-fatality rates of greater than 80...encoding the Ebola virus NP protein (12, 13) or with a control replicon encoding Lassa virus N (14). For booster vaccinations, animals 2660 VOL

  12. Fcgamma receptor-mediated suppression of human immunodeficiency virus type 1 replication in primary human macrophages.

    Science.gov (United States)

    Perez-Bercoff, Danielle; David, Annie; Sudry, Hugues; Barré-Sinoussi, Françoise; Pancino, Gianfranco

    2003-04-01

    Permissiveness of monocytes and macrophages to human immunodeficiency virus (HIV) infection is modulated by various stimuli. In this study we demonstrate that stimulation of primary monocytes and monocyte-derived macrophages (MDM) through the receptors for the Fc portion of immunoglobulin G (IgG) (FcgammaR) inhibits HIV type 1 (HIV-1) replication. Viral p24 production was decreased by 1.5 to 3 log units in MDM infected with both R5 and X4 HIV-1 strains upon stimulation by immobilized IgG but not upon stimulation by soluble IgG or by F(ab')(2) IgG fragments. Although MDM activation by immobilized IgG induced high levels of macrophage-derived chemokine secretion as well as a sustained down-regulation of CD4 and a transient decrease in CCR5 expression, these factors did not appear to play a major role in the suppression of HIV-1 replication. Single-cycle infection of FcgammaR-stimulated MDM with HIV-1 virions pseudotyped with either HIV-1 R5 or vesicular stomatitis virus G envelopes was inhibited, suggesting a postentry restriction of viral replication. PCR analyses of HIV-1 DNA intermediate replication forms suggested that reverse transcription is not affected by stimulation with immobilized human IgG, at least during the first replication cycle. The accumulation of PCR products corresponding to nuclear unintegrated two-long-terminal-repeat circles and the relative decrease of integrated HIV-1 DNA signals suggest an inhibition of proviral integration. Our data, showing that FcgammaR-mediated activation of MDM is a potent mechanism of HIV-1 suppression, raise the possibility that FcgammaR cross-linking by immune complexes may contribute to the control of viral replication in macrophages.

  13. Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation

    Directory of Open Access Journals (Sweden)

    Hsu Chen-Ming

    2009-08-01

    Full Text Available Abstract Background The pathogenesis of inflammation and fibrosis in chronic hepatitis C virus (HCV infection remains unclear. Transgenic mice with constitutive HCV core over-expression display steatosis only. While the reasons for this are unclear, it may be important that core protein production in these models begins during gestation, in contrast to human hepatitis C virus infection, which occurs post-natally and typically in adults. AIMS: To more realistically model the effect of core protein production in the adult liver, we developed a mouse with conditional expression of HCV core and examined the effect of core protein production in the adult liver. Methods Liver biopsy samples from transgenic mice with tetracycline(tet-regulated conditional core protein expression were evaluated immunohistologically. Microarray analysis of HCV core transgenic mice with steatohepatitis pointed to a role of the complement pathway. This was further explored by blocking complement activation by in vivo administration of CD55 (decay accelerating factor for complement, which inhibits activation of C3. Results Transgenic mice exhibited low, intermediate, or high HCV core protein expression when fed a permissive diet of standard chow. Aside from hepatic steatosis, hepatic inflammation and fibrosis were seen in mice with intermediate levels of core protein. Microarray analyses of inflamed liver demonstrated activation of both the complement (C3 up-regulation and coagulation pathways (fibrinogen B up-regulation. Administration of CD55 reduced hepatic inflammation. Conclusion Transgenic mice that conditionally express intermediate HCV core protein develop inflammation, steatosis, and fibrosis. These effects mediated by HCV core are reduced by administration of CD55, a regulator of the complement pathway. The model may be valuable in investigating the pathogenesis of liver inflammation in chronic hepatitis C.

  14. Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of lymphocystis disease virus.

    Science.gov (United States)

    Li, Qiong; Yue, Zhiqin; Liu, Hong; Liang, Chengzhu; Zheng, Xiaolong; Zhao, Yuran; Chen, Xiao; Xiao, Xizhi; Chen, Changfu

    2010-02-01

    A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of lymphocystis disease virus (LCDV). A set of five specific primers, two inner and two outer primers and a loop primer, were designed on the basis of the major capsid protein gene of LCDV. The reaction time and temperatures were optimized for 60 min at 63 degrees C, respectively. LAMP amplification products were detected by a ladder-like appearance on agarose gel electrophoresis or a naked-eye inspection of a color change in the reaction tube by addition of SYBR Green I. The assay was specific for LCDV, and there was no cross-reactivity with white spot syndrome virus (WSSV) or six other Iridoviridae viruses (epizootic hematopoietic necrosis virus, EHNV; tiger frog virus, TFV; Bohle iridovirus, BIV; soft-shelled turtle iridovirus, STIV; infectious spleen and kidney necrosis virus, ISKNV; red sea bream iridovirus, RSIV). The detection limit of the LAMP assay was 15 fg, which was similar to that of real-time quantitative polymerase chain reaction (PCR) and 10-fold higher than the conventional PCR. The LAMP assay was evaluated using 109 clinical samples, and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for detection of LCDV. The LCDV LAMP assay has potential for early diagnosis of LCDV infection.

  15. Inhibition of Rgs10 Expression Prevents Immune Cell Infiltration in Bacteria-induced Inflammatory Lesions and Osteoclast-mediated Bone Destruction

    Institute of Scientific and Technical Information of China (English)

    Sen Yang; Yi-Ping Li; Wei Chen; Liang Hao; Matthew McConnell; Xuedong Zhou; Min Wang; Yan Zhang; John D. Mountz; Michael Reddy; Paul D. Eleazer

    2013-01-01

    Regulator of G-protein Signaling 10 (Rgs10) plays an important function in osteoclast differentiation. However, the role of Rgs10 in immune cells and inflammatory responses, which activate osteoclasts in inflam-matory lesions, such as bacteria-induced periodontal disease lesions, remains largely unknown. In this study, we used an adeno-associated virus (AAV-) mediated RNAi (AAV-shRNA-Rgs10) knockdown approach to study Rgs10’s function in immune cells and osteoclasts in bacteria-induced inflammatory lesions in a mouse model of periodontal disease. We found that AAV-shRNA-Rgs10 mediated Rgs10 knockdown impaired osteoclastogenesis and osteoclast-mediated bone resorption, in vitro and in vivo. Interestingly, local injection of AAV-shRNA-Rgs10 into the periodontal tissues in the bacteria-induced inflammatory lesion greatly decreased the number of dendritic cells, T-cells and osteoclasts, and protected the periodontal tissues from local inflammatory damage and bone destruction. Importantly, AAV-mediated Rgs10 knockdown also reduced local expression of osteoclast markers and pro-inflammatory cytokines. Our results demonstrate that AAV-shRNA-Rgs10 knockdown in periodontal disease tissues can prevent bone resorption and inflammation simultaneously. Our data indicate that Rgs10 may regulate dendritic cell proliferation and maturation, as well as the subsequent stimulation of T-cell proliferation and maturation, and osteoclast differentiation and acti-vation. Our study suggests that AAV-shRNA-Rgs10 can be useful as a therapeutic treatment of periodontal disease.

  16. HELZ2 Is an IFN Effector Mediating Suppression of Dengue Virus

    Science.gov (United States)

    Fusco, Dahlene N.; Pratt, Henry; Kandilas, Stephen; Cheon, Scarlett Se Yun; Lin, Wenyu; Cronkite, D. Alex; Basavappa, Megha; Jeffrey, Kate L.; Anselmo, Anthony; Sadreyev, Ruslan; Yapp, Clarence; Shi, Xu; O'Sullivan, John F.; Gerszten, Robert E.; Tomaru, Takuya; Yoshino, Satoshi; Satoh, Tetsurou; Chung, Raymond T.

    2017-01-01

    Flaviviral infections including dengue virus are an increasing clinical problem worldwide. Dengue infection triggers host production of the type 1 IFN, IFN alpha, one of the strongest and broadest acting antivirals known. However, dengue virus subverts host IFN signaling at early steps of IFN signal transduction. This subversion allows unbridled viral replication which subsequently triggers ongoing production of IFN which, again, is subverted. Identification of downstream IFN antiviral effectors will provide targets which could be activated to restore broad acting antiviral activity, stopping the signal to produce endogenous IFN at toxic levels. To this end, we performed a targeted functional genomic screen for IFN antiviral effector genes (IEGs), identifying 56 IEGs required for antiviral effects of IFN against fully infectious dengue virus. Dengue IEGs were enriched for genes encoding nuclear receptor interacting proteins, including HELZ2, MAP2K4, SLC27A2, HSP90AA1, and HSP90AB1. We focused on HELZ2 (Helicase With Zinc Finger 2), an IFN stimulated gene and IEG which encodes a promiscuous nuclear factor coactivator that exists in two isoforms. The two unique HELZ2 isoforms are both IFN responsive, contain ISRE elements, and gene products increase in the nucleus upon IFN stimulation. Chromatin immunoprecipitation-sequencing revealed that the HELZ2 complex interacts with triglyceride-regulator LMF1. Mass spectrometry revealed that HELZ2 knockdown cells are depleted of triglyceride subsets. We thus sought to determine whether HELZ2 interacts with a nuclear receptor known to regulate immune response and lipid metabolism, AHR, and identified HELZ2:AHR interactions via co-immunoprecipitation, found that AHR is a dengue IEG, and that an AHR ligand, FICZ, exhibits anti-dengue activity. Primary bone marrow derived macrophages from HELZ2 knockout mice, compared to wild type controls, exhibit enhanced dengue infectivity. Overall, these findings reveal that IFN antiviral

  17. Gamma interferon augments Fc gamma receptor-mediated dengue virus infection of human monocytic cells.

    OpenAIRE

    Kontny, U.; Kurane, I; Ennis, F A

    1988-01-01

    It has been reported that anti-dengue antibodies at subneutralizing concentrations augment dengue virus infection of monocytic cells. This is due to the increased uptake of dengue virus in the form of virus-antibody complexes by cells via Fc gamma receptors. We analyzed the effects of recombinant human gamma interferon (rIFN-gamma) on dengue virus infection of human monocytic cells. U937 cells, a human monocytic cell line, were infected with dengue virus in the form of virus-antibody complexe...

  18. RIG-I mediates innate immune response in mouse neurons following Japanese encephalitis virus infection.

    Directory of Open Access Journals (Sweden)

    Arshed Nazmi

    Full Text Available BACKGROUND: Neuroinflammation associated with Japanese encephalitis (JE is mainly due to the activation of glial cells with subsequent release of proinflammatory mediators from them. The recognition of viral RNA, in part, by the pattern recognition receptor retinoic acid-inducible gene I (RIG-I has been indicated to have a role in such processes. Even though neurons are also known to express this receptor, its role after JE virus (JEV infections is yet to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Upon infecting murine neuroblastoma cells and primary cortical neurons with JEV the expression profile of key proinflammatory cyto/chemokines were analyzed by qRT-PCR and bead array, both before and after ablation of RIG-I. Immunoblotting was performed to evaluate the levels of key molecules downstream to RIG-I leading to production of proinflammatory mediators. Changes in the intracellular viral antigen expression were confirmed by intracellular staining and immunoblotting. JEV infection induced neuronal expression of IL-6, IL-12p70, MCP-1, IP-10 and TNF-α in a time-dependent manner, which showed significant reduction upon RIG-I ablation. Molecules downstream to RIG-I showed significant changes upon JEV-infection, that were modulated following RIG-I ablation. Ablation of RIG-I in neurons also increased their susceptibility to JEV. CONCLUSIONS/SIGNIFICANCE: In this study we propose that neurons are one of the potential sources of proinflammatory cyto/chemokines in JEV-infected brain that are produced via RIG-I dependent pathways. Ablation of RIG-I in neurons leads to increased viral load and reduced release of the cyto/chemokines.

  19. Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus

    DEFF Research Database (Denmark)

    Dukes, J.P.; King, D.P.; Alexandersen, Søren

    2006-01-01

    Speed is paramount in the diagnosis of foot-and-mouth disease (FMD) and simplicity is required if a test is to be deployed in the field. The development of a one-step, reverse transcription loop-mediated amplification (RT-LAMP) assay enables FMD virus (FMDV) to be detected in under an hour...... in a single tube without thermal cycling. A fragment of the 3D RNA polymerase gene of the virus is amplified at 65 degrees C in the presence of a primer mixture and both reverse transcriptase and Bst DNA polymerase. Compared with real-time RT-PCR, RT-LAMP was consistently faster, and ten copies of FMDV...... transcript were detected in twenty-two minutes. Amplification products were detected by visual inspection, agarose gel electrophoresis, or in real-time by the addition of a fluorescent dye. The specificity of the reaction was demonstrated by the absence of amplification of RNA from other viruses that cause...

