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Sample records for adeno-associated virus-mediated expression

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

    International Nuclear Information System (INIS)

    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. 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. 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. 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

  2. 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.

  3. Adeno-Associated Virus-Mediated Microdystrophin Expression Protects Young mdx Muscle from Contraction-Induced Injury

    OpenAIRE

    LIU, MINGJU; Yue, Yongping; Harper, Scott Q.; Grange, Robert W.; Jeffrey S. Chamberlain; Duan, Dongsheng

    2005-01-01

    Duchenne muscular dystrophy (DMD) is the most common inherited lethal muscle degenerative disease. Currently there is no cure. Highly abbreviated microdystrophin cDNAs were developed recently for adeno-associated virus (AAV)-mediated DMD gene therapy. Among these, a C-terminal-truncated ΔR4-R23/ΔC microgene (ΔR4/ΔC) has been considered as a very promising therapeutic candidate gene. In this study, we packaged a CMV.ΔR4/ΔC cassette in AAV-5 and evaluated the transduction and muscle contractile...

  4. Optimization of Recombinant Adeno-Associated Virus-Mediated Expression for Large Transgenes, Using a Synthetic Promoter and Tandem Array Enhancers.

    Science.gov (United States)

    Yan, Ziying; Sun, Xingshen; Feng, Zehua; Li, Guiying; Fisher, John T; Stewart, Zoe A; Engelhardt, John F

    2015-06-01

    The packaging capacity of recombinant adeno-associated viral (rAAV) vectors limits the size of the promoter that can be used to express the 4.43-kb cystic fibrosis transmembrane conductance regulator (CFTR) cDNA. To circumvent this limitation, we screened a set of 100-mer synthetic enhancer elements, composed of ten 10-bp repeats, for their ability to augment CFTR transgene expression from a short 83-bp synthetic promoter in the context of an rAAV vector designed for use in the cystic fibrosis (CF) ferret model. Our initial studies assessing transcriptional activity in monolayer (nonpolarized) cultures of human airway cell lines and primary ferret airway cells revealed that three of these synthetic enhancers (F1, F5, and F10) significantly promoted transcription of a luciferase transgene in the context of plasmid transfection. Further analysis in polarized cultures of human and ferret airway epithelia at an air-liquid interface (ALI), as well as in the ferret airway in vivo, demonstrated that the F5 enhancer produced the highest level of transgene expression in the context of an AAV vector. Furthermore, we demonstrated that increasing the size of the viral genome from 4.94 to 5.04 kb did not significantly affect particle yield of the vectors, but dramatically reduced the functionality of rAAV-CFTR vectors because of small terminal deletions that extended into the CFTR expression cassette of the 5.04-kb oversized genome. Because rAAV-CFTR vectors greater than 5 kb in size are dramatically impaired with respect to vector efficacy, we used a shortened ferret CFTR minigene with a 159-bp deletion in the R domain to construct an rAAV vector (AV2/2.F5tg83-fCFTRΔR). This vector yielded an ∼17-fold increase in expression of CFTR and significantly improved Cl(-) currents in CF ALI cultures. Our study has identified a small enhancer/promoter combination that may have broad usefulness for rAAV-mediated CF gene therapy to the airway. PMID:25763813

  5. 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.

  6. Adeno-Associated Virus-Mediated Cancer Gene Therapy: Current Status

    OpenAIRE

    Luo, Jingfeng; Luo, Yuxuan; Sun, Jihong; Zhou, Yurong; Zhang, Yajing; Yang, Xiaoming

    2014-01-01

    Gene therapy is one of the frontiers of modern medicine. Adeno-associated virus (AAV)-mediated gene therapy is becoming a promising approach to treat a variety of diseases and cancers. AAV-mediated cancer gene therapies have rapidly advanced due to their superiority to other gene-carrying vectors, such as the lack of pathogenicity, the ability to transfect both dividing and non-dividing cells, low host immune response, and long-term expression. This article reviews and provides up to date kno...

  7. Adeno-associated virus-mediated delivery of antiangiogenic factors as an antitumor strategy.

    Science.gov (United States)

    Nguyen, J T; Wu, P; Clouse, M E; Hlatky, L; Terwilliger, E F

    1998-12-15

    Antiangiogenic tumor therapies have recently attracted intense interest for their broad-spectrum action, low toxicity, and, in the case of direct endothelial targeting, an absence of drug resistance. To promote tumor regression and to maintain dormancy, antiangiogenic agents need to be chronically administered. Gene therapy offers a potential way to achieve sustained therapeutic release of potent antiangiogenic substances. As a step toward this goal, we have generated recombinant adeno-associated virus (rAAV) vectors that carry genes coding for angiostatin, endostatin, and an antisense mRNA species against vascular endothelial growth factor (VEGF). These rAAVs efficiently transduced three human tumor cell lines tested. Transduction with an rAAV-encoding antisense VEGF mRNA inhibited the production of endogenous tumor cell VEGF. Conditioned media from cells transduced with this rAAV or with rAAV-expressing endostatin or angiostatin inhibited capillary endothelial cell proliferation in vitro. Antiangiogenic rAAVs may offer a novel gene therapy approach to undermining tumor neovascularization and cancer progression. PMID:9865720

  8. Translational data from adeno-associated virus-mediated gene therapy of hemophilia B in dogs.

    Science.gov (United States)

    Nichols, Timothy C; Whitford, Margaret H; Arruda, Valder R; Stedman, Hansell H; Kay, Mark A; High, Katherine A

    2015-03-01

    Preclinical testing of new therapeutic strategies in relevant animal models is an essential part of drug development. The choice of animal models of disease that are used in these studies is driven by the strength of the translational data for informing about safety, efficacy, and success or failure of human clinical trials. Hemophilia B is a monogenic, X-linked, inherited bleeding disorder that results from absent or dysfunctional coagulation factor IX (FIX). Regarding preclinical studies of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B, dogs with severe hemophilia B (recombinant AAV vector are all feasible end points in these dogs. This review compares the preclinical studies of AAV vectors used to treat dogs with hemophilia B with the results obtained in subsequent human clinical trials using muscle- and liver-based approaches. PMID:25675273

  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. Adeno-associated virus-mediated heme oxygenase-1 gene transfer suppresses the progression of micronodular cirrhosis in rats

    Institute of Scientific and Technical Information of China (English)

    Tung-Yu Tsui; Chi-Keung Lau; Jian Ma; Gabriel Glockzin; Aiman Obed; Hans J Schlitt; Sheung-Tat Fan

    2006-01-01

    AIM: To test the hypothesis that enhancement of the activity of heme oxygenase can interfere with processes of fibrogenesis associated with recurrent liver injury, we investigated the therapeutic potential of over-expression of heme oxygense-1 in a CCl4-induced micronodular cirrhosis model.METHODS: Recombinant adeno-associated viruses carrying rat HO-1 or GFP gene were generated. 1x1012 vg of adeno-associated viruses were administered through portal injection at the time of the induction of liver fibrosis.RESULTS: Conditioning the rat liver with over-expression of HO-1 by rAAV/HO-1 significantly increased the HO enzymatic activities in a stable manner. The development of micronodular cirrhosis was significantly inhibited in rAAV/HO-1-transduced animals as compared to controls. Portal hypertension was markedly diminished in rAAV/HO-1-transduced animals as compared to controis, whereas there are no significant changes in systolic blood pressure. This finding was accompanied with improved liver biochemistry, less infiltrating macrophages and less activated hepatic stellate cells (HSCs) in rAAV/HO-1-transduced livers.CONCLUSIONS: Enhancement of HO activity in the livers suppresses the development of cirrhosis.

  11. Adeno-associated virus-mediated brain delivery of 5-lipoxygenase modulates the AD-like phenotype of APP mice

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    Chu Jin

    2012-01-01

    Full Text Available Abstract Background The 5-lipoxygenase (5LO enzymatic pathway is widely distributed within the central nervous system. Previous works showed that this protein is up-regulated in Alzheimer's disease (AD, and that its genetic absence results in a reduction of Amyloid beta (Aβ levels in the Tg2576 mice. Here by employing an adeno-associated viral (AAV vector system to over-express 5LO in the same mouse model, we examined its contribution to their cognitive impairments and brain AD-like amyloid pathology. Results Our results showed that compared with controls, 5LO-targeted gene brain over-expression in Tg2576 mice results in significant memory deficits. On the other hand, brain tissues had a significant elevation in the levels of Aβ peptides and deposition, no change in the steady state levels of amyloid-β precursor protein (APP, BACE-1 or ADAM-10, but a significant increase in PS1, nicastrin, and Pen-2, three major components of the γ-secretase complex. Additional data indicate that the transcription factor CREB was elevated and so were the mRNA levels for PS1, nicastrin and Pen-2. Conclusions These data demonstrate that neuronal 5LO plays a functional role in the pathogenesis of AD-like amyloidotic phenotype by modulating the γ-secretase pathway. They support the hypothesis that this enzyme is a novel therapeutic target for the treatment and prevention of AD.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. Adeno-associated virus-mediated bone morphogenetic protein-7 gene transfer induces C2C12 cell differentiation into osteoblast lineage cells

    Institute of Scientific and Technical Information of China (English)

    Min YANG; Qing-jun MA; Geng-ting DANG; Kang-tao MA; Ping CHEN; Chun-yan ZHOU

    2005-01-01

    Aim: To investigate the effects of bone morphogenetic protein-7 (BMP7)-expressing recombinant adeno-associated virus (AAV) vector on the differentiation of C2C12 cells. Methods: AAV-BMP7 was packaged by infecting the stable cell clone BHK-21 (integrated with recombinant AAV vector plasmid pSNAV-BMP7)with recombinant herpes simplex virus type 1, which expresses AAV-2 Rep and Cap and possesses AAV packaging functions. Following infection with AAVBMP7 at multiplicities of infection of 1× 105 vector genomes per cell and subsequent culture, C2C12 cells were assessed qualitatively for BMP7 production, alkaline phosphatase activity, osteocalcin production and Cbfal and MyoD expression.Results: C2C 12 cells transduced with AAV-BMP7 could produce BMP7 protein until d 28. Alkaline phosphatase in the cultured C2C12 cell lysate was elevated.Secreted osteocalcin in the culture medium was detectable at d 12 and Cbfal mRNA expression level was upregulated, coinciding with downregulation of MyoD in a temporal manner. Conclusion: The present in vitro study demonstrated that AAV-BMP7 could infect and efficiently convert C2C12 cells from myoblasts into osteoblast lineage cells.

  19. Recombinant adeno-associated virus-mediated gene transfer for the potential therapy of adenosine deaminase-deficient severe combined immune deficiency.

    Science.gov (United States)

    Silver, Jared N; Elder, Melissa; Conlon, Thomas; Cruz, Pedro; Wright, Amy J; Srivastava, Arun; Flotte, Terence R

    2011-08-01

    Severe combined immune deficiency due to adenosine deaminase (ADA) deficiency is a rare, potentially fatal pediatric disease, which results from mutations within the ADA gene, leading to metabolic abnormalities and ultimately profound immunologic and nonimmunologic defects. In this study, recombinant adeno-associated virus (rAAV) vectors based on serotypes 1 and 9 were used to deliver a secretory version of the human ADA (hADA) gene to various tissues to promote immune reconstitution following enzyme expression in a mouse model of ADA deficiency. Here, we report that a single-stranded rAAV vector, pTR2-CB-Igκ-hADA, (1) facilitated successful gene delivery to multiple tissues, including heart, skeletal muscle, and kidney, (2) promoted ectopic expression of hADA, and (3) allowed enhanced serum-based enzyme activity over time. Moreover, the rAAV-hADA vector packaged in serotype 9 capsid drove partial, prolonged, and progressive immune reconstitution in ADA-deficient mice. Overview Summary Gene therapies for severe combined immune deficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) over two decades have exclusively involved retroviral vectors targeted to lymphocytes and hematopoietic progenitor cells. These groundbreaking gene therapies represented an unprecedented revolution in clinical medicine but in most cases did not fully correct the immune deficiency and came with the potential risk of insertional mutagenesis. Alternatively, recombinant adeno-associated virus (rAAV) vectors have gained attention as valuable tools for gene transfer, having demonstrated no pathogenicity in humans, minimal immunogenicity, long-term efficacy, ease of administration, and broad tissue tropism (Muzyczka, 1992 ; Flotte et al., 1993 ; Kessler et al., 1996 ; McCown et al., 1996 ; Lipkowitz et al., 1999 ; Marshall, 2001 ; Chen et al., 2003 ; Conlon and Flotte, 2004 ; Griffey et al., 2005 ; Pacak et al., 2006 ; Stone et al., 2008 ; Liu et al., 2009 ; Choi et al., 2010

  20. Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors

    OpenAIRE

    Lai, Yi; Yue, Yongping; LIU, MINGJU; Ghosh, Arkasubhra; Engelhardt, John F.; Jeffrey S. Chamberlain; Duan, Dongsheng

    2005-01-01

    Although adeno-associated virus (AAV)-mediated gene therapy has been hindered by the small viral packaging capacity of the vector, trans-splicing AAV vectors are able to package twice the size of the vector genome. Unfortunately, the efficiency of current trans-splicing vectors is very low. Here we show that rational design of the gene splitting site has a profound influence on trans-splicing vector-mediated gene expression. Using mRNA accumulation as a guide, we generated a set of efficient ...

  1. 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.

  2. 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.

  3. Recombinant adeno-associated virus vector expressing angiostatin inhibits preretinal neovascularization in adult rats.

    Science.gov (United States)

    Lai, Chi-Chun; Wu, Wei-Chi; Chen, Show-Li; Sun, Ming-Hui; Xiao, Xiao; Ma, Lih; Lin, Keng-Kuo; Tsao, Yeou-Ping

    2005-01-01

    Clinically, preretinal neovascularization (PNV) induced by vessel occlusion is one of the leading causes to induce blindness. The present study was designed to determine if a recombinant adeno-associated viral vector expressing mouse angiostatin (rAAV-angiostatin) can inhibit experimental PNV in an adult Sprague-Dawley rat model. rAAV-angiostatin and rAAV-lacZ were delivered by intravitreal injections to the right and left eyes of rats. Transgenetic expression of angiostatin in the retina was determined by reverse-transcriptase polymerase chain reaction (RT-PCR). PNV was established by rose-bengal-assisted laser-induced retinal vein occlusion 21 days after the viral injections. The total number and sizes of the neovascular tufts were analyzed 14 days after venous occlusion using retinal flat mount by fluorescein-isothiocyanate-dextran angiography. Electroretinograms (ERGs) were recorded to study any possibility of retinal toxicity of rAAV-angiostatin 3 months after the injections. Angiostatin gene expression in the retina was detectable by RT-PCR, and ERG analysis showed no reduction of b-waves in the rAAV-angiostatin-injected eyes. The number and size of neovascular tufts were significantly lower in rAAV-angiostatin-injected eyes (p = 0.001) than controls. These findings indicated that rAAV-angiostatin successfully suppressed experimental PNV, and no retinal toxicity of the rAAV-angiostatin injection was observed according to ERG recordings. PMID:15637422

  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. 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

    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...

  6. Persistence, Localization, and External Control of Transgene Expression After Single Injection of Adeno-Associated Virus into Injured Joints

    OpenAIRE

    Lee, Hannah H.; O'Malley, Michael J.; Friel, Nicole A.; Payne, Karin A.; Qiao, Chunping; Xiao, Xiao(Institute for Strings, Cosmology and Astroparticle Physics (ISCAP) and Physics Department, Columbia University, 538 West 120th Street, New York, NY, 10027 U.S.A.); Chu, Constance R.

    2013-01-01

    A single intra-articular injection of adeno-associated virus (AAV) results in stable and controllable transgene expression in normal rat knees. Because undamaged joints are unlikely to require treatment, the study of AAV delivery in joint injury models is crucial to potential therapeutic applications. This study tests the hypotheses that persistent and controllable AAV-transgene expression are (1) highly localized to the cartilage when AAV is injected postinjury and (2) localized to the intra...

  7. 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.

  8. Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids.

    Science.gov (United States)

    Chamberlain, Kyle; Riyad, Jalish Mahmud; Weber, Thomas

    2016-02-01

    Recombinant adeno-associated virus vectors (rAAV) are being explored as gene delivery vehicles for the treatment of various inherited and acquired disorders. rAAVs are attractive vectors for several reasons: wild-type AAVs are nonpathogenic, and rAAVs can trigger long-term transgene expression even in the absence of genome integration-at least in postmitotic tissues. Moreover, rAAVs have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms. One limitation of rAAVs is that their genome-packaging capacity is only ∼5 kb. For most applications this is not of major concern because the median human protein size is 375 amino acids. Excluding the ITRs, for a protein of typical length, this allows the incorporation of ∼3.5 kb of DNA for the promoter, polyadenylation sequence, and other regulatory elements into a single AAV vector. Nonetheless, for certain diseases the packaging limit of AAV does not allow the delivery of a full-length therapeutic protein by a single AAV vector. Hence, approaches to overcome this limitation have become an important area of research for AAV gene therapy. Among the most promising approaches to overcome the limitation imposed by the packaging capacity of AAV is the use of dual-vector approaches, whereby a transgene is split across two separate AAV vectors. Coinfection of a cell with these two rAAVs will then-through a variety of mechanisms-result in the transcription of an assembled mRNA that could not be encoded by a single AAV vector because of the DNA packaging limits of AAV. The main purpose of this review is to assess the current literature with respect to dual-AAV-vector design, to highlight the effectiveness of the different methodologies and to briefly discuss future areas of research to improve the efficiency of dual-AAV-vector transduction. PMID:26757051

  9. Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression In mammalian cells and mice

    Directory of Open Access Journals (Sweden)

    Fussenegger Martin

    2007-11-01

    Full Text Available Abstract Background Adjustable gene expression is crucial in a number of applications such as de- or transdifferentiation of cell phenotypes, tissue engineering, various production processes as well as gene-therapy initiatives. Viral vectors, based on the Adeno-Associated Virus (AAV type 2, have emerged as one of the most promising types of vectors for therapeutic applications due to excellent transduction efficiencies of a broad variety of dividing and mitotically inert cell types and due to their unique safety features. Results We designed recombinant adeno-associated virus (rAAV vectors for the regulated expression of transgenes in different configurations. We integrated the macrolide-responsive E.REX systems (EON and EOFF into rAAV backbones and investigated the delivery and expression of intracellular as well as secreted transgenes for binary set-ups and for self- and auto-regulated one-vector configurations. Extensive quantitative analysis of an array of vectors revealed a high level of adjustability as well as tight transgene regulation with low levels of leaky expression, both crucial for therapeutical applications. We tested the performance of the different vectors in selected biotechnologically and therapeutically relevant cell types (CHO-K1, HT-1080, NHDF, MCF-7. Moreover, we investigated key characteristics of the systems, such as reversibility and adjustability to the regulating agent, to determine promising candidates for in vivo studies. To validate the functionality of delivery and regulation we performed in vivo studies by injecting particles, coding for compact self-regulated expression units, into mice and adjusting transgene expression. Conclusion Capitalizing on established safety features and a track record of high transduction efficiencies of mammalian cells, adeno- associated virus type 2 were successfully engineered to provide new powerful tools for macrolide-adjustable transgene expression in mammalian cells as well as

  10. 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

  11. Adeno-Associated Viral-Mediated Catalase Expression Suppresses Optic Neuritis in Experimental Allergic Encephalomyelitis

    Science.gov (United States)

    Guy, John; Qi, Xiaoping; Hauswirth, William W.

    1998-11-01

    Suppression of oxidative injury by viral-mediated transfer of the human catalase gene was tested in the optic nerves of animals with experimental allergic encephalomyelitis (EAE). EAE is an inflammatory autoimmune disorder of primary central nervous system demyelination that has been frequently used as an animal model for the human disease multiple sclerosis (MS). The optic nerve is a frequent site of involvement common to both EAE and MS. Recombinant adeno-associated virus containing the human gene for catalase was injected over the right optic nerve heads of SJL/J mice that were simultaneously sensitized for EAE. After 1 month, cell-specific catalase activity, evaluated by quantitation of catalase immunogold, was increased approximately 2-fold each in endothelia, oligodendroglia, astrocytes, and axons of the optic nerve. Effects of catalase on the histologic lesions of EAE were measured by computerized analysis of the myelin sheath area (for demyelination), optic disc area (for optic nerve head swelling), extent of the cellular infiltrate, extravasated serum albumin labeled by immunogold (for blood-brain barrier disruption), and in vivo H2O2 reaction product. Relative to control, contralateral optic nerves injected with the recombinant virus without a therapeutic gene, catalase gene inoculation reduced demyelination by 38%, optic nerve head swelling by 29%, cellular infiltration by 34%, disruption of the blood-brain barrier by 64%, and in vivo levels of H2O2 by 61%. Because the efficacy of potential treatments for MS are usually initially tested in the EAE animal model, this study suggests that catalase gene delivery by using viral vectors may be a therapeutic strategy for suppression of MS.

  12. A single injection of recombinant adeno-associated virus into the lumbar cistern delivers transgene expression throughout the whole spinal cord

    OpenAIRE

    Guo, Yansu; Wang, Dan; Qiao, Tao; Yang, Chunxing; Su, Qin; Gao, Guangping; Xu, Zuoshang

    2015-01-01

    The lack of methods to deliver transgene expression in spinal cord has hampered investigation of gene function and therapeutic targets for spinal cord diseases. Here we report that a single intrathecal injection of recombinant adeno-associated virus rhesus-10 (rAAVrh10) into the lumbar cistern led to transgene expression in sixty to ninety percent of the cells in the spinal cord. The transgene was expressed in all cell types, including neurons, glia, ependymal cells and endothelial cells. Add...

  13. Persistence, localization, and external control of transgene expression after single injection of adeno-associated virus into injured joints.

    Science.gov (United States)

    Lee, Hannah H; O'Malley, Michael J; Friel, Nicole A; Payne, Karin A; Qiao, Chunping; Xiao, Xiao; Chu, Constance R

    2013-04-01

    A single intra-articular injection of adeno-associated virus (AAV) results in stable and controllable transgene expression in normal rat knees. Because undamaged joints are unlikely to require treatment, the study of AAV delivery in joint injury models is crucial to potential therapeutic applications. This study tests the hypotheses that persistent and controllable AAV-transgene expression are (1) highly localized to the cartilage when AAV is injected postinjury and (2) localized to the intra-articular soft tissues when AAV is injected preinjury. Two AAV injection time points, postinjury and preinjury, were investigated in osteochondral defect and anterior cruciate ligament transection models of joint injury. Rats injected with AAV tetracycline response element (TRE)-luciferase received oral doxycycline for 7 days. Luciferase expression was evaluated longitudinally for 6 months. Transgene expression was persistent and controllable with oral doxycycline for 6 months in all groups. However, the location of transgene expression was different: postinjury AAV-injected knees had luciferase expression highly localized to the cartilage, while preinjury AAV-injected knees had more widespread signal from intra-articular soft tissues. The differential transgene localization between preinjury and postinjury injection can be used to optimize treatment strategies. Highly localized postinjury injection appears advantageous for treatments targeting repair cells. The more generalized and controllable reservoir of transgene expression following AAV injection before anterior cruciate ligament transection (ACLT) suggests an intriguing concept for prophylactic delivery of joint protective factors to individuals at high risk for early osteoarthritis (OA). Successful external control of intra-articular transgene expression provides an added margin of safety for these potential clinical applications. PMID:23496155

  14. Construction of genetically engineered macrophages expressing Smad6 and Smad7 genes with adeno-associated virus

    Institute of Scientific and Technical Information of China (English)

    黄云剑; 赵景宏; 杨唐俊; 范晓棠; 张金海; 蔡文琴

    2004-01-01

    Objective: To construct the genetically engineered macrophages expressing Smad6 and Smad7 genes with adeno-associated virus (AAV). Methods: The plasmids containing pcDNA3-Smad6/Flag and pcDNA3-Smad7/Flag were digested with BamH Ⅰ and Xho Ⅰ , respectively. Then the Smad6/Flag and Smad7/Flag gene segments obtained were cloned into plasmid pAAV-MCS respectively to construct the recombinant pAAV-Smad6/Flag and pAAV-Smad7/Flag plasmids. The resulting recombinant plasmids (pAAV-Smad6/Flag or pAAV-Smad7/Flag) or pAAV-LacZ plasmid were co-transfected into the HEK 293cells with pHelper and pAAV-RC by calcium-phosphate precipitation method. Recombinant AAV-2 viral particles were prepared from infected HEK293 cells and then were used to infect mouse macrophages. The expressions of Smad6and Smad7 in macrophages were detected by immunocytochemical staining and expression of b-galactosidase was evaluated by X-gal staining. Results: The recombinant AAV vector containing Smad6 or Smad7 genes was successfully constructed. More than 95% macrophage cells expressed X-gal and Smad6 and Smad7 genes at 72 h after infection. Conclusion: These results indicate that the genetically engineered macropbages can express Smad6 and Smad7 proteins effectively, laying the foundation for the studies of TGF-β-induced diseases in vivo and highlighting the feasibility of macrophage-based gene therapy.

  15. 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.

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

    Science.gov (United States)

    Vidović, Dragana; Gijsbers, Rik; Jimenez, Ana Quiles; Dooley, James; Van den Haute, Chris; Van der Perren, Anke; Liston, Adrian; Baekelandt, Veerle; Debyser, Zeger; Carlon, Marianne Sylvia

    2016-01-01

    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 approach for pulmonary gene transfer. Only limited clinical successes have been achieved for airway gene transfer so far, underscoring the need for further preclinical development of rAAV-based gene therapy for pulmonary disorders. We sought to determine the preclinical potential of an airway-tropic serotype, rAAV2/5, encoding reporter genes when delivered to mouse airways. Although several groups have assessed the stability of gene transfer using a nonintegrating rAAV in mouse airways, long-term stability for more than a year has not been reported. Additionally, an extensive quantitative analysis of the specific cell types targeted by rAAV2/5 using cell-specific markers is lacking. We obtained sustained gene expression in upper and lower airways up to 15 months after vector administration, a substantial proportion of the lifespan of a laboratory mouse. In addition, we demonstrated that readministration of rAAV2/5 to the airways is feasible and increases gene expression 14 months after primary vector administration, despite the presence of circulating neutralizing antibodies. Finally, identification of transduced cell types revealed different subpopulations being targeted by rAAV2/5, with 64% of β-galactosidase-positive cells being ciliated cells, 34% club cells in the conducting airways, and 75% alveolar type II cells in the alveoli at 1 month postinjection. This underscores the therapeutic potential of a nonintegrating rAAV vector to develop a gene therapeutic drug for a variety of pulmonary disorders, such as cystic fibrosis, primary ciliary dyskinesia, and surfactant deficiencies. PMID:26567984

  17. 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; Barry, Michael A

    2014-09-01

    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

  18. 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.

  19. 腺相关病毒介导重组血管抑素联合雷公藤红素对大鼠颅内C6胶质瘤的抗血管生成作用%Anti-angiogenesis effect of adeno-associated virus-mediated recombinant angiostatin combined with celastrol on intracranial C6 glioma in rats

    Institute of Scientific and Technical Information of China (English)

    王冠; 周洁; 冯珂珂; 田麒

    2011-01-01

    目的:腺相关病毒(adeno-associated virus,AAV)介导的重组血管抑素(angiostatin,AS)联合应用雷公藤红素( celastrol)治疗大鼠颅内C6胶质瘤,观察其对肿瘤体积、新生血管密度及肿瘤细胞凋亡的影响,探讨抗血管生成重组基因联合雷公藤红素对胶质瘤治疗的前景.方法:建立颅内原位荷C6脑胶质瘤大鼠模型,7d后随机分为4组,分别给予0.9%氯化钠溶液(作为对照)、AAV-AS、雷公藤红素及两者联合用药.每隔7d行头部强化MRI检查,计算肿瘤体积.于22 d后处死动物,检测AS蛋白表达、血管密度及肿瘤细胞凋亡情况.结果:联合治疗组及AAV-AS治疗组均检测到AS蛋白表达,证实基因转导成功.联合治疗组第22天时肿瘤体积、血管密度和凋亡指数均与对照组、雷公藤红素组及AAV-AS治疗组相比差异有统计学意义(P<0.05),联合治疗可以抑制肿瘤生长,降低新生血管密度,促进肿瘤细胞凋亡.结论:基因治疗联合雷公藤红素可通过抑制胶质瘤血管生成而抑制肿瘤生长;两者联合应用具有协同作用,可弥补两者单独应用的不足之处.%Objective: To examine the effects of therapeutic alliance of adeno-associated virus-mediated recombinant angiostatin (AAV-AS) combined with celastrol on tumor growth, microvessel density and apoptosis of intracranial glioma in rats, and to give a prospective of this therapeutic alliance. Methods: A rat intracranial C6 glioma model was established, and then the rats (n=40) were randomly assigned into four groups after 7 days, which were saline control group, AAV-AS group, celastrol group and therapeutic alliance group. The tumor growth was examined by magnetic resonance imaging (MRI) every 7 days, and the volume of tumor was calculated. The rats were killed after 22 days, and the expression of AS protein, the microvessel density and the apoptosis of tumor cells were detected. Results: The expression of AS protein was detectable in AAV

  20. 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...... as striatal input and output areas, including large parts of the cortex. In both species, rAAV5 resulted in a more widespread transgene expression compared to rAAV1. In neonatal rats, rAAV5 also transduced several other areas such as the olfactory bulbs, hippocampus, and septum. Phenotypic analysis of the GFP...

  1. 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...

  2. 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.

  3. 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...... to study effects of genetic manipulation in this non-primate large animal species. Finally, we generated an atlas of the Göttingen minipig brain for guiding future studies in this large animal species....

  4. High-efficiency transduction and specific expression of ChR2opt for optogenetic manipulation of primary cortical neurons mediated by recombinant adeno-associated viruses.

    Science.gov (United States)

    Jin, Lei; Lange, Wienke; Kempmann, Annika; Maybeck, Vanessa; Günther, Anne; Gruteser, Nadine; Baumann, Arnd; Offenhäusser, Andreas

    2016-09-10

    In recent years, optogenetic approaches have significantly advanced the experimental repertoire of cellular and functional neuroscience. Yet, precise and reliable methods for specific expression of optogenetic tools remain challenging. In this work, we studied the transduction efficiency of seven different adeno-associated virus (AAV) serotypes in primary cortical neurons and revealed recombinant (r) AAV6 to be the most efficient for constructs under control of the cytomegalovirus (CMV) promoter. To further specify expression of the transgene, we exchanged the CMV promoter for the human synapsin (hSyn) promoter. In primary cortical-glial mixed cultures transduced with hSyn promoter-containing rAAVs, expression of ChR2opt (a Channelrhodopsin-2 variant) was limited to neurons. In these neurons action potentials could be reliably elicited upon laser stimulation (473nm). The use of rAAV serotype alone to restrict expression to neurons results in a lower transduction efficiency than the use of a broader transducing serotype with specificity conferred via a restrictive promoter. Cells transduced with the hSyn driven gene expression were able to elicit action potentials with more spatially and temporally accurate illumination than neurons electrofected with the CMV driven construct. The hSyn promoter is particularly suited to use in AAVs due to its small size. These results demonstrate that rAAVs are versatile tools to mediate specific and efficient transduction as well as functional and stable expression of transgenes in primary cortical neurons. PMID:27416794

  5. 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.``

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Development of Recombinant Adeno-Associated Virus Serotype 2/8 Carrying Kringle Domains of Human Plasminogen for Sustained Expression and Cancer Therapy.

    Science.gov (United States)

    Kuo, Cheng-Hsiang; Chang, Bi-Ing; Lee, Fang-Tzu; Chen, Po-Ku; Lee, Jeng-Shin; Shi, Guey-Yueh; Wu, Hua-Lin

    2015-09-01

    Angiostatin and other plasminogen derivatives exhibit antitumor activities directly or indirectly, have demonstrated promising anticancer effects in preclinical studies, but have mostly failed in clinical trials partly due to their short serum half-lives. Our previous studies demonstrated that recombinant human plasminogen kringle 1-5 (K1-5) has superior antitumor activity compared with angiostatin. In addition, optimization of recombinant K1-5 with three amino acid substitutions enhances its antitumor effect. The current study was thus undertaken to evaluate prolonged expression of optimized K1-5 as cancer gene therapy. The recombinant adeno-associated virus (AAV) vector was used to express a secreted form of the optimized K1-5 (AAV-sK15tm) to improve its pharmacokinetic profile, which was considered to be the hurdle in angiostatin treatment of cancer. We successfully generated high-titer recombinant AAV vectors and observed sustained transgene expression for 567 days after a single injection of virus. The treated animals did not display any visible signs of abnormalities and showed normal serum biochemistry. The therapeutic potential of this treatment modality was demonstrated by both a strong inhibition of lung metastasis in the mouse B16F10 melanoma model and significant growth retardation of Lewis lung carcinoma xenografts in C57BL/6N mice as well as human A2058 melanoma xenografts in NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice. Taken together, our results suggested that AAV-sK15tm produced long-term suppressive effects on cancer growth in vivo and should warrant serious consideration for clinical development. PMID:25950911

  9. 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.