  20. Simultaneous presence of endogenous retrovirus and herpes virus antigens has profound effect on cell-mediated immune responses

    DEFF Research Database (Denmark)

    Brudek, Tomasz; Christensen, Tove; Hansen, Hans Jacob

    2004-01-01

    Retroviruses have been suggested as possible pathogenic factors in multiple sclerosis (MS), supported by the observation that endogenous retroviruses are activated in MS patients. Different members of the herpes family of which several are neurotropic have also been suggested as factors in MS...... pathogenesis. Further, interactions between retroviruses and herpes viruses have been implied in the development of MS. The objective of the study was investigation of cell-mediated immune responses of MS patients to retrovirus and herpes virus antigens, particularly antigen combinations, with analyses...... retrovirus HERV-H and herpes virus antigens resulted in highly increased cellular immune responses among both the MS patients and healthy subjects. The increase was synergistic in character in most samples. Very pronounced effects were obtained using HHV-6A and HSV-1 antigens. Blast transformation assays...

  1. Thrombocytopenia in Dengue: Interrelationship between Virus and the Imbalance between Coagulation and Fibrinolysis and Inflammatory Mediators

    Directory of Open Access Journals (Sweden)

    Elzinandes Leal de Azeredo

    2015-01-01

    Full Text Available Dengue is an infectious disease caused by dengue virus (DENV. In general, dengue is a self-limiting acute febrile illness followed by a phase of critical defervescence, in which patients may improve or progress to a severe form. Severe illness is characterized by hemodynamic disturbances, increased vascular permeability, hypovolemia, hypotension, and shock. Thrombocytopenia and platelet dysfunction are common in both cases and are related to the clinical outcome. Different mechanisms have been hypothesized to explain DENV-associated thrombocytopenia, including the suppression of bone marrow and the peripheral destruction of platelets. Studies have shown DENV-infected hematopoietic progenitors or bone marrow stromal cells. Moreover, anti-platelet antibodies would be involved in peripheral platelet destruction as platelets interact with endothelial cells, immune cells, and/or DENV. It is not yet clear whether platelets play a role in the viral spread. Here, we focus on the mechanisms of thrombocytopenia and platelet dysfunction in DENV infection. Because platelets participate in the inflammatory and immune response by promoting cytokine, chemokine, and inflammatory mediator secretion, their relevance as “immune-like effector cells” will be discussed. Finally, an implication for platelets in plasma leakage will be also regarded, as thrombocytopenia is associated with clinical outcome and higher mortality.

  2. Thrombocytopenia in Dengue: Interrelationship between Virus and the Imbalance between Coagulation and Fibrinolysis and Inflammatory Mediators.

    Science.gov (United States)

    de Azeredo, Elzinandes Leal; Monteiro, Robson Q; de-Oliveira Pinto, Luzia Maria

    2015-01-01

    Dengue is an infectious disease caused by dengue virus (DENV). In general, dengue is a self-limiting acute febrile illness followed by a phase of critical defervescence, in which patients may improve or progress to a severe form. Severe illness is characterized by hemodynamic disturbances, increased vascular permeability, hypovolemia, hypotension, and shock. Thrombocytopenia and platelet dysfunction are common in both cases and are related to the clinical outcome. Different mechanisms have been hypothesized to explain DENV-associated thrombocytopenia, including the suppression of bone marrow and the peripheral destruction of platelets. Studies have shown DENV-infected hematopoietic progenitors or bone marrow stromal cells. Moreover, anti-platelet antibodies would be involved in peripheral platelet destruction as platelets interact with endothelial cells, immune cells, and/or DENV. It is not yet clear whether platelets play a role in the viral spread. Here, we focus on the mechanisms of thrombocytopenia and platelet dysfunction in DENV infection. Because platelets participate in the inflammatory and immune response by promoting cytokine, chemokine, and inflammatory mediator secretion, their relevance as "immune-like effector cells" will be discussed. Finally, an implication for platelets in plasma leakage will be also regarded, as thrombocytopenia is associated with clinical outcome and higher mortality.

  3. Anti-tumor Immune Response Mediated by Newcastle Disease Virus HN Gene

    Institute of Scientific and Technical Information of China (English)

    PENG Li-ping; JIN Ning-yi; LI Xiao; SUN Li-li; WEN Zhong-mei; LIU Yan; GAO Peng; HUANG Hai-yan; PIAO Bing-guo; JIN Jing

    2011-01-01

    Hemagglutinin-neuramidinase(HN) is one of the most important surface structure proteins of the Newcastle disease virus(NDV). HN not only mediates receptor recognition but also possesses neuraminidase(NA) activity,which gives it the ability to cleave a component of those receptors, NAcneu. Previous studies have demonstrated that HN has interesting anti-neoplastic and immune-stimulating properties in mammalian species, including humans. To explore the application of the HN gene in cancer gene therapy, we constructed a Lewis lung carcinoma(LLC) solid tumor model using C57BL/6 mice. Mice were injected intratumorally with the recombinant adenovirus expressing HN gene(Ad-HN), and the effect of HN was explored by natural killer cell activity assay, cytotoxic lymphocyte activity assay, T cell subtype evaluation, and Thl/Th2 cytokines analysis. The results demonstrate that HN not only can elicit clonal expansion of both CD4+ and CD8+ T cell populations and cytotoxic T lymphocyte(CTL) and killer cell response, but also skews the immune response toward Thl. Thus, vaccination with Ad-HN may be a potential strategy for cancer gene therapy.

  4. Urea-mediated cross-presentation of soluble Epstein-Barr virus BZLF1 protein.

    Directory of Open Access Journals (Sweden)

    Sascha Barabas

    2008-11-01

    Full Text Available Soluble extracellular proteins usually do not enter the endogenous human leukocyte antigen (HLA I-dependent presentation pathway of antigen-presenting cells, strictly impeding their applicability for the re-stimulation of protein-specific CD8(+ cytotoxic T lymphocytes (CTL. Here we present for the Epstein-Barr virus (EBV BZLF1 a novel strategy that facilitates protein translocation into antigen-presenting cells by its solubilisation in high molar urea and subsequent pulsing of cells in presence of low molar urea. Stimulation of PBMC from HLA-matched EBV-seropositive individuals with urea-treated BZLF1 but not untreated BZLF1 induces an efficient reactivation of BZLF1-specific CTL. Urea-treated BZLF1 (uBZLF1 enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules. Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro. The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.

  5. Assembly of tobacco mosaic virus into fibrous and macroscopic bundled arrays mediated by surface aniline polymerization.

    Energy Technology Data Exchange (ETDEWEB)

    Niu, Z.; Bruckman, M.; Li, S.; Lee, A.; Lee, B.; Pingali, S.-V.; Thiyagarajan, P.; Wang, Q.; Univ. of South Carolina

    2007-06-05

    One-dimensional (1D) polyaniline/tobacco mosaic virus (TMV) composite nanofibers and macroscopic bundles of such fibers were generated via a self-assembly process of TMV assisted by in-situ polymerization of polyaniline on the surface of TMV. At near-neutral reaction pH, branched polyaniline formed on the surface of TMV preventing lateral association. Therefore, long 1D nanofibers were observed with high aspect ratios and excellent processibility. At a lower pH, transmission electron microscopy (TEM) analysis revealed that initially long nanofibers were formed which resulted in bundled structures upon long-time reaction, presumably mediated by the hydrophobic interaction because of the polyaniline on the surface of TMV. In-situ time-resolved small-angle X-ray scattering study of TMV at different reaction conditions supported this mechanism. This novel strategy to assemble TMV into 1D and 3D supramolecular composites could be utilized in the fabrication of advanced materials for potential applications including electronics, optics, sensing, and biomedical engineering.

  6. Direct detection of Marek's disease virus in poultry dust by loop-mediated isothermal amplification.

    Science.gov (United States)

    Woźniakowski, Grzegorz; Samorek-Salamonowicz, Elżbieta

    2014-11-01

    Marek's disease virus (MDV) is a serious concern for poultry production and represents a unique herpesvirus model. MDV can be shed by doubly infected chickens despite vaccination. The fully infectious MDV particles are produced in the feather follicle epithelium (FFE), and MDV remains infectious for many months in fine skin particles and feather debris. Molecular biology methods including PCR and real-time PCR have been shown to be valuable for the detection of MDV DNA in farm dust. Recently, loop-mediated isothermal amplification (LAMP) was found to be useful in the detection of MDV in feathers and internal organs of infected chickens. LAMP is also less affected by the inhibitors present in DNA samples. Taking into account the advantages of LAMP, direct detection of MDV DNA in poultry dust has been conducted in this research. The detection of MDV DNA was possible in 11 out of the 12 examined dust samples without DNA extraction. The DNA was retrieved from dust samples by dilution and incubation at 95 °C for 5 min. The direct detection of MDV DNA in the dust was possible within 30 min using a water bath and UV light. The results were confirmed by electrophoresis and melting curve analysis of the LAMP products. Our results show that LAMP may be used to test for the presence of virulent MDV in poultry farm dust without DNA extraction.

  7. Ebola Virus Uses Clathrin-Mediated Endocytosis as an Entry Pathway

    Science.gov (United States)

    2010-01-01

    Schmaljohn, A., Aman, M.J., 2002. Lipid raft microdomains: a gateway for compartmentalized trafficking of Ebola and Marburg viruses . J. Exp. Med. 195 (5...Ebola viruses . Cell 106 (1), 117–126. Chazal, N., Singer, G., Aiken, C., Hammarskjold, M.L., Rekosh, D., 2001. Human immunodeficiency virus type 1...taxonomic home for Marburg and Ebola viruses ? Intervirology 18 (1–2), 24–32. Kristiansen, J.E., Mortensen, I., 1987. Antibacterial effect of four

  8. Small molecule and peptide-mediated inhibition of Epstein-Barr virus nuclear antigen 1 dimerization

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun Young; Song, Kyung-A [Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Samsung Biomedical Research Institute (SBRI), Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Kieff, Elliott [Department of Medicine, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States); Kang, Myung-Soo, E-mail: mkang@skku.edu [Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Samsung Biomedical Research Institute (SBRI), Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Department of Medicine, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA 02115 (United States)

    2012-07-27

    Highlights: Black-Right-Pointing-Pointer Evidence that targeting EBNA1 dimer, an EBV onco-antigen, can be achievable. Black-Right-Pointing-Pointer A small molecule and a peptide as EBNA1 dimerization inhibitors identified. Black-Right-Pointing-Pointer Both inhibitors associated with EBNA1 and blocked EBNA1 DNA binding activity. Black-Right-Pointing-Pointer Also, prevented its dimerization, and repressed viral gene transcription. -- Abstract: Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)'s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation. In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459-607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-J{kappa} binding to the J{kappa} site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560-574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated

  9. Distinct domains of M-T2, the myxoma virus tumor necrosis factor (TNF) receptor homolog, mediate extracellular TNF binding and intracellular apoptosis inhibition.

    OpenAIRE

    Schreiber, M; Sedger, L; McFadden, G

    1997-01-01

    The myxoma virus tumor necrosis factor (TNF) receptor homolog, M-T2, is expressed both as a secreted glycoprotein that inhibits the cytolytic activity of rabbit TNF-alpha and as an endoglycosidase H-sensitive intracellular species that prevents myxoma virus-infected CD4+ T lymphocytes from undergoing apoptosis. To compare the domains of M-T2 mediating extracellular TNF inhibition and intracellular apoptosis inhibition, recombinant myxoma viruses expressing nested C-terminal truncations of M-T...

  10. Development of a Reverse Transcription Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Subtype H7N9 Avian Influenza Virus

    Directory of Open Access Journals (Sweden)

    Hongmei Bao

    2014-01-01

    Full Text Available A novel influenza A (H7N9 virus has emerged in China. To rapidly detect this virus from clinical samples, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP method for the detection of the H7N9 virus. The minimum detection limit of the RT-LAMP assay was 0.01 PFU H7N9 virus, making this method 100-fold more sensitive to the detection of the H7N9 virus than conventional RT-PCR. The H7N9 virus RT-LAMP assays can efficiently detect different sources of H7N9 influenza virus RNA (from chickens, pigeons, the environment, and humans. No cross-reactive amplification with the RNA of other subtype influenza viruses or of other avian respiratory viruses was observed. The assays can effectively detect H7N9 influenza virus RNA in drinking water, soil, cloacal swab, and tracheal swab samples that were collected from live poultry markets, as well as human H7N9 virus, in less than 30 min. These results suggest that the H7N9 virus RT-LAMP assays were efficient, practical, and rapid diagnostic methods for the epidemiological surveillance and diagnosis of influenza A (H7N9 virus from different resource samples.