  10. Neuronal tolerance to hypoxia-ischemia through recombinant adeno-associated viral vectors expressing neuronal and inducible nitric oxide synthase An in vivo study

    Institute of Scientific and Technical Information of China (English)

    Chunmei Chen; Weizhong Yang; Chunhua Wang; Songsheng Shi; Jianping Chen; Yong Huang; Dongsheng Cai

    2008-01-01

    BACKGROUND:Studies have confirmed that neuronal nitric oxide synthase(nNOS)mediates neurotoxic effects during the early stages of hypoxia-ischemia,while inducible nitric oxide synthase(iNOS)mediates delayed neurotoxicity during advanced stages of hypoxia-ischemia.OBJECTIVE:This study was designed to observe neuronal apoptosis and the expressions of nNOS,iNOS, p38 mitogen-activated protein kinase(MAPK),and caspase-3 mRNA following transfection of recombinant adeno-associated viral vectors separately expressing nNOS and iNOS antisense(rAAV-AsnNOS and rAVV-AsiNOS,respectively)into rat brains subjected to cerebral ischemia; to analyze mechanisms underlying elevated neuronal tolerance to hypoxia-ischemia.DESIGN:A randomized controlled in vivo experiment.SETTING:Fujian Institute of Neurosurgery & Department of Neurosurgery,Union Hospital,Fujian Medical University. MATERIALS:Eighty healthy adult male Sprague Dawley rats of clean grade were provided by the Zhejiang Laboratory Animal Center,China.The protocol was performed in accordance with ethical guidelines for the use and care of animals.The following vectors,rAAV-AsnNOS,rAAV-AsiNOS,and rAAV expressing the β-galactosidase gene(rAAV-LacZ),were successfully constructed by Fujian Institute of Neurosurgery. Rabbit anti-mouse nitrotyrosine(NT)monoclonal antibody(Zhongshan Jinqiao Biotechnology Co.,Ltd.,Beijing,China)and reverse transcription-polymerase chain reaction(RT-PCR)kit (two-step method)(Promega Company,USA)were used in this study.METHODS:This study was performed at the Fujian Institute of Neurosurgery in December 2003.Sixty rats were randomly divided into 3 groups,with 20 rats in each group:rAAV-AsnNOS group,rAAV-AsiNOS group,and rAAV-LacZ group.The remaining 20 rats served as controls.Pre-treated viral vectors (rAAV-AsnNOS,rAAV-AsiNOS,and rAAV-LacZ,respectively; each 50 μ L,virus titer of 2x109 viral particles/mL)were transfected into the cerebral cortex of the targeted.Phosphate buffer saline(50 μ L

  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. 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.

  13. 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

    Science.gov (United States)

    Zhu, Hongqing; Cao, Maohua; Mirandola, Leonardo; Figueroa, Jose A.; Cobos, Everardo; Chiriva-Internati, Maurizio; Hermonat, Paul L.

    2014-01-01

    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. PMID:24736312

  14. Regression of Schwannomas Induced by Adeno-Associated Virus-Mediated Delivery of Caspase-1

    OpenAIRE

    Prabhakar, Shilpa; Taherian, Mehran; Gianni, Davide; Conlon, Thomas J.; Fulci, Giulia; Brockmann, Jillian; Stemmer-Rachamimov, Anat; Sena-Esteves, Miguel; Breakefield, Xandra O.; Brenner, Gary J.

    2012-01-01

    Schwannomas are tumors formed by proliferation of dedifferentiated Schwann cells. Patients with neurofibromatosis 2 (NF2) and schwannomatosis develop multiple schwannomas in peripheral and cranial nerves. Although benign, these tumors can cause extreme pain and compromise sensory/motor functions, including hearing and vision. At present, surgical resection is the main treatment modality, but it can be problematic because of tumor inaccessibility and risk of nerve damage. We have explored gene...

  15. Adeno-associated virus for cystic fibrosis gene therapy

    Directory of Open Access Journals (Sweden)

    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.

  16. Enhancement of Muscle Gene Delivery with Pseudotyped Adeno-Associated Virus Type 5 Correlates with Myoblast Differentiation

    OpenAIRE

    Duan, Dongsheng; Yan, Ziying; Yue, Yongping; Ding, Wei; Engelhardt, John F.

    2001-01-01

    Adeno-associated virus (AAV)-based muscle gene therapy has achieved tremendous success in numerous animal models of human diseases. Recent clinical trials with this vector have also demonstrated great promise. However, to achieve therapeutic benefit in patients, large inocula of virus will likely be necessary to establish the required level of transgene expression. For these reasons, efforts aimed at increasing the efficacy of AAV-mediated gene delivery to muscle have the potential for improv...

  17. 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

  18. ADENO-ASSOCIATED SATELLITE VIRUS INTERFERENCE WITH THE REPLICATION OF ITS HELPER ADENOVIRUS

    Science.gov (United States)

    Parks, Wade P.; Casazza, Anna M.; Alcott, Judith; Melnick, Joseph L.

    1968-01-01

    Adeno-associated satellite virus type 4 interferes with the replication of its helper adenovirus. No interferon-like soluble substance could be detected in satellite-infected cultures and other DNA- and RNA-containing viruses were not inhibited by coinfection with satellite virus under conditions which reduced adenovirus yields by more than 90% in monkey cells. Altering the concentration of adenovirus in the presence of constant amounts of satellite resulted in a constant degree of interference over a wide range of adenovirus inocula and suggested that adenovirus concentration was not a significant factor in the observed interference. The interference with adenovirus replication was abolished by pretreating satellite preparations with specific antiserum, ultraviolet light or heating at 80°C for 30 min. This suggested that infectious satellite virus mediated the interference. Satellite virus concentration was found to be a determinant of interference and studies indicated that the amount of interference with adenovirus was directly proportional to the concentration of satellite virus. 8 hr after adenovirus infection, the replication of adenovirus was no longer sensitive to satellite interference. This was true even though the satellite virus was enhanced as effectively as if the cells were infected simultaneously with both viruses. Interference with adenovirus infectivity was accompanied by reduced yields of complement-fixing antigen and of virus particles which suggested that satellite virus interfered with the formation and not the function of adenovirus products. When cells were infected either with adenovirus alone or with adenovirus plus satellite, the same proportion of cells plated as adenovirus infectious centers. However, the number of plaque-forming units of adenovirus formed per cell in the satellite-infected cultures was reduced by approximately 90%, the same magnitude of reduction noted in whole cultures coinfected with satellite and adenovirus. This

  19. 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.

  20. 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.

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

    Institute of Scientific and Technical Information of China (English)

    Fan Dongsheng (樊东升); Shen Yang(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.

  2. 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.

  3. Novel Transcriptional Regulatory Signals in the Adeno-Associated Virus Terminal Repeat A/D Junction Element

    OpenAIRE

    Haberman, Rebecca P.; McCown, Thomas J.; Samulski, Richard Jude

    2000-01-01

    Adeno-associated virus (AAV) type 2 vectors transfer stable, long-term gene expression to diverse cell types in vivo. Many gene therapy applications require the control of long-term transgene expression, and AAV vectors, similar to other gene transfer systems, are being evaluated for delivery of regulated gene expression cassettes. Previously, we (R. P. Haberman, T. J. McCown, and R. J. Samulski, Gene Ther. 5:1604–1611, 1998) demonstrated the use of the tetracycline-responsive system for long...

  4. 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.

  5. Therapeutic Liabilities of in Vivo Viral Vector Tropism: Adeno-Associated Virus Vectors, NMDAR1 Antisense, and Focal Seizure Sensitivity

    OpenAIRE

    Haberman, Rebecca P.; Criswell, Hugh E.; Snowdy, Stephen; Ming, Zhen; Breese, George R.; Samulski, R. Jude; McCown, Thomas J.

    2002-01-01

    The N-methyl-d-aspartic acid (NMDA) receptor provides a potential target for gene therapy of focal seizure disorders. To test this approach, we cloned a 729-bp NMDA receptor (NMDAR1) cDNA fragment in the antisense orientation into adeno-associated virus (AAV) vectors, where expression was driven by either a tetracycline-off regulatable promoter (AAV-tTAK-NR1A) or a cytomegalovirus (CMV) promoter (AAV-CMV-NR1A). After infection of primary cultured cortical neurons with recombinant AAV-tTAK-NR1...

  6. Reduction of experimental diabetic vascular leakage by delivery of angiostatin with a recombinant adeno-associated virus vector

    OpenAIRE

    Shyong, Mong-Ping; Lee, Fenq-Lih; Kuo, Ping-Chang; Wu, Ai-Ching; Cheng, Huey-Chung; Chen, Show-Li; Tung, Tao-Hsin; Tsao, Yeou-Ping

    2007-01-01

    Purpose To evaluate the efficacy of recombinant adeno-associated virus (rAAV) vector expressing mouse angiostatin (Kringle domains 1 to 4) in reducing retinal vascular leakage in an experimental diabetic rat model. Methods rAAV-angiostatin was delivered by intravitreal injection to the right eyes of Sprague-Dawley rats. As a control, the contralateral eye received an intravitreal injection of rAAV-lacZ. Gene delivery was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). D...

  7. Structure of neurotropic adeno-associated virus AAVrh.8.

    Science.gov (United States)

    Halder, Sujata; Van Vliet, Kim; Smith, J Kennon; Duong, Thao Thi Phuong; McKenna, Robert; Wilson, James M; Agbandje-McKenna, Mavis

    2015-10-01

    Adeno-associated virus rhesus isolate 8 (AAVrh.8) is a leading vector for the treatment of neurological diseases due to its efficient transduction of neuronal cells and reduced peripheral tissue tropism. Toward identification of the capsid determinants for these properties, the structure of AAVrh.8 was determined by X-ray crystallography to 3.5 Å resolution and compared to those of other AAV isolates. The capsid viral protein (VP) structure consists of an αA helix and an eight-stranded anti-parallel β-barrel core conserved in parvoviruses, and large insertion loop regions between the β-strands form the capsid surface topology. The AAVrh.8 capsid exhibits the surface topology conserved in all AAVs: depressions at the icosahedral twofold axis and surrounding the cylindrical channel at the fivefold axis, and three protrusions around the threefold axis. A structural comparison to serotypes AAV2, AAV8, and AAV9, to which AAVrh.8 shares ∼ 84%, ∼ 91%, and ∼ 87% VP sequence identity, respectively, revealed differences in the surface loops known to affect receptor binding, transduction efficiency, and antigenicity. Consistent with this observation, biochemical assays showed that AAVrh.8 is unable to bind heparin and does not cross-react with conformational monoclonal antibodies and human donor serum directed against the other AAVs compared. This structure of AAVrh.8 thus identified capsid surface differences which can serve as template regions for rational design of vectors with enhanced transduction for specific tissues and escape pre-existing antibody recognition. These features are essential for the creation of an AAV vector toolkit that is amenable to personalized disease treatment. PMID:26334681

  8. 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.

  9. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  11. 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.

  12. 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.

  13. Adeno Associated Viral Vector Delivered RNAi for Gene Therapy of SOD1 Amyotrophic Lateral Sclerosis.

    Science.gov (United States)

    Stoica, Lorelei; Sena-Esteves, Miguel

    2016-01-01

    Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of upper and lower motor neurons. Mutations in superoxide dismutase 1 (SOD1) are a leading cause of ALS, responsible for up to 20% of familial cases. Although the exact mechanism by which mutant SOD1 causes disease remains unknown, multiple studies have shown that reduction of the mutant species leads to delayed disease onset and extension of lifespan of animal models. This makes SOD1 an ideal target for gene therapy coupling adeno associated virus vector (AAV) gene delivery with RNAi molecules. In this review we summarize the studies done thus far attempting to decrease SOD1 gene expression, using AAV vectors as delivery tools, and RNAi as therapeutic molecules. Current hurdles to be overcome, such as the need for widespread gene delivery through the entire central nervous system (CNS), are discussed. Continued efforts to improve current AAV delivery methods and capsids will accelerate the application of these therapeutics to the clinic. PMID:27531973

  14. CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox.

    Science.gov (United States)

    Senís, Elena; Fatouros, Chronis; Große, Stefanie; Wiedtke, Ellen; Niopek, Dominik; Mueller, Ann-Kristin; Börner, Kathleen; Grimm, Dirk

    2014-11-01

    Its remarkable ease and efficiency make the CRISPR (clustered regularly interspaced short palindromic repeats) DNA editing machinery highly attractive as a new tool for experimental gene annotation and therapeutic genome engineering in eukaryotes. Here, we report a versatile set of plasmids and vectors derived from adeno-associated virus (AAV) that allow robust and specific delivery of the two essential CRISPR components - Cas9 and chimeric g(uide)RNA - either alone or in combination. All our constructs share a modular design that enables simple and stringent guide RNA (gRNA) cloning as well as rapid exchange of promoters driving Cas9 or gRNA. Packaging into potent synthetic AAV capsids permits CRISPR delivery even into hard-to-transfect targets, as shown for human T-cells. Moreover, we demonstrate the feasibility to direct Cas9 expression to or away from hepatocytes, using a liver-specific promoter or a hepatic miRNA binding site, respectively. We also report a streamlined and economical protocol for detection of CRISPR-induced mutations in less than 3 h. Finally, we provide original evidence that AAV/CRISPR vectors can be exploited for gene engineering in vivo, as exemplified in the liver of adult mice. Our new tools and protocols should foster the broad application of CRISPR technology in eukaryotic cells and organisms, and accelerate its clinical translation into humans. PMID:25186301

  15. Systemic delivery of genes to striated muscles using adeno-associated viral vectors.

    Science.gov (United States)

    Gregorevic, Paul; Blankinship, Michael J; Allen, James M; Crawford, Robert W; Meuse, Leonard; Miller, Daniel G; Russell, David W; Chamberlain, Jeffrey S

    2004-08-01

    A major obstacle limiting gene therapy for diseases of the heart and skeletal muscles is an inability to deliver genes systemically to muscles of an adult organism. Systemic gene transfer to striated muscles is hampered by the vascular endothelium, which represents a barrier to distribution of vectors via the circulation. Here we show the first evidence of widespread transduction of both cardiac and skeletal muscles in an adult mammal, after a single intravenous administration of recombinant adeno-associated virus pseudotype 6 vectors. The inclusion of vascular endothelium growth factor/vascular permeability factor, to achieve acute permeabilization of the peripheral microvasculature, enhanced tissue transduction at lower vector doses. This technique enabled widespread muscle-specific expression of a functional micro-dystrophin in the skeletal muscles of dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy. We propose that these methods may be applicable for systemic delivery of a wide variety of genes to the striated muscles of adult mammals. PMID:15273747

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. 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.

  19. 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

  20. Productive life cycle of adeno-associated virus serotype 2 in the complete absence of a conventional polyadenylation signal.

    Science.gov (United States)

    Wang, Lina; Yin, Zifei; Wang, Yuan; Lu, Yuan; Zhang, Daniel; Srivastava, Arun; Ling, Changquan; Aslanidi, George V; Ling, Chen

    2015-09-01

    We showed that WT adeno-associated virus serotype 2 (AAV2) genome devoid of a conventional polyadenylation [poly(A)] signal underwent complete genome replication, encapsidation and progeny virion production in the presence of adenovirus. The infectivity of the progeny virion was also retained. Using recombinant AAV2 vectors devoid of a human growth hormone poly(A) signal, we also demonstrated that a subset of mRNA transcripts contained the inverted terminal repeat (ITR) sequence at the 3' end, which we designated ITR in RNA (ITRR). Furthermore, AAV replication (Rep) proteins were able to interact with the ITRR. Taken together, our studies suggest a new function of the AAV2 ITR as an RNA element to mediate transgene expression from poly(A)-deleted mRNA. PMID:26297494

  1. 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

  2. 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.

  3. Differential Cellular Tropism of Lentivirus and Adeno-Associated Virus in the Brain of Cynomolgus Monkey

    OpenAIRE

    An, Heeyoung; Cho, Doo-Wan; Lee, Seung Eun; Yang, Young-Su; Han, Su-Cheol; Lee, C. Justin

    2016-01-01

    Many researchers are using viruses to deliver genes of interest into the brains of laboratory animals. However, certain target brain cells are not easily infected by viruses. Moreover, the differential tropism of different viruses in monkey brain is not well established. We investigated the cellular tropism of lentivirus and adeno-associated virus (AAV) toward neuron and glia in the brain of cynomolgus monkeys (Macaca fascularis). Lentivirus and AAV were injected into putamen of the monkey br...

  4. Adeno-Associated Virus Serotype-9 Microdystrophin Gene Therapy Ameliorates Electrocardiographic Abnormalities in mdx Mice

    OpenAIRE

    Bostick, Brian; Yue, Yongping; Lai, Yi; Long, Chun; Li, Dejia; Duan, Dongsheng

    2008-01-01

    Adeno-associated virus (AAV)-mediated microdystrophin gene therapy holds great promise for treating Duchenne muscular dystrophy (DMD). Previous studies have revealed excellent skeletal muscle protection. Cardiac muscle is also compromised in DMD patients. Here we show that a single intravenous injection of AAV serotype-9 (AAV-9) microdystrophin vector efficiently transduced the entire heart in neonatal mdx mice, a dystrophin-deficient mouse DMD model. Furthermore, microdystrophin therapy norm...

  5. Rapid, simple and versatile manufacturing of recombinant adeno-associated virus vectors at scale

    OpenAIRE

    Lock, Martin; Alvira, Mauricio; Vandenberghe, Luk H.; Samanta, Arabinda; Toelen, Jaan; Debyser, Zeger; Wilson, James M

    2010-01-01

    Adeno-associated virus vector manufacturing at scale continues to hinder the application of AAV technology to gene therapy studies. While scalable systems based upon AAV-adenovirus, -herpesvirus and -baculovirus hybrids hold promise for clinical applications, they require time-consuming generation of reagents and are not highly suited to intermediate scale pre-clinical studies in large animals where several combinations of serotype and genome may need to be tested. Recently we observed that d...

  6. Feasibility of Generating Adeno-Associated Virus Packaging Cell Lines Containing Inducible Adenovirus Helper Genes

    OpenAIRE

    Qiao, Chunping; Li, Juan; Skold, Anna; Zhang, Xudong; Xiao, Xiao

    2002-01-01

    The adeno-associated virus (AAV) vector system is based on nonpathogenic and helper-virus-dependent parvoviruses. The vector system offers safe, efficient, and long-term in vivo gene transfer in numerous tissues. Clinical trials using AAV vectors have demonstrated vector safety as well as efficiency. The increasing interest in the use of AAV for clinical studies demands large quantities of vectors and hence a need for improvement in vector production. The commonly used transient-transfection ...

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

    OpenAIRE

    Yang Lin; Xiao Xiao

    2013-01-01

    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 evolutio...

  8. Highly Efficient Delivery of Adeno-Associated Viral Vectors to the Primate Retina.

    Science.gov (United States)

    Boye, Shannon E; Alexander, John J; Witherspoon, C Douglas; Boye, Sanford L; Peterson, James J; Clark, Mark E; Sandefer, Kristen J; Girkin, Chris A; Hauswirth, William W; Gamlin, Paul D

    2016-08-01

    Adeno-associated virus (AAV) has emerged as the preferred vector for targeting gene expression to the retina. Subretinally injected AAV can efficiently transduce retinal pigment epithelium and photoreceptors in primate retina. Inner and middle primate retina can be transduced by intravitreally delivered AAV, but with low efficiency. This is due to dilution of vector, potential neutralization of capsid because it is not confined to the immune-privileged retinal compartment, and the presence of the inner limiting membrane (ILM), a barrier separating the vitreous from the neural retina. We here describe a novel "subILM" injection method that addresses all three issues. Specifically, vector is placed in a surgically induced, hydrodissected space between the ILM and neural retina. In an initial experiment, we injected viscoelastic (Healon(®)), a substance we confirmed was biocompatible with AAV, to create a subILM bleb and subsequently injected AAV2-GFP into the bleb after irrigation with basic salt solution. For later experiments, we used a Healon-AAV mixture to place single, subILM injections. In all cases, subILM delivery of AAV was well tolerated-no inflammation or gross structural changes were observed by ophthalmological examination or optical coherence tomography. In-life fluorescence imaging revealed profound transgene expression within the area of the subILM injection bleb that persisted for the study duration. Uniform and extensive transduction of retinal ganglion cells (RGCs) was achieved in the areas beneath the subILM bleb. Transduction of Müller glia, ON bipolar cells, and photoreceptors was also observed. Robust central labeling from green fluorescent protein-expressing RGCs confirmed their continued survival, and was observed in the lateral geniculate nucleus, the superior colliculus, and the pretectum. Our results confirm that the ILM is a major barrier to transduction by AAV in primate retina and that, when it is circumvented, the efficiency and

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

    OpenAIRE

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

    2011-01-01

    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...

  10. Full Functional Rescue of a Complete Muscle (TA) in Dystrophic Hamsters by Adeno-Associated Virus Vector-Directed Gene Therapy

    OpenAIRE

    Xiao, Xiao; Li, Juan; Tsao, Yeou-Ping; Dressman, Devin; Hoffman, Eric P; Watchko, Jon F.

    2000-01-01

    Limb girdle muscular dystrophy (LGMD) 2F is caused by mutations in the δ-sarcoglycan (SG) gene. Previously, we have shown successful application of a recombinant adeno-associated virus (AAV) vector for genetic and biochemical rescue in the Bio14.6 hamster, a homologous animal model for LGMD 2F (J. Li et al., Gene Ther. 6:74–82, 1999). In this report, we show efficient and long-term δ-SG expression accompanied by nearly complete recovery of physiological function deficits after a single-dose A...

  11. 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.

  12. 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. PMID:25654275

  13. 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.

  14. Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin.

    Science.gov (United States)

    Jakobsen, Maria; Askou, Anne Louise; Stenderup, Karin; Rosada, Cecilia; Dagnæs-Hansen, Frederik; Jensen, Thomas G; Corydon, Thomas J; Mikkelsen, Jacob Giehm; Aagaard, Lars

    2015-08-01

    Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo. PMID:26204415

  15. 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.

  16. Stable transduction of large DNA by high-capacity adeno-associated virus/adenovirus hybrid vectors

    International Nuclear Information System (INIS)

    Viral vectors with high cloning capacity and host chromosomal integration ability are in demand for the efficient and permanent genetic modification of target cells with large DNA molecules. We have generated a hybrid gene transfer vehicle consisting of recombinant adeno-associated virus (AAV) replicative intermediates packaged in adenovirus (Ad) capsids. This arrangement allows cell cycle-independent nuclear delivery of recombinant AAV genomes with lengths considerably above the maximum size (i.e., 4.7 kb) that can be accommodated within AAV capsids. Here we show that high-capacity AAV/Ad hybrid vector gene transfer mediates cellular genomic integration of large fragments of foreign DNA and accomplishes stable long-term transgene expression in rapidly proliferating cells. Southern blot and polymerase chain reaction analyses of chromosomal DNA extracted from clones of stably transduced cells revealed that most of them contained a single copy of the full-length hybrid vector genome with AAV inverted terminal repeat (ITR) sequences at both ends. The high-capacity AAV/Ad hybrid vector system can thus be used for the transfer and expression of transgenes that cannot be delivered by conventional integrating viral vectors

  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. 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.

  19. Safe and bodywide muscle transduction in young adult Duchenne muscular dystrophy dogs with adeno-associated virus.

    Science.gov (United States)

    Yue, Yongping; Pan, Xiufang; Hakim, Chady H; Kodippili, Kasun; Zhang, Keqing; Shin, Jin-Hong; Yang, Hsiao T; McDonald, Thomas; Duan, Dongsheng

    2015-10-15

    The ultimate goal of muscular dystrophy gene therapy is to treat all muscles in the body. Global gene delivery was demonstrated in dystrophic mice more than a decade ago using adeno-associated virus (AAV). However, translation to affected large mammals has been challenging. The only reported attempt was performed in newborn Duchenne muscular dystrophy (DMD) dogs. Unfortunately, AAV injection resulted in growth delay, muscle atrophy and contracture. Here we report safe and bodywide AAV delivery in juvenile DMD dogs. Three ∼2-m-old affected dogs received intravenous injection of a tyrosine-engineered AAV-9 reporter or micro-dystrophin (μDys) vector at the doses of 1.92-6.24 × 10(14) viral genome particles/kg under transient or sustained immune suppression. DMD dogs tolerated injection well and their growth was not altered. Hematology and blood biochemistry were unremarkable. No adverse reactions were observed. Widespread muscle transduction was seen in skeletal muscle, the diaphragm and heart for at least 4 months (the end of the study). Nominal expression was detected in internal organs. Improvement in muscle histology was observed in μDys-treated dogs. In summary, systemic AAV gene transfer is safe and efficient in young adult dystrophic large mammals. This may translate to bodywide gene therapy in pediatric patients in the future. PMID:26264580

  20. Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2

    International Nuclear Information System (INIS)

    The two large Rep proteins, Rep78 and Rep68, from the adeno-associated virus type 2 (AAV-2) are required for AAV-2 DNA replication, site-specific integration, and for the regulation of viral gene expression. The study of their activities is dependent on the ability to deliver these proteins to the cells in a time and dose-dependent manner. We evaluated the ability of a protein transduction domain (PTD) derived from the human immunodeficiency virus 1 (HIV-1) TAT protein to drive the cellular internalization of exogenously delivered PTD-fused Rep68 proteins. This analysis unexpectedly revealed that recombinant Rep68 alone, in the absence of any PTD, could be endocytosed by the cells. Rep68 as the chimeric TAT-Rep68 proteins were internalized through endocytosis in clathrin-coated vesicles and retained in late endosomes/lysosomes with no detectable nuclear localization. In the presence of adenovirus, the Rep proteins could translocate into the nucleus where they displayed a biological activity. These findings support recent reports on the mechanism of entry of TAT-fused proteins and also revealed a new property of Rep68

  1. In vivo evaluation of adeno-associated virus gene transfer in airways of mice with acute or chronic respiratory infection.

    Science.gov (United States)

    Myint, Melissa; Limberis, Maria P; Bell, Peter; Somanathan, Suryanarayan; Haczku, Angela; Wilson, James M; Diamond, Scott L

    2014-11-01

    Patients with cystic fibrosis (CF) often suffer chronic lung infection with concomitant inflammation, a setting that may reduce the efficacy of gene transfer. While gene therapy development for CF often involves viral-based vectors, little is known about gene transfer in the context of an infected airway. In this study, three mouse models were established to evaluate adeno-associated virus (AAV) gene transfer in such an environment. Bordetella bronchiseptica RB50 was used in a chronic, nonlethal respiratory infection in C57BL/6 mice. An inoculum of ∼10(5) CFU allowed B. bronchiseptica RB50 to persist in the upper and lower respiratory tracts for at least 21 days. In this infection model, administration of an AAV vector on day 2 resulted in 2.8-fold reduction of reporter gene expression compared with that observed in uninfected controls. Postponement of AAV administration to day 14 resulted in an even greater (eightfold) reduction of reporter gene expression, when compared with uninfected controls. In another infection model, Pseudomonas aeruginosa PAO1 was used to infect surfactant protein D (SP-D) or surfactant protein A (SP-A) knockout (KO) mice. With an inoculum of ∼10(5) CFU, infection persisted for 2 days in the nasal cavity of either mouse model. Reporter gene expression was approximately ∼2.5-fold lower compared with uninfected mice. In the SP-D KO model, postponement of AAV administration to day 9 postinfection resulted in only a two fold reduction in reporter gene expression, when compared with expression seen in uninfected controls. These results confirm that respiratory infections, both ongoing and recently resolved, decrease the efficacy of AAV-mediated gene transfer. PMID:25144316

  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. 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...... for human gene therapy, primarily due to its lack of pathogenicity and low risk of insertional mutagenesis. However, the existing data pertaining to AAV transduction of MSCs is limited. The objective of this work was to examine the efficiency and kinetics of in vitro transduction using AAV serotype 2...... 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...

  4. Size does matter: overcoming the adeno-associated virus packaging limit

    Directory of Open Access Journals (Sweden)

    Flotte Terence R

    2000-07-01

    Full Text Available Abstract Recombinant adeno-associated virus (rAAV vectors mediate long-term gene transfer without any known toxicity. The primary limitation of rAAV has been the small size of the virion (20 nm, which only permits the packaging of 4.7 kilobases (kb of exogenous DNA, including the promoter, the polyadenylation signal and any other enhancer elements that might be desired. Two recent reports (D Duan et al: Nat Med 2000, 6:595-598; Z Yan et al: Proc Natl Acad Sci USA 2000, 97:6716-6721 have exploited a unique feature of rAAV genomes, their ability to link together in doublets or strings, to bypass this size limitation. This technology could improve the chances for successful gene therapy of diseases like cystic fibrosis or Duchenne muscular dystrophy that lead to significant pulmonary morbidity.

  5. Liver-Specific Allergen Gene Transfer by Adeno-Associated Virus Suppresses Allergic Airway Inflammation in Mice.

    Science.gov (United States)

    Chan, Cheng-Chi; Lai, Chin-Wen; Wu, Chia-Jen; Chen, Li-Chen; Tao, Mi-Hua; Kuo, Ming-Ling

    2016-08-01

    Allergic airway inflammation driven by T helper 2 (Th2)-type immunity is characterized by airway hyperresponsiveness, eosinophilic infiltration, and elevated IgE production. Various novel strategies for managing asthma have been explored, such as DNA vaccines, T-cell peptides, and allergen-specific immunotherapy. A principal goal of most immunotherapeutic approaches is active and long-term allergen-specific tolerance. Liver-specific gene transfer using adeno-associated virus (AAV) has been shown to favorably induce tolerogenic responses to therapeutic products in various experimental models. AAV8 has strong liver tropism and induces immune tolerance in mice. The present study aimed to determine whether hepatocyte-specific allergen expression by pseudotyped AAV2/8 alleviates asthmatic symptoms in ovalbumin (OVA)-sensitized mice. Mice were intravenously injected with AAV2/8 vector carrying membrane-bound OVA transgene under transcriptional control of a hepatocyte-specific alpha 1 antitrypsin promoter (AAV2/8-OVA) and then sensitized with OVA. AAV2/8-OVA specifically transduced the OVA transgene in the liver. Airway hyperresponsiveness, eosinophilia, mucus hypersecretion, and Th2 cytokines were significantly suppressed in both the lungs and secondary lymphoid organs of asthmatic mice infected with AAV2/8-OVA. Significant reduction of OVA-specific antibodies was detected in the bronchoalveolar lavage fluid from AAV2/8-OVA-treated mice. Moreover, AAV2/8-OVA treatment prominently promoted the expression of Foxp3, IL-10, and TGF-β in the liver. Enhanced Foxp3 expression was also detected in the lungs of asthmatic mice after AAV2/8-OVA treatment. Taken together, these results suggest that the induction of immune tolerance by hepatic AAV gene transfer may be beneficial for modulating allergic asthma. PMID:27178525

  6. 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…

  7. Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

    International Nuclear Information System (INIS)

    We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by ∼25-fold in WT MEFs, but only by ∼4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency ∼23-fold in WT MEFs, but only ∼4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, ∼59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only ∼28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene

  8. 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.

  9. Repair effects of co-expression of the VEGF and BMP genes via an adeno-associated viral vector on early steroid-in-duced avascular necrosis of the femoral head in rabbits%重组腺相关病毒介导VEGF和BMP双基因共表达对兔早期激素性股骨头坏死的修复作用

    Institute of Scientific and Technical Information of China (English)

    张晨; 李兴华; 李苗; 唐一仑; 时志斌; 党晓谦; 王坤正

    2014-01-01

    (AAV-VEGF/BMP)groups. The four group virus vectors were injected into core decompression region at the dose of 25μl/site after core decompression operation directly. Repair effects of rAAV vector on early SANFH in rabbits were evaluated by Western blot assay, HE staining, immunohistochemical staining, MRI, radionuclide bone scan, blood vessel counting detected by ink perfusion and fro-zen section, Micro-CT and biomechanical strength detection on the 12th week post-injection. Results Model success ratio was 73.33%. rAAV-hVEGF165-IRES-hBMP-7 virus vector efficiently expressed hVEGF165 and hBMP-7 genes on the 12th week after rAAV injection. hVEGF165 protein secreted in vivo promoted metabolism in core decompression region by increasing the quantity of new vessels and improving the blood supply;hBMP-7 protein secreted in vivo promoted new bone formation in core decompres-sion region by increasing bone mineral density and improving bone biomechanical strength. The AAV-VEGF/BMP group can pro-mote repair effects more effectively than AAV-VEGF group or AAV-BMP group. Conclusion The adeno-associated viral vectors co-expressing hVEGF165 and hBMP-7 can promote repair effects on early SANFH in rabbits by increasing the blood supply and strengthening the bone quality of femoral head.

  10. Gene therapy for choroideremia using an adeno-associated viral (AAV) vector.

    Science.gov (United States)

    Barnard, Alun R; Groppe, Markus; MacLaren, Robert E

    2015-03-01

    Choroideremia is an outer retinal degeneration with a characteristic clinical appearance that was first described in the nineteenth century. The disorder begins with reduction of night vision and gradually progresses to blindness by middle age. The appearance of the fundus in sufferers is recognizable by the characteristic pale color caused by the loss of the outer retina, retinal-pigmented epithelium, and choroidal vessels, leading to exposure of the underlying sclera. Choroideremia shows X-linked recessive inheritance and the choroideremia gene (CHM) was one of the first to be identified by positional cloning in 1990. Subsequent identification and characterization of the CHM gene, which encodes Rab escort protein 1 (REP1), has led to better comprehension of the disease and enabled advances in genetic diagnosis. Despite several decades of work to understand the exact pathogenesis, no established treatments currently exist to stop or even slow the progression of retinal degeneration in choroideremia. Encouragingly, several specific molecular and clinical features make choroideremia an ideal candidate for treatment with gene therapy. This work describes the considerations and challenges in the development of a new clinical trial using adeno-associated virus (AAV) encoding the CHM gene. PMID:25359548

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy.

    Science.gov (United States)

    Nance, Michael E; Duan, Dongsheng

    2015-12-01

    Duchenne muscular dystrophy (DMD) is a X-linked, progressive childhood myopathy caused by mutations in the dystrophin gene, one of the largest genes in the genome. It is characterized by skeletal and cardiac muscle degeneration and dysfunction leading to cardiac and/or respiratory failure. Adeno-associated virus (AAV) is a highly promising gene therapy vector. AAV gene therapy has resulted in unprecedented clinical success for treating several inherited diseases. However, AAV gene therapy for DMD remains a significant challenge. Hurdles for AAV-mediated DMD gene therapy include the difficulty to package the full-length dystrophin coding sequence in an AAV vector, the necessity for whole-body gene delivery, the immune response to dystrophin and AAV capsid, and the species-specific barriers to translate from animal models to human patients. Capsid engineering aims at improving viral vector properties by rational design and/or forced evolution. In this review, we discuss how to use the state-of-the-art AAV capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy. PMID:26414293

  13. Directed evolution of novel adeno-associated viruses for therapeutic gene delivery.

    Science.gov (United States)

    Bartel, M A; Weinstein, J R; Schaffer, D V

    2012-06-01

    Gene therapy vectors based on adeno-associated virus (AAV) are currently in clinical trials for numerous disease targets, such as muscular dystrophy, hemophilia, Parkinson's disease, Leber's congenital amaurosis and macular degeneration. Despite its considerable promise and emerging clinical success, several challenges impede the broader implementation of AAV gene therapy, including the prevalence of neutralizing antibodies in the human population, low transduction of a number of therapeutically relevant cell and tissue types, an inability to overcome physical and cellular barriers in vivo and a relatively limited carrying capacity. These challenges arise as the demands we place on AAV vectors are often different from or even at odds with the properties nature bestowed on their parent viruses. Viral-directed evolution-the iterative generation of large, diverse libraries of viral mutants and selection for variants with specific properties of interest-offers an approach to address these problems. Here we outline progress in creating novel classes of AAV variant libraries and highlight the successful isolation of variants with novel and advantageous in vitro and in vivo gene delivery properties. PMID:22402323

  14. Functional analysis of the putative integrin recognition motif on adeno-associated virus 9.

    Science.gov (United States)

    Shen, Shen; Berry, Garrett E; Castellanos Rivera, Ruth M; Cheung, Roland Y; Troupes, Andrew N; Brown, Sarah M; Kafri, Tal; Asokan, Aravind

    2015-01-16

    Adeno-associated viruses (AAVs) display a highly conserved NGR motif on the capsid surface. Earlier studies have established this tripeptide motif as being essential for integrin-mediated uptake of recombinant AAV serotype 2 (AAV2) in cultured cells. However, functional attributes of this putative integrin recognition motif in other recombinant AAV serotypes displaying systemic transduction in vivo remain unknown. In this study, we dissect the biology of an integrin domain capsid mutant derived from the human isolate AAV9 in mice. The AAV9/NGA mutant shows decreased systemic transduction in mice. This defective phenotype was accompanied by rapid clearance of mutant virions from the blood circulation and nonspecific sequestration by the spleen. Transient vascular hyperpermeability, induced by histamine coinjection, exacerbated AAV9/NGA uptake by the spleen but not the liver. However, such treatment did not affect AAV9 virions, suggesting a potential entry/post-entry defect for the mutant in different tissues. Further characterization revealed modestly decreased cell surface binding but a more pronounced defect in the cellular entry of mutant virions. These findings were corroborated by the observation that blocking multiple integrins adversely affected recombinant AAV9 transduction in different cell types, albeit with variable efficiencies. From a structural perspective, we observed that the integrin recognition motif is located in close proximity to the galactose binding footprint on AAV9 capsids and postulate that this feature could influence cell surface attachment, cellular uptake at the tissue level, and systemic clearance by the reticuloendothelial system. PMID:25404742

  15. Neutralizing Antibodies Against Adeno-Associated Viral Capsids in Patients with mut Methylmalonic Acidemia.

    Science.gov (United States)

    Harrington, Elizabeth A; Sloan, Jennifer L; Manoli, Irini; Chandler, Randy J; Schneider, Mark; McGuire, Peter J; Calcedo, Roberto; Wilson, James M; Venditti, Charles P

    2016-05-01

    Isolated methylmalonic acidemia (MMA), a group of autosomal recessive inborn errors of metabolism, is most commonly caused by complete (mut(0)) or partial (mut(-)) deficiency of the enzyme methylmalonyl-CoA mutase (MUT). The severe metabolic instability and increased mortality experienced by many affected individuals, especially those with mut(0) MMA, has led centers to use elective liver transplantation as a treatment for these patients. We have previously demonstrated the efficacy of systemic adeno-associated viral (AAV) gene delivery as a treatment for MMA in a murine model and therefore sought to survey AAV antibody titers against serotypes 2, 8, and 9 in a group of well-characterized MMA patients, accrued via a dedicated natural history study ( clinicaltrials.gov ID: NCT00078078). Plasma samples provided by 42 patients (8 mut(-) and 34 mut(0); 10 had received organ transplantation), who ranged in age between 2 and 31 years, were analyzed to examine AAV2 (n = 35), AAV8 (n = 41), and AAV9 (n = 42) antibody titers. In total, the seroprevalence of antibodies against AAV2, AAV8, or AAV9 was 20%, 22%, and 24%, respectively. We observed a lower-than-expected seropositivity rate (titers ≥1:20) in the pediatric MMA patients (2-18 years) for both AAV2 (p gene delivery as a treatment for mut MMA. PMID:26790480

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

    Directory of Open Access Journals (Sweden)

    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

  17. Recombinant adeno-associated viruses (rAAV2) facilitate the intraperitoneal gene delivery to cancer cells

    OpenAIRE

    Malecki, Maciej; PROCZKA, ROBERT; Chorostowska-Wynimko, Joanna; Swoboda, Paweł; DELBANI, ANNA; Pachecka, Jan

    2010-01-01

    Peritoneal dissemination of cancer cells is characteristic of advanced stages of ovarian, breast and lung cancers, and is associated with poor patient survival. The presence of cancer cells in effusions complicates treatment protocols, while cell eradication is seriously limited. One of the novel options available is cancer gene therapy with recombinant adeno-associated viruses. This combination represents the most promising gene delivery vehicles to neoplasmatic cells within serosal cavities...

  18. Long-term Rescue of a Lethal Murine Model of Methylmalonic Acidemia Using Adeno associated Viral Gene Therapy

    OpenAIRE

    Chandler, Randy J.; Venditti, Charles P

    2009-01-01

    Methylmalonic acidemia (MMA) is an organic acidemia caused by deficient activity of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). This disorder is associated with lethal metabolic instability and carries a poor prognosis for long-term survival. A murine model of MMA that replicates a severe clinical phenotype was used to examine the efficacy of recombinant adeno-associated virus (rAAV) serotype 8 gene therapy as a treatment for MMA. Lifespan extension, body weight, circulating meta...

  19. Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model

    OpenAIRE

    Wang, Bing; Li, Juan; Xiao, Xiao

    2000-01-01

    Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disorder, caused by recessive mutations in the dystrophin gene. One of every 3,500 males suffers from DMD, yet no treatment is currently available. Genetic therapeutic approaches, using primarily myoblast transplantation and adenovirus-mediated gene transfer, have met with limited success. Adeno-associated virus (AAV) vectors, although proven superior for muscle gene transfer, are too sm...

  20. Self-complementary adeno-associated viral vectors for gene therapy of hemophilia B: progress and challenges

    OpenAIRE

    Raj, Deepak; Davidoff, Andrew M.; Nathwani, Amit C.

    2011-01-01

    Therapies currently used for hemophilia involve injection of protein concentrates that are expensive, invasive and associated with side effects such as development of neutralizing antibodies (inhibitors) that diminish therapeutic efficacy. Gene transfer is an attractive alternative to circumvent these issues. However, until now, clinical trials using gene therapy to treat hemophilia have failed to demonstrate sustained efficacy, although a vector based on a self-complementary adeno-associated...

  1. 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.

  2. 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.

  3. 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.

  4. Secretory expression of PR39 following adeno-associated viral-encoding fusion gene transfer induces angiogenesis in hypoxia chick embryo%分泌表达PR39重组腺相关病毒对缺氧鸡胚促血管生成的影响

    Institute of Scientific and Technical Information of China (English)

    郝跃文; 孙立军; 刘莹; 王全颖; 杨广笑

    2009-01-01

    Objectives To investigate the impact of AAV-encoding NT4-TAT-His-PR39 fusion gene expression on HIF-1α level in ECV304 cultured under hypoxic condition ( 1% O2) and on angiogenesis in hypoxic chick embryo. Methods PR39 cDNA was connected with NT4, TAT, 6 x His cDNA by molecular biology methods. The recombinant AAV vector was obtained by three plasmid co-transfection in 293 cells. Then ECV304 were respectively infected with AAV-NT4-TAT-His-PR39, 6 x His expression and HIF-la level in ECV304 were detected by immunocytochemistry. The chicken embryos were randomized into the AAV-PR39, EV and PBS groups ( n = 10 each) subject to hypoxia (5% O2, n = 15 ) or normoxia environments (n = 15) , the vessel density of the chicken chorioallantoic membrane (CAM) were measured by Image Pro Plus (IPP) software. Results The expression of 6 x His protein was detected in AAV-PR39 infected ECV304 cells. HIF-1α protein activity was significantly increased in AAV-PR39 infected ECV304 underwent hypoxia compared to PBS and non-infected ECV304 groups ( P < 0. 05). The vessel density of chicken CAM in hypoxia environment but not in normoxia environment was also significantly higher in AAV-PR39 group than in EV group and PBS group (all P <0. 05). Conclusion AAV-encoding NT4-TAT-His-PR39 fusion gene expression significantly increased HIF-la level in ECV304 exposed to hypoxia and promoted angiogenesis in hypoxic chicken embryo.%目的 探讨腺相关病毒(AAV)介导的融合基因NT4-TAT-His-PR39对缺氧人脐静脉内皮细胞株ECV304内缺氧诱导因子1α(HIF-1α)表达的影响及其对缺氧环境鸡胚血管生成的影响.方法 PCR技术和T载体克降法克隆PR39基因,将编码PR39、穿膜肽TAT、6×His及NT4信号肽四个基因片段相连.磷酸钙沉淀法获得重组携带NT4-TAT-His-PR39的从V载体,然后AAV-PR39感染ECV304,免疫细胞化学检测ECV304胞内6×His表达情况.在1%O2条件培养ECV304,免疫细胞化学测定AAV-PR39组和PBS组胞内HIF-1

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

    Directory of Open Access Journals (Sweden)

    Maohua Cao

    Full Text Available 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.

  6. Human α7 Integrin Gene (ITGA7) Delivered by Adeno-Associated Virus Extends Survival of Severely Affected Dystrophin/Utrophin-Deficient Mice.

    Science.gov (United States)

    Heller, Kristin N; Montgomery, Chrystal L; Shontz, Kimberly M; Clark, K Reed; Mendell, Jerry R; Rodino-Klapac, Louise R

    2015-10-01

    Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. It is the most common, severe childhood form of muscular dystrophy. We investigated an alternative to dystrophin replacement by overexpressing ITGA7 using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal muscle that, like the dystrophin-glycoprotein complex, links the extracellular matrix to the internal actin cytoskeleton. ITGA7 is expressed in DMD patients and overexpression does not elicit an immune response to the transgene. We delivered rAAVrh.74.MCK.ITGA7 systemically at 5-7 days of age to the mdx/utrn(-/-) mouse deficient for dystrophin and utrophin, a severe mouse model of DMD. At 8 weeks postinjection, widespread expression of ITGA7 was observed at the sarcolemma of multiple muscle groups following gene transfer. The increased expression of ITGA7 significantly extended longevity and reduced common features of the mdx/utrn(-/-) mouse, including kyphosis. Overexpression of α7 expression protected against loss of force following contraction-induced damage and increased specific force in the diaphragm and EDL muscles 8 weeks after gene transfer. Taken together, these results further support the use of α7 integrin as a potential therapy for DMD. PMID:26076707

  7. 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.

  8. Virus-mediated EpoR76E Therapy Slows Optic Nerve Axonopathy in Experimental Glaucoma.

    Science.gov (United States)

    Bond, Wesley S; Hines-Beard, Jessica; GoldenMerry, Y Paul L; Davis, Mara; Farooque, Alma; Sappington, Rebecca M; Calkins, David J; Rex, Tonia S

    2016-02-01

    Glaucoma, a common cause of blindness, is currently treated by intraocular pressure (IOP)-lowering interventions. However, this approach is insufficient to completely prevent vision loss. Here, we evaluate an IOP-independent gene therapy strategy using a modified erythropoietin, EPO-R76E, which has reduced erythropoietic function. We used two models of glaucoma, the murine microbead occlusion model and the DBA/2J mouse. Systemic recombinant adeno-associated virus-mediated gene delivery of EpoR76E (rAAV.EpoR76E) was performed concurrent with elevation of IOP. Axon structure and active anterograde transport were preserved in both models. Vision, as determined by the flash visual evoked potential, was preserved in the DBA/2J. These results show that systemic EpoR76E gene therapy protects retinal ganglion cells from glaucomatous degeneration in two different models. This suggests that EPO targets a component of the neurodegenerative pathway that is common to both models. The efficacy of rAAV.EpoR76E delivered at onset of IOP elevation supports clinical relevance of this treatment. PMID:26502777

  9. The varicella-zoster virus-mediated delayed host shutoff: open reading frame 17 has no major function, whereas immediate-early 63 protein represses heterologous gene expression.

    Science.gov (United States)

    Desloges, Nathalie; Rahaus, Markus; Wolff, Manfred H

    2005-12-01

    We reported that varicella-zoster virus (VZV) causes a delayed host shutoff during its replicative cycle. VZV open reading frame 17 (ORF17) is the homologue of the herpes simplex virus (HSV) UL41 gene encoding the virion host shutoff (vhs) protein which is responsible for the shutoff effect observed in HSV-infected cells. In the present study, we demonstrated that ORF17 is expressed as a late protein during the VZV replicative cycle in different infected permissive cell lines which showed a delayed shutoff of cellular RNA. A cell line with stable expression of VZV ORF17 was infected with VZV. In these cells, VZV replication and delayed host shutoff remained unchanged when compared to normal infected cells. ORF17 was not capable of repressing the expression of the beta-gal reporter gene under the control of the human cytomegalovirus immediate-early gene promoter or to inhibit the expression of a CAT reporter gene under the control of the human GAPDH promoter, indicating that ORF17 has no major function in the VZV-mediated delayed host shutoff. To determine whether other viral factors are involved in the host shutoff, a series of cotransfection assays was performed. We found that the immediate-early 63 protein (IE63) was able to downregulate the expression of reporter genes under the control of the two heterologous promoters, indicating that this viral factor can be involved in the VZV-mediated delayed host shutoff. Other factors can be also implicated to modulate the repressing action of IE63 to achieve a precise balance between the viral and cellular gene expression.

  10. Adeno-Associated Virus Site-Specific Integration Is Mediated by Proteins of the Nonhomologous End-Joining Pathway▿

    OpenAIRE

    Daya, Shyam; Cortez, Nenita; Berns, Kenneth I.

    2009-01-01

    Adeno-associated virus type 2 (AAV 2) is the only eukaryotic virus capable of site-specific integration; the target site is at chromosome 19q13.4, a site termed AAVS1. The biology of AAV latency has been extensively studied in cell culture, yet the precise mechanism and the required cellular factors are not known. In this study, we assessed the relative frequencies of stable site-specific integration by characterization of cell clones containing integrated AAV vectors. By this assay, two prot...

  11. Adeno-associated Virus 9 Mediated FKRP Gene Therapy Restores Functional Glycosylation of α-dystroglycan and Improves Muscle Functions

    OpenAIRE

    Xu, Lei; Lu, Pei Juan; Wang, Chi-Hsien; Keramaris, Elizabeth; Qiao, Chunping; Xiao, Bin; Blake, Derek J.; Xiao, Xiao; Lu, Qi Long

    2013-01-01

    Mutations in the FKRP gene are associated with a wide range of muscular dystrophies from mild limb-girdle muscular dystrophy (LGMD) 2I to severe Walker–Warburg syndrome and muscle-eye-brain disease. The characteristic biochemical feature of these diseases is the hypoglycosylation of α-dystroglycan (α-DG). Currently there is no effective treatment available. In this study, we examined the adeno-associated virus serotype 9 vector (AAV9)-mediated gene therapy in the FKRP mutant mouse model with ...

  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. Adeno-associated virus 2-mediated antiangiogenic cancer gene therapy: long-term efficacy of a vector encoding angiostatin and endostatin over vectors encoding a single factor.

    Science.gov (United States)

    Ponnazhagan, Selvarangan; Mahendra, Gandham; Kumar, Sanjay; Shaw, Denise R; Stockard, Cecil R; Grizzle, William E; Meleth, Sreelatha

    2004-03-01

    Angiogenesis is characteristic of solid tumor growth and a surrogate marker for metastasis in many human cancers. Inhibition of tumor angiogenesis using antiangiogenic drugs and gene transfer approaches has suggested the potential of this form of therapy in controlling tumor growth. However, for long-term tumor-free survival by antiangiogenic therapy, the factors controlling tumor neovasculature need to be systemically maintained at stable therapeutic levels. Here we show sustained expression of the antiangiogenic factors angiostatin and endostatin as secretory proteins by recombinant adeno-associated virus 2 (rAAV)-mediated gene transfer. Both vectors provided significant protective efficacy in a mouse tumor xenograft model. Stable transgene persistence and systemic levels of both angiostatin and endostatin were confirmed by in situ hybridization of the vector-injected tissues and by serum ELISA measurements, respectively. Whereas treatment with rAAV containing either endostatin or angiostatin alone resulted in moderate to significant protection, the combination of endostatin and angiostatin gene transfer from a single vector resulted in a complete protection. These data suggest that AAV-mediated long-term expression of both endostatin and angiostatin may have clinical utility against recurrence of cancers after primary therapies and may represent rational adjuvant therapies in combination with radiation or chemotherapy. PMID:14996740

  14. 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.

  15. Production, purification, crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 5

    International Nuclear Information System (INIS)

    The production, purification, crystallization and preliminary crystallographic analysis of empty adeno-associated virus serotype 5 capsids are reported. Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Å resolution using synchrotron radiation and belong to the orthorhombic space group P212121, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Å. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress

  16. Production, purification, crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 5

    Energy Technology Data Exchange (ETDEWEB)

    DiMattia, Michael; Govindasamy, Lakshmanan; Levy, Hazel C.; Gurda-Whitaker, Brittney; Kalina, Amy [Department of Biochemistry and Molecular Biology, McKnight Brain Institute, Center for Structural Biology, University of Florida, Gainesville, FL 32610 (United States); Kohlbrenner, Erik [Division of Cell and Molecular Therapy, University of Florida, Gainesville, FL 32610 (United States); Chiorini, John A. [GTTB, NIDCR, National Institutes of Health, Bethesda, MD 20892 (United States); McKenna, Robert [Department of Biochemistry and Molecular Biology, McKnight Brain Institute, Center for Structural Biology, University of Florida, Gainesville, FL 32610 (United States); Muzyczka, Nicholas [Department of Molecular Genetics and Microbiology and Powell Gene Therapy Center, College of Medicine, University of Florida, Gainesville, FL 32610 (United States); Zolotukhin, Sergei [Division of Cell and Molecular Therapy, University of Florida, Gainesville, FL 32610 (United States); Agbandje-McKenna, Mavis, E-mail: mckenna@ufl.edu [Department of Biochemistry and Molecular Biology, McKnight Brain Institute, Center for Structural Biology, University of Florida, Gainesville, FL 32610 (United States)

    2005-10-01

    The production, purification, crystallization and preliminary crystallographic analysis of empty adeno-associated virus serotype 5 capsids are reported. Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Å resolution using synchrotron radiation and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Å. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress.

  17. 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.

  18. Pulmonary Targeting of Adeno-associated Viral Vectors by Next-generation Sequencing-guided Screening of Random Capsid Displayed Peptide Libraries.

    Science.gov (United States)

    Körbelin, Jakob; Sieber, Timo; Michelfelder, Stefan; Lunding, Lars; Spies, Elmar; Hunger, Agnes; Alawi, Malik; Rapti, Kleopatra; Indenbirken, Daniela; Müller, Oliver J; Pasqualini, Renata; Arap, Wadih; Kleinschmidt, Jürgen A; Trepel, Martin

    2016-06-01

    Vectors mediating strong, durable, and tissue-specific transgene expression are mandatory for safe and effective gene therapy. In settings requiring systemic vector administration, the availability of suited vectors is extremely limited. Here, we present a strategy to select vectors with true specificity for a target tissue from random peptide libraries displayed on adeno-associated virus (AAV) by screening the library under circulation conditions in a murine model. Guiding the in vivo screening by next-generation sequencing, we were able to monitor the selection kinetics and to determine the right time point to discontinue the screening process. The establishment of different rating scores enabled us to identify the most specifically enriched AAV capsid candidates. As proof of concept, a capsid variant was selected that specifically and very efficiently delivers genes to the endothelium of the pulmonary vasculature after intravenous administration. This technical approach of selecting target-specific vectors in vivo is applicable to any given tissue of interest and therefore has broad implications in translational research and medicine. PMID:27018516

  19. Delivery of human EV71 receptors by adeno-associated virus increases EV71 infection-induced local inflammation in adult mice.

    Science.gov (United States)

    Hsiao, Hung-Bo; Chou, Ai-Hsiang; Lin, Su-I; Lien, Shu-Pei; Liu, Chia-Chyi; Chong, Pele; Chen, Chih-Yeh; Tao, Mi-Hua; Liu, Shih-Jen

    2014-01-01

    Enterovirus71 (EV71) is now recognized as an emerging neurotropic virus in Asia and one major causative agent of hand-foot-mouth diseases (HFMD). However potential animal models for vaccine development are limited to young mice. In this study, we used an adeno-associated virus (AAV) vector to introduce the human EV71 receptors P-selectin glycoprotein ligand-1 (hPSGL1) or a scavenger receptor class-B member-2 (hSCARB2) into adult ICR mice to change their susceptibility to EV71 infection. Mice were administered AAV-hSCARB2 or AAV-hPSGL1 through intravenous and oral routes. After three weeks, expression of human SCARB2 and PSGL1 was detected in various organs. After infection with EV71, we found that the EV71 viral load in AAV-hSCARB2- or AAV-hPSGL1-transduced mice was higher than that of the control mice in both the brain and intestines. The presence of EV71 viral particles in tissues was confirmed using immunohistochemistry analysis. Moreover, inflammatory cytokines were induced in the brain and intestines of AAV-hSCARB2- or AAV-hPSGL1-transduced mice after EV71 infection but not in wild-type mice. However, neurological disease was not observed in these animals. Taken together, we successfully infected adult mice with live EV71 and induced local inflammation using an AAV delivery system.

  20. Expression and inheritance of hypersensitive resistance to rice hoja blanca virus mediated by the viral nucleocapsid protein gene in transgenic rice.

    Science.gov (United States)

    Lentini, Z; Lozano, I; Tabares, E; Fory, L; Domínguez, J; Cuervo, M; Calvert, L

    2003-04-01

    Rice hoja blanca virus (RHBV) is a major virus disease of economic importance affecting rice in northern South America, Central America and the Caribbean. This is the first report of transgenic resistance to RHBV and the transformation of an indica rice variety from Latin America. Rice transformed with the RHBV nucleocapsid protein ( N) gene had a significant reduction in disease development. Several reactions were observed that ranged from susceptible to completely resistant plants (immunity). The resistant reactions were characterized by the production of local lesions like a hypersensitive reaction or a recovery phenotype with the emergence of symptom-less new leaves. These transgenic RHBV-resistant rice lines expressed the N gene RNA at low levels that were below the detection limit by Northern blots and only resolved by RT-PCR. The nucleocapsid protein could not be detected in any of the transgenic plants either by Western or ELISA tests. These results suggest that the resistance encoded by the N gene in these plants appears to be mediated by RNA. When challenged with RHBV, the resistant transgenic lines showed a significant increased performance for important agronomic traits including the number of tillers, the number of grains per plant and the yield as compared to the susceptible control. Furthermore, upon inoculation some of the most-resistant transgenic lines showed agronomic traits similar to the uninoculated non-transgenic Cica 8 control. Using both agronomic traits and disease severity as criteria, several of the most-resistant lines were followed through the R(4) generation and demonstrated that the N gene and RHBV resistance was inherited in a stable manner. These transgenic rice lines could become a new genetic resource in developing RHBV-resistant cultivars. PMID:12671749

  1. 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.

  2. 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.

  3. Effect of nuclear factor κB inhibition on serotype 9 adeno-associated viral (AAV9) minidystrophin gene transfer to the mdx mouse.

    Science.gov (United States)

    Reay, Daniel P; Niizawa, Gabriela A; Watchko, Jon F; Daood, Molly; Reay, Ja'Nean C; Raggi, Eugene; Clemens, Paula R

    2012-01-01

    Gene therapy studies for Duchenne muscular dystrophy (DMD) have focused on viral vector-mediated gene transfer to provide therapeutic protein expression or treatment with drugs to limit dystrophic changes in muscle. The pathological activation of the nuclear factor (NF)-κB signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy. Furthermore, activation of NF-κB may inhibit gene transfer by promoting inflammation in response to the transgene or vector. Therefore, we hypothesized that inhibition of pathological NF-κB activation in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD. Systemic gene transfer using serotype 9 adeno-associated viral (AAV9) vectors is promising for treatment of preclinical models of DMD because of vector tropism to cardiac and skeletal muscle. In quadriceps of C57BL/10ScSn-Dmd(mdx)/J (mdx) mice, the addition of octalysine (8K)-NF-κB essential modulator (NEMO)-binding domain (8K-NBD) peptide treatment to AAV9 minidystrophin gene delivery resulted in increased levels of recombinant dystrophin expression suggesting that 8K-NBD treatment promoted an environment in muscle tissue conducive to higher levels of expression. Indices of necrosis and regeneration were diminished with AAV9 gene delivery alone and to a greater degree with the addition of 8K-NBD treatment. In diaphragm muscle, high-level transgene expression was achieved with AAV9 minidystoophin gene delivery alone; therefore, improvements in histological and physiological indices were comparable in the two treatment groups. The data support benefit from 8K-NBD treatment to complement gene transfer therapy for DMD in muscle tissue that receives incomplete levels of transduction by gene transfer, which may be highly significant for clinical applications of muscle gene delivery. PMID:22231732

  4. 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.

  5. 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.

  6. Adeno-Associated Virus (AAV) Mediated Dystrophin Gene Transfer Studies and Exon Skipping Strategies for Duchenne Muscular Dystrophy (DMD).

    Science.gov (United States)

    Kawecka, Klaudia; Theodoulides, Michael; Hasoglu, Yalin; Jarmin, Susan; Kymalainen, Hanna; Le-Heron, Anita; Popplewell, Linda; Malerba, Alberto; Dickson, George; Athanasopoulos, Takis

    2015-01-01

    Duchenne muscular dystrophy (DMD), an X-linked inherited musclewasting disease primarily affecting young boys with prevalence of between1:3,500- 1:5,000, is a rare genetic disease caused by defects in the gene for dystrophin. Dystrophin protein is critical to the stability of myofibers in skeletal and cardiac muscle. There is currently no cure available to ameliorate DMD and/or its patho-physiology. A number of therapeutic strategies including molecular-based therapeutics that replace or correct the missing or nonfunctional dystrophin protein have been devised to correct the patho-physiological consequences induced by dystrophin absence. We will review the current in vivo experimentation status (including preclinical models and clinical trials) for two of these approaches, namely: 1) Adeno-associated virus (AAV) mediated (micro) dystrophin gene augmentation/ supplementation and 2) Antisense oligonucleotide (AON)-mediated exon skipping strategies. PMID:26159373

  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. 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.

  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. 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. 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.

  12. 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.

  13. 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

  14. Adeno-Associated Virus Type 5-Mediated Intraarticular Administration of Tumor Necrosis Factor Small Interfering RNA Improves Collagen-Induced Arthritis

    NARCIS (Netherlands)

    M. Khoury; G. Courties; S. Fabre; C. Bouffi; C.A. Seemayer; M.J. Vervoordeldonk; P.P. Tak; C. Jorgensen; F. Apparailly

    2010-01-01

    Objective. RNA interference (RNAi) is a powerful tool for sequence-specific gene silencing, and interest in its application in human diseases is growing. Given the success of recent strategies for administering gene therapy in rheumatoid arthritis using recombinant vectors such as adeno-associated v

  15. 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

  16. Recombinant adeno-associated virus 2-mediated transfer of the human superoxide-dismutase gene does not confer radioresistance on HeLa cervical carcinoma cells

    International Nuclear Information System (INIS)

    Background and purpose: The success rate of any therapeutic approach depends on the therapeutic window, which can be increased by either raising the resistance of the normal tissue without protecting the tumor cells or by sensitizing the tumor cells but not the normal cells. Two promising candidate genes for normal tissue protection against radiation-induced damage may be the copper-zinc (CuZnSOD) and manganese superoxide-dismutase genes (MnSOD). The recombinant adeno-associated virus 2 (rAAV-2) offers attractive advantages over other vector systems: low immunogenicity, ability to infect dividing and non-dividing tissues and a low chance of insertional mutagenesis, due to extra-chromosomal localization. We report the production of novel rAAV-2-SOD vectors and the investigation of their modulating effects on HeLa-RC cells after irradiation. Material and methods: rAAV-2 vectors were cloned containing the human CuZnSOD or MnSOD as transgene and vector stocks were produced. In the initial experiments human cervix carcinoma (HeLa-RC) cells were chosen for their susceptibility to rAAV-2. On day 0, cells were seeded and transduced with the rAAV-2-SOD vectors. On day 3, cells were harvested, irradiated (0.5-8 Gy) and reseeded in different assays (FACS, SOD, MTT and colony assays). Results: Although >70% of all cells expressed SOD and significant amounts of functional SOD protein were detected, no radioprotective effect of SOD was observed after transduction of HeLa-RC cells. Conclusions: Novel rAAV-2-SOD vectors that could be produced at high titer, were able to efficiently infect cells and express the SOD genes. The absence of a radioprotective effect in HeLa-RC cancer cells indicates an additional safety feature and suggests that rAAV-mediated MnSOD overexpression might contribute to increasing the therapeutic index when applied for normal tissue protection

  17. A Rapid, Cost-Effective Method to Prepare Recombinant Adeno-Associated Virus for Efficient Gene Transfer to the Developing Mouse Inner Ear.

    Science.gov (United States)

    Gomes, Michelle M; Wang, Lingyan; Jiang, Han; Kahl, Christoph A; Brigande, John V

    2016-01-01

    There is keen interest to define gene therapies aimed at restoration of auditory and vestibular function in the diseased or damaged mammalian inner ear. A persistent limitation of regenerative medical strategies that seek to correct or modify gene expression in the sensory epithelia of the inner ear involves efficacious delivery of a therapeutic genetic construct. Our approach is to define methodologies that enable fetal gene transfer to the developing mammalian inner ear in an effort to correct defective gene expression during formation of the sensory epithelia or during early postnatal life. Conceptually, the goal is to atraumatically introduce the genetic construct into the otocyst-staged mouse inner ear and transfect otic progenitors that give rise to sensory hair cells and supporting cells. Our long-term goal is to define therapeutic interventions for congenital deafness and balance disorders with the expectation that the approach may also be exploited for therapeutic intervention postnatally.In the inaugural volume of this series, we introduced electroporation-mediated gene transfer to the developing mouse inner ear that encompassed our mouse survival surgery and transuterine microinjection protocols (Brigande et al., Methods Mol Biol 493:125-139, 2009). In this chapter, we first briefly update our use of sodium pentobarbital anesthesia, our preferred anesthetic for mouse ventral laparotomy, in light of its rapidly escalating cost. Next, we define a rapid, cost-effective method to produce recombinant adeno-associated virus (rAAV) for efficient gene transfer to the developing mouse inner ear. Our immediate goal is to provide a genetic toolkit that will permit the definition and validation of gene therapies in mouse models of human deafness and balance disorders. PMID:27259920

  18. Light-Activated Nuclear Translocation of Adeno-Associated Virus Nanoparticles Using Phytochrome B for Enhanced, Tunable, and Spatially Programmable Gene Delivery.

    Science.gov (United States)

    Gomez, Eric J; Gerhardt, Karl; Judd, Justin; Tabor, Jeffrey J; Suh, Junghae

    2016-01-26

    Gene delivery vectors that are activated by external stimuli may allow improved control over the location and the degree of gene expression in target populations of cells. Light is an attractive stimulus because it does not cross-react with cellular signaling networks, has negligible toxicity, is noninvasive, and can be applied in space and time with unparalleled precision. We used the previously engineered red (R)/far-red (FR) light-switchable protein phytochrome B (PhyB) and its R light dependent interaction partner phytochrome interacting factor 6 (PIF6) from Arabidopsis thaliana to engineer an adeno-associated virus (AAV) platform whose gene delivery efficiency is controlled by light. Upon exposure to R light, AAV engineered to display PIF6 motifs on the capsid bind to PhyB tagged with a nuclear localization sequence (NLS), resulting in significantly increased translocation of viruses into the host cell nucleus and overall gene delivery efficiency. By modulating the ratio of R to FR light, the gene delivery efficiency can be tuned to as little as 35% or over 600% of the unengineered AAV. We also demonstrate spatial control of gene delivery using projected patterns of codelivered R and FR light. Overall, our successful use of light-switchable proteins in virus capsid engineering extends these important optogenetic tools into the adjacent realm of nucleic acid delivery and enables enhanced, tunable, and spatially controllable regulation of viral gene delivery. Our current light-triggered viral gene delivery prototype may be broadly useful for genetic manipulation of cells ex vivo or in vivo in transgenic model organisms, with the ultimate prospect of achieving dose- and site-specific gene expression profiles for either therapeutic (e.g., regenerative medicine) or fundamental discovery research efforts. PMID:26618393

  19. Intracerebral adeno-associated virus gene delivery of apolipoprotein E2 markedly reduces brain amyloid pathology in Alzheimer's disease mouse models.