  11. Cell-mediated immunity to HIV-1 in Walter Reed stages 1-6 individuals: correlation with virus burden.

    Science.gov (United States)

    Trauger, R J; Giermakowska, W K; Ferre, F; Duffy, P C; Wallace, M R; Lewis, D E; Beecham, H J; Burnett, K G; Jensen, F C; Carlo, D J

    1993-01-01

    Cell-mediated immunity (CMI) to human immunodeficiency virus-1 (HIV-1) was assessed in a blinded fashion for a patient group (n = 79) representing Walter Reed (WR) stages 1-6. At the same time, viral load was quantitatively measured by two different methods, specifically, virus isolation and HIV viral DNA copy number as measured by the polymerase chain reaction (PCR). After unblinding it was determined that the ability to generate a lymphoproliferative response to an inactivated gp120-depleted HIV (HIV-ag) and tetanus toxoid diminished with advancing WR staging, with complete anergy to HIV-ag and tetanus at stage 6. As a group, individuals whose peripheral blood mononuclear cells (PBMC) proliferated to HIV-ag were either virus isolation negative or produced low levels of virus as measured by p24 antigen (< 250 pg p24) on day 7. Similarly, HIV DNA copy number in the HIV-ag responders was low (< 200 copies/4 x 10(5) PBMC). In contrast, antigen proliferation to tetanus toxoid did not correlate with virus load. Thus, clinical progression as defined by the WR staging system appears to coincide with a loss of CMI to HIV. More importantly, the low viral load measured in HIV-ag responders suggests a link between viral burden and CMI to HIV which might be exploited in the design of immunotherapies for HIV-infected individuals. PMID:8495981

  12. Rapid detection of peste des petits ruminants virus by a reverse transcription loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Li, Lin; Bao, Jingyue; Wu, Xiaodong; Wang, Zhiliang; Wang, Junwei; Gong, Mingxia; Liu, Chunju; Li, Jinming

    2010-12-01

    Peste des petits ruminants virus (PPRV) is the causative agent of peste des petits ruminants (PPR), an economically important viral disease of small ruminants. In this report, a one-step, single-tube, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PPRV. A set of six LAMP primers were designed based on the matrix gene sequence of PPRV to amplify the target RNA by incubation at 63°C for 60min with Bst DNA polymerase and reverse transcriptase. The amplified products could be observed by the naked eye. The specificity of the RT-LAMP assay was validated by amplifying eight strains of PPRV isolated in different geographical areas. No cross-reactivity with other related viruses, including rinderpest virus, canine distemper virus and measles virus, was detected. The sensitivity of the assay was similar to that of real-time reverse transcription polymerase chain reaction (RT-PCR) and 10-fold higher than that of conventional RT-PCR. Twenty clinical samples were evaluated by the RT-LAMP assay, and the results were consistent with those of real-time RT-PCR. As a simple, rapid and accurate detection method, this RT-LAMP assay has important potential applications in the clinical diagnosis of PPR and the surveillance of PPRV. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. Can Viruses be Modified to Achieve Sustained Gene Transfer?

    Directory of Open Access Journals (Sweden)

    Hildegund CJ Ertl

    2011-07-01

    Full Text Available It is very easy to replace a faulty gene in an immunocompromised mouse. First, one takes a well-characterized virus, such as an adenovirus or an adeno-associated virus, and incorporates the correct version of the faulty gene together with some regulatory sequences into the genome. Then, one transduces the recombinant genome into helper cells, which will add the viral capsid. At last, one injects the resulting viral vector into the sick mouse, and the mouse is cured. It is not that easy in an immunocompetent mouse, let alone in a human, as over the eons the immune system evolved to eliminate viruses regardless if they penetrate as dangerous pathogens or are injected by a well-meaning gene therapist. Here we offer our perspective on the potential of how viral vectors achieve sustained gene transfer in the face of a hostile immune system.

  14. Breakdown of blood-brain barrier function in the murine lymphocytic choriomeningitis virus infection mediated by virus-specific CD8+ T cells

    DEFF Research Database (Denmark)

    Andersen, I H; Marker, O; Thomsen, Allan Randrup

    1991-01-01

    -amino-[1-14C]isobutyric acid we now demonstrate an uncompromised BBB in i.c. infected T cell-deficient nu/nu mice, but serious dysfunction in heterozygous littermates. Transfer experiments were used to characterize and compare the cell subset(s) involved in inducing BBB dysfunction and fatal disease......Intracerebral inoculation of lymphocytic choriomeningitis virus (LCMV) generally results in a fatal T cell-mediated meningitis. In a previous study we have demonstrated a compromised blood-brain barrier (BBB) under such conditions. Using semi-quantitative radiography and the low molecular tracer 2...

  15. Cordyceps Militaris Alleviates Severity of Murine Acute Lung Injury Through miRNAs-Mediated CXCR2 Inhibition

    Directory of Open Access Journals (Sweden)

    Sheng Liu

    2015-07-01

    Full Text Available Background/Aims: Acute lung injury (ALI and acute respiratory distress syndrome (ARDS are lethal diseases in humans, and the current treatments have limited therapeutic effects. Cordyceps militaris (CM is a caterpillar-grown traditional medicinal mushroom, and has been used as a natural invigorant for longevity, endurance, and vitality in China. Recently, purified extracts from CM have been shown to have beneficial effects on various diseases including cancer. Nevertheless, a role of CM in ALI has not been examined previously. Methods: Here, we used a bleomycin-induced ALI model to study the effects of CM on the severity of ALI in mice. The levels of CXCR2, a receptor for Interleukin 8 (IL-8 in pulmonary microvascular endothelial cells, were examined in different experimental groups. The levels of microRNA (miR-1321 and miR-3188 were also examined in lung samples and in CM. Adeno-associated viruses carrying miR-1321 and miR-3188 were injected into bleomycin-treated mice for evaluation their effects on the severity of ALI. Results: CM treatment significantly alleviated the severity of bleomycin-induced ALI in mice. The increases in lung CXCR2 by bleomycin were significantly reduced by CM at protein level, but not at mRNA level. CM contained high levels of 2 miRNAs (miR-1321 and miR-3188 that target 3'-UTR of CXCR2 mRNA to inhibit its expression. Overexpression of miR-1321 and miR-3188 in mouse lung through AAV-mediated gene therapy mimicked the effects of CM. Conclusion: CM may alleviate severity of murine ALI through miRNAs-mediated CXCR2 inhibition.

  16. Clathrin-mediated endocytosis in living host cells visualized through quantum dot labeling of infectious hematopoietic necrosis virus.

    Science.gov (United States)

    Liu, Haibin; Liu, Yi; Liu, Shulin; Pang, Dai-Wen; Xiao, Gengfu

    2011-07-01

    Infectious hematopoietic necrosis virus (IHNV) is an important fish pathogen that infects both wild and cultured salmonids. As a species of the genus Novirhabdovirus, IHNV is a valuable model system for exploring the host entry mechanisms of rhabdoviruses. In this study, quantum dots (QDs) were used as fluorescent labels for sensitive, long-term tracking of IHNV entry. Using live-cell fluorescence microscopy, we found that IHNV is internalized through clathrin-coated pits after the virus binds to host cell membranes. Pretreatment of host cells with chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and clathrin light chain (LCa) depletion using RNA interference both resulted in a marked reduction in viral entry. We also visualized transport of the virus via the cytoskeleton (i.e., actin filaments and microtubules) in real time. Actin polymerization is involved in the transport of endocytic vesicles into the cytosol, whereas microtubules are required for the trafficking of clathrin-coated vesicles to early endosomes, late endosomes, and lysosomes. Disrupting the host cell cytoskeleton with cytochalasin D or nocodazole significantly impaired IHNV infectivity. Furthermore, infection was significantly affected by pretreating the host cells with bafilomycin A1, a compound that inhibits the acidification of endosomes and lysosomes. Strong colocalizations of IHNV with endosomes indicated that the virus is internalized into these membrane-bound compartments. This is the first report in which QD labeling is used to visualize the dynamic interactions between viruses and endocytic structures; the results presented demonstrate that IHNV enters host cells via clathrin-mediated endocytic, cytoskeleton-dependent, and low-pH-dependent pathways.

  17. Exosome-Mediated Intercellular Communication between Hepatitis C Virus-Infected Hepatocytes and Hepatic Stellate Cells.

    Science.gov (United States)

    Devhare, Pradip B; Sasaki, Reina; Shrivastava, Shubham; Di Bisceglie, Adrian M; Ray, Ranjit; Ray, Ratna B

    2017-03-15

    Fibrogenic pathways in the liver are principally regulated by activation of hepatic stellate cells (HSC). Fibrosis is associated with chronic hepatitis C virus (HCV) infection, although the mechanism is poorly understood. HSC comprise the major population of nonparenchymal cells in the liver. Since HCV does not replicate in HSC, we hypothesized that exosomes secreted from HCV-infected hepatocytes activate HSC. Primary or immortalized human hepatic stellate (LX2) cells were exposed to exosomes derived from HCV-infected hepatocytes (HCV-exo), and the expression of fibrosis-related genes was examined. Our results demonstrated that HCV-exo internalized to HSC and increased the expression of profibrotic markers. Further analysis suggested that HCV-exo carry miR-19a and target SOCS3 in HSC, which in turn activates the STAT3-mediated transforming growth factor β (TGF-β) signaling pathway and enhances fibrosis marker genes. The higher expression of miR-19a in exosomes was also observed from HCV-infected hepatocytes and in sera of chronic HCV patients with fibrosis compared to healthy volunteers and non-HCV-related liver disease patients with fibrosis. Together, our results demonstrated that miR-19a carried through the exosomes from HCV-infected hepatocytes activates HSC by modulating the SOCS-STAT3 axis. Our results implicated a novel mechanism of exosome-mediated intercellular communication in the activation of HSC for liver fibrosis in HCV infection.IMPORTANCE HCV-associated liver fibrosis is a critical step for end-stage liver disease progression. However, the molecular mechanisms for hepatic stellate-cell activation by HCV-infected hepatocytes are underexplored. Here, we provide a role for miR-19a carried through the exosomes in intercellular communication between HCV-infected hepatocytes and HSC in fibrogenic activation. Furthermore, we demonstrate the role of exosomal miR-19a in activation of the STAT3-TGF-β pathway in HSC. This study contributes to the

  18. Association of interleukin-10 with hepatitis B virus (HBV mediated disease progression in Indian population

    Directory of Open Access Journals (Sweden)

    Roli Saxena

    2014-01-01

    Full Text Available Background & objectives: Interleukin (IL-10, an anti-inflammatory Th2 cytokine, is one of the key coordinators of the inflammatory responses involved. The present study was designed to evaluate the impact of IL-10 (-819/-592 genotypes, haplotypes, mRNA and the protein levels with risk for hepatitis B virus (HBV related hepatocellular carcinoma (HCC development in India. Methods: A total of 390 subjects (145 controls, 62 inactive HBV-carriers, 64 chronic-active HBV patients, 60 HBV related cirrhotics and 59 HBV- HCC subjects were enrolled in the study. Allele specific (AS-PCR, ELISA and RT-PCR methods were used for assessing polymorphism, spontaneous blood levels and the mRNA expression, respectively of IL-10. Results: The study revealed that the CC/TA genotype acted as a risk factor for cirrhosis (OR a =2.02; P<0.05 and the subsequent HCC development (OR a =2.20; P<0.05, with controls as reference. However, no significant association was found between the two haplotypes (CC and TA observed and HCC risk. Moreover, the IL-10 protein and mRNA levels in peripheral blood mono nuclear cells (PBMCs showed a significant elevation as the disease progressed to cirrhosis. But, no variation was observed in the IL-10 levels in subjects with different IL-10 genotypes. Interpretation & conclusions: These preliminary results suggest a strong association of IL10 (-819/-592 with the HBV infection mediated disease progression, from inactive carrier state to malignancy, in Indian population.

  19. Method of inhibiting plant virus pathogen infections by crispr/cas9-mediated interference

    KAUST Repository

    Mahfouz, Magdy Mahmoud

    2016-11-24

    A genetically modified tobacco plant or tomato plant resistant to at least one pathogenic geminiviridae virus species is provided. The plant comprises a heterologous CRISPR/Cas9 system and at least one heterologous nucleotide sequence that is capable of hybridizing to a nucleotide sequence of the pathogenic virus and that directs inactivation of the pathogenic virus species or plurality of viral species by the CRISPR/Cas9 system. The heterologous nucleotide sequence can be complementary to, but not limited to an Intergenic Region (IR) of the Tomato Yellow Leaf Curl Virus (TYLCV), Further provided are methods of generating a genetically modified plant that is resistant to a virus pathogen by a heterologous CRISPR/Cas9 system and expression of a gRNA specifically targeting the virus.

  20. CRISPR/Cas9-Mediated Genome Editing as a Therapeutic Approach for Leber Congenital Amaurosis 10.

    Science.gov (United States)

    Ruan, Guo-Xiang; Barry, Elizabeth; Yu, Dan; Lukason, Michael; Cheng, Seng H; Scaria, Abraham

    2017-02-01

    As the most common subtype of Leber congenital amaurosis (LCA), LCA10 is a severe retinal dystrophy caused by mutations in the CEP290 gene. The most frequent mutation found in patients with LCA10 is a deep intronic mutation in CEP290 that generates a cryptic splice donor site. The large size of the CEP290 gene prevents its use in adeno-associated virus (AAV)-mediated gene augmentation therapy. Here, we show that targeted genomic deletion using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system represents a promising therapeutic approach for the treatment of patients with LCA10 bearing the CEP290 splice mutation. We generated a cellular model of LCA10 by introducing the CEP290 splice mutation into 293FT cells and we showed that guide RNA pairs coupled with SpCas9 were highly efficient at removing the intronic splice mutation and restoring the expression of wild-type CEP290. In addition, we demonstrated that a dual AAV system could effectively delete an intronic fragment of the Cep290 gene in the mouse retina. To minimize the immune response to prolonged expression of SpCas9, we developed a self-limiting CRISPR/Cas9 system that minimizes the duration of SpCas9 expression. These results support further studies to determine the therapeutic potential of CRISPR/Cas9-based strategies for the treatment of patients with LCA10. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  1. Homologous recombination mediates functional recovery of dysferlin deficiency following AAV5 gene transfer.