    Science.gov (United States)

    Zhao, Lingzhi; Gottesdiener, Andrew J; Parmar, Mayur; Li, Mingjie; Kaminsky, Stephen M; Chiuchiolo, Maria J; Sondhi, Dolan; Sullivan, Patrick M; Holtzman, David M; Crystal, Ronald G; Paul, Steven M

    2016-08-01

    The common apolipoprotein E alleles (ε4, ε3, and ε2) are important genetic risk factors for late-onset Alzheimer's disease, with the ε4 allele increasing risk and reducing the age of onset and the ε2 allele decreasing risk and markedly delaying the age of onset. Preclinical and clinical studies have shown that apolipoprotein E (APOE) genotype also predicts the timing and amount of brain amyloid-β (Aβ) peptide deposition and amyloid burden (ε4 >ε3 >ε2). Using several administration protocols, we now report that direct intracerebral adeno-associated virus (AAV)-mediated delivery of APOE2 markedly reduces brain soluble (including oligomeric) and insoluble Aβ levels as well as amyloid burden in 2 mouse models of brain amyloidosis whose pathology is dependent on either the expression of murine Apoe or more importantly on human APOE4. The efficacy of APOE2 to reduce brain Aβ burden in either model, however, was highly dependent on brain APOE2 levels and the amount of pre-existing Aβ and amyloid deposition. We further demonstrate that a widespread reduction of brain Aβ burden can be achieved through a single injection of vector via intrathalamic delivery of AAV expressing APOE2 gene. Our results demonstrate that AAV gene delivery of APOE2 using an AAV vector rescues the detrimental effects of APOE4 on brain amyloid pathology and may represent a viable therapeutic approach for treating or preventing Alzheimer's disease especially if sufficient brain APOE2 levels can be achieved early in the course of the disease. PMID:27318144

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells.

    Science.gov (United States)

    Emmerling, Verena V; Pegel, Antje; Milian, Ernest G; Venereo-Sanchez, Alina; Kunz, Marion; Wegele, Jessica; Kamen, Amine A; Kochanek, Stefan; Hoerer, Markus

    2016-02-01

    Viral vectors used for gene and oncolytic therapy belong to the most promising biological products for future therapeutics. Clinical success of recombinant adeno-associated virus (rAAV) based therapies raises considerable demand for viral vectors, which cannot be met by current manufacturing strategies. Addressing existing bottlenecks, we improved a plasmid system termed rep/cap split packaging and designed a minimal plasmid encoding adenoviral helper function. Plasmid modifications led to a 12-fold increase in rAAV vector titers compared to the widely used pDG standard system. Evaluation of different production approaches revealed superiority of processes based on anchorage- and serum-dependent HEK293T cells, exhibiting about 15-fold higher specific and volumetric productivity compared to well-established suspension cells cultivated in serum-free medium. As for most other viral vectors, classical stirred-tank bioreactor production is thus still not capable of providing drug product of sufficient amount. We show that manufacturing strategies employing classical surface-providing culture systems can be successfully transferred to the new fully-controlled, single-use bioreactor system Integrity(TM) iCELLis(TM) . In summary, we demonstrate substantial bioprocess optimizations leading to more efficient and scalable production processes suggesting a promising way for flexible large-scale rAAV manufacturing. PMID:26284700

  6. Adeno-associated viral-mediated LARGE gene therapy rescues the muscular dystrophic phenotype in mouse models of dystroglycanopathy.

    Science.gov (United States)

    Yu, Miao; He, Yonglin; Wang, Kejian; Zhang, Peng; Zhang, Shengle; Hu, Huaiyu

    2013-03-01

    Dystroglycanopathies are a group of congenital muscular dystrophies (CMD) often caused by mutations in genes encoding glycosyltransferases that lead to hypoglycosylation of α-dystroglycan (α-DG) and reduce its extracellular matrix-binding activity. Overexpressing LARGE (formerly known as like-glycosyltransferase) generates an extracellular matrix-binding carbohydrate epitope in cells with CMD-causing mutations in not only LARGE but also other glycosyltransferases, including POMT1, POMGnT1, and fukutin, creating the possibilities of a one-for-all gene therapy. To determine the feasibility of LARGE gene therapy, a serotype 9 adeno-associated viral vector for overexpressing LARGE (AAV9-LARGE) was injected intracardially into newborns of two mouse models of CMD: the natural LARGE mutant Large(myd) mice and protein O-mannose N-acetylglucosaminyltransferase 1 (POMGnT1) knockout mice. AAV9-LARGE virus treatment yielded partial restoration of α-DG glycosylation and ligand-binding activity. The muscular dystrophy phenotype in skeletal muscles was ameliorated as revealed by significantly reduced fibrosis, necrosis, and numbers of centrally located nuclei with improved motor function. These results indicate that LARGE overexpression in vivo by AAV9-mediated gene therapy is effective at restoring functional glycosylation of α-DG and rescuing the muscular dystrophy phenotype in deficiency of not only LARGE but also POMGnT1, providing evidence that in vivo LARGE gene therapy may be broadly useful in dystroglycanopathies. PMID:23379513

  7. 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. PMID:24435020

  8. 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.

  9. Development of a rapid, robust, and universal picogreen-based method to titer adeno-associated vectors.

    Science.gov (United States)

    Piedra, Jose; Ontiveros, Maria; Miravet, Susana; Penalva, Cristina; Monfar, Mercè; Chillon, Miguel

    2015-02-01

    Recombinant adeno-associated viruses (rAAVs) are promising vectors in preclinical and clinical assays for the treatment of diseases with gene therapy strategies. Recent technological advances in amplification and purification have allowed the production of highly purified rAAV vector preparations. Although quantitative polymerase chain reaction (qPCR) is the current method of choice for titrating rAAV genomes, it shows high variability. In this work, we report a rapid and robust rAAV titration method based on the quantitation of encapsidated DNA with the fluorescent dye PicoGreen®. This method allows detection from 3×10(10) viral genome/ml up to 2.4×10(13) viral genome/ml in a linear range. Contrasted with dot blot or qPCR, the PicoGreen-based assay has less intra- and interassay variability. Moreover, quantitation is rapid, does not require specific primers or probes, and is independent of the rAAV pseudotype analyzed. In summary, development of this universal rAAV-titering method may have substantive implications in rAAV technology.

  10. Production, Purification, Crystallization and Preliminary X-ray Structural Studies of Adeno-Associated Virus Serotype 5

    Energy Technology Data Exchange (ETDEWEB)

    DiMattia,M.; Govindasamy, L.; Levy, H.; Whitaker-Gurda, B.; Kohlbrenner, E.; Chiorini, J.; McKenna, R.; Muzyczka, N.; Zolotukhin, S.; Agbandje-McKenna, M.

    2005-01-01

    Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Angstroms resolution using synchrotron radiation and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Angstroms. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress.

  11. 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.

  12. 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.

  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. Adeno-associated viral vector serotype 5 poorly transduces liver in rat models.

    Directory of Open Access Journals (Sweden)

    Paula S Montenegro-Miranda

    Full Text Available Preclinical studies in mice and non-human primates showed that AAV serotype 5 provides efficient liver transduction and as such seems a promising vector for liver directed gene therapy. An advantage of AAV5 compared to serotype 8 already shown to provide efficient correction in a phase 1 trial in patients suffering from hemophilia B, is its lower seroprevalence in the general population. Our goal is liver directed gene therapy for Crigler-Najjar syndrome type I, inherited severe unconjugated hyperbilirubinemia caused by UGT1A1 deficiency. In a relevant animal model, the Gunn rat, we compared the efficacy of AAV 5 and 8 to that of AAV1 previously shown to be effective. Ferrying a construct driving hepatocyte specific expression of UGT1A1, both AAV8 and AAV1 provided an efficient correction of hyperbilirubinemia. In contrast to these two and to other animal models AAV5 failed to provide any correction. To clarify whether this unexpected finding was due to the rat model used or due to a problem with AAV5, the efficacy of this serotype was compared in a mouse and two additional rat strains. Administration of an AAV5 vector expressing luciferase under the control of a liver specific promoter confirmed that this serotype poorly performed in rat liver, rendering it not suitable for proof of concept studies in this species.

  15. Capsid Mutated Adeno-Associated Virus Delivered to the Anterior Chamber Results in Efficient Transduction of Trabecular Meshwork in Mouse and Rat.

    Directory of Open Access Journals (Sweden)

    Barbara Bogner

    Full Text Available Adeno associated virus (AAV is well known for its ability to deliver transgenes to retina and to mediate improvements in animal models and patients with inherited retinal disease. Although the field is less advanced, there is growing interest in AAV's ability to target cells of the anterior segment. The purpose of our study was to fully articulate a reliable and reproducible method for injecting the anterior chamber (AC of mice and rats and to investigate the transduction profiles of AAV2- and AAV8-based capsid mutants containing self-complementary (sc genomes in the anterior segment of the eye.AC injections were performed in C57BL/6 mice and Sprague Dawley rats. The cornea was punctured anterior of the iridocorneal angle. To seal the puncture site and to prevent reflux an air bubble was created in the AC. scAAVs expressing GFP were injected and transduction was evaluated by immunohistochemistry. Both parent serotype and capsid modifications affected expression. scAAV2- based vectors mediated efficient GFP-signal in the corneal endothelium, ciliary non-pigmented epithelium (NPE, iris and chamber angle including trabecular meshwork, with scAAV2(Y444F and scAAV2(triple being the most efficient.This is the first study to semi quantitatively evaluate transduction of anterior segment tissues following injection of capsid-mutated AAV vectors. scAAV2- based vectors transduced corneal endothelium, ciliary NPE, iris and trabecular meshwork more effectively than scAAV8-based vectors. Mutagenesis of surface-exposed tyrosine residues greatly enhanced transduction efficiency of scAAV2 in these tissues. The number of Y-F mutations was not directly proportional to transduction efficiency, however, suggesting that proteosomal avoidance alone may not be sufficient. These results are applicable to the development of targeted, gene-based strategies to investigate pathological processes of the anterior segment and may be applied toward the development of gene

  16. 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.

  17. Optimized adeno-associated viral vector-mediated striatal DOPA delivery restores sensorimotor function and prevents dyskinesias in a model of advanced Parkinson's disease.

    Science.gov (United States)

    Björklund, Tomas; Carlsson, Thomas; Cederfjäll, Erik Ahlm; Carta, Manolo; Kirik, Deniz

    2010-02-01

    Viral vector-mediated gene transfer utilizing adeno-associated viral vectors has recently entered clinical testing as a novel tool for delivery of therapeutic agents to the brain. Clinical trials in Parkinson's disease using adeno-associated viral vector-based gene therapy have shown the safety of the approach. Further efforts in this area will show if gene-based approaches can rival the therapeutic efficacy achieved with the best pharmacological therapy or other, already established, surgical interventions. One of the strategies under development for clinical application is continuous 3,4-dihydroxyphenylalanine delivery. This approach has been shown to be efficient in restoring motor function and reducing established dyskinesias in rats with a partial lesion of the nigrostriatal dopamine projection. Here we utilized high purity recombinant adeno-associated viral vectors serotype 5 coding for tyrosine hydroxylase and its co-factor synthesizing enzyme guanosine-5'-triphosphate cyclohydrolase-1, delivered at an optimal ratio of 5 : 1, to show that the enhanced 3,4-dihydroxyphenylalanine production obtained with this optimized delivery system results in robust recovery of function in spontaneous motor tests after complete dopamine denervation. We found that the therapeutic efficacy was substantial and could be maintained for at least 6 months. The tyrosine hydroxylase plus guanosine-5'-triphosphate cyclohydrolase-1 treated animals were resistant to developing dyskinesias upon peripheral l-3,4-dihydroxyphenylalanine drug challenge, which is consistent with the interpretation that continuous dopamine stimulation resulted in a normalization of the post-synaptic response. Interestingly, recovery of forelimb use in the stepping test observed here was maintained even after a second lesion depleting the serotonin input to the forebrain, suggesting that the therapeutic efficacy was not solely dependent on dopamine synthesis and release from striatal serotonergic terminals

  18. Construction of a recombinant human parvovirus B19: adeno-associated virus 2 (AAV) DNA inverted terminal repeats are functional in an AAV-B19 hybrid virus.

    OpenAIRE

    Srivastava, C H; Samulski, R J; L. Lu; Larsen, S H; A Srivastava

    1989-01-01

    To facilitate genetic analysis of the human pathogenic parvovirus B19, we constructed a hybrid B19 viral genome in which the defective B19 inverted terminal repeats were replaced with the full-length inverted terminal repeats from a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). The hybrid AAV-B19 genome was rescued from a recombinant plasmid and then the DNA was replicated upon transfection into adenovirus 2-infected human KB cells in the presence of AAV genes coding for...

  19. 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

  20. 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

  1. Longitudinal follow-up and characterization of a robust rat model for Parkinson's disease based on overexpression of alpha-synuclein with adeno-associated viral vectors.

    Science.gov (United States)

    Van der Perren, Anke; Toelen, Jaan; Casteels, Cindy; Macchi, Francesca; Van Rompuy, Anne-Sophie; Sarre, Sophie; Casadei, Nicolas; Nuber, Silke; Himmelreich, Uwe; Osorio Garcia, Maria Isabel; Michotte, Yvette; D'Hooge, Rudi; Bormans, Guy; Van Laere, Koen; Gijsbers, Rik; Van den Haute, Chris; Debyser, Zeger; Baekelandt, Veerle

    2015-03-01

    Testing of new therapeutic strategies for Parkinson's disease (PD) is currently hampered by the lack of relevant and reproducible animal models. Here, we developed a robust rat model for PD by injection of adeno-associated viral vectors (rAAV2/7) encoding α-synuclein into the substantia nigra, resulting in reproducible nigrostriatal pathology and behavioral deficits in a 4-week time period. Progressive dopaminergic dysfunction was corroborated by histopathologic and biochemical analysis, motor behavior testing and in vivo microdialysis. L-DOPA treatment was found to reverse the behavioral phenotype. Non-invasive positron emission tomography imaging and magnetic resonance spectroscopy allowed longitudinal monitoring of neurodegeneration. In addition, insoluble α-synuclein aggregates were formed in this model. This α-synuclein rat model shows improved face and predictive validity, and therefore offers the possibility to reliably test novel therapeutics. Furthermore, it will be of great value for further research into the molecular pathogenesis of PD and the importance of α-synuclein aggregation in the disease process. PMID:25599874

  2. Adeno-associated virus type 2 rep protein inhibits human papillomavirus type 16 E2 recruitment of the transcriptional coactivator p300.

    Science.gov (United States)

    Marcello, A; Massimi, P; Banks, L; Giacca, M

    2000-10-01

    Infection by human adeno-associated virus type 2 (AAV2) is a possible protective factor in the development of cervical carcinomas associated with human papillomaviruses (HPV). The replicative proteins of AAV2 (Rep) have been implicated in the inhibition of papillomavirus replication and transforming activities, although the molecular events underlying these effects are poorly understood. We observed that each of the four forms of AAV2 Rep inhibited the E1- and E2-driven replication of oncogenic HPV type 16 (HPV16). Rep40, corresponding to the C-terminal domain of all Rep proteins, inhibited both HPV DNA replication and HPV16 E2-mediated transactivation. Rep40 specifically bound the N-terminal transactivation domain of HPV16 E2 both in vitro and in vivo. This interaction was found to specifically disrupt the binding of E2 to the cellular transcriptional coactivator p300. Accordingly, the inhibitory effect of Rep on HPV16 E2 transactivation was rescued by the overexpression of p300. These data indicate a novel role of Rep in the down-regulation of papillomaviruses through inhibition of complex formation between the HPV16 E2 transcriptional activator and its cellular coactivator, p300. PMID:10982355

  3. 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.

  4. Trans-Splicing Adeno-Associated Viral Vector-Mediated Gene Therapy Is Limited by the Accumulation of Spliced mRNA but Not by Dual Vector Coinfection Efficiency

    OpenAIRE

    XU, ZHUPING; Yue, Yongping; Lai, Yi; Ye, Chaoyang; Qiu, Jianming; Pintel, David J.; Duan, Dongsheng

    2004-01-01

    Therapeutic application of recombinant adeno-associated virus (AAV) has been limited by its small carrying capacity. To overcome this limitation trans-splicing vectors were developed recently. However, the transduction efficiency of trans-splicing vectors is considerably lower than that of a single intact vector in skeletal muscle. To improve trans-splicing vectors for skeletal muscle gene therapy, we examined whether coinfection efficiency is a rate-limiting factor in the mdx mouse, a model ...

  5. [Establishment of hepatitis B virus (HBV) chronic infection mouse model by in vivo transduction with a recombinant adeno-associated virus 8 carrying 1. 3 copies of HBV genome (rAAN8-1. 3HBV)].

    Science.gov (United States)

    Dong, Xiao-Yan; Yu, Chi-Jie; Wang, Gang; Tian, Wen-Hong; Lu, Yue; Zhang, Feng-Wei; Wang, Wen; Wang, Yue; Tan, Wen-Jie; Wu, Xiao-Bing

    2010-11-01

    In this report, we developed a HBV infection model in C57BL/6 mouse line by in vivo injection of a recombinant adeno-associated virus 8 vector carrying 1. 3 copies of HBV genome (ayw subtype) (rAAV8-1. 3HBV). We firstly prepared and purified the rAAV8-1. 3HBV and then injected it into three C57BL/6 mice with the dose of 2 x 10e11vg, respectively. HBsAg and HBeAg were assayed in sera collected at different time points post injection. Ten weeks post injection, the three mice were sacrificed and blood and liver tissue were taken for assay. Copies of HBV DNA were detected by real time PCR and the way of HBV DNA replication was identified by PCR. Subsequently, detection of HBV antigen by immunohistochemistry and pathology analysis of liver tissue of mice were performed. The results suggested that expression of HBsAg and HBeAg lasted for at least 10 weeks in mice sera. Among mice injected with rAAV8-1. 3HBV, HBsAg levels were showed an 'increasing-decreasing-increasing' pattern (the lowest level at the 4th week post injection), while HBeAg levels were kept high and relatively stable. HBV DNA copies were 4.2 x 10(3), 3.6 x 10(3), 2.5 x 10(3) copies/mL in sera and 8.0 x 10(6), 5.7 x 10(6), 2.6 x 10(6) copies/g in hepatic tissues of three mice, respectively. We found that the linear 1. 3HBV DNA in the rAAV8-1. 3HBV could self form into circular HBV genome and replicate in livers of HBV transfected mice. HBsAg and HBcAg were both positive in liver tissue of mice injected with rAAV8-1. 3HBV and no obvious pathological characters were found in liver of mice injected with rAAV8-1. 3HBV. In conclusion, we successfully developed a HBV chronic infection model in C57BL/6 mouse line by in vivo transduction with the recombinant virus rAAV8-1. 3HBV, in which HBV genes could be continuously expressed and replicated over 10 weeks, and paved a way for further characterization of the human chronic hepatitis B virus infection and evaluation of vaccine and anti-HBV agents. PMID:21344744

  6. 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.

  7. Partial correction of the CFTR-dependent ABPA mouse model with recombinant adeno-associated virus gene transfer of truncated CFTR gene.

    Science.gov (United States)

    Mueller, Christian; Torrez, Daniel; Braag, Sofia; Martino, Ashley; Clarke, Tracy; Campbell-Thompson, Martha; Flotte, Terence R

    2008-01-01

    Recently, we have developed a model of airway inflammation in a CFTR knockout mouse utilizing Aspergillus fumigatus crude protein extract (Af-cpe) to mimic allergic bronchopulmonary aspergillosis (ABPA) 1, an unusual IgE-mediated hypersensitivity syndrome seen in up to 15% of cystic fibrosis (CF) patients and rarely elsewhere. We hypothesized that replacement of CFTR via targeted gene delivery to airway epithelium would correct aberrant epithelial cytokine signaling and ameliorate the ABPA phenotype in CFTR-deficient (CFTR 489X - /-, FABP-hCFTR + / +) mice. CFTR knockout mice underwent intra-tracheal (IT) delivery of recombinant adeno-associated virus serotype 5 (rAAV5Delta-264CFTR) or rAAV5-GFP at 2.58 x 10(12) viral genomes/mouse. All mice were then sensitized with two serial injections (200 microg) of crude Af antigen via the intra-peritoneal (IP) route. Untreated mice were sensitized without virus exposure. Challenges were performed 2 weeks after final sensitization, using a 0.25% solution containing Aspergillus fumigatus crude protein extract delivered by inhalation on three consecutive days. The rAAV5Delta-264CFTR-treated mice had lower total serum IgE levels (172513 ng/ml +/- 1312) than rAAV5-GFP controls (26 892 ng/ml +/- 3715) (p = 0.037) and non-treated, sensitized controls (24 816 +/- 4219 ng/ml). Serum IgG1 levels also were lower in mice receiving the CFTR vector. Interestingly, splenocytes from rAAV5Delta-264CFTR-treated mice secreted less IL-13, INFg, TNFa, RANTES and GM-CSF after ConA stimulation. Gene therapy with rAAV5Delta-264CFTR attenuated the hyper-IgE response in this reproducible CF mouse model of ABPA, with systemic effects also evident in the cytokine response of stimulated splenocytes. PMID:18023072

  8. Delivery of basic fibroblast growth factor gene to healing tendon by adeno-associated virus 2 vector and tissue reactions of adeno-associated virus vectors%腺相关病毒载体转导基因至愈合肌腱及载体组织反应的研究

    Institute of Scientific and Technical Information of China (English)

    朱蓓; 汤锦波; 曹怡; 陈传好

    2008-01-01

    目的 探讨腺相关病毒(AAV)载体转导碱性成纤维细胞牛长因子(bFGF)基因对肌腱愈合的影响,并观察腺病毒、AAV以及脂质体一质粒三种基因治疗载体应用于肌腱所产生的组织反应.方法 取13只成年白色来亨鸡的双侧巾趾趾深屈肌腱(26根),随机分为实验组和对照组,每组13根,实验组肌腱完全切断后注射AAV2-bFGF并以改良Kessler法修复,对照组不注射AAV2-bFGF,仪以改良Kessler法修复.第4周未行免疫组织化学染色,第8周术测定趾屈曲功.将6只成年新西兰白兔的趾深屈肌腱(36根)分成二组,每组12根,分别注射10μL的腺病毒、AAV2和脂质体.质粒载体,术后第3、7、14天分别取肌腱,进行石蜡切片、HE染色.结果 AAV2-bFGF可以在术后4周显著地提高肌腱bFGF的表达,而且不增加趾屈曲阻力(粘连形成).所测屈曲功第8周末实验组为(0.052±0.031)J,对照组为(0.049±0.035)J,两组问差异无统计学意义(t=0.31266,P=0.8984).脂质体-质粒载体组肌腱组织反廊重于腺病毒载体组,AAV2载体组肌腱组织反应最轻,在腱外膜处有组织反应,而腱内膜区域几乎无组织反应.结论 用AAV2载体转bFGF基因至肌腱能有效增加愈合肌腱的bFGF.在三种所研究的载体中,腺病毒和AAV2载体引起的组织反应比脂质体.质粒载体轻.AAV2引起的组织反应最轻.AAV2可能会成为肌腱的转基因良好载体.%Objective To explore the effect of basic fibroblast growth factor (bFGF) gene transferred by adeno-associated virus (AAV) 2 vectors on tendon healing and to observe tissue reactions of adenovirus, AAV and liposome-plasmid vectors in tendons, Methods Twenty-six flexor digitorum profundus (FDP) tendons from bilateral long toes of 13 chickens were randomly divided into equal 2 groups. Tendons in the experimental group were cut completely and treated with AAV2-bFGF before repair by the modified Kessler method. Tendons as controls were not treated with

  9. 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

  10. Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.

    Science.gov (United States)

    Lock, Martin; Alvira, Mauricio R; Chen, Shu-Jen; Wilson, James M

    2014-04-01

    Accurate titration of adeno-associated viral (AAV) vector genome copies is critical for ensuring correct and reproducible dosing in both preclinical and clinical settings. Quantitative PCR (qPCR) is the current method of choice for titrating AAV genomes because of the simplicity, accuracy, and robustness of the assay. However, issues with qPCR-based determination of self-complementary AAV vector genome titers, due to primer-probe exclusion through genome self-annealing or through packaging of prematurely terminated defective interfering (DI) genomes, have been reported. Alternative qPCR, gel-based, or Southern blotting titering methods have been designed to overcome these issues but may represent a backward step from standard qPCR methods in terms of simplicity, robustness, and precision. Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. Here we compare a ddPCR-based AAV genome titer assay with a standard and an optimized qPCR assay for the titration of both single-stranded and self-complementary AAV genomes. We demonstrate absolute quantification of single-stranded AAV vector genomes by ddPCR with up to 4-fold increases in titer over a standard qPCR titration but with equivalent readout to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-, 1.9-, and 2.3-fold higher than those determined by standard qPCR, optimized qPCR, and agarose gel assays, respectively. Droplet digital PCR-based genome titering was superior to qPCR in terms of both intra- and interassay precision and is more resistant to PCR inhibitors, a desirable feature for in-process monitoring of early-stage vector production and for vector genome biodistribution

  11. 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

  12. 重组8型腺相关病毒介导HBV急性感染树鼩模型建立%Establishment of a tree shrew model of acute hepatitis B virus infection by transduction with a recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome

    Institute of Scientific and Technical Information of China (English)

    曾扬; 吴小红; 胡靓雅; 刘晨风; 于虹; 郭彦; 周勇; 孙世惠; 周育森

    2013-01-01

    目的 利用重组8型腺相关病毒介导1.3拷贝HBV基因组(1.3HBV,ayw亚型)在树鼩肝脏表达,建立HBV急性感染树鼩模型.方法 通过大腿内侧静脉注射将携带有1.3 HBV的重组8型腺相关病毒(recombinant adeno-associated virus 8,rAAV8-1.3HBV)导入树鼩肝脏,通过ELISA检测树鼩血清中HBsAg、HBeAg、HBsAb、HBeAb、HBcAb,荧光定量PCR检测树鼩肝脏和血清中HBV DNA,全自动生化分析仪检测血清中ALT水平,并观察感染后肝脏的病变情况.结果 HBV感染主要血清标志物1~2周内均检测阳性;30 d后肝组织仍可检测到病毒抗原阳性细胞;55 d时肝组织HBV DNA拷贝数仍可达到104~105;树鼩血清中HBV DNA拷贝数持续一个月高于正常组;肝组织炎细胞略增多,血清ALT水平持续升高.结论 rAAV8所携带的HBV基因组高效专一导入树鼩肝细胞并复制表达,成功建立HBV急性感染树鼩模型,为进一步探索rAAV8树鼩慢性感染模型打下一定的基础.%Objective To establish a tree shrew model of acute hepatitis B virus infection by injection of a recombinant adeno-associated virus 8 vector carrying 1.3 copies of HBV genome (ayw subtype) (rAAV8-1.3 HBV)into the liver of tree shrews.Methods Serum and liver tissues were collected at indicated times after i.v.injection of rAAV8-1.3 HBV into the tree shrews.The HBsAg,BeAg,HBsAb,HBeAb,HBcAb,ALT and HBV virus load were examined by ELISA and real-time PCR,respectively.The expression of HBcAg and pathological changes in the liver were also observed after the rAAV8-1.3 HBV infection.Results Markers of serum HBV were all positive 2 weeks after and HBcAg-positive hepatocytes were even detected in the liver 55 days after rAAV8-1.3 HBV injection.The copies of HBV DNA in liver reached 104-105 at 55 days after rAAV8-1.3HBV injection.Serum HBV DNA could be detected for over one month.Mild pathological changes with elevated ALT were observed after rAAV8-1.3 HBV injection.Conclusions A tree shrew

  13. Neuritin Up-regulates Kv4.2 α-Subunit of Potassium Channel Expression and Affects Neuronal Excitability by Regulating the Calcium-Calcineurin-NFATc4 Signaling Pathway*

    Science.gov (United States)

    Yao, Jin-jing; Zhao, Qian-Ru; Liu, Dong-Dong; Chow, Chi-Wing; Mei, Yan-Ai

    2016-01-01

    Neuritin is an important neurotrophin that regulates neural development, synaptic plasticity, and neuronal survival. Elucidating the downstream molecular signaling is important for potential therapeutic applications of neuritin in neuronal dysfunctions. We previously showed that neuritin up-regulates transient potassium outward current (IA) subunit Kv4.2 expression and increases IA densities, in part by activating the insulin receptor signaling pathway. Molecular mechanisms of neuritin-induced Kv4.2 expression remain elusive. Here, we report that the Ca2+/calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) c4 axis is required for neuritin-induced Kv4.2 transcriptional expression and potentiation of IA densities in cerebellum granule neurons. We found that neuritin elevates intracellular Ca2+ and increases Kv4.2 expression and IA densities; this effect was sensitive to CaN inhibition and was eliminated in Nfatc4−/− mice but not in Nfatc2−/− mice. Stimulation with neuritin significantly increased nuclear accumulation of NFATc4 in cerebellum granule cells and HeLa cells, which expressed IR. Furthermore, NFATc4 was recruited to the Kv4.2 gene promoter loci detected by luciferase reporter and chromatin immunoprecipitation assays. More importantly, data obtained from cortical neurons following adeno-associated virus-mediated overexpression of neuritin indicated that reduced neuronal excitability and increased formation of dendritic spines were abrogated in the Nfatc4−/− mice. Together, these data demonstrate an indispensable role for the CaN/NFATc4 signaling pathway in neuritin-regulated neuronal functions. PMID:27307045

  14. Neuritin Up-regulates Kv4.2 α-Subunit of Potassium Channel Expression and Affects Neuronal Excitability by Regulating the Calcium-Calcineurin-NFATc4 Signaling Pathway.

    Science.gov (United States)

    Yao, Jin-Jing; Zhao, Qian-Ru; Liu, Dong-Dong; Chow, Chi-Wing; Mei, Yan-Ai

    2016-08-12

    Neuritin is an important neurotrophin that regulates neural development, synaptic plasticity, and neuronal survival. Elucidating the downstream molecular signaling is important for potential therapeutic applications of neuritin in neuronal dysfunctions. We previously showed that neuritin up-regulates transient potassium outward current (IA) subunit Kv4.2 expression and increases IA densities, in part by activating the insulin receptor signaling pathway. Molecular mechanisms of neuritin-induced Kv4.2 expression remain elusive. Here, we report that the Ca(2+)/calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) c4 axis is required for neuritin-induced Kv4.2 transcriptional expression and potentiation of IA densities in cerebellum granule neurons. We found that neuritin elevates intracellular Ca(2+) and increases Kv4.2 expression and IA densities; this effect was sensitive to CaN inhibition and was eliminated in Nfatc4(-/-) mice but not in Nfatc2(-/-) mice. Stimulation with neuritin significantly increased nuclear accumulation of NFATc4 in cerebellum granule cells and HeLa cells, which expressed IR. Furthermore, NFATc4 was recruited to the Kv4.2 gene promoter loci detected by luciferase reporter and chromatin immunoprecipitation assays. More importantly, data obtained from cortical neurons following adeno-associated virus-mediated overexpression of neuritin indicated that reduced neuronal excitability and increased formation of dendritic spines were abrogated in the Nfatc4(-/-) mice. Together, these data demonstrate an indispensable role for the CaN/NFATc4 signaling pathway in neuritin-regulated neuronal functions. PMID:27307045

  15. Adeno-associated virus type-2 expression of pigmented epithelium-derived factor or Kringles 1–3 of angiostatin reduce retinal neovascularization

    OpenAIRE

    Raisler, Brian J.; Berns, Kenneth I.; Grant, Maria B.; Beliaev, Denis; Hauswirth, William W.

    2002-01-01

    Neovascular diseases of the retina include age-related macular degeneration and diabetic retinopathy, and together they comprise the leading causes of adult-onset blindness in developed countries. Current surgical, pharmaceutical, and laser therapies for age-related macular degeneration (AMD) rarely result in improved vision, do not significantly prevent neovascularization (NV), and often result in at least some vision loss. To address this therapeutic gap, we determined the efficacy of recom...

  16. 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.

  17. Potential for cellular stress response to hepatic factor VIII expression from AAV vector

    Science.gov (United States)

    Zolotukhin, Irene; Markusic, David M; Palaschak, Brett; Hoffman, Brad E; Srikanthan, Meera A; Herzog, Roland W

    2016-01-01

    Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII) or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein. Further, it has been reported that over-expression of the FVIII protein induces endoplasmic reticulum stress and activates the unfolded protein response pathway both in vitro and in hepatocytes in vivo, presumably due to retention of misfolded FVIII protein within the endoplasmic reticulum. Engineering of the F8 transgene, including removal of the B domain (BDD-FVIII) and codon optimization, now allows for the generation of adeno-associated virus vectors capable of expressing therapeutic levels of FVIII. Here we sought to determine if the risks of inducing the unfolded protein response in murine hepatocytes extend to adeno-associated virus gene transfer. Although our data show a mild activation of unfolded protein response markers following F8 gene delivery at a certain vector dose in C57BL/6 mice, it was not augmented upon further elevated dosing, did not induce liver pathology or apoptosis, and did not impact FVIII immunogenicity. PMID:27738644

  18. Novel qPCR strategy for quantification of recombinant adeno-associated virus serotype 2 vector genome-titer%测定重组腺相关病毒基因组滴度的qPCR新方法

    Institute of Scientific and Technical Information of China (English)

    蒙青林; 张彬彬; 张春

    2013-01-01

    Adeno-associated virus (AAV) has many advantages for gene therapy over other vector systems. However, after the production of recombinant AAV (Raav) vectors, the biological titration of rAAV stocks is still cumbersome. Different investigators used laboratory-specific methods or internal reference standards that may limit preclinical and clinical applications. The inverted terminal repeats (ITR) sequences are the only cw-regulated viral elements required for rAAV packaging and remain within viral vector genomes. ITR is the excellent target sequences for qPCR quantification of rAAV titer. In this study, we developed a novel qPCR strategy to quantify rAAVs' vector genome titer via targeting the ITR2 or ITR2-CMV element. In conclusion, the method is fast and accurate for the titration of rAAV2-derived vector genomes. It will promote the standardization of rAAV titration in the future.%腺相关病毒(Adeno-associated virus,AAV)在基因治疗应用中具有很多优势,但是其生物学滴度的测定仍很繁琐,不同实验室使用各自的方法和参照,这些都影响了重组腺相关病毒(rAAV)载体在临床前和临床上的应用.反向末端重复序列(Inverted terminal repeats,ITR)是重组腺相关病毒载体中不可或缺的顺式作用元件,针对ITR2以及ITR2-CMV设计的qPCR检测方法可以快速、准确地得到rAAV2的基因组滴度,由于该方法可以广泛适用,因此对推动AAV滴度检测的标准化有重要意义.

  19. 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.

  20. 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.

    OpenAIRE

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

    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 B1...

  1. 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

  2. Effect of Nuclear Factor κB Inhibition on Serotype 9 Adeno-Associated Viral (AAV9) Minidystrophin Gene Transfer to the mdx Mouse

    OpenAIRE

    Reay, Daniel P.; Niizawa, Gabriela A; Watchko, Jon F; Daood, Molly; Reay, Ja’Nean C; Raggi, Eugene; Clemens, Paula R

    2012-01-01

    Gene therapy studies for Duchenne muscular dystrophy (DMD) have focused on viral vector-mediated gene transfer to provide therapeutic protein expression or treatment with drugs to limit dystrophic changes in muscle. The pathological activation of the nuclear factor (NF)-κB signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy. Furthermore, activation of NF-κB may inhibit gene transfer by promoting inflammation in response to the transgene or ve...

  3. Inhibition of infectious bursal disease virus replication in chicken embryos by miRNAs delivered by recombinant avian adeno-associated viral vector%重组禽腺联病毒介导的miRNA抑制传染性法氏囊病病毒在鸡胚内的复制

    Institute of Scientific and Technical Information of China (English)

    沈鹏鹏; 王永娟; 孙怀昌; 张鑫宇; 夏晓莉

    2011-01-01

    [Objective]We studied the inhibition of infectious bursal disease virus ( IBDV ) replication in chicken embryos by recombinant avian adeno-associated virus (AAAV)-delivered VP1- and VP2-specific microRNAs (miRNAs).[Methods and Results]We co-transfected AAV-293 cells with the VP1- or VP2 gene-specific miRNA expression vector pAITR-RFPmiVP1 or AITR-RFPmiVP2E, AAAV packaging vector pcDNA-ARC and adenovirus helper vector pHelper, resulting in recombinant virus rAAAV-RFPmiVP1 or rAAAV-RFPmiVP2E.We also generated the recombinant viruses rAAAV-RFP (without miRNA expression cassette) and rAAAV-RFPmiVP2con ( expressing control miRNA ) using the same method as the control purpose.Electron microscopy showed that the recombinant viruses had a typical morphology of AAV.We confirmed the presence of miRNA expression cassette in the recombinant viral genomes by using PCR.Our poly (A)-tailed RT-PCR showed correct expression of the miRNAs in the rAAAV-transduced DF-1 cells.We inoculated the recombinant viruses individually into 8-day-old SPF chicken embryos and then challenged them using Lukert strain IBDV on day 2 after inoculation.Our IBDV titration assay showed that the 50% tissue culture infectious dose ( TCID50) of rAAAV-RFP- or rAAAV-RFPmiVP2con-inoculated group was 8.0 log10, whereas the TCID50 of rAAAV-RFPmiVP1-inoculated group decreased to 1.0 and 0.8 log10 on day 3 and 6 after challenge, respectively.Similarly, the TCID50 of rAAAV-RFPmiVP2E-inoculated group decreased to 1.5 and 2.0 log10, respectively.[Conclusion]These data suggest that rAAAV can transduce efficiently chicken embryos and the expressed VP1- and VP2-specific miRNAs can inhibit the replication of IBDV efficiently.%[目的]在鸡胚水平上探索VP1和VP2基因特异miRNA抑制传染性法氏囊病病毒(infectious bursaldisease virus,IBDV)复制的可行性.[方法与结果]将表达VP1基因特异miRNA重组载体pAITR-RFPmiVP1或VP2基因特异miRNA重组载体pAITR-RFPmiVP2E

  4. Sonic hedgehog elevates N-myc gene expression in neural stem cells★

    OpenAIRE

    Liu, Dongsheng; Wang, Shouyu; Cui, Yan; Shen, Lun; Du, Yanping; Li, Guilin; Zhang, Bo; Wang, Renzhi

    2012-01-01

    Proliferation of neural stem cells is regulated by the secreted signaling molecule sonic hedgehog. In this study, neural stem cells were infected with recombinant adeno-associated virus expressing sonic hedgehog-N-enhanced green fluorescent protein. The results showed that overexpression of sonic hedgehog in neural stem cells induced the increased expression of Gli1 and N-myc, a target gene of sonic hedgehog. These findings suggest that N-myc is a direct downstream target of the sonic hedgeho...

  5. Ribosomal DNA Integrating rAAV-rDNA Vectors Allow for Stable Transgene Expression

    OpenAIRE

    Lisowski, Leszek; Lau, Ashley; Wang, Zhongya; Zhang, Yue(Walter Burke Institute for Theoretical Physics, California Institute of Technology, Pasadena, CA, 91125, U.S.A.); Zhang, Feijie; Grompe, Markus; Kay, Mark A

    2012-01-01

    Although recombinant adeno-associated virus (rAAV) vectors are proving to be efficacious in clinical trials, the episomal character of the delivered transgene restricts their effectiveness to use in quiescent tissues, and may not provide lifelong expression. In contrast, integrating vectors enhance the risk of insertional mutagenesis. In an attempt to overcome both of these limitations, we created new rAAV-rDNA vectors, with an expression cassette flanked by ribosomal DNA (rDNA) sequences cap...

  6. 重组腺相关病毒2型/人凝血因子IX的质量研究%Quality control of recombinant adeno-associated virus type 2/human blood coagulation factor IX

    Institute of Scientific and Technical Information of China (English)

    高凯; 王军志; 饶春明; 吴小兵

    2003-01-01

    目的研究并建立重组腺相关病毒2型/人凝血因子IX(recombinant adeno-associated virus type 2/human blood coagulation factor IX,rAAV-2/hFIX)的质量标准.方法采用PCR法确认病毒所携带的重组核酸结构和测定辅助病毒(helper virus)和野生型腺相关病毒(wtAAV)的残留片段.SDS-PAGE电泳测定病毒外壳蛋白分子量及纯度,TSK gel SP-NPR阳离子交换柱系统测定病毒颗粒纯度.以斑点杂交法测定病毒颗粒数.一期法于IX因子基因剔除小鼠体内测定rAAV-2/hFIX生物学活性,并通过ELISA法测定感染BHK-21细胞后hFIX的表达量.结果 PCR法确证病毒的重组核酸结构与构建预期相同;在1×107 VG的rAAV-2/hFIX颗粒中,残留辅助病毒的基因片段数少于1个拷贝;在1×108 VG的rAAV-2/hFIX颗粒中,野生型AAV-2基因片段数少于1个拷贝.病毒颗粒及外壳蛋白纯度均大于98%,病毒颗粒数大于1.0×1015 VG*L-1(virus genome*L-1).IX因子剔除小鼠肌肉注射病毒后21 d,小鼠血液中人凝血因子IX活性达到大于正常人因子IX活性的15%,IX因子的体外表达水平大于20.0 μg*L-1.其他各项检测指标均符合规定.结论建立了rAAV-2/hFIX的质量标准,用于控制产品质量.

  7. Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta.

    Science.gov (United States)

    Brodzik, R; Bandurska, K; Deka, D; Golovkin, M; Koprowski, H

    2005-12-16

    Plant viruses show great potential for production of pharmaceuticals in plants. Such viruses can harbor a small antigenic peptide(s) as a part of their coat proteins (CP) and elicit an antigen-specific immune response. Here, we report the high yield and consistency in production of recombinant alfalfa mosaic virus (AlMV) particles for specific presentation of the small loop 15 amino acid epitope from domain-4 of the Bacillus anthracis protective antigen (PA-D4s). The epitope was inserted immediately after the first 25 N-terminal amino acids of AlMV CP to retain genome activation and binding of CP to viral RNAs. Recombinant AlMV particles were efficiently produced in tobacco, easily purified for immunological analysis, and exhibited extended stability and systemic proliferation in planta. Intraperitional injections of mice with recombinant plant virus particles harboring the PA-D4s epitope elicited a distinct immune response. Western blotting and ELISA analysis showed that sera from immunized mice recognized both native PA antigen and the AlMV CP. PMID:16236249

  8. Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta

    International Nuclear Information System (INIS)

    Plant viruses show great potential for production of pharmaceuticals in plants. Such viruses can harbor a small antigenic peptide(s) as a part of their coat proteins (CP) and elicit an antigen-specific immune response. Here, we report the high yield and consistency in production of recombinant alfalfa mosaic virus (AlMV) particles for specific presentation of the small loop 15 amino acid epitope from domain-4 of the Bacillus anthracis protective antigen (PA-D4s). The epitope was inserted immediately after the first 25 N-terminal amino acids of AlMV CP to retain genome activation and binding of CP to viral RNAs. Recombinant AlMV particles were efficiently produced in tobacco, easily purified for immunological analysis, and exhibited extended stability and systemic proliferation in planta. Intraperitional injections of mice with recombinant plant virus particles harboring the PA-D4s epitope elicited a distinct immune response. Western blotting and ELISA analysis showed that sera from immunized mice recognized both native PA antigen and the AlMV CP

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

    OpenAIRE

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

    2012-01-01

    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...

  10. Expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in cultured glomerular mesangial cells (GMC) by high glucose and interventional effects of rAAV-AS%高糖环境下VEGF与PEDF在GMC中表达及rAAV-AS基因的干预作用

    Institute of Scientific and Technical Information of China (English)

    赵猛; 李伟

    2011-01-01

    Objective To explore the effect of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in cultured glomerular mesangial cells (GMCs) and intervention of recombinant adeno-associated virus mediated angiostatin gene.Methods Cultured HBZY21 rat GMCs were divided into 6 groups ( glucose as a stimulated factor, rAAV-AS as an intervenor): low glucose, high glucose, high glucose and rAAV, high glucose and rAAV-AS.The expressions of PEDF and VEGF of each group were measured by immunohistochemistry and ELISA.Results ( 1 ) The level of VEGF protein was remarkably increased and the level of PEDF protein was decreased by high glucose.(2) High glucose could increase the expression of VEGF and decrease significantly the expression of PEDF.rAAV-AS could prevent this change induced by high glucose.Conclusions rAAV-AS might play an important role in preventing diabetes nephropathy by partly compensating the unbalanced expressions of VEGF and PEDF induced by hyperglycemia.%目的 探讨高糖对大鼠肾小球系膜细胞(GMC)色素上皮细胞衍生因子(PEDF) 和血管内皮生长因子(VEGF)表达影响以及腺相关病毒介导血管抑素(rAAV-AS)基因的干预作用.方法 将培养的大鼠GMC株分为6组,高糖作为刺激因素,rAAV-AS 作为干预因素.分别设低糖组、高糖组、高糖加腺相关病毒(rAAV)组及高糖加rAAV-AS 治疗组.用免疫细胞化学及ELISA法测定各组系膜细胞PEDF 以及VEGF蛋白表达.结果 高糖可下调GMC中PEDF蛋白的表达,上调GMC中VEGF蛋白的表达.rAAV-AS可逆转高糖导致GMC的VEGF蛋白表达的增加及PEDF蛋白表达的降低.结论 rAAV-AS可以一定程度改善高糖诱导的VEGF和PEDF表达失衡而发挥对糖尿病肾病(DN)的保护作用.

  11. Improved Function of the Failing Rat Heart by Regulated Expression of Insulin-Like Growth Factor I via Intramuscular Gene Transfer

    OpenAIRE

    Lai, N. Chin; Tang, Tong; Gao, Mei Hua; Saito, Miho; Miyanohara, Atsushi; Hammond, H. Kirk

    2011-01-01

    Current methods of gene transfer for heart disease include injection into heart muscle or intracoronary coronary delivery, approaches that typically provide limited expression and are cumbersome to apply. To circumvent these problems, we selected a transgene, insulin-like growth factor-I (IGF-I), which may, in theory, have favorable effects on heart function when secreted from a remote site. We examined the feasibility and efficacy of skeletal muscle injection of adeno-associated virus 5 enco...

  12. Study of adeno-associated virus carrying the HGFK1 gene(AAV-HGFK1) in treating rat hepatocellular carcinoma%腺相关病毒介导的HGFK1对大鼠肝细胞癌的治疗作用研究

    Institute of Scientific and Technical Information of China (English)

    顾春荣; 郭跃武; 赵晖; 孙元珏; 姚阳; 沈赞; 林李家宓

    2009-01-01

    -angiogenesis molecule than angiostatin. In this study, we observed the effects and mechanisms of HGFK1 gene on the HCC. Methods: A recombinant adeno-associated vires carrying the HGFK1 gene (rAAV-HGFK1) was constructed.HCC of rat was induced by McA-RH7777. rAAV-HGFK1 was used to treat the rat, median survival time and metastasis rate were observed. Results: Ten days after tumor cell inoculation, surgery were performed to confirm the tumor formation, PBS, rAAV-EGFP or rAAV-HGFK1 was injected directly into the tumor nodule followed by portal vein injection. Results from our study demonstrated that rAAV-HGFK1 treatment significantly prolonged the median survival time of the HCC bearing rats from 30 days (PBS and rAAV-EGFP groups) to 49 days (rAAV-HGFK1 group). More importantly rAAV-HGFK1 inhibited tumor growth and completely prevented liver, lung and peritoneal metastasis. In the controlled PBS and AAV-EGFP group, liver and peritoneal metastasis rate were both 100%, and lung metastasis rate was 100% and 83%, respectively. While there was no metastasis found in treatment group, with only 33% of ascites happened. This was most possibly due to the primary tumor in liver but not due to the metastasis. Moreover, at a higher magnification (1000×), it was clear that the HGFK1 protein was expressed mainly in the cytoplasma of liver cells. In parallel, IHC staining of CD31 also demonstrated a significantly lower level of microvessel density (MVD) (6.21±1.6) in the liver tumor of the AAV-HGFK1 treatment group, as compared to the two control PBS and AAV-EGFP groups (25.1±2.1 and 26.8±2.5, respectively, P<0.01). HE staining showed that AAV-HGFK1 treatment induced large areas of necrosis in the tumor tissues, while minimal areas of necrosis were observed in the tumor tissue in the control groups. In addition, no toxicity appeared when high dosage (4.8× 1012 vg/rat) of rAAV-HGFK1 was administered in rats. Conclusion: Results from this study demonstrated that HGFK1 inhibited the growth and

  13. 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 tools for studying diabetes, pancreatitis and pancreatic cancer. Nevertheless, the use of Cre/loxP systems to conditionally activate or inactivate the expression of genes in a cell type- and/or temporal-specific manner is not applicable to cell tracing and/or gene manipulations in more than one lineage at a time. Here we report a technique that allows efficient delivery of dyes for cell tagging into the mouse pancreas through the duct system, and that also delivers viruses carrying transgenes or siRNA under a specific promoter. When this technique is applied in genetically modified mice, it enables the investigator to perform either double lineage tracing or cell lineage tracing combined with gene manipulation in a second lineage. The technique requires <40 min.

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

    International Nuclear Information System (INIS)

    Highlights: → For the first time the role of CD137 in dengue virus (DENV) infection. → Induction of DENV-mediated apoptosis by CD137 signaling. → Sensitization to CD137-mediated apoptosis by dengue virus capsid protein (DENV C). → 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.

  15. 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.

  16. Hyaluronic acid pretreatment for Sendai virus-mediated cochlear gene transfer.

    Science.gov (United States)

    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.

  17. High-Resolution Labeling and Functional Manipulation of Specific Neuron Types in Mouse Brain by Cre-Activated Viral Gene Expression

    OpenAIRE

    Kuhlman, Sandra J.; Huang, Z. Josh

    2008-01-01

    We describe a method that combines Cre-recombinase knockin mice and viral-mediated gene transfer to genetically label and functionally manipulate specific neuron types in the mouse brain. We engineered adeno-associated viruses (AAVs) that express GFP, dsRedExpress, or channelrhodopsin (ChR2) upon Cre/loxP recombination-mediated removal of a transcription-translation STOP cassette. Fluorescent labeling was sufficient to visualize neuronal structures with synaptic resolution in vivo, and ChR2 e...

  18. 两种不同病毒载体携带靶向大鼠金属蛋白酶组织抑制因子(TIMP)-1小干扰RNA抗肝纤维化作用的比较%Comparison between the antifibrotic effects of adeno-associated virus and lentivirus carrying small interfering RNA of TIMP-1 in rat liver fibrosis

    Institute of Scientific and Technical Information of China (English)

    马雪梅; 张群; 庞国进; 丛敏

    2013-01-01

    Objective To construct recombinant adeno-associated virus and lentivirus carrying siRNA of TIMP-1 and to investigate their antifibrotic effects on CCl4-induced liver fibrosis in rats.Methods One pair of siRNA which could effectively inhibit expression of the TIMP-1 gene in HSC-T6 was screened and cloned into AAV vector and lentiviral vector to construct the recombinant AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1.AAV/EGFP and Lenti/EGFP as negative control were also obtained.Fifty-eight male Wistar rats were randomly divided into six groups:control group (n =8),CCl4 group,AAV/EGFP,Lenti/EGFP,AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1 groups (all n =10).After the administration of CCl4 for four weeks,liver samples were collected for the immunohistochemical staining and detection of TIMP-1 expression.Results Livers from the control rats showed normal lobular structure around vessels (HE and Masson staining).In contrast,livers from the model,AAV/EGFP and Lenti/EGFP groups showed severe fibrosis,including septal fibrosis,extensive bridging,and fatty degeneration.The expressions of TIMP-1 mRNA and protein were also elevated in the livers from these groups.Compared with the fibrosis model group,the AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1 groups showed good preservation of liver lobular architecture and only mild bridging fibrosis,accompanied by decreased expression of TIMP-1 mRNA and protein.Semi-quantitative analysis of the fibrosis stage indicated that most rats in the model,AAV/EGFP and Lenti/EGFP groups were of S3 and S4 (80%),while 20% of the rats were of S5.In contrast,most rats (90%) in the AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1 groups were of stages S2 and S3,with only one rat of S4.There was no significant difference between these recombinant virus therapy groups.Conclusions Both AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1 can suppress the expression of TIMP-1 in rat fibrotic liver,playing an effective antifibrotic role in the rat liver.%目的 观察以腺相关病

  19. PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina.

    Science.gov (United States)

    Hickmott, Jack W; Chen, Chih-Yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

    2016-01-01

    Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia. PMID:27556059

  20. PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina

    Science.gov (United States)

    Hickmott, Jack W; Chen, Chih-yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

    2016-01-01

    Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia. PMID:27556059

  1. Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes

    OpenAIRE

    Fine, Eli J; Appleton, Caleb M.; Douglas E. White; Brown, Matthew T.; Harshavardhan Deshmukh; Kemp, Melissa L.; Gang Bao

    2015-01-01

    CRISPR/Cas9 systems have been used in a wide variety of biological studies; however, the large size of CRISPR/Cas9 presents challenges in packaging it within adeno-associated viruses (AAVs) for clinical applications. We identified a two-cassette system expressing pieces of the S. pyogenes Cas9 (SpCas9) protein which splice together in cellula to form a functional protein capable of site-specific DNA cleavage. With specific CRISPR guide strands, we demonstrated the efficacy of this system in c...

  2. 9型重组腺相关病毒介导抗核转录因子-κB核酶基因体外转染大鼠心肌细胞及对核转录因子-κB活性的影响%Transfection of rats H9C2 cells with recombinant adeno-associated virus Serotype 9 mediated AntiNF-κB ribozyme in vitro and effects on nuclear factor-κB activity

    Institute of Scientific and Technical Information of China (English)

    高霞; 马依彤; 杨毅宁; 向阳; 陈邦党; 刘芬

    2010-01-01

    Objective To evaluate the transfection efficiency using recombinant adeno-associated virus serotype 9 (rAAV9) mediated anti-nuclear factor-κB (NF) -κB ribozyme and enhanced green fluorescent protein (rAAV9-EGFP-R65) to rats H9C2 cells and the effect on NF-κB activity. Methods rAAV9EGFP-R65 was transfected into H9C2 ceils at multiplicities of infection ( MOI = 1 x 106 v. g./cell). EGFP expression in the cells was observed under an inverted fluorescence microscope, and the percentage of EGFP positive cells was determined by flow cytometry. Alamar Blue assay was used to assess the proliferation of the transfected cells. H9C2 ceils were treated with tumor necrosis factor (TNF)-α, rAAV9-EGFP-R65 and PDTC. The DNA binding activity of NF-KF-κB was examined by electrophoretic mobility shift assay (EMSA). Results The cells began to exhibit EGFP expression one day after transfection. The fluorescence intensity was increased with the time of transfection. EGFP expression reached the maximum on the day 5, at the point of which the transduction efficiency was (32.27 + 3.19)%. Alamar Blue assay did not reveal significant difference in the absorbance between the transfected cells and the control cells. TNF-α could activate NF-κB, and rAAV9-EGFP-R65 and PDCT could efficiently decrease NF-κB activation in rats H9C2 cells. Conclusion rAAV9-EGFP-R65 can be stably and efficiently expressed in H9C2 cells without causing cell growth inhibition, rAAVg-EGFP-R65 can availably inhibit NF-κB activation in rats H9C2 cells in vitro.%目的 观察9型重组腺相关病毒(rAAV9)介导抗核转录因子-κB(NF-κB)核酶基因(rAAV9-ECFP-R65)对大鼠心肌H9C2细胞的转染及对NF-κB活性的影响.方法 rAAV9-EGFP-R65按转染复数(MOI)1×106v.g./cell转染H9C2细胞,在倒置荧光显微镜下观察增强型绿色荧光蛋白(EGFP)阳性表达,采用流式细胞仪检测转染效率.Alamar Blue法检测rAAV9-EGFP-R65对H9C2细胞增殖影响.肿瘤坏死因子-α(TNF-α)、rAAV9

  3. 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.

  4. Stanniocalcin-2 overexpression reduces atherosclerosis in hypercholesterolemic mice

    DEFF Research Database (Denmark)

    Steffensen, Lasse B; Conover, Cheryl A; Bjørklund, Martin M;

    2016-01-01

    lesion development. We then used adeno-associated virus-mediated expression of STC2 to increase the fraction of PAPP-A present in the inhibited state and found that it decreased the development of atherosclerosis by 47% (P = 0.0005) in apolipoprotein E-deficient mice challenged with a Western type diet...... compared to controls. CONCLUSIONS: This study is the first to suggest the involvement of STC2 in regulating PAPP-A activity during the development of atherosclerosis. Furthermore, we demonstrate that lesion development can be inhibited in an experimental model by driving the balance towards inhibited PAPP-A....

  5. Viral expression cassette elements to enhance transgene target specificity and expression in gene therapy.

    Science.gov (United States)

    Powell, Sara Kathleen; Rivera-Soto, Ricardo; Gray, Steven James

    2015-01-01

    Over the last five years, the number of clinical trials involving AAV (adeno-associated virus) and lentiviral vectors continue to increase by about 150 trials each year. For continued success, AAV and lentiviral expression cassettes need to be designed to meet each disease's specific needs. This review discusses how viral vector expression cassettes can be engineered with elements to enhance target specificity and increase transgene expression. The key differences relating to target specificity between ubiquitous and tissue-specific promoters are discussed, as well as how endogenous miRNAs and their target sequences have been used to restrict transgene expression. Specifically, relevant studies indicating how cis-acting elements such as introns, WPRE, polyadenylation signals, and the CMV enhancer are highlighted to show their utility for enhancing transgene expression in gene therapy applications. All discussion bears in mind that expression cassettes have space constraints. In conclusion, this review can serve as a menu of vector genome design elements and their cost in terms of space to thoughtfully engineer viral vectors for gene therapy. PMID:25636961

  6. Expression of recombinant adeno-associated virus/human coagulant factor IX gene in human/rat colon carcimaa cells%重组腺相关病毒介导的人凝血因子IX基因在人/鼠结肠癌上皮细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    马泳泳; 陈方平; 杜建伟; 陈焱; 彭建强; 吴小兵; 解勤之

    2008-01-01

    目的:探讨重组腺相关病毒/人凝血因子Ix基因(rAAV-2/hFIX)在人/鼠结肠癌上皮细胞(SW480/C26细胞株)中的表达.方法:重组腺相关病毒/绿色荧光蛋白(rhhV-2/GFP)感染SW480/C26细胞,在荧光显微镜下观察GFP的表达,计算转染率.rAAV-2/hHX感染SW480/C26细胞,检测hFIX基因、FIXAg量及hFIX活性的表达.结果:SW480细胞株/C26细胞株GFP48hr阳性率分别为(57.0±8.5) %和(48.0土5.7)%;转导组14 d和21 d均可见外源hFIX的cDNA片断.SW480细胞转导了rAAV-2/hFIX后在第2天和第2l天ELISA法测hFIX抗原量分别为(98.0士4.3)和(30.9±6.5)ng·106cell-1·24h-1.未转导组为(2.7±0.1)ng·106cell-1·24h-1.C26细胞转导了rhAV-2/hFIX后在第2天测hFIX抗原量为(78土5.12)ng·106cell-1·24h-1.未转导组为(2.9士0.1)ng·108cell-1·24h-1.hFIX活性与hFIX抗原浓度明显相关.较对照组亦显著增高.结论:rAAV.2/GFP能有效地转导SW480/C26细胞.rhAV-2/hFIX能有效地转导SW480/C26细胞并表达具有凝血活性的功能蛋白hFIX.

  7. Neuronal expression of glucosylceramide synthase in central nervous system regulates body weight and energy homeostasis.

    Directory of Open Access Journals (Sweden)

    Viola Nordström

    Full Text Available Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase. As a major mechanism of central nervous system (CNS metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV-mediated Ugcg delivery to the arcuate nucleus (Arc significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis.

  8. The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells.

    Science.gov (United States)

    Duan, Yajian; Ma, Gaoen; Huang, Xionggao; D'Amore, Patricia A; Zhang, Feng; Lei, Hetian

    2016-07-29

    The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR.

  9. Enhanced viral production and virus-mediated mortality of bacterioplankton in a natural iron-fertilized bloom event above the Kerguelen Plateau

    Directory of Open Access Journals (Sweden)

    A. Malits

    2014-07-01

    Full Text Available Above the Kerguelen Plateau in the Southern Ocean natural iron fertilization sustains a large phytoplankton bloom over three months during austral summer. During the KEOPS1 project (KErguelen Ocean and Plateau compared Study1 we sampled this phytoplankton bloom during its declining phase along with the surrounding HNLC waters to study the effect of natural iron fertilization on the role of viruses in the microbial food web. Bacterial and viral abundances were 1.7 and 2.1 times, respectively, higher within the bloom than in HNLC waters. Viral production and virus-mediated mortality of bacterioplankton was 4.1 and 4.9 times, respectively, higher in the bloom, while the fraction of infected cells (FIC and the fraction of lysogenic cells (FLC showed no significant differences between environments. The present study suggests viruses to be more important for bacterial mortality within the bloom and dominate over protozoan grazing during the late bloom phase. As a consequence, at least at a late bloom stage, viral lysis shunts part of the photosynthetically fixed carbon in iron-fertilized regions into the dissolved organic matter (DOM pool with potentially less particulate organic carbon transfered to larger members of the food web or exported.

  10. Enhanced viral production and virus-mediated mortality of bacterioplankton in a natural iron-fertilized bloom event above the Kerguelen Plateau

    Science.gov (United States)

    Malits, A.; Christaki, U.; Obernosterer, I.; Weinbauer, M. G.

    2014-12-01

    Above the Kerguelen Plateau in the Southern Ocean natural iron fertilization sustains a large phytoplankton bloom over 3 months during austral summer. During the KEOPS1 project (KErguelen Ocean and Plateau compared Study1) we sampled this phytoplankton bloom during its declining phase along with the surrounding high-nutrient-low-chlorophyll (HNLC) waters to study the effect of natural iron fertilization on the role of viruses in the microbial food web. Bacterial and viral abundances were 1.7 and 2.1 times, respectively, higher within the bloom than in HNLC waters. Viral production and virus-mediated mortality of bacterioplankton were 4.1 and 4.9 times, respectively, higher in the bloom, while the fraction of infected cells (FIC) and the fraction of lysogenic cells (FLC) showed no significant differences between environments. The present study suggests viruses to be more important for bacterial mortality within the bloom and dominate over grazing of heterotrophic nanoflagellates (HNFs) during the late bloom phase. As a consequence, at least at a late bloom stage, viral lysis shunts part of the photosynthetically fixed carbon in iron-fertilized regions into the dissolved organic matter (DOM) pool with potentially less particulate organic carbon transferred to larger members of the food web or exported.

  11. Virus-Mediated Metalloproteinase 1 Induction Revealed by Transcriptome Profiling of Bovine Herpesvirus 4-Infected Bovine Endometrial Stromal Cells.

    Science.gov (United States)

    Tebaldi, Giulia; Jacca, Sarah; Montanini, Barbara; Capra, Emanuele; Rosamilia, Alfonso; Sala, Arianna; Stella, Alessandra; Castiglioni, Bianca; Ottonello, Simone; Donofrio, Gaetano

    2016-07-01

    Viral infections can cause genital tract disorders (including abortion) in cows, and bovine herpesvirus 4 (BoHV-4) is often present in endometritis-affected animals. A major problem with cattle uterine viral infections in general, and BoHV-4 in particular, is our limited understanding of the pathogenic role(s) that these infections play in the endometrium. A similar lack of knowledge holds for the molecular mechanisms utilized, and the host cell pathways affected, by BoHV-4. To begin to fill these gaps, we set up optimized conditions for BoHV-4 infection of a pure population of bovine endometrial stromal cells (BESCs) to be used as source material for RNA sequencing-based transcriptome profiling. Many genes were found to be upregulated (417) or downregulated (181) after BoHV-4 infection. As revealed by enrichment functional analysis on differentially expressed genes, BoHV-4 infection affects various pathways related to cell proliferation and cell surface integrity, at least three of which were centered on upregulation of matrix metalloproteinase 1 (MMP1) and interleukin 8 (IL8). This was confirmed by reverse transcription PCR, real-time PCR, Western-immunoblot analysis, and a luciferase assay with a bovine MMP1-specific promoter reporter construct. Further, it was found that MMP1 transcription was upregulated by the BoHV-4 transactivator IE2/RTA, leading to abnormally high metalloproteinase tissue levels, potentially leading to defective endometrium healing and unresolved inflammation. Based on these findings, we propose a new model for BoHV-4 action centered on IE2-mediated MMP1 upregulation and novel therapeutic interventions based on IFN gamma-mediated MMP1 downregulation. PMID:27281703

  12. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  13. Transfection of brain-derived neurotrophic factor gene by recombinant adeno-associated virus vector in retinal ganglion cells in vitro%腺伴随病毒介导的脑源性神经营养因子对体外培养的鼠视网膜神经节细胞转染及生长特性的影响

    Institute of Scientific and Technical Information of China (English)

    李海燕; 赵家良; 张华

    2008-01-01

    incubated for 7 days.Total RNA were extracted from rAAV-BDNF transfected cells using Trizol reagent.The gene expression of BDNF gene in RGCs waft analyzed by reverse transcription polymerase-chain reaction(RT-PCR).(3)Supernatant of the rAAV-BDNF transfected cells was collected at 7 days and 14 days after transfection.The protein expression of BDNF in the cell supernatant Was examined with ELISA assay.(4)The survival and apoptosis of rAAV-BDNF transfected cells, untransfeeted cells and the cells with addition of BDNF in culture medium were evaluated bv MTT colorimetric assay and flow cytometry with Annexin V-FITC staining.respectively.Results (1)RT-PCR analysis showed that mRNA expression of BDNF gene could be detected in transfected ceHs but not in untransfected ceHs.(2)The concentrations of BDNF protein in the conditioned medium of the rAAV-BDNF transfected cells were(616.1±40.0)ng/L and(1075.1±48.7)ng/L 7 days and 14 days after the transfection.respectively.(3)MTF colorimetric assay showed that the OD values of rAAV.BDNF transfected cells and untransfected cells were similar at the time of 3 and 6 days after transfection(t=1.084 and 1.582. P=0.284 and 0.120).The OD value of transfected cells was higher than that of untransfected ceHs 9 days after the transfection(t=4.854,P=0.001).(4)The apoptosis rate in rAAV-BDNF transfected cells and the cells with BDNF exposure was lower than that of the untransfected cells(P=0.015,0.017).Conclusions Rat RGCs are abie to be transfected by rAAV-BDNF in vitro.The transfected ceHs Can express BDNF gene at the level of beth mRNA and protein.Apoptosis rate is lOW in the transfected cells.This study indicates that rAAV-BDNF transfection Can be used for the potential gene tllerapy in glaucoma neuroprotection.

  14. CONSTRUCTION AND EXPRESSION OF ADENOASSOCIATED VIRUS- BASED PLASMID EXPRESSING VECTORS CONTAINING hIL- 2 GENE OR mIFN-γ GENE

    Institute of Scientific and Technical Information of China (English)

    张景迎; 梁宏立; 陈诗书

    2000-01-01

    Objective To improve the plasmid vectors in gene therapy, adeno - associated virus (AA V) based plasmid expressing vectors containing hIL-2 gene or mIFN-γ gene were constructed and its expression in transfected cells was studied. Methods By means of step to step cloning, promoter CMVp was placed at the downstream of 5' inverted terminal repeat from AA V (AA V- ITR) of pAP, hIL- 2 gene or mIFN- γ gene inserted into pAC between CMVp and poly A. Then intron A was inserted into pAC- hIL - 2 or pAC- mIFN- γ between CMVp and IL - 2 gene or IFNγ gene to construct pAI- hIL - 2 or pAI- mIFN - γ. Liposome -plasmid complexes were formed by mixing Dosper with these AAV-based plasmids containing hIL-2 gene or mIFN-γgene. Results High biological activities of IL - 2 or IFN- γ could be detected in the supernatants of NIH3T3 and MM45T. Li cells after transfection. Insertion of intron A into pAC-hIL-2 or pAC-mIFN-γ improved the expression of IL- 2 or IFN- γ. Conclusion These data demonstrated that the constructed AA V- based plasmid expressing vectors could efficiently express therapeutic genes in cultured cells and could be used as a nonviral gene transfer system in human gene therapy.