    Directory of Open Access Journals (Sweden)

    William E Grose

    Full Text Available The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9. Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes.

  2. Involvement of Noxa in mediating cellular ER stress responses to lytic virus infection

    OpenAIRE

    2011-01-01

    Noxa is a Bcl-2 homology domain-containing pro-apoptotic mitochondrial protein. Noxa mRNA and protein expression are upregulated by dsRNA or virus, and ectopic Noxa expression enhances cellular sensitivity to virus or dsRNA-induced apoptosis. Here we demonstrate that Noxa null baby mouse kidney (BMK) cells are deficient in normal cytopathic response to lytic viruses, and that reconstitution of the knockout cells with wild type Noxa restored normal cytopathic responses. Noxa regulation by viru...

  3. Dual Function of Ccr5 during Langat Virus Encephalitis: Reduction in Neutrophil-Mediated Central Nervous System Inflammation and Increase in T Cell-Mediated Viral Clearance.

    Science.gov (United States)

    Michlmayr, Daniela; Bardina, Susana V; Rodriguez, Carlos A; Pletnev, Alexander G; Lim, Jean K

    2016-06-01

    Tick-borne encephalitis virus (TBEV) is a vector-transmitted flavivirus that causes potentially fatal neurologic infection. There are thousands of cases reported annually, and despite the availability of an effective vaccine, the incidence of TBEV is increasing worldwide. Importantly, up to 30% of affected individuals develop long-term neurologic sequelae. We investigated the role of chemokine receptor Ccr5 in a mouse model of TBEV infection using the naturally attenuated tick-borne flavivirus Langat virus (LGTV). Ccr5-deficient mice presented with an increase in viral replication within the CNS and decreased survival during LGTV encephalitis compared with wild-type controls. This enhanced susceptibility was due to the temporal lag in lymphocyte migration into the CNS. Adoptive transfer of wild-type T cells, but not Ccr5-deficient T cells, significantly improved survival outcome in LGTV-infected Ccr5-deficient mice. Concomitantly, a significant increase in neutrophil migration into the CNS in LGTV-infected Ccr5(-/-) mice was documented at the late stage of infection. Ab-mediated depletion of neutrophils in Ccr5(-/-) mice resulted in a significant improvement in mortality, a decrease in viral load, and a decrease in overall tissue damage in the CNS compared with isotype control-treated mice. Ccr5 is crucial in directing T cells toward the LGTV-infected brain, as well as in suppressing neutrophil-mediated inflammation within the CNS.

  4. Exponential enhancement of oncolytic vesicular stomatitis virus potency by vector-mediated suppression of inflammatory responses in vivo.

    Science.gov (United States)

    Altomonte, Jennifer; Wu, Lan; Chen, Li; Meseck, Marcia; Ebert, Oliver; García-Sastre, Adolfo; Fallon, John; Woo, Savio L C

    2008-01-01

    Oncolytic virotherapy is a promising strategy for treatment of malignancy, although its effectiveness is hampered by host antiviral inflammatory responses. The efficacy of treatment of oncolytic vesicular stomatitis virus (VSV) in rats bearing multifocal hepatocellular carcinoma (HCC) can be substantially elevated by antibody-mediated depletion of natural killer (NK) cells. In order to test the hypothesis that the oncotyic potency of VSV can be exponentially elevated by evasion of inflammatory responses in vivo, we constructed a recombinant VSV vector expressing equine herpes virus-1 glycoprotein G, which is a broad-spectrum viral chemokine binding protein (rVSV-gG). Infusion of rVSV-gG via the hepatic artery into immune-competent rats bearing syngeneic and multifocal HCC in their livers, resulted in a reduction of NK and NKT cells in the tumors and a 1-log enhancement in intratumoral virus titer in comparison with a reference rVSV vector. The treatment led to increased tumor necrosis and substantially prolonged animal survival without toxicities. These results indicate that rVSV-gG has the potential to be developed as an effective and safe oncolytic agent to treat patients with advanced HCC. Furthermore, the novel concept that oncolytic potency can be substantially enhanced by vector-mediated suppression of host antiviral inflammatory responses could have general applicability in the field of oncolytic virotherapy for cancer.

  5. High abundances of viruses in a deep-sea hydrothermal vent system indicates viral mediated microbial mortality

    Science.gov (United States)

    Ortmann, Alice C.; Suttle, Curtis A.

    2005-08-01

    Little is known about the distribution and abundance of viruses at deep-sea hydrothermal vents. Based on estimates made using epifluorescence microscopy and the dye YoPro-1, much higher viral abundances were observed at active hydrothermal vents than in the surrounding deep sea. This indicates that viral production was occurring and that viruses were a source of microbial mortality. Samples collected from three actively venting sites (Clam Bed, S&M and Salut) within the Endeavour Ridge system off the west coast of North America had viral abundances ranging from 1.45×10 5 to 9.90×10 7 ml -1, while the abundances of prokaryotes ranged from 1.30×10 5 to 4.46×10 6 ml -1. The abundances of viruses and prokaryotes in samples collected along the neutrally buoyant plume associated with the Main Endeavour Field were lower than at actively venting sites, with a mean of 5.3×10 5 prokaryotes ml -1 (s.d. 2.9×10 5, n=64) and 3.50×10 6 viruses ml -1 (s.d. 1.89×10 6, n=64), but were higher than non-plume samples (2.7×10 5 prokaryotes ml -1, s.d. 5.0×10 4, n=15 and 2.94×10 6 viruses ml -1, s.d. 1.08×10 6, n=15). Prokaryotic and viral abundances in non-hydrothermal regions were as much as 10-fold higher than found in previous studies, in which sample fixation likely resulted in underestimates. This suggests that viral infection may be a greater source of prokaryotic mortality throughout the deep sea than previously recognized. Overall, our results indicate that virus-mediated mortality of prokaryotes at these hydrothermal-vent environments is significant and may reduce energy flow to higher trophic levels.

  6. NK cells are strongly activated by Lassa and Mopeia virus-infected human macrophages in vitro but do not mediate virus suppression.

    Science.gov (United States)

    Russier, Marion; Reynard, Stéphanie; Tordo, Noël; Baize, Sylvain

    2012-07-01

    Lassa virus (LASV) and Mopeia virus (MOPV) are closely related Arenaviruses. LASV causes hemorrhagic fever, whereas MOPV is not pathogenic. Both viruses display tropism for APCs such as DCs and macrophages. During viral infections, NK cells are involved in the clearance of infected cells and promote optimal immune responses by interacting with APCs. We used an in vitro model of human NK and APC coculture to study the role of NK cells and to characterize their interactions with APCs during LASV and MOPV infections. As expected, NK cells alone were neither infected nor activated by LASV and MOPV, and infected DCs did not activate NK cells. By contrast, LASV- and MOPV-infected macrophages activated NK cells, as shown by the upregulation of CD69, NKp30, and NKp44, the downregulation of CXCR3, and an increase in NK-cell proliferation. NK cells acquired enhanced cytotoxicity, as illustrated by the increase in granzyme B (GrzB) expression and killing of K562 targets, but did not produce IFN-γ. Contact between NK cells and infected macrophages and type I IFNs were essential for activation; however, NK cells could not kill infected cells and control infection. Overall, these findings show that MOPV- as well as pathogenic LASV-infected macrophages mediate NK-cell activation.

  7. Rapid detection of Piper yellow mottle virus and Cucumber mosaic virus infecting black pepper (Piper nigrum) by loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Bhat, A I; Siljo, A; Deeshma, K P

    2013-10-01

    The loop-mediated isothermal amplification (LAMP) assay for Piper yellow mottle virus and the reverse transcription (RT) LAMP assay for Cucumber mosaic virus each consisted of a set of five primers designed against the conserved sequences in the viral genome. Both RNA and DNA isolated from black pepper were used as a template for the assay. The results were assessed visually by checking turbidity, green fluorescence and pellet formation in the reaction tube and also by gel electrophoresis. The assay successfully detected both viruses in infected plants whereas no cross-reactions were recorded with healthy plants. Optimum conditions for successful amplification were determined in terms of the concentrations of magnesium sulphate and betaine, temperature, and duration. The detection limit for both LAMP and RT-LAMP was up to 100 times that for conventional PCR and up to one-hundredth of that for real-time PCR. The optimal conditions arrived at were validated by testing field samples of infected vines of three species from different regions.

  8. A cell-permeable hairpin peptide inhibits hepatitis C viral nonstructural protein 5A-mediated translation and virus production.

    Science.gov (United States)

    Khachatoorian, Ronik; Arumugaswami, Vaithilingaraja; Ruchala, Piotr; Raychaudhuri, Santanu; Maloney, Eden M; Miao, Edna; Dasgupta, Asim; French, Samuel W

    2012-06-01

    NS5A is a key regulator of the hepatitis C virus (HCV) life cycle including RNA replication, assembly, and translation. We and others have shown that NS5A augments HCV internal ribosomal entry site (IRES)-mediated translation. Furthermore, Quercetin treatment and heat shock protein (HSP) 70 knockdown inhibit the NS5A-driven augmentation of IRES-mediated translation and infectious virus production. We have also coimmunoprecipitated HSP70 with NS5A and demonstrated cellular colocalization, leading to the hypothesis that the NS5A/HSP70 complex formation is important for IRES-mediated translation. Here, we have identified the NS5A region responsible for complex formation through in vitro deletion analyses. Deletion of NS5A domains II and III failed to reduce HSP70 binding, whereas domain I deletion eliminated complex formation. NS5A domain I alone also bound HSP70. Deletion mapping of domain I identified the C-terminal 34 amino acids (C34) as the interaction site. Furthermore, addition of C34 to domains II and III restored complex formation. C34 expression significantly reduced intracellular viral protein levels, in contrast to same-size control peptides from other NS5A domains. C34 also competitively inhibited NS5A-augmented IRES-mediated translation, whereas controls did not. Triple-alanine scan mutagenesis determined that an exposed beta-sheet hairpin in C34 was primarily responsible for NS5A-augmented IRES-mediated translation. Moreover, treatment with a 10-amino acid peptide derivative of C34 suppressed NS5A-augmented IRES-mediated translation and significantly inhibited intracellular viral protein synthesis, with no associated cytotoxicity. These results support the hypothesis that the NS5A/HSP70 complex augments viral IRES-mediated translation, identify a sequence-specific hairpin element in NS5A responsible for complex formation, and demonstrate the functional significance of C34 hairpin-mediated NS5A/HSP70 interaction. Identification of this element may allow

  9. Advances with using CRISPR/Cas-mediated gene editing to treat infections with hepatitis B virus and hepatitis C virus.

    Science.gov (United States)

    Moyo, Buhle; Bloom, Kristie; Scott, Tristan; Ely, Abdullah; Arbuthnot, Patrick

    2017-01-10

    Chronic infections with hepatitis B and hepatitis C viruses (HBV and HCV) account for the majority of cases of cirrhosis and hepatocellular carcinoma. Current therapies for the infections have limitations and improved efficacy is necessary to prevent complications in carriers of the viruses. In the case of HBV persistence, the replication intermediate comprising covalently closed circular DNA (cccDNA) is particularly problematic. Licensed therapies have little effect on cccDNA and HBV replication relapses following treatment withdrawal. Disabling cccDNA is thus key to curing HBV infections and application of gene editing technology, such as harnessing the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, has curative potential. Several studies have reported good efficacy when employing CRISPR/Cas technologies to disable HBV replication in cultured cells and in hydrodynamically injected mice. Recent advances with HCV drug development have revolutionized treatment of the infection. Nevertheless, individuals may be refractory to treatment. Targeting RNA from HCV with CRISPR/Cas isolated from Francisella novicida may have therapeutic utility. Although preclinical work shows that CRISPR/Cas technology has potential to overcome infection with HBV and HCV, significant challenges need to be met. Ensuring specificity for viral targets and efficient delivery of the gene editing sequences to virus-infected cells are particularly important. The field is at an interesting stage and the future of curative antiviral drug regimens, particularly for treatment of chronic HBV infection, may well entail use of combinations that include derivatives of CRISPR/Cas.

  10. An African horse sickness virus serotype 4 recombinant canarypox virus vaccine elicits specific cell-mediated immune responses in horses.