  15. 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

  16. Successful transgene expression with serial doses of aerosolized rAAV2 vectors in rhesus macaques.

    Science.gov (United States)

    Fischer, Anne C; Beck, Suzanne E; Smith, Carolina I; Laube, Beth L; Askin, Frederic B; Guggino, Sandra E; Adams, Robert J; Flotte, Terence R; Guggino, William B

    2003-12-01

    Bronchoscopic microspraying of recombinant adeno-associated viral (rAAV) vectors targets high doses of vector directly to pulmonary epithelium. Single-dose endobronchial gene therapy trials have been accomplished in cystic fibrosis patients; however, repeated dosing strategies are likely essential for lifetime correction. These studies address whether serial redosing with rAAV2 vectors results in an antiserotypic response and, furthermore, whether it triggers an inflammatory response prohibitive to transgene expression. Serial redosing of 9 x 10(11) infectious units of aerosolized rAAV2 vectors to rhesus macaques resulted in successful gene transfer by quantitative PCR (1.43 x 10(9) copies/g tissue) and transgene expression. Additionally, confocal microscopy and immunohistochemical analysis demonstrated in situ expression localized to the pulmonary epithelium. Although serial redosing did induce a heightened anti-neutralizing antibody response in sera, gene transfer prevailed with resultant expression. This study is the first to demonstrate successful gene transfer subsequent to repeated aerosolized doses of rAAV2 in immunocompetent nonhuman primates without associated inflammatory responses prohibitive to transgene expression. PMID:14664794

  17. CYP46A1, the rate-limiting enzyme for cholesterol degradation, is neuroprotective in Huntington's disease.

    Science.gov (United States)

    Boussicault, Lydie; Alves, Sandro; Lamazière, Antonin; Planques, Anabelle; Heck, Nicolas; Moumné, Lara; Despres, Gaëtan; Bolte, Susanne; Hu, Amélie; Pagès, Christiane; Galvan, Laurie; Piguet, Francoise; Aubourg, Patrick; Cartier, Nathalie; Caboche, Jocelyne; Betuing, Sandrine

    2016-03-01

    Huntington's disease is an autosomal dominant neurodegenerative disease caused by abnormal polyglutamine expansion in huntingtin (Exp-HTT) leading to degeneration of striatal neurons. Altered brain cholesterol homeostasis has been implicated in Huntington's disease, with increased accumulation of cholesterol in striatal neurons yet reduced levels of cholesterol metabolic precursors. To elucidate these two seemingly opposing dysregulations, we investigated the expression of cholesterol 24-hydroxylase (CYP46A1), the neuronal-specific and rate-limiting enzyme for cholesterol conversion to 24S-hydroxycholesterol (24S-OHC). CYP46A1 protein levels were decreased in the putamen, but not cerebral cortex samples, of post-mortem Huntington's disease patients when compared to controls. Cyp46A1 mRNA and CYP46A1 protein levels were also decreased in the striatum of the R6/2 Huntington's disease mouse model and in SThdhQ111 cell lines. In vivo, in a wild-type context, knocking down CYP46A1 expression in the striatum, via an adeno-associated virus-mediated delivery of selective shCYP46A1, reproduced the Huntington's disease phenotype, with spontaneous striatal neuron degeneration and motor deficits, as assessed by rotarod. In vitro, CYP46A1 restoration protected SThdhQ111 and Exp-HTT-expressing striatal neurons in culture from cell death. In the R6/2 Huntington's disease mouse model, adeno-associated virus-mediated delivery of CYP46A1 into the striatum decreased neuronal atrophy, decreased the number, intensity level and size of Exp-HTT aggregates and improved motor deficits, as assessed by rotarod and clasping behavioural tests. Adeno-associated virus-CYP46A1 infection in R6/2 mice also restored levels of cholesterol and lanosterol and increased levels of desmosterol. In vitro, lanosterol and desmosterol were found to protect striatal neurons expressing Exp-HTT from death. We conclude that restoring CYP46A1 activity in the striatum promises a new therapeutic approach in

  18. Adeno-associated virus–targeted disruption of the CFTR gene in cloned ferrets

    OpenAIRE

    Sun, Xingshen; Yan, Ziying; Yi, Yaling; Li, Ziyi; Lei, Diana; Rogers, Christopher S.; Chen, Juan; Zhang, Yulong; Welsh, Michael J.; Leno, Gregory H.; Engelhardt, John F.

    2008-01-01

    Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene–deficient model...

  19. Gene replacement therapies for Duchenne muscular dystrophy using adeno-associated viral vectors

    OpenAIRE

    Seto, Jane T.; Ramos, Julian N.; Muir, Lindsey; Jeffrey S. Chamberlain; Odom, Guy L.

    2012-01-01

    The muscular dystrophies collectively represent a major health challenge, as few significant treatment options currently exist for any of these disorders. Recent years have witnessed a proliferation of novel approaches to therapy, spanning increased testing of existing and new pharmaceuticals, DNA delivery (both anti-sense oligonucleotides and plasmid DNA), gene therapies and stem cell technologies. While none of these has reached the point of being used in clinical practice, all show promise...

  20. Restriction Factors Against Recombinant Adeno-associated Virus Vectormediated Gene Transfer in Dystrophin-deficient Muscles.

    Science.gov (United States)

    Dupont, Jean-Baptiste

    2016-01-01

    Despite the unprecedented beneficial effects of rAAV gene therapy in animal models of Duchenne muscular dystrophy (DMD), the need to inject large amounts of vector in vivo to improve phenotype raises obvious biosafety concerns. While rAAV vectors generally exhibit a good safety profile, specific pathological phenotypes such as those observed in dystrophin-deficient muscles may promote immunotoxic/genotoxic effects. Increasing the therapeutic index of rAAV in DMD muscles by reducing the effective dose could be a pivotal means of ensuring efficient clinical translation. This requires a comprehensive understanding of the rAAV transduction process, which is almost always studied in non-pathological tissues or in vitro. In this review, we focus on the molecular fate of rAAV after injection, and how the individual stages of transduction could be affected in the context of DMD. PMID:27121109

  1. Interference Between Two Adeno-associated Satellite Viruses: a Three-Component System

    Science.gov (United States)

    Torikai, K.; Mayor, H. D.

    1969-01-01

    Adenovirus-associated satellite viruses interfere with the replication of their helper adenoviruses. According to a previous report, this interference is not mediated by interferon. A three-component system comprising simian adenovirus SV15 and satellites types 1 and 4 was studied to determine whether satellite viruses also interfere with one another. Satellite type 1 interfered with the replication of type 4 and vice versa. The degree of interference was directly proportional to the dose of interfering satellite. The events leading to mutual satellite interference were operative during the first 12 hr of replication, the period associated with active synthesis of viral deoxyribonucleic acid. PMID:5786177

  2. Generation of Insulin-Producing Human Mesenchymal Stem Cells Using Recombinant Adeno-Associated Virus

    OpenAIRE

    Kim, Jeong Hwan; Park, Si-Nae; Suh, Hwal

    2007-01-01

    The purpose of current experiment is the generation of insulin-producing human mesenchymal stem cells as therapeutic source for the cure of type 1 diabetes. Type 1 diabetes is generally caused by insulin deficiency accompanied by the destruction of islet β-cells. In various trials for the treatment of type 1 diabetes, cell-based gene therapy using stem cells is considered as one of the most useful candidate for the treatment. In this experiment, human mesenchymal stem cells were transduced wi...

  3. Systemic delivery of genes to striated muscles using adeno-associated viral vectors

    OpenAIRE

    Gregorevic, Paul; Blankinship, Michael J; Allen, James M.; Robert W Crawford; Meuse, Leonard; Miller, Daniel G.; Russell, David W.; Jeffrey S. Chamberlain

    2004-01-01

    A major obstacle limiting gene therapy for diseases of the heart and skeletal muscles is an inability to deliver genes systemically to muscles of an adult organism. Systemic gene transfer to striated muscles is hampered by the vascular endothelium, which represents a barrier to distribution of vectors via the circulation. Here we show the first evidence of widespread transduction of both cardiac and skeletal muscles in an adult mammal, after a single intravenous administration of recombinant ...

  4. Pax4 Expression does not Transduce Pancreatic Alpha Cells to Beta Cells

    Directory of Open Access Journals (Sweden)

    Ling Chen

    2015-07-01

    Full Text Available Background/Aims: The lack of available beta cells greatly limits the use of beta cell transplantation as a therapy for diabetes. Thus, generation of beta cells from other sources is substantially required. Pax4 has been shown to induce reprograming of alpha cells into beta cells during embryogenesis. Nevertheless, whether expression of Pax4 in adult alpha cells could trigger this alpha-to-beta cell reprogramming is unknown. Methods: Here we generated an adeno-associated virus carrying Pax4 and GFP under a CMV promoter (AAV-Pax4. We used AAV-Pax4 to transduce a mouse alpha cell line in vitro, and to transduce primary alpha cells in diabetic mice. Reprogramming was examined by double immunostaining and by changes in beta cell number. The effects on blood glucose were evaluated by fasting blood glucose and glucose response. Results: In vitro, Pax4 overexpression neither induced insulin expression, nor suppressed glucagon expression in alpha cells. In vivo, Pax4 overexpression failed to increase beta cell number, and did not alter hyperglycemia and glucose response in diabetic mice. Conclusion: Pax4 expression is not sufficient to transduce pancreatic alpha cells into beta cells. Overexpression of Pax4 in alpha cells may not increase functional beta cell number in diabetic patients.

  5. Restoration of visual function by expression of a light-gated mammalian ion channel in retinal ganglion cells or ON-bipolar cells

    OpenAIRE

    Gaub, Benjamin M.; Berry, Michael H.; Holt, Amy E.; Reiner, Andreas; Kienzler, Michael A; Dolgova, Natalia; Nikonov, Sergei; Aguirre, Gustavo D.; Beltran, William A.; Flannery, John G.; Isacoff, Ehud Y.

    2014-01-01

    We restored visual function to animal models of human blindness using a chemical compound that photosensitizes a mammalian ion channel. Virus-mediated expression of this light sensor in surviving retinal cells of blind mice restored light responses in vitro, reanimated innate light avoidance, and enabled learned visually guided behavior. The treatment also restored light responses to the retina of blind dogs. Patients that might benefit from this treatment would need to have intact ganglion c...

  6. Recombinant AAV-mediated Expression of Human BDNF Protects Neurons against Cell Apoptosis in Aβ-induced Neuronal Damage Model

    Institute of Scientific and Technical Information of China (English)

    LIU Zhaohui; MA Dongliang; FENG Gaifeng; MA Yanbing; HU Haitao

    2007-01-01

    The human brain-derived neurotrophic factor (hBDNF) gene was cloned by polymerase chain reaction and the recombinant adeno-associated viral vector inserted with hBDNF gene (AAV-hBDNF) was constructed. Cultured rat hippocampal neurons were treated with Aβ25-35 and serued as the experimental Aβ-induced neuronal damage model (AD model), and the AD model was infected with AAV-hBDNF to explore neuroprotective effects of expression of BDNF. Cell viability was assayed by MTT. The expression of bcl-2 anti-apoptosis protein was detected by immunocytochemical staining. The change of intracellular free Ca ion ([Ca2+]i) was measured by laser scanning confocal microscopy. The results showed that BDNF had protective effects against Aβ-induced neuronal damage. The expression of the bcl-2 anti-apoptosis protein was raised significantly and the balance of [Ca2+]i was maintained in the AAV-hBDNF treatment group as compared with AD model group. These data suggested that recombinant AAV mediated a stable expression of hBDNF in cultured hippocampal neurons and resulted in significant neuron protective effects in AD model. The BDNF may reduce neuron apoptosis through increasing the expression of the bcl-2 anti-apoptosis protein and inhibiting intracellular calcium overload. The viral vector-mediated gene expression of BDNF may pave the way of a novel therapeutic strategy for the treatment of neurodegenerative diseases such as Alzheimer's disease.

  7. Cell-type specific oxytocin gene expression from AAV delivered promoter deletion constructs into the rat supraoptic nucleus in vivo.

    Directory of Open Access Journals (Sweden)

    Raymond L Fields

    Full Text Available The magnocellular neurons (MCNs in the hypothalamus selectively express either oxytocin (OXT or vasopressin (AVP neuropeptide genes, a property that defines their phenotypes. Here we examine the molecular basis of this selectivity in the OXT MCNs by stereotaxic microinjections of adeno-associated virus (AAV vectors that contain various OXT gene promoter deletion constructs using EGFP as the reporter into the rat supraoptic nucleus (SON. Two weeks following injection of the AAVs, immunohistochemical assays of EGFP expression from these constructs were done to determine whether the EGFP reporter co-localizes with either the OXT- or AVP-immunoreactivity in the MCNs. The results show that the key elements in the OT gene promoter that regulate the cell-type specific expression the SON are located -216 to -100 bp upstream of the transcription start site. We hypothesize that within this 116 bp domain a repressor exists that inhibits expression specifically in AVP MCNs, thereby leading to the cell-type specific expression of the OXT gene only in the OXT MCNs.

  8. Generation of expression plasmids for angiostatin, endostatin and TIMP-2 for cancer gene therapy.

    Science.gov (United States)

    Indraccolo, S; Minuzzo, S; Gola, E; Habeler, W; Carrozzino, F; Noonan, D; Albini, A; Santi, L; Amadori, A; Chieco-Bianchi, L

    1999-01-01

    Antiangiogenic therapy may represent a promising approach to cancer treatment. Indeed, the efficacy of endogenous angiogenesis inhibitors, including angiostatin, endostatin and TIMPs, has been demonstrated in many types of solid tumors in animal models. In view of the possible problems associated with long-term administration of inhibitors as recombinant proteins, we propose their delivery as nucleic acids through a gene therapy approach. To this end, eukaryotic expression constructs for murine angiostatin and endostatin as well as human TIMP-2 were generated, and characterized in vitro. All constructs carry the relevant cDNAs under the control of the strong HCMV promoter/enhancer, and cleavable leader signals to allow protein secretion. Expression of the angiogenesis inhibitors was detected by in vitro transcription/translation experiments as well as transfection of 293T cells, followed by Western blotting (WB) or radioimmunoprecipitation analysis of both cell lysates and supernatants (SNs). These constructs might be used for in vivo intramuscular delivery of plasmid DNA and as a set of reagents for the development of retroviral as well as adeno-associated viral (AAV) vectors expressing angiogenesis inhibitors. PMID:10669955

  9. Vector-Mediated In Vivo Antibody Expression.

    Science.gov (United States)

    Schnepp, Bruce C; Johnson, Philip R

    2014-08-01

    This article focuses on a novel vaccine strategy known as vector-mediated antibody gene transfer, with a particular focus on human immunodeficiency virus (HIV). This strategy provides a solution to the problem of current vaccines that fail to generate neutralizing antibodies to prevent HIV-1 infection and AIDS. Antibody gene transfer allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein. This approach uses recombinant adeno-associated viral (rAAV) vectors, which have been shown to transduce muscle with high efficiency and direct the long-term expression of a variety of transgenes, to deliver the gene encoding a broadly neutralizing antibody into the muscle. Following rAAV vector gene delivery, the broadly neutralizing antibodies are endogenously synthesized in myofibers and passively distributed to the circulatory system. This is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. Vector-mediated gene transfer studies in mice and monkeys with anti-HIV and simian immunodeficiency virus (SIV)-neutralizing antibodies demonstrated long-lasting neutralizing activity in serum with complete protection against intravenous challenge with virulent HIV and SIV. These results indicate that existing potent anti-HIV antibodies can be rapidly moved into the clinic. However, this methodology need not be confined to HIV. The general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets such as hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis. PMID:26104192

  10. Long-term increased carnitine palmitoyltransferase 1A expression in ventromedial hypotalamus causes hyperphagia and alters the hypothalamic lipidomic profile.

    Directory of Open Access Journals (Sweden)

    Paula Mera

    Full Text Available Lipid metabolism in the ventromedial hypothalamus (VMH has emerged as a crucial pathway in the regulation of feeding and energy homeostasis. Carnitine palmitoyltransferase (CPT 1A is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation and it has been proposed as a crucial mediator of fasting and ghrelin orexigenic signalling. However, the relationship between changes in CPT1A activity and the intracellular downstream effectors in the VMH that contribute to appetite modulation is not fully understood. To this end, we examined the effect of long-term expression of a permanently activated CPT1A isoform by using an adeno-associated viral vector injected into the VMH of rats. Peripherally, this procedure provoked hyperghrelinemia and hyperphagia, which led to overweight, hyperglycemia and insulin resistance. In the mediobasal hypothalamus (MBH, long-term CPT1AM expression in the VMH did not modify acyl-CoA or malonyl-CoA levels. However, it altered the MBH lipidomic profile since ceramides and sphingolipids increased and phospholipids decreased. Furthermore, we detected increased vesicular γ-aminobutyric acid transporter (VGAT and reduced vesicular glutamate transporter 2 (VGLUT2 expressions, both transporters involved in this orexigenic signal. Taken together, these observations indicate that CPT1A contributes to the regulation of feeding by modulating the expression of neurotransmitter transporters and lipid components that influence the orexigenic pathways in VMH.

  11. Long-term controlled GDNF over-expression reduces dopamine transporter activity without affecting tyrosine hydroxylase expression in the rat mesostriatal system.

    Science.gov (United States)

    Barroso-Chinea, Pedro; Cruz-Muros, Ignacio; Afonso-Oramas, Domingo; Castro-Hernández, Javier; Salas-Hernández, Josmar; Chtarto, Abdelwahed; Luis-Ravelo, Diego; Humbert-Claude, Marie; Tenenbaum, Liliane; González-Hernández, Tomás

    2016-04-01

    The dopamine (DA) transporter (DAT) is a plasma membrane glycoprotein expressed in dopaminergic (DA-) cells that takes back DA into presynaptic neurons after its release. DAT dysfunction has been involved in different neuro-psychiatric disorders including Parkinson's disease (PD). On the other hand, numerous studies support that the glial cell line-derived neurotrophic factor (GDNF) has a protective effect on DA-cells. However, studies in rodents show that prolonged GDNF over-expression may cause a tyrosine hydroxylase (TH, the limiting enzyme in DA synthesis) decline. The evidence of TH down-regulation suggests that another player in DA handling, DAT, may also be regulated by prolonged GDNF over-expression, and the possibility that this effect is induced at GDNF expression levels lower than those inducing TH down-regulation. This issue was investigated here using intrastriatal injections of a tetracycline-inducible adeno-associated viral vector expressing human GDNF cDNA (AAV-tetON-GDNF) in rats, and doxycycline (DOX; 0.01, 0.03, 0.5 and 3mg/ml) in the drinking water during 5weeks. We found that 3mg/ml DOX promotes an increase in striatal GDNF expression of 12× basal GDNF levels and both DA uptake decrease and TH down-regulation in its native and Ser40 phosphorylated forms. However, 0.5mg/ml DOX promotes a GDNF expression increase of 3× basal GDNF levels with DA uptake decrease but not TH down-regulation. The use of western-blot under non-reducing conditions, co-immunoprecipitation and in situ proximity ligation assay revealed that the DA uptake decrease is associated with the formation of DAT dimers and an increase in DAT-α-synuclein interactions, without changes in total DAT levels or its compartmental distribution. In conclusion, at appropriate GDNF transduction levels, DA uptake is regulated through DAT protein-protein interactions without interfering with DA synthesis. PMID:26777664

  12. Skeletal muscle-specific expression of human blood coagulation factor Ⅸ rescues factor Ⅸ deficiency mouse by AAV-mediated gene transfer

    Institute of Scientific and Technical Information of China (English)

    赖立辉; 陈立; 卢大儒; 王琪; 高啸波; 邱信芳; Jerry; L.Hsueh; 薛京伦; 王健民; 周虹

    1999-01-01

    The efficacy of recombinant adeno-associated virus (AAV) vector to deliver and express human blood clotting factor DC (hFIX) gene in skeletal muscle of coagulation factor IX deficiency mouse strain (FactorIX-knockout) is e-valuated. The muscle creatine kinase enhancer (MCK) and βactin promoter ((3A) were used to drive the hFIX minigene (hFIXml), which was flanked by AAV inverted terminal repeats (ITRs). Following intramuscular injection of high liter (2.5 x 1011 vector genomes/mL) of AAV, increased hFIX expression (256 ng/mL of plasma) was achieved. The time course of hFIX expression demonstrated that the expression level gradually increased over a period of two weeks before anti-hFIX antibodies developed in mouse circulating plasma. Those results provided a promising evidence that rAAV-me-diated gene transfer and skeletal muscle-specific expression of hFIX is a feasible strategy for treating patients for hemophilia B.

  13. Intracranial injection of AAV expressing NEP but not IDE reduces amyloid pathology in APP+PS1 transgenic mice.

    Directory of Open Access Journals (Sweden)

    Nikisha Carty

    Full Text Available The accumulation of β-amyloid peptides in the brain has been recognized as an essential factor in Alzheimer's disease pathology. Several proteases, including Neprilysin (NEP, endothelin converting enzyme (ECE, and insulin degrading enzyme (IDE, have been shown to cleave β-amyloid peptides (Aβ. We have previously reported reductions in amyloid in APP+PS1 mice with increased expression of ECE. In this study we compared the vector-induced increased expression of NEP and IDE. We used recombinant adeno-associated viral vectors expressing either native forms of NEP (NEP-n or IDE (IDE-n, or engineered secreted forms of NEP (NEP-s or IDE (IDE-s. In a six-week study, immunohistochemistry staining for total Aβ was significantly decreased in animals receiving the NEP-n and NEP-s but not for IDE-n or IDE-s in either the hippocampus or cortex. Congo red staining followed a similar trend revealing significant decreases in the hippocampus and the cortex for NEP-n and NEP-s treatment groups. Our results indicate that while rAAV-IDE does not have the same therapeutic potential as rAAV-NEP, rAAV-NEP-s and NEP-n are effective at reducing amyloid loads, and both of these vectors continue to have significant effects nine months post-injection. As such, they may be considered reasonable candidates for gene therapy trials in AD.

  14. Optogenetics reveals a role for accumbal medium spiny neurons expressing dopamine D2 receptors in cocaine-induced behavioral sensitization.

    Science.gov (United States)

    Song, Shelly Sooyun; Kang, Byeong Jun; Wen, Lei; Lee, Hyo Jin; Sim, Hye-Ri; Kim, Tae Hyong; Yoon, Sehyoun; Yoon, Bong-June; Augustine, George J; Baik, Ja-Hyun

    2014-01-01

    Long-lasting, drug-induced adaptations within the nucleus accumbens (NAc) have been proposed to contribute to drug-mediated addictive behaviors. Here we have used an optogenetic approach to examine the role of NAc medium spiny neurons (MSNs) expressing dopamine D2 receptors (D2Rs) in cocaine-induced behavioral sensitization. Adeno-associated viral vectors encoding channelrhodopsin-2 (ChR2) were delivered into the NAc of D2R-Cre transgenic mice. This allowed us to selectively photostimulate D2R-MSNs in NAc. D2R-MSNs form local inhibitory circuits, because photostimulation of D2R-MSN evoked inhibitory postsynaptic currents (IPSCs) in neighboring MSNs. Photostimulation of NAc D2R-MSN in vivo affected neither the initiation nor the expression of cocaine-induced behavioral sensitization. However, photostimulation during the drug withdrawal period attenuated expression of cocaine-induced behavioral sensitization. These results show that D2R-MSNs of NAc play a key role in withdrawal-induced plasticity and may contribute to relapse after cessation of drug abuse. PMID:25352792

  15. Optogenetics reveals a role for accumbal medium spiny neurons expressing dopamine D2 receptors in cocaine-induced behavioral sensitization.

    Science.gov (United States)

    Song, Shelly Sooyun; Kang, Byeong Jun; Wen, Lei; Lee, Hyo Jin; Sim, Hye-Ri; Kim, Tae Hyong; Yoon, Sehyoun; Yoon, Bong-June; Augustine, George J; Baik, Ja-Hyun

    2014-01-01

    Long-lasting, drug-induced adaptations within the nucleus accumbens (NAc) have been proposed to contribute to drug-mediated addictive behaviors. Here we have used an optogenetic approach to examine the role of NAc medium spiny neurons (MSNs) expressing dopamine D2 receptors (D2Rs) in cocaine-induced behavioral sensitization. Adeno-associated viral vectors encoding channelrhodopsin-2 (ChR2) were delivered into the NAc of D2R-Cre transgenic mice. This allowed us to selectively photostimulate D2R-MSNs in NAc. D2R-MSNs form local inhibitory circuits, because photostimulation of D2R-MSN evoked inhibitory postsynaptic currents (IPSCs) in neighboring MSNs. Photostimulation of NAc D2R-MSN in vivo affected neither the initiation nor the expression of cocaine-induced behavioral sensitization. However, photostimulation during the drug withdrawal period attenuated expression of cocaine-induced behavioral sensitization. These results show that D2R-MSNs of NAc play a key role in withdrawal-induced plasticity and may contribute to relapse after cessation of drug abuse.

  16. Normalizing Dopamine D2 Receptor-Mediated Responses in D2 Null Mutant Mice by Virus-Mediated Receptor Restoration: Comparing D2L and D2S

    OpenAIRE

    Neve, Kim A.; Ford, Christopher P.; Buck, David C; Grandy, David K; Neve, Rachael L.; Phillips, Tamara J.

    2013-01-01

    D2 receptor null mutant (Drd2−/−) mice have altered responses to the rewarding and locomotor effects of psychostimulant drugs, which is evidence of a necessary role for D2 receptors in these behaviors. Furthermore, work with mice that constitutively express only the D2 receptor short form (D2S), as a result of genetic deletion of the long form (D2L), provides the basis for a current model in which D2L is thought to be the postsynaptic D2 receptor on medium spiny neurons in the basal forebrain...

  17. An amplified promoter system for targeted expression of calcium indicator proteins in the cerebellar cortex

    Directory of Open Access Journals (Sweden)

    Bernd eKuhn

    2012-07-01

    Full Text Available Recording of identified neuronal network activity using genetically encoded calcium indicators (GECIs requires labeling that is cell type-specific and bright enough for the detection of functional signals. However, specificity and strong expression are often not achievable using the same promoter. Here we present a combinatorial approach for targeted expression and single-cell-level quantification in which a weak promoter is used to drive trans-amplification under a strong general promoter. We demonstrated this approach using recombinant adeno-associated viruses (rAAVs to deliver the sequence of the GECI D3cpv in the mouse cerebellar cortex. Direct expression under the human synapsin promoter (hSYN led to high levels of expression (50-100 µM in five interneuron types of the cerebellar cortex but not in Purkinje cells (PCs (≤10 μM, yielding sufficient contrast to allow functional signals to be recorded from somata and processes in awake animals using two-photon microscopy. When the hSYN promoter was used to drive expression of the tetracycline transactivator (tTA, a second rAAV containing the bidirectional TET promoter (Ptetbi could drive strong D3cpv expression in PCs (10-300 µM, enough to allow reliable complex spike detection in the dendritic arbor. An amplified approach should be of use in monitoring neural processing in selected cell types and boosting expression of optogenetic probes. Additionally, we overcome cell toxicity associated with rAAV injection and/or local GECI overexpression by combining the virus injection with systemic pre-injection of hyperosmotic D-mannitol, and by this double the time window for functional imaging.

  18. RNA interference improves myopathic phenotypes in mice over-expressing FSHD region gene 1 (FRG1).

    Science.gov (United States)

    Wallace, Lindsay M; Garwick-Coppens, Sara E; Tupler, Rossella; Harper, Scott Q

    2011-11-01

    Muscular dystrophies, and other diseases of muscle, arise from recessive and dominant gene mutations. Gene replacement strategies may be beneficial for the former, while gene silencing approaches may provide treatment for the latter. In the last two decades, muscle-directed gene therapies were primarily focused on treating recessive disorders. This disparity at least partly arose because feasible mechanisms to silence dominant disease genes lagged behind gene replacement strategies. With the discovery of RNA interference (RNAi) and its subsequent development as a promising new gene silencing tool, the landscape has changed. In this study, our objective was to demonstrate proof-of-principle for RNAi therapy of a dominant myopathy in vivo. We tested the potential of adeno-associated viral (AAV)-delivered therapeutic microRNAs, targeting the human Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1), to correct myopathic features in mice expressing toxic levels of human FRG1 (FRG1(-high) mice). We found that FRG1 gene silencing improved muscle mass, strength, and histopathological abnormalities associated with muscular dystrophy in FRG1(-high) mice, thereby demonstrating therapeutic promise for treatment of dominantly inherited myopathies using RNAi. This approach potentially applies to as many as 29 different gene mutations responsible for myopathies inherited as dominant disorders. PMID:21730972

  19. A viral vector expressing hypoxia-inducible factor 1 alpha inhibits hippocampal neuronal apoptosis

    Institute of Scientific and Technical Information of China (English)

    Xiqing Chai; Weina Kong; Lingyun Liu; Wenguo Yu; Zhenqing Zhang; Yimin Sun

    2014-01-01

    Hypoxia-inducible factor 1 (HIF-1) attenuates amyloid-beta protein neurotoxicity and decreases apoptosis induced by oxidative stress or hypoxia in cortical neurons. In this study, we construct-ed a recombinant adeno-associated virus (rAAV) vector expressing the human HIF-1αgene (rAAV-HIF-1α), and tested the assumption that rAAV-HIF-1αrepresses hippocampal neuronal apoptosis induced by amyloid-beta protein. Our results conifrmed that rAAV-HIF-1αsigniifcant-ly reduces apoptosis induced by amyloid-beta protein in primary cultured hippocampal neurons. Direct intracerebral rAAV-HIF-1αadministration also induced robust and prolonged HIF-1αproduction in rat hippocampus. Single rAAV-HIF-1αadministration resulted in decreased apoptosis of hippocampal neurons in an Alzheimer’s disease rat model established by intrace-rebroventricular injection of aggregated amyloid-beta protein (25-35). Our in vitro and in vivo ifndings demonstrate that HIF-1 has potential for attenuating hippocampal neuronal apoptosis induced by amyloid-beta protein, and provides experimental support for treatment of neurode-generative diseases using gene therapy.

  20. AC2 and AC4 proteins of Tomato yellow leaf curl China virus and Tobacco curly shoot virus mediate suppression of RNA silencing

    Institute of Scientific and Technical Information of China (English)

    CUI Xiaofeng; ZHOU Xueping

    2004-01-01

    Tomato yellow leaf curl China virus Y10 isolate (TYLCCNV-Y10) alone could systemically infect host plants such as Nicotiana benthamiana without symptoms. In contrast, Tobacco curly shoot virus Y35 isolate (TbCSV-Y35) alone induces leaf curl symptoms in N. benthamiana. When inoculated into transgenic N. benthamiana plants expressing GFP gene (line 16c), TYLCCNV-Y10 neither reverses the established GFP silencing nor blocks the onset of GFP silencing. In contrast, TbCSV-Y35 can partially reverse the established GFP silencing and block the onset of GFP silencing in new leaves. In the patch co-infiltration assays, the AC2 and AC4 proteins of TYLCCNV-Y10 and TbCSV-Y35 could suppress local GFP silencing and delay systemic GFP silencing, suggesting that they are suppressors of RNA silencing. Comparison of the accumulation levels of GFP mRNA in the co-infiltration patches showed that Y10 AC2 and Y35 AC2 proteins had similar efficiency for suppression of RNA silencing. However, Y35 AC4 protein functioned as a stronger suppressor of RNA silencing than Y10 AC4 protein. Therefore, the pathogenicity difference between TbCSV-Y35 and TYLCCNV-Y10 may be related to the functional difference in their AC4 proteins.

  1. 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. PMID:26318517

  2. Selective optical control of synaptic transmission in the subcortical visual pathway by activation of viral vector-expressed halorhodopsin.

    Directory of Open Access Journals (Sweden)

    Katsuyuki Kaneda

    Full Text Available The superficial layer of the superior colliculus (sSC receives visual inputs via two different pathways: from the retina and the primary visual cortex. However, the functional significance of each input for the operation of the sSC circuit remains to be identified. As a first step toward understanding the functional role of each of these inputs, we developed an optogenetic method to specifically suppress the synaptic transmission in the retino-tectal pathway. We introduced enhanced halorhodopsin (eNpHR, a yellow light-sensitive, membrane-targeting chloride pump, into mouse retinal ganglion cells (RGCs by intravitreously injecting an adeno-associated virus serotype-2 vector carrying the CMV-eNpHR-EYFP construct. Several weeks after the injection, whole-cell recordings made from sSC neurons in slice preparations revealed that yellow laser illumination of the eNpHR-expressing retino-tectal axons, putatively synapsing onto the recorded cells, effectively inhibited EPSCs evoked by electrical stimulation of the optic nerve layer. We also showed that sSC spike activities elicited by visual stimulation were significantly reduced by laser illumination of the sSC in anesthetized mice. These results indicate that photo-activation of eNpHR expressed in RGC axons enables selective blockade of retino-tectal synaptic transmission. The method established here can most likely be applied to a variety of brain regions for studying the function of individual inputs to these regions.