    Science.gov (United States)

    El Garch, H; Crafford, J E; Amouyal, P; Durand, P Y; Edlund Toulemonde, C; Lemaitre, L; Cozette, V; Guthrie, A; Minke, J M

    2012-09-15

    A recombinant canarypox virus vectored vaccine co-expressing synthetic genes encoding outer capsid proteins, VP2 and VP5, of African horse sickness virus (AHSV) serotype 4 (ALVAC(®)-AHSV4) has been demonstrated to fully protect horses against homologous challenge with virulent field virus. Guthrie et al. (2009) detected weak and variable titres of neutralizing antibody (ranging from horses received two vaccinations twenty-eight days apart and three horses remained unvaccinated. The detection of VP2/VP5 specific IFN-γ responses was assessed by enzyme linked immune spot (ELISpot) assay and clearly demonstrated that all ALVAC(®)-AHSV4 vaccinated horses developed significant IFN-γ production compared to unvaccinated horses. More detailed immune responses obtained by flow cytometry demonstrated that ALVAC(®)-AHSV4 vaccinations induced immune cells, mainly CD8(+) T cells, able to recognize multiple T-epitopes through all VP2 and only the N-terminus sequence of VP5. Neither VP2 nor VP5 specific IFN-γ responses were detected in unvaccinated horses. Overall, our data demonstrated that an experimental recombinant canarypox based vaccine induced significant CMI specific for both VP2 and VP5 proteins of AHSV4.

  11. Simultaneous detection and differentiation of dengue virus serotypes 1-4, Japanese encephalitis virus, and West Nile virus by a combined reverse-transcription loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Yin Jianhua

    2011-07-01

    Full Text Available Abstract Background Rapid identification and differentiation of mosquito-transmitted flaviviruses in acute-phase sera of patients and field-caught vector mosquitoes are important for the prediction and prevention of large-scale epidemics. Results We developed a flexible reverse-transcription loop-mediated isothermal amplification (RT-LAMP unit for the detection and differentiation of dengue virus serotypes 1-4 (DENV1-4, Japanese encephalitis virus (JEV, and West Nile virus (WNV. The unit efficiently amplified the viral genomes specifically at wide ranges of viral template concentrations, and exhibited similar amplification curves as monitored by a real-time PCR engine. The detection limits of the RT-LAMP unit were 100-fold higher than that of RT-PCR in 5 of the six flaviviruses. The results on specificity indicated that the six viruses in the assay had no cross-reactions with each other. By examining 66 viral strains of DENV1-4 and JEV, the unit identified the viruses with 100% accuracy and did not cross-react with influenza viruses and hantaviruses. By screening a panel of specimens containing sera of 168 patients and 279 pools of field-caught blood sucked mosquitoes, results showed that this unit is high feasible in clinical settings and epidemiologic field, and it obtained results 100% correlated with real-time RT-PCR. Conclusions The RT-LAMP unit developed in this study is able to quickly detect and accurately differentiate the six kinds of flaviviruses, which makes it extremely feasible for screening these viruses in acute-phase sera of the patients and in vector mosquitoes without the need of high-precision instruments.

  12. Pseudorabies virus US3 protein kinase mediates actin stress fiber breakdown

    NARCIS (Netherlands)

    Minnebruggen, van G.; Favoreel, H.W.; Jacobs, L.; Nauwynck, H.J.

    2003-01-01

    Disruption of specific components of the host cytoskeleton has been reported for several viruses and is thought to be beneficial for viral replication and spread. Our previous work demonstrated that infection of swine kidney (SK-6) cells with pseudorabies virus (PRV), a swine alphaherpesvirus, induc

  13. Cellular Nuclear Export Factors TAP and Aly Are Required for HDAg-L-mediated Assembly of Hepatitis Delta Virus.

    Science.gov (United States)

    Huang, Hsiu-Chen; Lee, Chung-Pei; Liu, Hui-Kang; Chang, Ming-Fu; Lai, Yu-Heng; Lee, Yu-Ching; Huang, Cheng

    2016-12-09

    Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus (HBV). HDV genome encodes two forms of hepatitis delta antigen (HDAg), small HDAg (HDAg-S), which is required for viral replication, and large HDAg (HDAg-L), which is essential for viral assembly. HDAg-L is identical to HDAg-S except that it bears a 19-amino acid extension at the C terminus. Both HDAgs contain a nuclear localization signal (NLS), but only HDAg-L contains a CRM1-independent nuclear export signal at its C terminus. The nuclear export activity of HDAg-L is important for HDV particle formation. However, the mechanisms of HDAg-L-mediated nuclear export of HDV ribonucleoprotein are not clear. In this study, the host cellular RNA export complex TAP-Aly was found to form a complex with HDAg-L, but not with an export-defective HDAg-L mutant, in which Pro(205) was replaced by Ala. HDAg-L was found to colocalize with TAP and Aly in the nucleus. The C-terminal domain of HDAg-L was shown to directly interact with the N terminus of TAP, whereas an HDAg-L mutant lacking the NLS failed to interact with full-length TAP. In addition, small hairpin RNA-mediated down-regulation of TAP or Aly reduced nuclear export of HDAg-L and assembly of HDV virions. Furthermore, a peptide, TAT-HDAg-L(198-210), containing the 10-amino acid TAT peptide and HDAg-L(198-210), inhibited the interaction between HDAg-L and TAP and blocked HDV virion assembly and secretion. These data demonstrate that formation and release of HDV particles are mediated by TAP and Aly. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. In-cell infection: a novel pathway for Epstein-Barr virus infection mediated by cell-in-cell structures.

    Science.gov (United States)

    Ni, Chao; Chen, Yuhui; Zeng, Musheng; Pei, Rongjuan; Du, Yong; Tang, Linquan; Wang, Mengyi; Hu, Yazhuo; Zhu, Hanyu; He, Meifang; Wei, Xiawei; Wang, Shan; Ning, Xiangkai; Wang, Manna; Wang, Jufang; Ma, Li; Chen, Xinwen; Sun, Qiang; Tang, Hong; Wang, Ying; Wang, Xiaoning

    2015-07-01

    Epstein-Barr virus (EBV) can infect both susceptible B lymphocytes and non-susceptible epithelial cells (ECs). Viral tropism analyses have revealed two intriguing means of EBV infection, either by a receptor-mediated infection of B cells or by a cell-to-cell contact-mediated infection of non-susceptible ECs. Herein, we report a novel "in-cell infection" mechanism for EBV infection of non-susceptible ECs through the formation of cell-in-cell structures. Epithelial CNE-2 cells were invaded by EBV-infected Akata B cells to form cell-in-cell structures in vitro. Such unique cellular structures could be readily observed in the specimens of nasopharyngeal carcinoma. Importantly, the formation of cell-in-cell structures led to the autonomous activation of EBV within Akata cells and subsequent viral transmission to CNE-2 cells, as evidenced by the expression of viral genes and the presence of virion particles in CNE-2 cells. Significantly, EBV generated from in-cell infected ECs displayed altered tropism with higher infection efficacy to both B cells and ECs. In addition to CNE-2 tumor cells, cell-in-cell structure formation could also mediate EBV infection of NPEC1-Bmi1 cells, an immortalized nasopharyngeal epithelial cell line. Furthermore, efficient infection by this mechanism involved the activation of the PI3K/AKT signaling pathway. Thus, our study identified "in-cell infection" as a novel mechanism for EBV infection. Given the diversity of virus-infected cells and the prevalence of cell-in-cell structures during chronic infection, we speculate that "in-cell infection" is likely a general mechanism for EBV and other viruses to infect non-susceptible ECs.

  15. Vaccinia virus-mediated intra-tumoral expression of matrix metalloproteinase 9 enhances oncolysis of PC-3 xenograft tumors

    Directory of Open Access Journals (Sweden)

    Schäfer Simon

    2012-08-01

    Full Text Available Abstract Background Oncolytic viruses, including vaccinia virus (VACV, are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, we enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9-mediated degradation of proteins of the tumoral extracellular matrix (ECM, leading to increased viral distribution within the tumors. Methods For this study, the oncolytic vaccinia virus GLV-1h255, containing the mmp-9 gene, was constructed and used to treat PC-3 tumor-bearing mice, achieving an intra-tumoral over-expression of MMP-9. The intra-tumoral MMP-9 content was quantified by immunohistochemistry in tumor sections. Therapeutic efficacy of GLV-1h255 was evaluated by monitoring tumor growth kinetics and intra-tumoral virus titers. Microenvironmental changes mediated by the intra-tumoral MMP-9 over-expression were investigated by microscopic quantification of the collagen IV content, the blood vessel density (BVD and the analysis of lymph node metastasis formation. Results GLV-1h255-treatment of PC-3 tumors led to a significant over-expression of intra-tumoral MMP-9, accompanied by a marked decrease in collagen IV content in infected tumor areas, when compared to GLV-1h68-infected tumor areas. This led to considerably elevated virus titers in GLV-1h255 infected tumors, and to enhanced tumor regression. The analysis of the BVD, as well as the lumbar and renal lymph node volumes, revealed lower BVD and significantly smaller lymph nodes in both GLV-1h68- and GLV-1h255- injected mice compared to those injected with PBS, indicating that MMP-9 over-expression does not alter the metastasis-reducing effect of oncolytic VACV. Conclusions Taken together, these results indicate that a GLV-1h255-mediated intra-tumoral over-expression of MMP-9 leads to a degradation of collagen IV

  16. Psoralen-mediated virus photoinactivation in platelet concentrates: enhanced specificity of virus kill in the absence of shorter UVA wavelengths

    Energy Technology Data Exchange (ETDEWEB)

    Margolis-Nunno, Henrietta; Robinson, Richard; Horowitz, Bernard; Ben-Hur, Ehud [New York Blood Center, NY (United States); Geacintov, N.E. [New York Univ., NY (United States). Dept. of Chemistry

    1995-11-01

    Treatments with psoralens and long-wavelength ultraviolet radiation (UVA, 320-400 nm; PUVA) have shown efficacy for virus sterilization of platelet concentrates (PC). We have employed the psoralen derivative 4`-aminomethyl-4,5`,8-trimethylpsoralen (AMT), and have found that platelet integrity is best preserved when rutin, a flavonoid that quenches multiple reactive oxygen species, is present during AMT/UVA treatment of PC. In this report, we examine the effects of different UVA spectra under our standard PC treatment conditions (i.e. 50 {mu}g/mL AMT, 0.35 mM rutin and 38 J/cm{sup 2} UVA). Added vesicular stomatitis virus (VSV; {>=} 5.5 log{sub 10}) was completely inactivated with the simultaneous maintenance of the platelet aggregation response (> 90% of control) when a UVA light source with transmission mainly between 360 and 370 nm (narrow UVA1) was used. In contrast, with a broad-band UVA (320-400 nm; broad UVA) light source, the aggregation response was greatly compromised (< 50% of control) with only a minor increase in the rate of VSV kill. With this lamp, platelet function could be improved to about 75% of the control by adding a long-pass filter, which reduced the transmission of shorter ({<=} 345 nm) UVA wavelengths (340-400 nm; UVA1). At equivalent levels of virus kill, aggregation function was always best preserved when narrow UVA1 was used for PUVA treatment. Even in the absence of AMT, and with or without rutin present, narrow UVA1 irradiation was better tolerated by platelets than was broad UVA. (author).

  17. Influenza Virus-Mediated Membrane Fusion: Determinants of Hemagglutinin Fusogenic Activity and Experimental Approaches for Assessing Virus Fusion

    Directory of Open Access Journals (Sweden)

    Susan Daniel

    2012-07-01

    Full Text Available Hemagglutinin (HA is the viral protein that facilitates the entry of influenza viruses into host cells. This protein controls two critical aspects of entry: virus binding and membrane fusion. In order for HA to carry out these functions, it must first undergo a priming step, proteolytic cleavage, which renders it fusion competent. Membrane fusion commences from inside the endosome after a drop in lumenal pH and an ensuing conformational change in HA that leads to the hemifusion of the outer membrane leaflets of the virus and endosome, the formation of a stalk between them, followed by pore formation. Thus, the fusion machinery is an excellent target for antiviral compounds, especially those that target the conserved stem region of the protein. However, traditional ensemble fusion assays provide a somewhat limited ability to directly quantify fusion partly due to the inherent averaging of individual fusion events resulting from experimental constraints. Inspired by the gains achieved by single molecule experiments and analysis of stochastic events, recently-developed individual virion imaging techniques and analysis of single fusion events has provided critical information about individual virion behavior, discriminated intermediate fusion steps within a single virion, and allowed the study of the overall population dynamics without the loss of discrete, individual information. In this article, we first start by reviewing the determinants of HA fusogenic activity and the viral entry process, highlight some open questions, and then describe the experimental approaches for assaying fusion that will be useful in developing the most effective therapies in the future.

  18. Humoral and cell-mediated immune responses in DNA immunized mink challenged with wild-type canine distemper virus

    DEFF Research Database (Denmark)

    Nielsen, Line; Søgaard, Mette; Karlskov-Mortensen, Peter

    2009-01-01

    The aim of the study was to investigate the different phases of the immune response after DNA immunization with the hemagglutinin and nucleoprotein genes from canine distemper virus (CDV). Although attenuated live CDV vaccines have effectively reduced the incidence of disease, canine distemper...... is still a problem worldwide. The broad host range of CDV creates a constant viral reservoir among wildlife animals. Our results demonstrated early humoral and cell-mediated immune responses (IFN-gamma) in DNA vaccinated mink compared to mock-vaccinated mink after challenge with a Danish wild-type CDV...

  19. Hepatitis C Virus Core Protein Abrogates the DDX3 Function That Enhances IPS-1-Mediated IFN–Beta Induction

    OpenAIRE

    2010-01-01

    The DEAD box helicase DDX3 assembles IPS-1 (also called Cardif, MAVS, or VISA) in non-infected human cells where minimal amounts of the RIG-I-like receptor (RLR) protein are expressed. DDX3 C-terminal regions directly bind the IPS-1 CARD-like domain as well as the N-terminal hepatitis C virus (HCV) core protein. DDX3 physically binds viral RNA to form IPS-1-containing spots, that are visible by confocal microscopy. HCV polyU/UC induced IPS-1-mediated interferon (IFN)-beta promoter activation,...

  20. TBK1 Mediates Critical Effects of Measles Virus Nucleocapsid Protein (MVNP) on Pagetic Osteoclast Formation

    Science.gov (United States)

    Sun, Quanhong; Sammut, Bénédicte; Wang, Feng-Ming; Kurihara, Noriyoshi; Windle, Jolene J.; Roodman, G. David; Galson, Deborah L.

    2013-01-01

    Paget’s disease of bone (PDB) is characterized by abnormal osteoclasts with unique characteristics that include: increased sensitivity of osteoclast progenitors to 1,25(OH)2D3, RANKL and TNF-α, increased osteoclast numbers, increased expression of IL-6 and several transcription factors. We recently reported that measles virus nucleocapsid protein (MVNP) plays a key role in the development of these abnormal osteoclasts. MVNP can induce the pagetic osteoclast phenotype in vitro and in vivo in TRAP-MVNP transgenic mice. However, the molecular mechanisms by which MVNP generates pagetic osteoclasts have not been determined. TANK-binding kinase 1 (TBK1) and IκB kinase-ɛ (IKKɛ) are IKK family members which complex with MVNP and activate both IRF3 and NF-κB pathways. MVNP increases the amount of TBK1 protein in bone marrow monocytes (BMM). Interestingly, we found that RANKL increased TBK1 and IKKɛ early in osteoclast differentiation, suggesting a possible role in normal osteoclastogenesis. However, only TBK1 is further increased in osteoclasts formed by TRAP-MVNP BMM due to increased TBK1 protein stability. TBK1 over-expression induced IL6 promoter reporter activity, and elevated endogenous IL6 mRNA and p65 NF-κB, TAF12 and ATF7 proteins in several cell lines. Over-expression of TBK1 was insufficient to induce pagetic osteoclasts from WT BMM, but synergized with MVNP to increase pagetic osteoclast formation from TRAP-MVNP BMM. BX795 inhibition of TBK1 impaired MVNP-induced IL-6 expression in both NIH3T3 cells and BMM, and shRNA knockdown of Tbk1 in NIH3T3 cells impaired IL-6 secretion induced by MVNP and decreased TAF12 and ATF7, factors involved in 1,25(OH)2D3 hypersensitivity of pagetic osteoclasts. Similarly, Tbk1 knockdown in BMM from TRAP-MVNP and WT mice specifically impaired development of the MVNP-induced osteoclast pagetic phenotype. These results demonstrate that TBK1 plays a critical role in mediating the effects of MVNP on osteoclast differentiation

  1. [Colorimetric detection of human influenza A H1N1 virus by reverse transcription loop mediated isothermal amplification].

    Science.gov (United States)

    Nie, Kai; Wang, Da-Yan; Qin, Meng; Gao, Rong-Bao; Wang, Miao; Zou, Shu-Mei; Han, Feng; Zhao, Xiang; Li, Xi-Yan; Shu, Yue-Long; Ma, Xue-Jun

    2010-03-01

    A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.

  2. Recombinant vesicular stomatitis virus vector mediates postexposure protection against Sudan Ebola hemorrhagic fever in nonhuman primates.

    Science.gov (United States)

    Geisbert, Thomas W; Daddario-DiCaprio, Kathleen M; Williams, Kinola J N; Geisbert, Joan B; Leung, Anders; Feldmann, Friederike; Hensley, Lisa E; Feldmann, Heinz; Jones, Steven M

    2008-06-01

    Recombinant vesicular stomatitis virus (VSV) vectors expressing homologous filoviral glycoproteins can completely protect rhesus monkeys against Marburg virus when administered after exposure and can partially protect macaques after challenge with Zaire ebolavirus. Here, we administered a VSV vector expressing the Sudan ebolavirus (SEBOV) glycoprotein to four rhesus macaques shortly after exposure to SEBOV. All four animals survived SEBOV challenge, while a control animal that received a nonspecific vector developed fulminant SEBOV hemorrhagic fever and succumbed. This is the first demonstration of complete postexposure protection against an Ebola virus in nonhuman primates and provides further evidence that postexposure vaccination may have utility in treating exposures to filoviruses.

  3. AAV-mediated knock-down of HRC exacerbates transverse aorta constriction-induced heart failure.

    Directory of Open Access Journals (Sweden)

    Chang Sik Park

    Full Text Available Histidine-rich calcium binding protein (HRC is located in the lumen of sarcoplasmic reticulum (SR that binds to both triadin (TRN and SERCA affecting Ca(2+ cycling in the SR. Chronic overexpression of HRC that may disrupt intracellular Ca(2+ homeostasis is implicated in pathogenesis of cardiac hypertrophy. Ablation of HRC showed relatively normal phenotypes under basal condition, but exhibited a significantly increased susceptibility to isoproterenol-induced cardiac hypertrophy. In the present study, we characterized the functions of HRC related to Ca(2+ cycling and pathogenesis of cardiac hypertrophy using the in vitro siRNA- and the in vivo adeno-associated virus (AAV-mediated HRC knock-down (KD systems, respectively.AAV-mediated HRC-KD system was used with or without C57BL/6 mouse model of transverse aortic constriction-induced failing heart (TAC-FH to examine whether HRC-KD could enhance cardiac function in failing heart (FH. Initially we expected that HRC-KD could elicit cardiac functional recovery in failing heart (FH, since predesigned siRNA-mediated HRC-KD enhanced Ca(2+ cycling and increased activities of RyR2 and SERCA2 without change in SR Ca(2+ load in neonatal rat ventricular cells (NRVCs and HL-1 cells. However, AAV9-mediated HRC-KD in TAC-FH was associated with decreased fractional shortening and increased cardiac fibrosis compared with control. We found that phospho-RyR2, phospho-CaMKII, phospho-p38 MAPK, and phospho-PLB were significantly upregulated by HRC-KD in TAC-FH. A significantly increased level of cleaved caspase-3, a cardiac cell death marker was also found, consistent with the result of TUNEL assay.Increased Ca(2+ leak and cytosolic Ca(2+ concentration due to a partial KD of HRC could enhance activity of CaMKII and phosphorylation of p38 MAPK, causing the mitochondrial death pathway observed in TAC-FH. Our results present evidence that down-regulation of HRC could deteriorate cardiac function in TAC-FH through

  4. Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells.

    Science.gov (United States)

    Palella, T D; Silverman, L J; Schroll, C T; Homa, F L; Levine, M; Kelley, W N

    1988-01-01

    The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.

  5. Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells

    Energy Technology Data Exchange (ETDEWEB)

    Palella, T.D.; Silverman, L.J.; Schroll, C.T.; Homa, F.L.; Levine, M.; Kelley, W.N.

    1988-01-01

    The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.

  6. Complement-mediated neutralization of dengue virus requires mannose-binding lectin

    DEFF Research Database (Denmark)

    Avirutnan, Panisadee; Hauhart, Richard E; Marovich, Mary A

    2011-01-01

    -dependent activation of the complement cascade neutralized insect cell-derived West Nile virus (WNV) in cell culture and restricted pathogenesis in mice. Here, we investigated the antiviral activity of MBL in infection by dengue virus (DENV), a related flavivirus. Using a panel of naïve sera from mouse strains...... with lower levels. Our studies suggest that allelic variation of MBL in humans may impact complement-dependent control of DENV pathogenesis. IMPORTANCE Dengue virus (DENV) is a mosquito-transmitted virus that causes a spectrum of clinical disease in humans ranging from subclinical infection to dengue...... hemorrhagic fever and dengue shock syndrome. Four serotypes of DENV exist, and severe illness is usually associated with secondary infection by a different serotype. Here, we show that mannose-binding lectin (MBL), a pattern recognition molecule that initiates the lectin pathway of complement activation...

  7. Jasmonic acid-related resistance in tomato mediates interactions between whitefly and whitefly-transmitted virus

    National Research Council Canada - National Science Library

    Yan-Chun Sun; Li-Long Pan; Feng-Ze Ying; Ping Li; Xiao-Wei Wang; Shu-Sheng Liu

    2017-01-01

      The indirect interactions between insect vectors, such as whiteflies, and the viruses they transmit, such as begomoviruses, via host plants may produce a range of outcome depending on the species...

  8. A non-classical phase diagram for virus-bacterial co-evolution mediated by CRISPR

    Science.gov (United States)

    Han, Pu; Deem, Michael

    CRISPR is a newly discovered prokaryotic immune system. Bacteria and archaea with this system incorporate genetic material from invading viruses into their genomes, providing protection against future infection by similar viruses. Due to the cost of CRISPR, bacteria can lose the acquired immunity. We will show an intriguing phase diagram of the virus extinction probability, which when the rate of losing the acquired immunity is small, is more complex than that of the classic predator-prey model. As the CRISPR incorporates genetic material, viruses are under pressure to evolve to escape the recognition by CRISPR, and this co-evolution leads to a non-trivial phase structure that cannot be explained by the classical predator-prey model.

  9. Neuraminidase-mediated haemagglutination of recent human influenza A(H3N2) viruses is determined by arginine 150 flanking the neuraminidase catalytic site.

    Science.gov (United States)

    Mögling, Ramona; Richard, Mathilde J; Vliet, Stefan van der; Beek, Ruud van; Schrauwen, Eefje J A; Spronken, Monique I; Rimmelzwaan, Guus F; Fouchier, Ron A M

    2017-06-01

    Over the last decade, an increasing proportion of circulating human influenza A(H3N2) viruses exhibited haemagglutination activity that was sensitive to neuraminidase inhibitors. This change in haemagglutination as compared to older circulating A(H3N2) viruses prompted an investigation of the underlying molecular basis. Recent human influenza A(H3N2) viruses were found to agglutinate turkey erythrocytes in a manner that could be blocked with either oseltamivir or neuraminidase-specific antisera, indicating that agglutination was driven by neuraminidase, with a low or negligible contribution of haemagglutinin. Using representative virus recombinants it was shown that the haemagglutinin of a recent A(H3N2) virus indeed had decreased activity to agglutinate turkey erythrocytes, while its neuraminidase displayed increased haemagglutinating activity. Viruses with chimeric and mutant neuraminidases were used to identify the amino acid substitution histidine to arginine at position 150 flanking the neuraminidase catalytic site as the determinant of this neuraminidase-mediated haemagglutination. An analysis of publicly available neuraminidase gene sequences showed that viruses with histidine at position 150 were rapidly replaced by viruses with arginine at this position between 2005 and 2008, in agreement with the phenotypic data. As a consequence of neuraminidase-mediated haemagglutination of recent A(H3N2) viruses and poor haemagglutination via haemagglutinin, haemagglutination inhibition assays with A(H3N2) antisera are no longer useful to characterize the antigenic properties of the haemagglutinin of these viruses for vaccine strain selection purposes. Continuous monitoring of the evolution of these viruses and potential consequences for vaccine strain selection remains important.

  10. Mediatization

    DEFF Research Database (Denmark)

    Hjarvard, Stig

    2017-01-01

    Mediatization research shares media effects studies' ambition of answering the difficult questions with regard to whether and how media matter and influence contemporary culture and society. The two approaches nevertheless differ fundamentally in that mediatization research seeks answers...... to these general questions by distinguishing between two concepts: mediation and mediatization. The media effects tradition generally considers the effects of the media to be a result of individuals being exposed to media content, i.e. effects are seen as an outcome of mediated communication. Mediatization...... research is concerned with long-term structural changes involving media, culture, and society, i.e. the influences of the media are understood in relation to how media are implicated in social and cultural changes and how these processes come to create new conditions for human communication and interaction...

  11. In vitro generation of human cytotoxic lymphocytes by virus. Viral glycoproteins induce nonspecific cell-mediated cytotoxicity without release of interferon

    OpenAIRE

    1981-01-01

    Purified hemagglutinin and fusion glycoproteins of measles virus either in soluble form or inserted in artifical membranes bind to human peripheral blood lymphocytes and induce cell-mediated cytotoxicity (CMC) in a dose-response fashion. Both autologous and heterologous noninfected target cells are lysed in vitro. The expression of CMC is not inhibited by anti-measles virus antibody added to lymphocytes previously exposed to viral glycoproteins. THe killer lymphocytes are Fc receptor positive...

  12. One-step reverse transcription loop-mediated isothermal amplification for the rapid detection of cucumber green mottle mosaic virus.