  3. AAVPG: A vigilant vector where transgene expression is induced by p53

    Energy Technology Data Exchange (ETDEWEB)

    Bajgelman, Marcio C.; Medrano, Ruan F.V.; Carvalho, Anna Carolina P.V.; Strauss, Bryan E., E-mail: bstrauss@usp.br

    2013-12-15

    Using p53 to drive transgene expression from viral vectors may provide on demand expression in response to physiologic stress, such as hypoxia or DNA damage. Here we introduce AAVPG, an adeno-associated viral (AAV) vector where a p53-responsive promoter, termed PG, is used to control transgene expression. In vitro assays show that expression from the AAVPG-luc vector was induced specifically in the presence of functional p53 (1038±202 fold increase, p<0.001). The AAVPG-luc vector was an effective biosensor of p53 activation in response to hypoxia (4.48±0.6 fold increase in the presence of 250 µM CoCl{sub 2}, p<0.001) and biomechanical stress (2.53±0.4 fold increase with stretching, p<0.05). In vivo, the vigilant nature of the AAVPG-luc vector was revealed after treatment of tumor-bearing mice with doxorubicin (pre-treatment, 3.4×10{sup 5}±0.43×10{sup 5} photons/s; post-treatment, 6.6×10{sup 5}±2.1×10{sup 5} photons/s, p<0.05). These results indicate that the AAVPG vector is an interesting option for detecting p53 activity both in vitro and in vivo. - Highlights: • AAV vector where transgene expression is controlled by the tumor suppressor p53. • The new vector, AAVPG, shown to function as a biosensor of p53 activity, in vitro and in vivo. • The p53 activity monitored by the AAVPG vector is relevant to cancer and other diseases. • AAVPG reporter gene expression was activated upon DNA damage, hypoxia and mechanical stress.

  4. High-resolution labeling and functional manipulation of specific neuron types in mouse brain by Cre-activated viral gene expression.

    Directory of Open Access Journals (Sweden)

    Sandra J Kuhlman

    Full Text Available We describe a method that combines Cre-recombinase knockin mice and viral-mediated gene transfer to genetically label and functionally manipulate specific neuron types in the mouse brain. We engineered adeno-associated viruses (AAVs that express GFP, dsRedExpress, or channelrhodopsin (ChR2 upon Cre/loxP recombination-mediated removal of a transcription-translation STOP cassette. Fluorescent labeling was sufficient to visualize neuronal structures with synaptic resolution in vivo, and ChR2 expression allowed light activation of neuronal spiking. The structural dynamics of a specific class of neocortical neuron, the parvalbumin-containing (Pv fast-spiking GABAergic interneuron, was monitored over the course of a week. We found that although the majority of Pv axonal boutons were stable in young adults, bouton additions and subtractions on axonal shafts were readily observed at a rate of 10.10% and 9.47%, respectively, over 7 days. Our results indicate that Pv inhibitory circuits maintain the potential for structural re-wiring in post-adolescent cortex. With the generation of an increasing number of Cre knockin mice and because viral transfection can be delivered to defined brain regions at defined developmental stages, this strategy represents a general method to systematically visualize the structure and manipulate the function of different cell types in the mouse brain.

  5. LIGHT induces distinct signals to clear an AAV-expressed persistent antigen in the mouse liver and to induce liver inflammation.

    Directory of Open Access Journals (Sweden)

    Michael L Washburn

    Full Text Available BACKGROUND: Infection with adeno-associated virus (AAV vector with liver tropism leads to persistent expression of foreign antigens in the mouse liver, with no significant liver inflammation or pathology. This provides a model to investigate antigen persistence in the liver and strategies to modulate host immunity to reduce or clear the foreign antigen expressed from AAV vector in the liver. METHODS/PRINCIPAL FINDINGS: We showed that expressing LIGHT with an adenovirus vector (Ad in mice with established AAV in the liver led to clearance of the AAV. Ad-LIGHT enhanced CD8 effector T cells in the liver, correlated with liver inflammation. LTbetaR-Ig proteins blocked Ad-LIGHT in clearing AAV. Interestingly, in LTbetaR-null mice, Ad-LIGHT still cleared AAV but caused no significant liver inflammation. CONCLUSIONS/SIGNIFICANCE: Our data suggest that LIGHT interaction with the LTbetaR plays a critical role in liver inflammation but is not required for LIGHT-mediated AAV clearance. These findings will shed light on developing novel immuno-therapeutics in treating people chronically infected with hepato-tropic viruses.

  6. High-Resolution Labeling and Functional Manipulation of Specific Neuron Types in Mouse Brain by Cre-Activated Viral Gene Expression

    Science.gov (United States)

    Kuhlman, Sandra J.; Huang, Z. Josh

    2008-01-01

    We describe a method that combines Cre-recombinase knockin mice and viral-mediated gene transfer to genetically label and functionally manipulate specific neuron types in the mouse brain. We engineered adeno-associated viruses (AAVs) that express GFP, dsRedExpress, or channelrhodopsin (ChR2) upon Cre/loxP recombination-mediated removal of a transcription-translation STOP cassette. Fluorescent labeling was sufficient to visualize neuronal structures with synaptic resolution in vivo, and ChR2 expression allowed light activation of neuronal spiking. The structural dynamics of a specific class of neocortical neuron, the parvalbumin-containing (Pv) fast-spiking GABAergic interneuron, was monitored over the course of a week. We found that although the majority of Pv axonal boutons were stable in young adults, bouton additions and subtractions on axonal shafts were readily observed at a rate of 10.10% and 9.47%, respectively, over 7 days. Our results indicate that Pv inhibitory circuits maintain the potential for structural re-wiring in post-adolescent cortex. With the generation of an increasing number of Cre knockin mice and because viral transfection can be delivered to defined brain regions at defined developmental stages, this strategy represents a general method to systematically visualize the structure and manipulate the function of different cell types in the mouse brain. PMID:18414675

  7. The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing.

    Science.gov (United States)

    de Solis, Christopher A; Ho, Anthony; Holehonnur, Roopashri; Ploski, Jonathan E

    2016-01-01

    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 1 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. PMID:27587996

  8. 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.

  9. Effects of myostatin propeptide gene tranfection on glucose metabolism in cultured C2C12 cells

    Institute of Scientific and Technical Information of China (English)

    张莎莎

    2014-01-01

    Objective To investigate the effects of recombinant adeno-associated virus-mediated myostatin propeptide(MPRO)on uptake and oxidation of glucose,and glycogen synthesis in C2C12 myotubes,as well as the associated molecular mechanism.Methods Mature C2C12myotubes were assigned to the following 6 groups:control,insulin,green fluorescent protein(GFP),insulin+

  10. Trichomonas vaginalis virus-mediated expression of EGFP in the parasitic protozoan Trichomonas vagi-nails%阴道毛滴虫病毒介导的EGFP在阴道毛滴虫细胞内的表达

    Institute of Scientific and Technical Information of China (English)

    郭瑞娟; 李淑红; 张西臣; 李建华; 宫鹏涛; 杨举; 张国才

    2010-01-01

    目的 研究阴道毛滴虫病毒(Trichomonas vaginalis virus,TVV)介导外源基因进入阴道毛滴虫体内表达的能力,探索TVV作为双链RNA病毒转染载体的可能性.方法 根据TVV基因组的序列特征,用绿色荧光蛋白(EG-FP)编码基因替换TVV的全部或部分基因编码区,构建TVV与增强型EGFP编码基因的嵌合体pTVV-EGFP,其体外转录体经电穿孔方法转染携病毒阴道毛滴虫株,RT-PCR及SDS-PAGE方法检测EGFP的表达情况.结果 电穿孔转染后培养的虫体在荧光显微镜下观察到绿色荧光信号,且续传15代后仍然存在;RT-PCR检测到EGFP的mRNA,SDS-PAGE检测到转染后虫体及培养上清中有分子质量单位为27 ku的蛋白,与已知EGFP的分子质量相符.结论 TVV能成功介导外源性EGFP编码基因在阴道毛滴虫体内表达.

  11. Construction of small interfering RNA vector targeting Atp6i and T cell immune response cDNA7 and preparation of its recombinant adeno-associated virus particle%靶向Atp6i和T细胞免疫应答cDNA7的小干扰RNA载体构建及其腺相关病毒重组颗粒的制备

    Institute of Scientific and Technical Information of China (English)

    张丽杰

    2012-01-01

    Objective To construct the recombinant RNA interference vector targeting Atp6i and T cell immune response cDNA7 (TIRC7),and to prepare its recombinant AAV particle in order to provide evidence for gene therapy for diseases using the recombinant RNA interference vector in vivo and in vitro.Methods Sequences of small interference iRNA targeting the common region of Atp6i and TIRC7 genes were designed and connected to the pAAV.H1 vector linearized by enzyme digestion of Xba Ⅰ and Bgl Ⅱ and then transformed into DH5α where plasmid was identified using enzyme digestion for sequencing analysis.The identified siRNA sequences,pAAV-RC and pHelper were used to transfect AAV-293 cells by calcium phosphate precipitation method for recombinant AAV.H1Atp6i particle,with phosphate buffer solution(PBS)used in blank control group and AAV.H1Luc in negative control group.The transfection efficiency was then observed by fluorescence microscopy.Western blotting was used to detect the effects of the recombinant AAV.H1Atp6i particle on TIRC7 protein.Results The recombinant siRNA targeting Atp6i and TIRC7 was successfully constructed.Furthermore,AAV-293 cells were successfully transfected by the recombinant particle,pAAV-RC and pHelper,with the transfection efficiency being almost 100%.And the recombinant AAV.H1Atp6i particle was successfully packed and obtained.Western blotting showed the expressions of TIRC7 protein in blank control,negative control and AAV.H1Atp6i treatment groups,with the molecular weight being 75 000.Nevertheless,the TIRC7 expression was decreased by 80% in AAV.H1Atp6i treatment group as compared with that in blank control and negative control groups (0.271±0.072 vs 0.988±0.042 and 0.992±0.053,all P<0.05).Conclusion The recombinant siRNA targeting Atp6i and TIRC7 and recombinant AAV.H1Atp6i particle are both successfully obtained,which may exhibit interference on TIRC7 expression.%目的 构建靶向Atp6i和T细胞免疫应答cDNA7(TIRC7)的RNA干扰

  12. Adeno-associated virus Rep78 restricts adenovirus E1B55K-mediated p53 nuclear exportation

    Institute of Scientific and Technical Information of China (English)

    Jingjing Wang; Wenjuan Li; Ran Wang; Jinglun Xue; Jinzhong Chen

    2013-01-01

    Inactivation of p53 is needed during adenovirus type 5 DNA replication.E1B55K,an adenovirus early protein,has been reported to interact with p53 and inhibit p53 transactivation.Previous studies have shown that adenoassociated virus (AAV) type 2 could reduce the transforming potential of adenovirus by rescuing p53 from adenovirus-mediated degradation,but the details are not clear yet.We detected the Rep78-p53 interaction by co-immunoprecipitation assay.The co-localization assay revealed that Rep78 inhibits E1B55K-mediated p53 nuclear exportation.However,Rep78 did not detectably influence p53 stability and could not relieve the transcriptional inactivation of p53,as E1B55K could not be replaced from the p53-E1B55K complex by Rep78.Our results reveal a new possible mechanism that AAV-2 Rep78 inhibits adenovirus 5 by relocalizing p53 in the nucleus,which may shed some light on the regulatory mechanism of AAV-2 on its helper virus,adenovirus.

  13. Adeno-Associated Viral-Mediated LARGE Gene Therapy Rescues the Muscular Dystrophic Phenotype in Mouse Models of Dystroglycanopathy

    OpenAIRE

    Yu, Miao; He, Yonglin; Wang, Kejian; Zhang, Peng; Zhang, Shengle; Hu, Huaiyu

    2013-01-01

    Dystroglycanopathies are a group of congenital muscular dystrophies (CMD) often caused by mutations in genes encoding glycosyltransferases that lead to hypoglycosylation of α-dystroglycan (α-DG) and reduce its extracellular matrix-binding activity. Overexpressing LARGE (formerly known as like-glycosyltransferase) generates an extracellular matrix-binding carbohydrate epitope in cells with CMD-causing mutations in not only LARGE but also other glycosyltransferases, including POMT1, POMGnT1, an...

  14. Exploiting high-throughput screens to optimize Adeno-Associated Viral Vectors for gene transfer into primary human keratinocytes

    OpenAIRE

    Sallach, Jessica

    2014-01-01

    Chronic non-healing wounds such as diabetic ulcers or burns represent a devastating health problem with significant clinical, physical and social implications. The healing can be frustrating and painful for patients. The difficult healing process requires advanced therapeutic strategies such as the use of primary human keratinocytes (HK) as autologous transplants, which may be considered for clinical use. To improve engraftment or to introduce therapeutic genes into primary HK, efficient and ...

  15. Transient B cell depletion or improved transgene expression by codon optimization promote tolerance to factor VIII in gene therapy.

    Directory of Open Access Journals (Sweden)

    Brandon K Sack

    Full Text Available The major complication in the treatment of hemophilia A is the development of neutralizing antibodies (inhibitors against factor VIII (FVIII. The current method for eradicating inhibitors, termed immune tolerance induction (ITI, is costly and protracted. Clinical protocols that prevent rather than treat inhibitors are not yet established. Liver-directed gene therapy hopes to achieve long-term correction of the disease while also inducing immune tolerance. We sought to investigate the use of adeno-associated viral (serotype 8 gene transfer to induce tolerance to human B domain deleted FVIII in hemophilia A mice. We administered an AAV8 vector with either human B domain deleted FVIII or a codon-optimized transgene, both under a liver-specific promoter to two strains of hemophilia A mice. Protein therapy or gene therapy was given either alone or in conjunction with anti-CD20 antibody-mediated B cell depletion. Gene therapy with a low-expressing vector resulted in sustained near-therapeutic expression. However, supplementary protein therapy revealed that gene transfer had sensitized mice to hFVIII in a high-responder strain but not in mice of a low-responding strain. This heightened response was ameliorated when gene therapy was delivered with anti-murine CD20 treatment. Transient B cell depletion prevented inhibitor formation in protein therapy, but failed to achieve a sustained hypo-responsiveness. Importantly, use of a codon-optimized hFVIII transgene resulted in sustained therapeutic expression and tolerance without a need for B cell depletion. Therefore, anti-CD20 may be beneficial in preventing vector-induced immune priming to FVIII, but higher levels of liver-restricted expression are preferred for tolerance.

  16. Fukutin is prerequisite to ameliorate muscular dystrophic phenotype by myofiber-selective LARGE expression.

    Science.gov (United States)

    Ohtsuka, Yoshihisa; Kanagawa, Motoi; Yu, Chih-Chieh; Ito, Chiyomi; Chiyo, Tomoko; Kobayashi, Kazuhiro; Okada, Takashi; Takeda, Shin'ichi; Toda, Tatsushi

    2015-01-01

    α-Dystroglycanopathy (α-DGP) is a group of muscular dystrophy characterized by abnormal glycosylation of α-dystroglycan (α-DG), including Fukuyama congenital muscular dystrophy (FCMD), muscle-eye-brain disease, Walker-Warburg syndrome, and congenital muscular dystrophy type 1D (MDC1D), etc. LARGE, the causative gene for MDC1D, encodes a glycosyltransferase to form [-3Xyl-α1,3GlcAβ1-] polymer in the terminal end of the post-phosphoryl moiety, which is essential for α-DG function. It has been proposed that LARGE possesses the great potential to rescue glycosylation defects in α-DGPs regardless of causative genes. However, the in vivo therapeutic benefit of using LARGE activity is controversial. To explore the conditions needed for successful LARGE gene therapy, here we used Large-deficient and fukutin-deficient mouse models for MDC1D and FCMD, respectively. Myofibre-selective LARGE expression via systemic adeno-associated viral gene transfer ameliorated dystrophic pathology of Large-deficient mice even when intervention occurred after disease manifestation. However, the same strategy failed to ameliorate the dystrophic phenotype of fukutin-conditional knockout mice. Furthermore, forced expression of Large in fukutin-deficient embryonic stem cells also failed to recover α-DG glycosylation, however coexpression with fukutin strongly enhanced α-DG glycosylation. Together, our data demonstrated that fukutin is required for LARGE-dependent rescue of α-DG glycosylation, and thus suggesting new directions for LARGE-utilizing therapy targeted to myofibres. PMID:25661440

  17. Role of Hypothalamic VGF in Energy Balance and Metabolic Adaption to Environmental Enrichment in Mice.

    Science.gov (United States)

    Foglesong, Grant D; Huang, Wei; Liu, Xianglan; Slater, Andrew M; Siu, Jason; Yildiz, Vedat; Salton, Stephen R J; Cao, Lei

    2016-03-01

    Environmental enrichment (EE), a housing condition providing complex physical, social, and cognitive stimulation, leads to improved metabolic health and resistance to diet-induced obesity and cancer. One underlying mechanism is the activation of the hypothalamic-sympathoneural-adipocyte axis with hypothalamic brain-derived neurotrophic factor (BDNF) as the key mediator. VGF, a peptide precursor particularly abundant in the hypothalamus, was up-regulated by EE. Overexpressing BDNF or acute injection of BDNF protein to the hypothalamus up-regulated VGF, whereas suppressing BDNF signaling down-regulated VGF expression. Moreover, hypothalamic VGF expression was regulated by leptin, melanocortin receptor agonist, and food deprivation mostly paralleled to BDNF expression. Recombinant adeno-associated virus-mediated gene transfer of Cre recombinase to floxed VGF mice specifically decreased VGF expression in the hypothalamus. In contrast to the lean and hypermetabolic phenotype of homozygous germline VGF knockout mice, specific knockdown of hypothalamic VGF in male adult mice led to increased adiposity, decreased core body temperature, reduced energy expenditure, and impaired glucose tolerance, as well as disturbance of molecular features of brown and white adipose tissues without effects on food intake. However, VGF knockdown failed to block the EE-induced BDNF up-regulation or decrease of adiposity indicating a minor role of VGF in the hypothalamic-sympathoneural-adipocyte axis. Taken together, our results suggest hypothalamic VGF responds to environmental demands and plays an important role in energy balance and glycemic control likely acting in the melanocortin pathway downstream of BDNF. PMID:26730934

  18. AAV9-mediated gene transfer of desmin ameliorates cardiomyopathy in desmin-deficient mice.

    Science.gov (United States)

    Heckmann, M B; Bauer, R; Jungmann, A; Winter, L; Rapti, K; Strucksberg, K-H; Clemen, C S; Li, Z; Schröder, R; Katus, H A; Müller, O J

    2016-08-01

    Mutations of the human desmin (DES) gene cause autosomal dominant and recessive myopathies affecting skeletal and cardiac muscle tissue. Desmin knockout mice (DES-KO), which develop progressive myopathy and cardiomyopathy, mirror rare human recessive desminopathies in which mutations on both DES alleles lead to a complete ablation of desmin protein expression. Here, we investigated whether an adeno-associated virus-mediated gene transfer of wild-type desmin cDNA (AAV-DES) attenuates cardiomyopathy in these mice. Our approach leads to a partial reconstitution of desmin protein expression and the de novo formation of the extrasarcomeric desmin-syncoilin network in cardiomyocytes of treated animals. This finding was accompanied by reduced fibrosis and heart weights and improved systolic left-ventricular function when compared with control vector-treated DES-KO mice. Since the re-expression of desmin protein in cardiomyocytes of DES-KO mice restores the extrasarcomeric desmin-syncoilin cytoskeleton, attenuates the degree of cardiac hypertrophy and fibrosis, and improves contractile function, AAV-mediated desmin gene transfer may be a novel and promising therapeutic approach for patients with cardiomyopathy due to the complete lack of desmin protein expression. PMID:27101257

  19. Recombinant AAV Vectors for Enhanced Expression of Authentic IgG.

    Directory of Open Access Journals (Sweden)

    Sebastian P Fuchs

    Full Text Available Adeno-associated virus (AAV has become a vector of choice for the treatment of a variety of genetic diseases that require safe and long-term delivery of a missing protein. Muscle-directed gene transfer for delivery of protective antibodies against AIDS viruses and other pathogens has been used experimentally in mice and monkeys. Here we examined a number of variations to AAV vector design for the ability to produce authentic immunoglobulin G (IgG molecules. Expression of rhesus IgG from a single single-stranded AAV (ssAAV vector (one vector approach was compared to expression from two self-complementary AAV (scAAV vectors, one for heavy chain and one for light chain (two vector approach. Both the one vector and the two vector approaches yielded considerable levels of expressed full-length IgG. A number of modifications to the ssAAV expression system were then examined for their ability to increase the efficiency of IgG expression. Inclusion of a furin cleavage sequence with a linker peptide just upstream of the 2A self-cleaving sequence from foot-and-mouth disease virus (F2A increased IgG expression approximately 2 fold. Inclusion of these sequences also helped to ensure a proper sequence at the C-terminal end of the heavy chain. Inclusion of the post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE further increased IgG expression 1.5-2.0 fold. IgG1 versions of the two rhesus IgGs that were examined consistently expressed better than the IgG2 forms. In contrast to what has been reported for AAV2-mediated expression of other proteins, introduction of capsid mutations Y445F and Y731F did not increase ssAAV1-mediated expression of IgG as determined by transduction experiments in cell culture. Our findings provide a rational basis for AAV vector design for expression of authentic IgG.

  20. Recombinant AAV Vectors for Enhanced Expression of Authentic IgG.

    Science.gov (United States)

    Fuchs, Sebastian P; Martinez-Navio, José M; Gao, Guangping; Desrosiers, Ronald C

    2016-01-01

    Adeno-associated virus (AAV) has become a vector of choice for the treatment of a variety of genetic diseases that require safe and long-term delivery of a missing protein. Muscle-directed gene transfer for delivery of protective antibodies against AIDS viruses and other pathogens has been used experimentally in mice and monkeys. Here we examined a number of variations to AAV vector design for the ability to produce authentic immunoglobulin G (IgG) molecules. Expression of rhesus IgG from a single single-stranded AAV (ssAAV) vector (one vector approach) was compared to expression from two self-complementary AAV (scAAV) vectors, one for heavy chain and one for light chain (two vector approach). Both the one vector and the two vector approaches yielded considerable levels of expressed full-length IgG. A number of modifications to the ssAAV expression system were then examined for their ability to increase the efficiency of IgG expression. Inclusion of a furin cleavage sequence with a linker peptide just upstream of the 2A self-cleaving sequence from foot-and-mouth disease virus (F2A) increased IgG expression approximately 2 fold. Inclusion of these sequences also helped to ensure a proper sequence at the C-terminal end of the heavy chain. Inclusion of the post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE) further increased IgG expression 1.5-2.0 fold. IgG1 versions of the two rhesus IgGs that were examined consistently expressed better than the IgG2 forms. In contrast to what has been reported for AAV2-mediated expression of other proteins, introduction of capsid mutations Y445F and Y731F did not increase ssAAV1-mediated expression of IgG as determined by transduction experiments in cell culture. Our findings provide a rational basis for AAV vector design for expression of authentic IgG. PMID:27332822

  1. The use of a viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein (eGFP) in vitro and in vivo

    Science.gov (United States)

    Lewis, Jo E.; Brameld, John M.; Hill, Phil; Barrett, Perry; Ebling, Francis J.P.; Jethwa, Preeti H.

    2015-01-01

    Introduction The viral 2A sequence has become an attractive alternative to the traditional internal ribosomal entry site (IRES) for simultaneous over-expression of two genes and in combination with recombinant adeno-associated viruses (rAAV) has been used to manipulate gene expression in vitro. New method To develop a rAAV construct in combination with the viral 2A sequence to allow long-term over-expression of the vgf gene and fluorescent marker gene for tracking of the transfected neurones in vivo. Results Transient transfection of the AAV plasmid containing the vgf gene, viral 2A sequence and eGFP into SH-SY5Y cells resulted in eGFP fluorescence comparable to a commercially available reporter construct. This increase in fluorescent cells was accompanied by an increase in VGF mRNA expression. Infusion of the rAAV vector containing the vgf gene, viral 2A sequence and eGFP resulted in eGFP fluorescence in the hypothalamus of both mice and Siberian hamsters, 32 weeks post infusion. In situ hybridisation confirmed that the location of VGF mRNA expression in the hypothalamus corresponded to the eGFP pattern of fluorescence. Comparison with old method The viral 2A sequence is much smaller than the traditional IRES and therefore allowed over-expression of the vgf gene with fluorescent tracking without compromising viral capacity. Conclusion The use of the viral 2A sequence in the AAV plasmid allowed the simultaneous expression of both genes in vitro. When used in combination with rAAV it resulted in long-term over-expression of both genes at equivalent locations in the hypothalamus of both Siberian hamsters and mice, without any adverse effects. PMID:26300182

  2. Inhibitive effects of lenti virus-mediated hTERT RNA interference and TMPyP4-PDT on cervix cancer SiHa cells%慢病毒介导的hTERT-shRNA联合光动力治疗对宫颈癌细胞SiHa的影响

    Institute of Scientific and Technical Information of China (English)

    王颉; 张友忠; 冯进波; 刘洪丽; 宗丽菊

    2015-01-01

    proliferative activity of SiHa cells was assessed by Cell Counting Kit (CCK-8)assay;the expressions of hTERT,p53,HPV-16 E6,and Caspase-8 mRNA were detected by real-time PCR,and the hTERT pro-tein was tested by immunofluorescence (IF);the apoptosis was examined by AnnexinV-PE/7-AAD double dye kit;the ability of cellular invasion was assessed by Transwell experiment.Results Compared with the blank control group,the other three groups had decreased the expressions of hTERT,HPV-16 E6,and Caspase-8 mRNA,but increased the ex-pression of p53 mRNA.The down-regulation of hTERT protein was the most significant in the combined treatment group (P<0.05).In the combined treatment group,the cellular survivability and invasion ability of SiHa cells were in-hibited more markedly than in the other groups (P<0.05 ),while the apoptosis rate increased more significantly (P<0.05).Conclusion Lenti virus-mediated hTERT RNA interference combined with photodynamic therapy of TMPyP4 can suppress the abilities of proliferation and invasion,and enhance the apoptosis rate of cervix cancer SiHa cells.

  3. Evaluation of lateral spread of transgene expression following subretinal AAV-mediated gene delivery in dogs.

    Directory of Open Access Journals (Sweden)

    Ashlee R Bruewer

    Full Text Available Dog models with spontaneously occurring mutations in retinal dystrophy genes are an invaluable resource for preclinical development of retinal gene therapy. Adeno-associated virus (AAV vectors have been most successful; to target the outer retina and RPE they are delivered by subretinal injection, causing a temporary retinal detachment with some potential for retinal morbidity. A recent reporter gene study using an AAV2/8 vector in dogs reported transgene expression beyond the boundary of the subretinal bleb. This could be a desirable feature which increases the area of retina treated while minimizing the retinal detachment and any associated morbidity. We performed a detailed study of the lateral spread of transgene expression beyond the subretinal injection site following subretinally delivered AAV vectors in normal dogs. Vectors expressed green fluorescent protein (GFP using a small chicken beta-actin promoter. AAV2/2 (quadruple tyrosine to phenylalanine (Y-F capsid mutant, self-complementary (sc AAV2/8 (single Y-F capsid mutant and a scAAV2/5 were used. We found that in all eyes GFP expression involved retina beyond the initial post-injection subretinal bleb boundary. In all eyes there was post-injection spread of the retinal detachment within the first 3 days post procedure and prior to retinal reattachment. In 11/16 eyes this accounted for the entire "lateral spread" of GFP expression while in 5/16 eyes a very slight extension of GFP expression beyond the final boundary of the subretinal bleb could be detected. All 3 AAV constructs induced GFP expression in the nerve fiber layer with spread to the optic nerve. Patients treated by subretinal injection should be monitored for possible expansion of the subretinal injection bleb prior to reattachment. Injections in the para-foveal region may expand to lead to a foveal detachment that may be undesirable. Cell-specific promoters may be required to limit spread of expressed transgene to the brain

  4. AAV-mediated overexpression of neuroserpin in the hippocampus decreases PSD-95 expression but does not affect hippocampal-dependent learning and memory.

    Directory of Open Access Journals (Sweden)

    Vicky W K Tsang

    Full Text Available Neuroserpin is a serine protease inhibitor, or serpin, that is expressed in the nervous system and inhibits the protease tissue plasminogen activator (tPA. Neuroserpin has been suggested to play a role in learning and memory but direct evidence for such a role is lacking. Here we have used an adeno-associated virus (AAV vector expression system to investigate the effect of neuroserpin on hippocampal-dependent learning and memory in the young adult rat. A FLAG-tagged neuroserpin construct was initially characterized by in vitro transcription/translation and transfection into HEK293 cells and shown to interact with tPA and be targeted to the secretory pathway. Targeted injection of a chimeric AAV1/2 vector expressing FLAG-neuroserpin resulted in localized overexpression in the dorsal hippocampus. Neuroserpin overexpression led to the appearance of an unstable neuroserpin:tPA complex in zymographic assays consistent with interaction with endogenous tPA in vivo. Rats overexpressing neuroserpin also showed a significant decrease in the levels of postsynaptic density protein 95, a major postsynaptic scaffolding protein. Three weeks after injection, a range of behavioural tests was performed to measure spatial and associative learning and memory, as well as innate and acquired fear. These tests provided no evidence of a role for neuroserpin in hippocampal-dependent learning and memory. In summary this study does not support a role for neuroserpin in hippocampal-dependent learning and memory in young adult rats but does suggest an involvement of neuroserpin in hippocampal synaptic plasticity.

  5. Potent Dystrophin knock-Down in Vitro and in Vivo Using RNAi Technonlogy and Expression Signature of Myotubes with Dystrophin knocked Down: Attempts at Unravelling the Mystery

    Directory of Open Access Journals (Sweden)

    MM Ghahramani Seno

    2005-10-01

    Full Text Available Duchenne Muscular Dystrophy (DMD is one of a group of genetically heterogeneous muscular dystrophies that are characterized by progressive weakness and wasting of skeletal muscle. Loss of myofibres occurs in response to a deficiency of dystrophin, a protein which is believed to be responsible for myofibre maintenance and integrity. Dystrophin forms a link between the cytoskeleton and the membrane-spanning dystrophin-associated glycoprotein complex (DAPC, indicative of a structural role for dystrophin. The application of gene therapy protocols for DMD still presents many daunting challenges due partly to intrinsic features of the dystrophin gene. Hence, improvement in the understanding of the underlying primary molecular events leading to a dystrophic pathology might pave the way for the discovery of new starting points. Here we present a strategy to use RNAi technology to study the events occurring in muscle cell development due to dystrophin deficiency. RNAi has been proven to be a powerful technology to study molecular effects due to knockdown of single genes. We have used a series of siRNAs to target and knock down the expression of dystrophin in primary cultures of mouse muscle, and subsequently used transcriptomic array analysis to identify genes whose expression were affected in response to dystrophin deficiency. The data obtained from this experiment, which include some very interesting potential new targets, are currently being analysed. We have also developed a recombinant adeno-associated virus (rAAV vector expressing an shRNA targeting dystrophin. The use of such rAAV-shDNA vectors enables us to target dystrophin in vivo to obtain a better and potentially curative insight into the pathophysiology of DMD.

  6. Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease

    DEFF Research Database (Denmark)

    Kozlitina, Julia; Smagris, Eriks; Stender, Stefan;

    2014-01-01

    × 10(-7): two in PNPLA3, an established locus for NAFLD, and one (encoding p.Glu167Lys) in TM6SF2, a gene of unknown function. The TM6SF2 variant encoding p.Glu167Lys was also associated with higher circulating levels of alanine transaminase, a marker of liver injury, and with lower levels of low......-density lipoprotein-cholesterol (LDL-C), triglycerides and alkaline phosphatase in 3 independent populations (n > 80,000). When recombinant protein was expressed in cultured hepatocytes, 50% less Glu167Lys TM6SF2 protein was produced relative to wild-type TM6SF2. Adeno-associated virus-mediated short hairpin RNA......Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease. To elucidate the molecular basis of NAFLD, we performed an exome-wide association study of liver fat content. Three variants were associated with higher liver fat levels at the exome-wide significance level of 3.6...

  7. Vector-induced NT-3 expression in rats promotes collateral growth of injured corticospinal tract axons far rostral to a spinal cord injury.

    Science.gov (United States)

    Weishaupt, N; Mason, A L O; Hurd, C; May, Z; Zmyslowski, D C; Galleguillos, D; Sipione, S; Fouad, K

    2014-07-11

    Rewiring the injured corticospinal tract (CST) by promoting connections between CST axons and spared neurons is a strategy being explored experimentally to achieve improved recovery of motor function after spinal cord injury (SCI). Reliable interventions to promote and direct growth of collaterals from injured CST axons are in high demand to promote functionally relevant detour pathways. A promising tool is neurotrophin-3 (NT-3), which has shown growth-stimulating and chemo-attractive effects for spared CST axons caudal to a CST lesion. Yet, efforts to promote growth of injured CST axons rostral to a SCI with NT-3 have been less successful to date. Evidence indicates that immune activation in the local growth environment, either intrinsic or induced by the endotoxin lipopolysaccharide (LPS), can play a decisive role in the CST's responsiveness to NT-3. Here, we test the potential of NT-3 as a tool to enhance and direct collateral growth from the injured CST rostral to a SCI (1) using long-term expression of NT-3 by adeno-associated viral vectors, (2) with and without stimulating the immune system with LPS. Our results indicate that inducing a growth response from injured CST axons into a region of vector-mediated NT-3 expression is possible in the environment of the spinal cord rostral to a SCI, but seems dependent on the distance between the responding axon and the source of NT-3. Our findings also suggest that injured CST axons do not increase their growth response to NT-3 after immune activation with LPS in this environment. In conclusion, this is to our knowledge the first demonstration that NT-3 can be effective at promoting growth of injured CST collaterals far rostral to a SCI. Making NT-3 available in close proximity to CST target axons may be the key to success when using NT-3 to rewire the injured CST in future investigations.