    Science.gov (United States)

    Li, Jin-yu; Wei, Qi-wei; Liu, Yong; Tan, Xin-qiu; Zhang, Wen-na; Wu, Jian-yan; Charimbu, Miriam Karwitha; Hu, Bai-shi; Cheng, Zhao-bang; Yu, Cui; Tao, Xiao-rong

    2013-11-01

    Cucumber green mottle mosaic virus (CGMMV) has caused serious damage to Cucurbitaceae crops worldwide. The virus is considered one of the most serious Cucurbitaceae quarantine causes in many countries. In this study, a highly efficient and practical one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for the detection of CGMMV. The total RNA or crude RNA extracted from watermelon plants or seeds could be detected easily by this RT-LAMP assay. The RT-LAMP assay was conducted in isothermal (63°C) conditions within 1h. The amplified products of CGMMV could be detected as ladder-like bands using agarose gel electrophoresis or visualized in-tube under UV light with the addition of a fluorescent dye. The RT-LAMP amplification was specific to CGMMV, as no cross-reaction was observed with other viruses. The RT-LAMP assay was 100-fold more sensitive than that of reverse-transcription polymerase chain reaction (RT-PCR). This is the first report of the application of the RT-LAMP assay to detect CGMMV. The sensitive, specific and rapid RT-LAMP assay developed in this study can be applied widely in laboratories, the field and quarantine surveillance of CGMMV.

  13. Plaque identification of strand-forming canine distemper virus by staphylococcal protein A-mediated reverse passive haemadsorption.

    Science.gov (United States)

    Johnson, G C; Fulks, K; Krakowka, S

    1985-02-01

    The R252 neurotropic isolate of canine distemper virus (CDV) produces cytopathic effects (CPE) dominated by strand formation rather than by the formation of multinucleate giant cells. The lack of well-defined CPE and consequent rapid spread of infection throughout the cell monolayer has hindered plaque purification of this virus by conventional methods. However, the use of an immunological detection system which utilizes binding of hyperimmune dog serum to virus-infected cells, followed by the identification of those sites by staphylococcal Protein A-coupled sheep red blood cells (reverse passive haemadsorption) allowed infected foci in cell monolayers to be detected as early as 4 days after infection, coincident with the appearance of the first immunofluorescently identified viral foci. Foci of haemadsorption were specific to sites of CDV infection as demonstrated by blocking experiments. Material recovered from the plaques was successful in infecting Vero cells. Thus, immunologically mediated adsorption of Protein A coupled red blood cells can be used to identify and isolate foci of viral infection which exhibit minimal or no viral CPE without destroying viral replicative ability.

  14. Development of a Rapid Detection Method for Potato virus X by Reverse Transcription Loop-Mediated Isothermal Amplification

    Directory of Open Access Journals (Sweden)

    Joojin Jeong

    2015-09-01

    Full Text Available The primary step for efficient control of viral diseases is the development of simple, rapid, and sensitive virus detection. Reverse transcription loop-mediated isothermal amplification (RT-LAMP has been used to detect viral RNA molecules because of its simplicity and high sensitivity for a number of viruses. RT-LAMP for the detection of Potato virus X (PVX was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR to demonstrate its advantages over RT-PCR. RT-LAMP reactions were conducted with or without a set of loop primers since one out of six primers showed PVX specificity. Based on real-time monitoring, RT-LAMP detected PVX around 30 min, compared to 120 min for RT-PCR. By adding a fluorescent reagent during the reaction, the extra step of visualization by gel electrophoresis was not necessary. RT-LAMP was conducted using simple inexpensive instruments and a regular incubator to evaluate whether RNA could be amplified at a constant temperature instead of using an expensive thermal cycler. This study shows the potential of RT-LAMP for the diagnosis of viral diseases and PVX epidemiology because of its simplicity and rapidness compared to RT-PCR.

  15. IL-17 response mediates acute lung injury induced by the 2009 Pandemic Influenza A(H1N1)Virus

    Institute of Scientific and Technical Information of China (English)

    Chenggang Li; Chen Wang; Zhongwei Chen; Li Xing; Chong Tang; Xiangwu Ju; Feng Guo; Jiejie Deng; Yan Zhao; Peng Yang; Jun Tang; Penghui Yang; Huanling Wang; Zhongpeng Zhao; Zhinan Yin; Bin Cao; Xiliang Wang; Chengyu Jiang; Yang Sun; Taisheng Li; Chen Wang; Zhong Wang; Zhen Zou; Yiwu Yan; Wei Wang

    2012-01-01

    The 2009 flu pandemic involved the emergence of a new strain of a swine-origin H1N1 influenza virus(S-OIV H1N1)that infected almost every country in the world.Most infections resulted in respiratory illness and some severe cases resulted in acute lung injury.In this report,we are the first to describe a mouse model of S-OIV virus infection with acute lung injury and immune responses that reflect human clinical disease.The clinical efficacy of the antiviral oseltamivir(Tamiflu)administered in the early stages of S-OIV H1N1 infection was confirmed in the mouse model.Moreover,elevated levels of IL-17,Th-17 mediators and IL-17-responsive cytokines were found in serum samples of S-OIV-infected patients in Beijing.IL-17 deficiency or treatment with monoclonal antibodies against IL-17-ameliorated acute lung injury induced by the S-OIV H1N1 virus in mice.These results suggest that IL-17 plays an important role in S-OIV-induced acute lung injury and that monoclonal antibodies against IL-17 could be useful as a potential therapeutic remedy for future S-OIV H1N1 pandemics.

  16. Varicella-zoster virus (VZV) mediates a delayed host shutoff independent of open reading frame (ORF) 17 expression.

    Science.gov (United States)

    Waterboer, Tim; Rahaus, Markus; Wolff, Manfred H

    2002-01-01

    Varicella-zoster virus (VZV) open reading frame 17 (ORF 17) is the gene corresponding to Herpes simplex-virus (HSV) UL41. The UL41 gene encodes the virion host shutoff factor (vhs), a RNase that has been the object of detailed studies. In contrast to HSV, knowledge about VZV mediated shutoff effects and the role of ORF 17 is poor. We investigated the ORF 17 expression in infected cells and analyzed shutoff effects. ORF 17 expression could not be proven in infected human fibroblast cell lines and melanoma (MeWo) cells. Only after induction by Phorbol 12-myristate 13-acetate an ORF 17 expression became detectable in MeWo cells. Nevertheless, using stable expressed GAPDH mRNA as a marker for mRNA degradation, a VZV mediated shutoff, independent of ORF 17 expression, became measurable. Transfection experiments demonstrated that transient ORF 17 expression did not decrease the cellular GAPDH mRNA level. We examined whether the VZV shutoff factor is a tegument protein causing an early shutoff or whether it needs to be expressed (delayed shutoff). The GAPDH mRNA level in Actinomycin D pretreated and infected MeWo cells did not decrease even faster than the theoretical decay rate based on a half-life of 24 h. These findings lead to the conclusion that the VZV shutoff factor is not a mature protein localized in the virion and that VZV causes a delayed virion host shutoff effect.

  17. Human immunodeficiency virus type 1-mediated syncytium formation is compatible with adenovirus replication and facilitates efficient dispersion of viral gene products and de novo-synthesized virus particles.

    Science.gov (United States)

    Li, H; Haviv, Y S; Derdeyn, C A; Lam, J; Coolidge, C; Hunter, E; Curiel, D T; Blackwell, J L

    2001-12-10

    Conditionally replicative adenovirus (CRAd) vectors are designed for specific oncolytic replication in tumor tissues with concomitant sparing of normal cells. As such, CRAds offer an unprecedented level of anticancer potential for malignancies that have been refractory to previous cancer gene therapy interventions. CRAd efficacy may, however, be compromised by inefficient dispersion of the replicating vector within the tumor tissue. To address this issue, we evaluated the utility of a fusogenic membrane glycoprotein (FMG), which induces the fusion of neighboring cellular membranes to form multinucleated syncytia. We hypothesized that the FMG-mediated syncytia would facilitate dispersion of the adenovirus (Ad) gene products and viral progeny. To test this, human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, which induce syncytia in the presence of CD4+ target cells, were expressed by an Ad (Ad5HIVenv) in permissive (CD4-positive) and nonpermissive (CD4-negative) cell lines. After validating this Ad-FMG model, the efficiency of Ad replication in the presence or absence of syncytia was evaluated. The results demonstrated that syncytium formation was compatible with Ad replication and dramatically increased the dispersion of virus gene products within the cytoplasm of the syncytia as well as viral particles in the nuclei of the syncytial mass. Moreover, progeny virions were released more efficiently from syncytia compared with nonsyncytial cells. These data demonstrate the utility of FMGs as a dispersion agent and suggest that FMGs can improve the efficacy of CRAd gene therapy.

  18. Small interfering RNAs targeting peste des petits ruminants virus M mRNA increase virus-mediated fusogenicity and inhibit viral replication in vitro.

    Science.gov (United States)

    Liu, Fuxiao; Wu, Xiaodong; Zou, Yanli; Li, Lin; Liu, Shan; Chi, Tianying; Wang, Zhiliang

    2015-11-01

    Peste des petits ruminants (PPR), caused by peste des petits ruminants virus (PPRV), is an acute or subacute, highly contagious and economically important disease of small ruminants. The PPRV is classified into the genus Morbillivirus in the family Paramyxoviridae. The PPRV matrix (M) protein possesses an intrinsic ability to bind to lipid membranes, and plays a crucial role in viral assembly and further budding. In this study, three different small interfering RNAs (siRNA) were designed on the basis of translated region for PPRV Nigeria 75/1M mRNA, and were subsequently synthesized for their transfection into Vero-SLAM cells, followed by infection with PPRVs. The results showed that two out of three siRNAs robustly induced cell-to-cell fusion as early as 36h post-infection with PPRVs, effectively suppressed expression of the M protein by interference for the M mRNA, and eventually inhibited viral replication in vitro. These findings led us to speculate that siRNA-mediated knockdown of the M protein would alter its interaction with viral glycoproteins, thus exacerbating intercellular fusion but hampering virus release. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Host-Mediated Effects of Semipersistently Transmitted Squash Vein Yellowing Virus on Sweetpotato Whitefly (Hemiptera: Aleyrodidae) Behavior and Fitness.

    Science.gov (United States)

    Shrestha, Deepak; McAuslane, Heather J; Adkins, Scott T; Smith, Hugh A; Dufault, Nicholas; Colee, James; Webb, Susan E

    2017-08-01

    Plant viruses may indirectly affect insect vector behavior and fitness via a shared host plant. Here, we evaluated the host-mediated effects of Squash vein yellowing virus (SqVYV) on the behavior and fitness of its whitefly vector, Bemisia tabaci (Gennadius) Middle East-Asia Minor 1, formerly biotype B. Alighting, settling, and oviposition behavioral assays were conducted on infected and mock-inoculated squash (Cucurbita pepo L.) and watermelon [Citrullus lanatus (Thunb) Matsum and Nakai] plants. Developmental time of immature stages, adult longevity, and fecundity were measured on infected and mock-inoculated squash plants. For adult longevity and fecundity, whiteflies were reared on infected and mock-inoculated squash plants to determine the effects of nymphal rearing host on the adult stage. More whiteflies alighted and remained settled on infected squash than on mock-inoculated squash 0.25, 1, 8, and 24 h after release. No such initial preference was observed on watermelon plants, but by 8 h after release, more whiteflies were found on mock-inoculated watermelon plants than on infected plants. Whiteflies laid approximately six times more eggs on mock-inoculated watermelon than on infected watermelon; however, no differences were observed on squash. Development from egg to adult emergence was 3 d shorter on infected than mock-inoculated squash plants. Females lived 25% longer and had higher fecundity on infected squash plants than on mock-inoculated plants, regardless of infection status of the rearing host. The host-mediated effects of SqVYV infection on whitefly behavior differ on two cucurbit host plants, suggesting the potential for more rapid spread of the virus within watermelon fields. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. Application of a Real-time Reverse Transcription Loop Mediated Amplification Method to the Detection of Rabies Virus in Arctic Foxes in Greenland

    DEFF Research Database (Denmark)

    Wakeley, Philip; Johnson, Nicholas; Rasmussen, Thomas Bruun

    Reverse transcription loop mediated amplification (RT-LAMP) offers a rapid, isothermal method for amplification of virus RNA. In this study a panel of positive rabies virus samples originally prepared from arctic fox brain tissue was assessed for the presence of rabies viral RNA using a real time...... RT-LAMP. The method had previously been shown to work with samples from Ghana which clustered with cosmopolitan lineage rabies viruses but the assay had not been assessed using samples from animals infected with rabies from the arctic region. The assay is designed to amplify both cosmopolitan strains...... virus of arctic origin virus can be detected using RT-LAMP and the method reported is more rapid than the real-time RT-PCR. Further arctic fox samples are under analysis in order to confirm these findings....

  1. Development of a reverse transcription loop-mediated isothermal amplification assay for the rapid diagnosis of avian influenza A (H7N9) virus infection.

    Science.gov (United States)

    Nakauchi, Mina; Takayama, Ikuyo; Takahashi, Hitoshi; Tashiro, Masato; Kageyama, Tsutomu

    2014-08-01

    A genetic diagnosis system for detecting avian influenza A (H7N9) virus infection using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) technology was developed. The RT-LAMP assay showed no cross-reactivity with seasonal influenza A (H3N2 and H1N1pdm09) or influenza B viruses circulating in humans or with avian influenza A (H5N1) viruses. The sensitivity of the RT-LAMP assay was 42.47 copies/reaction. Considering the high specificity and sensitivity of the assay for detecting the avian influenza A (H7N9) virus and that the reaction was completed within 30 min, the RT-LAMP assay developed in this study is a promising rapid diagnostic tool for avian influenza A (H7N9) virus infection.

  2. Novel plant virus-based vaccine induces protective cytotoxic T-lymphocyte-mediated antiviral immunity through dendritic cell maturation.

    Science.gov (United States)

    Lacasse, Patrick; Denis, Jérôme; Lapointe, Réjean; Leclerc, Denis; Lamarre, Alain

    2008-01-01

    Currently used vaccines protect mainly through the production of neutralizing antibodies. However, antibodies confer little or no protection for a majority of chronic viral infections that require active involvement of cytotoxic T lymphocytes (CTLs). Virus-like particles (VLPs) have been shown to be efficient inducers of cell-mediated immune responses, but administration of an adjuvant is generally required. We recently reported the generation of a novel VLP system exploiting the self-assembly property of the papaya mosaic virus (PapMV) coat protein. We show here that uptake of PapMV-like particles by murine splenic dendritic cells (DCs) in vivo leads to their maturation, suggesting that they possess intrinsic adjuvant-like properties. DCs pulsed with PapMV-like particles displaying the lymphocytic choriomeningitis virus (LCMV) p33 immunodominant CTL epitope (PapMV-p33) efficiently process and cross-present the viral epitope to p33-specific transgenic T cells. Importantly, the CTL epitope is also properly processed and presented in vivo, since immunization of p33-specific T-cell receptor transgenic mice with PapMV-p33 induces the activation of large numbers of specific CTLs. C57BL/6 mice immunized with PapMV-p33 VLPs in the absence of adjuvant develop p33-specific effector CTLs that rapidly expand following LCMV challenge and protect vaccinated mice against LCMV infection in a dose-dependent manner. These results demonstrate the efficiency of this novel plant virus-based vaccination platform in inducing DC maturation leading to protective CTL responses.

  3. Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus.

    Directory of Open Access Journals (Sweden)

    Vidya P Nair

    2016-04-01

    Full Text Available Hepatitis E virus (HEV causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4. Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp, X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1 and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient

  4. Promising MS2 mediated virus-like particle vaccine against foot-and-mouth disease.

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    Dong, Yan-mei; Zhang, Guo-guang; Huang, Xiao-jun; Chen, Liang; Chen, Hao-tai

    2015-05-01

    Foot-and-mouth disease (FMD) has caused severe economic losses to millions of farmers worldwide. In this work, the coding genes of 141-160 epitope peptide (EP141-160) of VP1 were inserted into the coat protein (CP) genes of MS2 in prokaryotic expression vector, and the recombinant protein self-assembled into virus-like particles (VLP). Results showed that the CP-EP141-160 VLP had a strong immunoreaction with the FMD virus (FMDV) antigen in vitro, and also had an effective immune response in mice. Further virus challenge tests were carried out on guinea pigs and swine, high-titer neutralizing antibodies were produced and the CP-EP141-160 VLP vaccine could protect most of the animals against FMDV.

  5. Hyaluronic acid pretreatment for Sendai virus-mediated cochlear gene transfer.

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    Kurioka, T; Mizutari, K; Niwa, K; Fukumori, T; Inoue, M; Hasegawa, M; Shiotani, A

    2016-02-01

    Gene therapy with viral vectors is one of the most promising strategies for sensorineural hearing loss. However, safe and effective administration of the viral vector into cochlear tissue is difficult because of the anatomical isolation of the cochlea. We investigated the efficiency and safety of round window membrane (RWM) application of Sendai virus, one of the most promising non-genotoxic vectors, after pretreatment with hyaluronic acid (HA) on the RWM to promote efficient viral translocation into the cochlea. Sendai virus expressing the green fluorescent protein reporter gene was detected throughout cochlear tissues following application combined with HA pretreatment. Quantitative analysis revealed that maximum expression was reached 3 days after treatment. The efficiency of transgene expression was several 100-fold greater with HA pretreatment than that without. Furthermore, unlike the conventional intracochlear delivery methods, this approach did not cause hearing loss. These findings reveal the potential utility of gene therapy with Sendai virus and HA for treatment of sensorineural hearing loss.

  6. Transgene-mediated suppression of the RNA interference pathway in Aedes aegypti interferes with gene silencing and enhances Sindbis virus and dengue virus type 2 replication.

    Science.gov (United States)

    Khoo, C C H; Doty, J B; Heersink, M S; Olson, K E; Franz, A W E

    2013-02-01

    RNA interference (RNAi) is the major innate antiviral pathway in Aedes aegypti that responds to replicating arboviruses such as dengue virus (DENV) and Sindbis virus (SINV). On the one hand, the mosquito's RNAi machinery is capable of completely eliminating DENV2 from Ae. aegypti. On the other, transient silencing of key genes of the RNAi pathway increases replication of SINV and DENV2, allowing the viruses to temporally overcome dose-dependent midgut infection and midgut escape barriers (MEB) more efficiently. Here we expressed Flock house virus B2 (FHV-B2) from the poly-ubiquitin (PUb) promoter in Ae. aegypti using the ΦC31 site-directed recombination system to investigate the impact of transgene-mediated RNAi pathway suppression on infections with SINV-TR339eGFP and DENV2-QR94, the latter of which has been shown to be confronted with a strong MEB in Ae. aegypti. FHV-B2 was constitutively expressed in midguts of sugar- and blood-fed mosquitoes of transgenic line PUbB2 P61. B2 over-expression suppressed RNA silencing of carboxypeptidase A-1 (AeCPA-1) in midgut tissue of PUbB2 P61 mosquitoes. Following oral challenge with SINV-TR339eGFP or DENV2-QR94, mean titres in midguts of PUbB2 P61 females were significantly higher at 7 days post-bloodmeal (pbm) than in those of nontransgenic control mosquitoes. At 14 days pbm, infection rates of carcasses were significantly increased in PubB2 P61 mosquitoes infected with SINV-TR339eGFP. Following infection with DENV2-QR94, midgut infection rates were significantly increased in the B2-expressing mosquitoes at 14 days pbm. However, B2 expression in PUbB2 P61 did not increase the DENV2-QR94 dissemination rate, indicating that the infection phenotype was not primarily controlled by RNAi. © 2013 Royal Entomological Society.

  7. Transovarial transmission of a plant virus is mediated by vitellogenin of its insect vector.

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    Yan Huo

    2014-03-01

    Full Text Available Most plant viruses are transmitted by hemipteroid insects. Some viruses can be transmitted from female parent to offspring usually through eggs, but the mechanism of this transovarial transmission remains unclear. Rice stripe virus (RSV, a Tenuivirus, transmitted mainly by the small brown planthopper (Laodelphax striatellus, is also spread to the offspring through the eggs. Here, we used the RSV-planthopper system as a model to investigate the mechanism of transovarial transmission and demonstrated the central role of vitellogenin (Vg of L. striatellus in the process of virus transmission into the eggs. Our data showed Vg can bind to pc3 in vivo and in vitro and colocalize in the germarium. RSV filamentous ribonucleoprotein particles (RNPs only accumulated in the terminal filaments and pedicel areas prior to Vg expression and was not present in the germarium until Vg was expressed, where RSV RNPs and Vg had colocalized. Observations by immunoelectron microscopy (IEM also indicated that these two proteins colocalized in nurse cells. Knockdown of Vg expression due to RNA interference resulted in inhibition of the invasion of ovarioles by RSV. Together, the data obtained indicated that RSV RNPs may enter the nurse cell of the germarium via endocytosis through binding with Vg. Finally, the virus enters the oocytes through nutritive cords, using the same route as for Vg transport. Our results show that the Vg of L. striatellus played a critical role in transovarial transmission of RSV and shows how viruses can use existing transovarial transportation systems in insect vectors for their own purposes.

  8. Bordetella pertussis infection exacerbates influenza virus infection through pertussis toxin-mediated suppression of innate immunity.

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    Victor I Ayala

    Full Text Available Pertussis (whooping cough is frequently complicated by concomitant infections with respiratory viruses. Here we report the effect of Bordetella pertussis infection on subsequent influenza virus (PR8 infection in mouse models and the role of pertussis toxin (PT in this effect. BALB/c mice infected with a wild-type strain of B. pertussis (WT and subsequently (up to 14 days later infected with PR8 had significantly increased pulmonary viral titers, lung pathology and mortality compared to mice similarly infected with a PT-deficient mutant strain (ΔPT and PR8. Substitution of WT infection by intranasal treatment with purified active PT was sufficient to replicate the exacerbating effects on PR8 infection in BALB/c and C57/BL6 mice, but the effects of PT were lost when toxin was administered 24 h after virus inoculation. PT had no effect on virus titers in primary cultures of murine tracheal epithelial cells (mTECs in vitro, suggesting the toxin targets an early immune response to increase viral titers in the mouse model. However, type I interferon responses were not affected by PT. Whole genome microarray analysis of gene expression in lung tissue from PT-treated and control PR8-infected mice at 12 and 36 h post-virus inoculation revealed that PT treatment suppressed numerous genes associated with communication between innate and adaptive immune responses. In mice depleted of alveolar macrophages, increase of pulmonary viral titers by PT treatment was lost. PT also suppressed levels of IL-1β, IL-12, IFN-γ, IL-6, KC, MCP-1 and TNF-α in the airways after PR8 infection. Furthermore PT treatment inhibited early recruitment of neutrophils and NK cells to the airways. Together these findings demonstrate that infection with B. pertussis through PT activity predisposes the host to exacerbated influenza infection by countering protective innate immune responses that control virus titers.

  9. In Vivo Zinc Finger Nuclease-mediated Targeted Integration of a Glucose-6-phosphatase Transgene Promotes Survival in Mice With Glycogen Storage Disease Type IA.

    Science.gov (United States)

    Landau, Dustin J; Brooks, Elizabeth Drake; Perez-Pinera, Pablo; Amarasekara, Hiruni; Mefferd, Adam; Li, Songtao; Bird, Andrew; Gersbach, Charles A; Koeberl, Dwight D

    2016-04-01

    Glycogen storage disease type Ia (GSD Ia) is caused by glucose-6-phosphatase (G6Pase) deficiency in association with severe, life-threatening hypoglycemia that necessitates lifelong dietary therapy. Here we show that use of a zinc-finger nuclease (ZFN) targeted to the ROSA26 safe harbor locus and a ROSA26-targeting vector containing a G6PC donor transgene, both delivered with adeno-associated virus (AAV) vectors, markedly improved survival of G6Pase knockout (G6Pase-KO) mice compared with mice receiving the donor vector alone (P Ia, as compared with normal littermates, at 8 months following vector administration (P Ia.

  10. Heterosubtype neutralizing responses to influenza A (H5N1 viruses are mediated by antibodies to virus haemagglutinin.

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    Jean-Michel Garcia

    Full Text Available BACKGROUND: It is increasingly clear that influenza A infection induces cross-subtype neutralizing antibodies that may potentially confer protection against zoonotic infections. It is unclear whether this is mediated by antibodies to the neuraminidase (NA or haemagglutinin (HA. We use pseudoviral particles (H5pp coated with H5 haemagglutinin but not N1 neuraminidase to address this question. In this study, we investigate whether cross-neutralizing antibodies in persons unexposed to H5N1 is reactive to the H5 haemagglutinin. METHODOLOGY/PRINCIPAL FINDINGS: We measured H5-neutralization antibody titers pre- and post-vaccination using the H5N1 micro-neutralization test (MN and H5pp tests in subjects given seasonal vaccines and in selected sera from European elderly volunteers in a H5N1 vaccine trial who had detectable pre-vaccination H5N1 MN antibody titers. We found detectable (titer > or = 20 H5N1 neutralizing antibodies in a minority of pre-seasonal vaccine sera and evidence of a serological response to H5N1 in others after seasonal influenza vaccination. There was excellent correlation in the antibody titers between the H5N1 MN and H5pp tests. Similar correlations were found between MN and H5pp in the pre-vaccine sera from the cohort of H5N1 vaccine trial recipients. CONCLUSIONS/SIGNIFICANCE: Heterosubtype neutralizing antibody to H5N1 in healthy volunteers unexposed to H5N1 is mediated by cross-reaction to the H5 haemagglutinin.

  11. Pancreatic cell tracing, lineage tagging and targeted genetic manipulations in multiple cell types using pancreatic ductal infusion of adeno-associated viral vectors and/or cell-tagging dyes.

    Science.gov (United States)

    Xiao, Xiangwei; Guo, Ping; Prasadan, Krishna; Shiota, Chiyo; Peirish, Lauren; Fischbach, Shane; Song, Zewen; Gaffar, Iljana; Wiersch, John; El-Gohary, Yousef; Husain, Sohail Z; Gittes, George K

    2014-12-01

    Genetic manipulations, with or without lineage tracing for specific pancreatic cell types, are very powerful t