  8. Elevated expression of serotonin 5-HT2A receptors in the rat ventral tegmental area enhances vulnerability to the behavioral effects of cocaine

    Directory of Open Access Journals (Sweden)

    David V. Herin

    2013-02-01

    Full Text Available The dopamine mesocorticoaccumbens pathway which originates in the ventral tegmental area (VTA and projects to the nucleus accumbens and prefrontal cortex is a circuit important in mediating the actions of psychostimulants. The function of this circuit is modulated by the actions of serotonin (5-HT at 5-HT2A receptors (5-HT2AR localized to the VTA. In the present study, we tested the hypothesis that virally-mediated overexpression of 5-HT2AR in the VTA would increase cocaine-evoked locomotor activity in the absence of alterations in basal locomotor activity. A plasmid containing the gene for the 5-HT2AR linked to a synthetic marker peptide (Flag was created and the construct was packaged in an adeno-associated virus vector (rAAV-5-HT2AR-Flag. This viral vector (2 µl; 109-10 transducing units/ml was unilaterally infused into the VTA of male rats, while control animals received an intra-VTA infusion of Ringer’s solution. Virus-pretreated rats exhibited normal spontaneous locomotor activity measured in a modified open-field apparatus at 7, 14, and 21 days following infusion. After an injection of cocaine (15 mg/kg, ip, both horizontal hyperactivity and rearing were significantly enhanced in virus-treated rats (p<0.05. Immunohistochemical analysis confirmed expression of Flag and overexpression of the 5-HT2AR protein. These data indicate that the vulnerability of adult male rats to hyperactivity induced by cocaine is enhanced following increased levels of expression of the 5-HT2AR in the VTA and suggest that the 5-HT2AR receptor in the VTA plays a role in regulation of responsiveness to cocaine.

  9. Identification of an Alternative Splicing Product of the Otx2 Gene Expressed in the Neural Retina and Retinal Pigmented Epithelial Cells

    Science.gov (United States)

    Kole, Christo; Berdugo, Naomi; Da Silva, Corinne; Aït-Ali, Najate; Millet-Puel, Géraldine; Pagan, Delphine; Blond, Frédéric; Poidevin, Laetitia; Ripp, Raymond; Fontaine, Valérie; Wincker, Patrick; Zack, Donald J.; Sahel, José-Alain; Poch, Olivier; Léveillard, Thierry

    2016-01-01

    To investigate the complexity of alternative splicing in the retina, we sequenced and analyzed a total of 115,706 clones from normalized cDNA libraries from mouse neural retina (66,217) and rat retinal pigmented epithelium (49,489). Based upon clustering the cDNAs and mapping them with their respective genomes, the estimated numbers of genes were 9,134 for the mouse neural retina and 12,050 for the rat retinal pigmented epithelium libraries. This unique collection of retinal of messenger RNAs is maintained and accessible through a web-base server to the whole community of retinal biologists for further functional characterization. The analysis revealed 3,248 and 3,202 alternative splice events for mouse neural retina and rat retinal pigmented epithelium, respectively. We focused on transcription factors involved in vision. Among the six candidates suitable for functional analysis, we selected Otx2S, a novel variant of the Otx2 gene with a deletion within the homeodomain sequence. Otx2S is expressed in both the neural retina and retinal pigmented epithelium, and encodes a protein that is targeted to the nucleus. OTX2S exerts transdominant activity on the tyrosinase promoter when tested in the physiological environment of primary RPE cells. By overexpressing OTX2S in primary RPE cells using an adeno associated viral vector, we identified 10 genes whose expression is positively regulated by OTX2S. We find that OTX2S is able to bind to the chromatin at the promoter of the retinal dehydrogenase 10 (RDH10) gene. PMID:26985665

  10. 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.

  11. dsRNA介导的番木瓜环斑病毒(PRSV)的抗病性研究%Virus-resistance of Papaya Ringspot Virus Mediated by Double-Strand RNA

    Institute of Scientific and Technical Information of China (English)

    张帆; 姜玲

    2011-01-01

    Tobacco ( Nicotiana tabacum) , Arabidopsis thaliana, and host papaya( Carica papaya L. )of PRSV were used as model plant materials to testing whether RNAi could mediate resistance to papaya ringspot virus (PRSV). The agrobacterium was harbored with pHellsgate12-CPIR containing structured inverted sequences of the CP gene of PRSV,where were transformed into the tobacco and Arabidopsis. We also conducted an Agrobacterium-mediated transient expression experiment by infiltration in papaya. The three transgenic plants inoculated with PRSV were tested for resistance to PRSV. After PRSV challenge-inoculation for 3 to 7 days, leaves of the wild type plants displayed symptoms of necrosis, wrinkling and/or chlorosis, and the scale of the abnormal leaves in the transgenic plants was significantly lower than that of the wild type when the PRSV were inoculated in papaya and Arabidopsis separately.However, the difference in symptoms was not significant in transgenic tobacco and the control wild plant. We carried out a RT-PCR experiment on the three kinds of plants. The expression of CP mRNA was detected in the wild type plants infected with PRSV, but not in the transgenic plants. These results suggest that the transgenic plants may induce the RNAi process to resist PRSV.%以模式植物拟南芥(Arabidopsis thaliana)和烟草(Nicotiana tabacum)及PRSV寄主植物番木瓜(Carica papaya L.)作为试验材料,开展了番木瓜环斑病毒外壳蛋白基因dsRNA介导的PRSV病原抗性的研究.利用农杆菌介导法将番木瓜环斑病毒外壳蛋白CP基因反向重复表达载体pHellsgate12-CPIR(简称PHG12-CPIR)分别转化到烟草和拟南芥中,获得阳性植株,并利用渗透法和农杆菌介导的瞬时表达体系将pHG12-CPIR载体导入到番木瓜中.对转基因植株进行攻毒试验并分析了其抗病性.在接种3~7 d内,在拟南芥和番木瓜上转基因植株的发病情况较轻,而野生型植株叶片与转基因植株相比,均表现出不同

  12. Combined expression of CTGF and tissue inhibitor of metalloprotease-1 promotes synthesis of proteoglycan and collagen type Ⅱ in rhesus monkey lumbar intervertebral disc cells in vitro

    Institute of Scientific and Technical Information of China (English)

    LIU Yong; KONG Jie; CHEN Bo-hua; HU You-gu

    2010-01-01

    Background Low back pain has emerged as a widespread disease often caused by intervertebral disc degeneration.This study aimed to establish an in vitro cell culture model of rhesus monkey lumbar intervertebral discs and to investigate the effect of combined connective tissue growth factor (CTGF) and tissue inhibitor of metalloprotease-1(TIMP-1) expression mediated by adeno-associated virus (AAV) on collagen type Ⅱ and proteoglycan levels.The purpose of these investigations was to explore potential methods for relieving the degeneration of lumbar intervertebral disc cells.Methods Rhesus monkey lumbar intervertebral disc nucleus pulposus cells (NPCs) were isolated by enzyme digestion,cultured, and transduced with rAAV2-CTGF-IRES-TIMP-1, rAAV2-CTGF, or rAAV2-TIMP-1 at a multiplicity of infection (MOl) of 106.The expression of collagen type Ⅱ and proteoglycan was measured using RT-PCR and Western blotting.The synthetic rate of proteoglycan was measured using 35S incorporation.Results Rhesus monkey lumbar intervertebral disc NPCs were transduced with rAAV2-CTGF-IRES-TIMP-1,rAAV2-CTGF, and rAAV2-TIMP-1 and the transduced genes were expressed and detected.Compared to the control,CTGF promoted the synthesis of collagen type Ⅱ and proteoglycan.TIMP-1 showed an enhancing effect on the expression of proteoglycan but no effect on collagen type Ⅱ.Expression of both genes in rhesus monkey lumbar intervertebral disc NPCs significantly enhances the synthesis of proteoglycan and collagen type Ⅱ.Conclusions Single gene transduction of CTGF or TIMP-1 can enhanced synthesis of proteoglycan.CTGF expression can also enhance collagen type Ⅱ protein synthesis.Combined transduction of both CTGF and TIMP1 can significantly promote the expression of proteoglycan and collagen type Ⅱ to levels greater than transduction of a single gene alone.Our study provides a good basis for multi-gene therapy to treat lumbar intervertebral disc degeneration.

  13. Global gene expression profiling of pancreatic islets in mice during streptozotocin-induced β-cell damage and pancreatic Glp-1 gene therapy

    Directory of Open Access Journals (Sweden)

    Jason M. Tonne

    2013-09-01

    Streptozotocin (STZ, a glucosamine-nitrosourea compound, has potent genotoxic effects on pancreatic β-cells and is frequently used to induce diabetes in experimental animals. Glucagon-like peptide-1 (GLP-1 has β-cell protective effects and is known to preserve β-cells from STZ treatment. In this study, we analyzed the mechanisms of STZ-induced diabetes and GLP-1-mediated β-cell protection in STZ-treated mice. At 1 week after multiple low-dose STZ administrations, pancreatic β-cells showed impaired insulin expression, while maintaining expression of nuclear Nkx6.1. This was accompanied by significant upregulation of p53-responsive genes in islets, including a mediator of cell cycle arrest, p21 (also known as Waf1 and Cip1. STZ treatment also suppressed expression of a wide range of genes linked with key β-cell functions or diabetes development, such as G6pc2, Slc2a2 (Glut2, Slc30a8, Neurod1, Ucn3, Gad1, Isl1, Foxa2, Vdr, Pdx1, Fkbp1b and Abcc8, suggesting global β-cell defects in STZ-treated islets. The Tmem229B, Prss53 and Ttc28 genes were highly expressed in untreated islets and strongly suppressed by STZ, suggesting their potential roles in β-cell function. When a pancreas-targeted adeno-associated virus (AAV vector was employed for long-term Glp-1 gene delivery, pancreatic GLP-1 expression protected mice from STZ-induced diabetes through preservation of the β-cell mass. Despite its potent β-cell protective effects, however, pancreatic GLP-1 overexpression showed limited effects on the global gene expression profiles in the islets. Network analysis identified the programmed-cell-death-associated pathways as the most relevant network in Glp-1 gene therapy. Upon pancreatic GLP-1 expression, upregulation of Cxcl13 and Nptx2 was observed in STZ-damaged islets, but not in untreated normal islets. Given the pro-β-cell-survival effects of Cxcl12 (Sdf-1 in inducing GLP-1 production in α-cells, pancreatic GLP-1-mediated Cxcl13 induction might also play a

  14. VEGF-expressing human umbilical cord mesenchymal stem cells, an improved therapy strategy for Parkinson's disease.

    Science.gov (United States)

    Xiong, N; Zhang, Z; Huang, J; Chen, C; Zhang, Z; Jia, M; Xiong, J; Liu, X; Wang, F; Cao, X; Liang, Z; Sun, S; Lin, Z; Wang, T

    2011-04-01

    The umbilical cord provides a rich source of primitive mesenchymal stem cells (human umbilical cord mesenchymal stem cells (HUMSCs)), which have the potential for transplantation-based treatments of Parkinson's Disease (PD). Our pervious study indicated that adenovirus-associated virus-mediated intrastriatal delivery of human vascular endothelial growth factor 165 (VEGF 165) conferred molecular protection to the dopaminergic system. As both VEGF and HUMSCs displayed limited neuroprotection, in this study we investigated whether HUMSCs combined with VEGF expression could offer enhanced neuroprotection. HUMSCs were modified by adenovirus-mediated VEGF gene transfer, and subsequently transplanted into rotenone-lesioned striatum of hemiparkinsonian rats. As a result, HUMSCs differentiated into dopaminergic neuron-like cells on the basis of neuron-specific enolase (NSE) (neuronal marker), glial fibrillary acidic protein (GFAP) (astrocyte marker), nestin (neural stem cell marker) and tyrosine hydroxylase (TH) (dopaminergic marker) expression. Further, VEGF expression significantly enhanced the dopaminergic differentiation of HUMSCs in vivo. HUMSC transplantation ameliorated apomorphine-evoked rotations and reduced the loss of dopaminergic neurons in the lesioned substantia nigra (SNc), which was enhanced significantly by VEGF expression in HUMSCs. These findings present the suitability of HUMSC as a vector for gene therapy and suggest that stem cell engineering with VEGF may improve the transplantation strategy for the treatment of PD.

  15. Intravenous injection of AAVrh10-GALC after the neonatal period in twitcher mice results in significant expression in the central and peripheral nervous systems and improvement of clinical features.

    Science.gov (United States)

    Rafi, Mohammad A; Rao, Han Zhi; Luzi, Paola; Luddi, Alice; Curtis, Mark T; Wenger, David A

    2015-03-01

    Globoid cell leukodystrophy (GLD) or Krabbe disease is an autosomal recessive disorder resulting from the defective lysosomal enzyme galactocerebrosidase (GALC). The lack of GALC enzyme leads to severe neurological symptoms. While most human patients are infants who do not survive beyond 2 years of age, older patients are also diagnosed. In addition to human patients, several naturally occurring animal models, including dog, mouse, and monkey, have also been identified. The mouse model of Krabbe disease, twitcher (twi) mouse has been used for many treatment trials including gene therapy. Using the combination of intracerebroventricular, intracerebellar, and intravenous (iv) injection of the adeno-associated virus serotype rh10 (AAVrh10) expressing mouse GALC in neonate twi mice we previously have demonstrated a significantly extended normal life and exhibition of normal behavior in treated mice. In spite of the prolonged healthy life of these treated mice and improved myelination, it is unlikely that using multiple injection sites for viral administration will be approved for treatment of human patients. In this study, we have explored the outcome of the single iv injection of viral vector at post-natal day 10 (PND10). This has resulted in increased GALC activity in the central nervous system (CNS) and high GALC activity in the peripheral nervous system (PNS). As we have shown previously, an iv injection of AAVrh10 at PND2 results in a small extension of life beyond the typical lifespan of the untreated twi mice (~40 days). In this study, we report that mice receiving a single iv injection at PND10 had no tremor and continued to gain weight until a few weeks before they died. On average, they lived 20-25 days longer than untreated mice. We anticipate that this strategy in combination with other therapeutic options may be beneficial and applicable to treatment of human patients. PMID:25533112

  16. 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

  17. 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

  18. 口服重组腺相关病毒基因药物%Oral recombinant adeno-associated virus gene medicine

    Institute of Scientific and Technical Information of China (English)

    刁勇; 许瑞安

    2009-01-01

    重组腺相关病毒(rAAV)载体介导的口服基冈药物引起业界广泛的重视.尽管经口服给药后转基因的有效表达面临许多障碍,但该技术的有效性已得到大量实验证实.本文总结了口服rAAV基冈药物的临床前研究结果,重点阐述了该类型药物的传递、吸收、分布和基冈转导等药动学特点.已证实rAAV基因药物对人体的安全性高,但口服rAAV基因药物的临床应用仍需对其作用机制和生物约剂学特征进行深入和广泛的研究.

  19. Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications

    OpenAIRE

    Friedland, Ari E.; Baral, Reshica; Singhal, Pankhuri; Loveluck, Katherine; Shen, Shen; Sanchez, Minerva; Marco, Eugenio; Gotta, Gregory M.; Maeder, Morgan L.; Kennedy, Edward M.; Kornepati, Anand V. R.; Sousa, Alexander; Collins, McKensie A.; Jayaram, Hari; Cullen, Bryan R.

    2015-01-01

    Background CRISPR-Cas systems have been broadly embraced as effective tools for genome engineering applications, with most studies to date utilizing the Streptococcus pyogenes Cas9. Here we characterize and manipulate the smaller, 1053 amino acid nuclease Staphylococcus aureus Cas9. Results We find that the S. aureus Cas9 recognizes an NNGRRT protospacer adjacent motif (PAM) and cleaves target DNA at high efficiency with a variety of guide RNA (gRNA) spacer lengths. When directed against geno...

  20. Evolutionary Relationships among Parvoviruses: Virus-Host Coevolution among Autonomous Primate Parvoviruses and Links between Adeno-Associated and Avian Parvoviruses

    OpenAIRE

    Lukashov, Vladimir V.; Goudsmit, Jaap

    2001-01-01

    The current classification of parvoviruses is based on virus host range and helper virus dependence, while little data on evolutionary relationships among viruses are available. We identified and analyzed 472 sequences of parvoviruses, among which there were (virtually) full-length genomes of all 41 viruses currently recognized as individual species within the family Parvoviridae. Our phylogenetic analysis of full-length genomes as well as open reading frames distinguished three evolutionary ...

  1. microRNA-155 Regulates Alpha-Synuclein-Induced Inflammatory Responses in Models of Parkinson Disease.

    Science.gov (United States)

    Thome, Aaron D; Harms, Ashley S; Volpicelli-Daley, Laura A; Standaert, David G

    2016-02-24

    Increasing evidence points to inflammation as a chief mediator of Parkinson's disease (PD), a progressive neurodegenerative disorder characterized by loss of dopamine neurons in the substantia nigra pars compacta (SNpc) and widespread aggregates of the protein α-synuclein (α-syn). Recently, microRNAs, small, noncoding RNAs involved in regulating gene expression at the posttranscriptional level, have been recognized as important regulators of the inflammatory environment. Using an array approach, we found significant upregulation of microRNA-155 (miR-155) in an in vivo model of PD produced by adeno-associated-virus-mediated expression of α-syn. Using a mouse with a complete deletion of miR-155, we found that loss of miR-155 reduced proinflammatory responses to α-syn and blocked α-syn-induced neurodegeneration. In primary microglia from miR-155(-/-) mice, we observed a markedly reduced inflammatory response to α-syn fibrils, with attenuation of major histocompatibility complex class II (MHCII) and proinflammatory inducible nitric oxide synthase expression. Treatment of these microglia with a synthetic mimic of miR-155 restored the inflammatory response to α-syn fibrils. Our results suggest that miR-155 has a central role in the inflammatory response to α-syn in the brain and in α-syn-related neurodegeneration. These effects are at least in part due to a direct role of miR-155 on the microglial response to α-syn. These data implicate miR-155 as a potential therapeutic target for regulating the inflammatory response in PD. PMID:26911687

  2. Transduction of brain dopamine neurons by adenoviral vectors is modulated by CAR expression: rationale for tropism modified vectors in PD gene therapy.

    Directory of Open Access Journals (Sweden)

    Travis B Lewis

    Full Text Available BACKGROUND: Gene-based therapy is a new paradigm for the treatment of Parkinson disease (PD and offers considerable promise for precise targeting and flexibility to impact multiple pathobiological processes for which small molecule agents are not available. Some success has been achieved utilizing adeno-associated virus for this approach, but it is likely that the characteristics of this vector system will ultimately create barriers to progress in clinical therapy. Adenovirus (Ad vector overcomes limitations in payload size and targeting. The cellular tropism of Ad serotype 5 (Ad5-based vectors is regulated by the Ad attachment protein binding to its primary cellular receptor, the coxsackie and adenovirus receptor (CAR. Many clinically relevant tissues are refractory to Ad5 infection due to negligible CAR levels but can be targeted by tropism-modified, CAR-independent forms of Ad. Our objective was to evaluate the role of CAR protein in transduction of dopamine (DA neurons in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Ad5 was delivered to the substantia nigra (SN in wild type (wt and CAR transgenic animals. Cellular tropism was assessed by immunohistochemistry (IHC in the SN and striatal terminals. CAR expression was assessed by western blot and IHC. We found in wt animals, Ad5 results in robust transgene expression in astrocytes and other non-neuronal cells but poor infection of DA neurons. In contrast, in transgenic animals, Ad5 infects SNc neurons resulting in expression of transduced protein in their striatal terminals. Western blot showed low CAR expression in the ventral midbrain of wt animals compared to transgenic animals. Interestingly, hCAR protein localizes with markers of post-synaptic structures, suggesting synapses are the point of entry into dopaminergic neurons in transgenic animals. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate that CAR deficiency limits infection of wild type DA neurons by Ad5 and provide a rationale for the

  3. Transduction of Brain Dopamine Neurons by Adenoviral Vectors Is Modulated by CAR Expression: Rationale for Tropism Modified Vectors in PD Gene Therapy

    OpenAIRE

    Lewis, Travis B.; Glasgow, Joel N.; Glandon, Anya M.; Curiel, David T.; Standaert, David G.

    2010-01-01

    BACKGROUND: Gene-based therapy is a new paradigm for the treatment of Parkinson disease (PD) and offers considerable promise for precise targeting and flexibility to impact multiple pathobiological processes for which small molecule agents are not available. Some success has been achieved utilizing adeno-associated virus for this approach, but it is likely that the characteristics of this vector system will ultimately create barriers to progress in clinical therapy. Adenovirus (Ad) vector ove...

  4. Successful Regional Delivery and Long-term Expression of a Dystrophin Gene in Canine Muscular Dystrophy: A Preclinical Model for Human Therapies

    OpenAIRE

    Wang, Zejing; Storb, Rainer; Halbert, Christine L.; Banks, Glen B.; Butts, Tiffany M.; Finn, Eric E.; Allen, James M.; Miller, A. Dusty; Jeffrey S. Chamberlain; Tapscott, Stephen J.

    2012-01-01

    Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle disease caused by mutations in the dystrophin gene. Adeno-associated viral (AAV) vector-mediated gene replacement strategies hold promise as a treatment. Studies in animal models and human trials suggested that immune responses to AAV capsid proteins and transgene products prevented efficient gene therapy. In this study, we used widespread intramuscular (i.m.) injection to deliver AAV6-canine micro-dystrophin (c-µdys) throughout a ...

  5. 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.

  6. AAV vector-mediated secretion of chondroitinase provides a sensitive tracer for axonal arborisations

    NARCIS (Netherlands)

    Alves, João Nuno; Muir, Elizabeth M; Andrews, Melissa R; Ward, Anneliese; Michelmore, Nicholas; Dasgupta, Debayan; Verhaagen, J.; Moloney, Elizabeth B; Keynes, Roger J; Fawcett, James W; Rogers, John H

    2014-01-01

    As part of a project to express chondroitinase ABC (ChABC) in neurons of the central nervous system, we have inserted a modified ChABC gene into an adeno-associated viral (AAV) vector and injected it into the vibrissal motor cortex in adult rats to determine the extent and distribution of expression

  7. Expressing Constitutively Active Rheb in Adult Dorsal Root Ganglion Neurons Enhances the Integration of Sensory Axons that Regenerate Across a Chondroitinase-Treated Dorsal Root Entry Zone Following Dorsal Root Crush.

    Science.gov (United States)

    Wu, Di; Klaw, Michelle C; Kholodilov, Nikolai; Burke, Robert E; Detloff, Megan R; Côté, Marie-Pascale; Tom, Veronica J

    2016-01-01

    While the peripheral branch of dorsal root ganglion neurons (DRG) can successfully regenerate after injury, lesioned central branch axons fail to regrow across the dorsal root entry zone (DREZ), the interface between the dorsal root and the spinal cord. This lack of regeneration is due to the limited regenerative capacity of adult sensory axons and the growth-inhibitory environment at the DREZ, which is similar to that found in the glial scar after a central nervous system (CNS) injury. We hypothesized that transduction of adult DRG neurons using adeno-associated virus (AAV) to express a constitutively-active form of the GTPase Rheb (caRheb) will increase their intrinsic growth potential after a dorsal root crush. Additionally, we posited that if we combined that approach with digestion of upregulated chondroitin sulfate proteoglycans (CSPG) at the DREZ with chondroitinase ABC (ChABC), we would promote regeneration of sensory axons across the DREZ into the spinal cord. We first assessed if this strategy promotes neuritic growth in an in vitro model of the glial scar containing CSPG. ChABC allowed for some regeneration across the once potently inhibitory substrate. Combining ChABC treatment with expression of caRheb in DRG significantly improved this growth. We then determined if this combination strategy also enhanced regeneration through the DREZ after dorsal root crush in adult rats in vivo. After unilaterally crushing C4-T1 dorsal roots, we injected AAV5-caRheb or AAV5-GFP into the ipsilateral C5-C8 DRGs. ChABC or PBS was injected into the ipsilateral dorsal horn at C5-C8 to digest CSPG, for a total of four animal groups (caRheb + ChABC, caRheb + PBS, GFP + ChABC, GFP + PBS). Regeneration was rarely observed in PBS-treated animals, whereas short-distance regrowth across the DREZ was observed in ChABC-treated animals. No difference in axon number or length between the ChABC groups was observed, which may be related to intraganglionic inflammation induced by the

  8. BPI700-Fcγ1700 chimeric gene expression and its protective effect in a mice model of the lethal E. coli infection

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background Infections caused by gram-negative bacteria (GNB) often lead to high mortality in common clinical settings. The effect of traditional antibiotic therapy is hindered by drug-resistant bacteria and unneutralizable endotoxin. Few effective methods can protect high risk patients from bacterial infection. This study explored the protection of adeno-associated virus 2 (AAV2)-bacteriacidal permeability increasing protein 700 (BPI700) -fragment crystallizable gamma one 700 (Fcγ1700) chimeric gene transferred mice against the minimal lethal dose (MLD) of E.coli and application of gene therapy for bacterial infection.Methods After AAV2-BPI700-Fcγ1700 virus transfection,dot blotting and Western blotting were used to detect the target gene products in Chinese hamster ovary-K1 cells (CHO-K1cells). Reverse transcription-polymerase chain reaction and immunohistochemical assay were carried out to show the target gene expression in mice. Modified BPI-enzyme linked immunosorbent assay was used to identify the target gene products in murine serum. The protection of BPI700-Fcγ1700 gene transferred mice was examined by survival rate after MLD E. coli challenge. Colony forming unit (CFU) count, limulus amebocyte lysate kit and cytokine kit were used to quantify the bacteria, the level of endotoxin, and proinflammatory cytokine.Results BPI1-199-Fc(1 protein was identified in the CHO-K1 cell culture supernatant, injected muscles and serum of the gene transferred mice. After MLD E. coli challenge, the survival rate of AAV2-BPI700-Fc(1700 gene transferred mice (36.7%) was significantly higher than that of AAV2-enhanced green fluorescent protein (AAV2- EGFP) gene transferred mice (3.3%) and PBS control mice (5.6%). The survival rate of AAV2-BPI700-Fc(1700 gene transferred mice treated with cefuroxime sodium was 65.0%. The bacterium number in main viscera, the levels of endotoxin and proinflammatory cytokine (tumor necrcsis factor-α and interleukin-1β) in serum of the AAV2-BPI

  9. Enhanced transduction of mouse salivary glands with AAV5-based vectors

    NARCIS (Netherlands)

    H. Katano; M.R. Kok; A.P. Cotrim; S. Yamano; M. Schmidt; S. Afione; B.J. Baum; J.A. Chiorini

    2006-01-01

    We previously demonstrated that recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in salivary gland cells in vitro and in vivo. However, it is not known how other rAAV serotypes perform when infused into salivary glands. The capsids of serotypes 4

  10. Evaluation of β-globin gene therapy constructs in single-copy transgenic mice.

    NARCIS (Netherlands)

    J. Ellis (James); K.C. Tan-Un; P. Pasceri; A. Harper; X. Wu; P.J. Fraser (Peter); F.G. Grosveld (Frank)

    1997-01-01

    textabstractEffective gene therapy constructs based on retrovirus or adeno-associated virus vectors will require regulatory elements that direct expression of genes transduced at single copy. Most beta-globin constructs designed for therapy of beta-thalassemias are regulated by the 5'HS2 component o

  11. Imbalances in prefrontal cortex CC-Homer1 versus CC-Homer2 expression promote cocaine preference.

    Science.gov (United States)

    Ary, Alexis W; Lominac, Kevin D; Wroten, Melissa G; Williams, Amy R; Campbell, Rianne R; Ben-Shahar, Osnat; von Jonquieres, Georg; Klugmann, Matthias; Szumlinski, Karen K

    2013-05-01

    Homer postsynaptic scaffolding proteins regulate forebrain glutamate transmission and thus, are likely molecular candidates mediating hypofrontality in addiction. Protracted withdrawal from cocaine experience increases the relative expression of Homer2 versus Homer1 isoforms within medial prefrontal cortex (mPFC). Thus, this study used virus-mediated gene transfer strategies to investigate the functional relevance of an imbalance in mPFC Homer1/2 expression as it relates to various measures of sensorimotor, cognitive, emotional and motivational processing, as well as accompanying alterations in extracellular glutamate in C57BL/6J mice. mPFC Homer2b overexpression elevated basal glutamate content and blunted cocaine-induced glutamate release within the mPFC, whereas Homer2b knockdown produced the opposite effects. Despite altering mPFC glutamate, Homer2b knockdown failed to influence cocaine-elicited conditioned place preferences, nor did it produce consistent effects on any other behavioral measures. In contrast, elevating the relative expression of Homer2b versus Homer1 within mPFC, by overexpressing Homer2b or knocking down Homer1c, shifted the dose-response function for cocaine-conditioned reward to the left, without affecting cocaine locomotion or sensitization. Intriguingly, both these transgenic manipulations produced glutamate anomalies within the nucleus accumbens (NAC) of cocaine-naive animals that are reminiscent of those observed in cocaine experienced animals, including reduced basal extracellular glutamate content, reduced Homer1/2 and glutamate receptor expression, and augmented cocaine-elicited glutamate release. Together, these data provide novel evidence in support of opposing roles for constitutively expressed Homer1 and Homer2 isoforms in regulating mPFC glutamate transmission in vivo and support the hypothesis that cocaine-elicited increases in the relative amount of mPFC Homer2 versus Homer1 signaling produces abnormalities in NAC glutamate

  12. Gene expression

    International Nuclear Information System (INIS)

    We prepared probes for isolating functional pieces of the metallothionein locus. The probes enabled a variety of experiments, eventually revealing two mechanisms for metallothionein gene expression, the order of the DNA coding units at the locus, and the location of the gene site in its chromosome. Once the switch regulating metallothionein synthesis was located, it could be joined by recombinant DNA methods to other, unrelated genes, then reintroduced into cells by gene-transfer techniques. The expression of these recombinant genes could then be induced by exposing the cells to Zn2+ or Cd2+. We would thus take advantage of the clearly defined switching properties of the metallothionein gene to manipulate the expression of other, perhaps normally constitutive, genes. Already, despite an incomplete understanding of how the regulatory switch of the metallothionein locus operates, such experiments have been performed successfully

  13. Viral vector mediated expression of mutant huntingtin in the dorsal raphe produces disease-related neuropathology but not depressive-like behaviors in wildtype mice.

    Science.gov (United States)

    Pitzer, Mark; Lueras, Jordan; Warden, Anna; Weber, Sydney; McBride, Jodi

    2015-05-22

    depressive-like behaviors. Wildtype mice were injected with an adeno-associated virus (AAV2/1) encoding HTT containing either a pathogenic (N171-82Q) or control (N171-16Q) CAG repeat length into the ventral DRN and depressive-like behaviors and motor behaviors were assessed for 12 weeks post-surgery. Quantitative PCR and immunohistochemistry (IHC) verified positive transduction in the ventral aspects of the DRN, including the ventral sub-nucleus (DRv) and interfascicular sub-nucleus (DRif). IHC demonstrated microgliosis in and around the injection site and mHTT-positive inclusions in serotonin-producing neurons and a small percentage of astrocytes in animals injected with N171-82Q compared to controls. Moreover, N171-82Q injected mice showed a 75% reduction in cells that stained positive for the serotonin synthesis enzyme, tryptophan hydroxylase-2 (TPH2) compared to controls (pdisease-related cellular neuropathology but is not sufficient to elicit depressive-like behaviors. Ongoing studies are investigating whether a larger injection volume that transfects a larger percentage of the DRN and/or a longer time course of mHTT expression might elicit depressive-like behaviors. Moreover, mHTT expression in other regions of the brain, such as the hippocampal dentate gyrus and/or the frontal cortex might be necessary to elicit HD depression. Together, these results may prove helpful in addressing which therapeutic and/or pharmacological strategies might be most efficacious when treating depressive symptomology in patients suffering from HD. PMID:25732261

  14. Neuropeptide Y gene transfection inhibits post-epileptic hippocampal synaptic reconstruction

    Institute of Scientific and Technical Information of China (English)

    Fan Zhang; Wenqing Zhao; Wenling Li; Changzheng Dong; Xinying Zhang; Jiang Wu; Na Li; Chuandong Liang

    2013-01-01

    Exogenous neuropeptide Y has antiepileptic effects; however, the underlying mechanism and optimal administration method for neuropeptide Y are still unresolved. Previous studies have used intracerebroventricular injection of neuropeptide Y into animal models of epilepsy. In this study, a recombinant adeno-associated virus expression vector carrying the neuropeptide Y gene was injected into the lateral ventricle of rats, while the ipsilateral hippocampus was injected with kainic acid to establish the epileptic model. After transfection of neuropeptide Y gene, mossy fiber sprouting in the hippocampal CA3 region of epileptic rats was significantly suppressed, hippocampal synaptophysin (p38) mRNA and protein expression were inhibited, and epileptic seizures were reduced. These experimental findings indicate that a recombinant adeno-associated virus expression vector carrying the neuropeptide Y gene reduces mossy fiber sprouting and inhibits abnormal synaptophysin expression, thereby suppressing post-epileptic synaptic reconstruction.

  15. Induction of brain region-specific forms of obesity by agouti.

    OpenAIRE

    Kas, M. J. H.; Tiesjema, B; Dijk, G. van; Garner, KM; Barsh, GS; ter Brake, O; Verhaagen, J.; Adan, RAH

    2004-01-01

    Disruption of melanocortin ( MC) signaling, such as by ectopic Agouti overexpression, leads to an obesity syndrome with hyperphagia, obesity, and accelerated body weight gain during high-fat diet. To investigate where in the brain disruption of MC signaling results in obesity, long-term Agouti expression was induced after local injections of recombinant adeno-associated viral particles in selected brain nuclei of adult rats. Agouti expression in the paraventricular nucleus, a hypothalamic reg...

  16. 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)

    SAI; JiaMing; HU; YouGu; WANG; DeChun

    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.

  17. Gene therapy for mucopolysaccharidosis

    OpenAIRE

    Ponder, Katherine P.; Haskins, Mark E.

    2007-01-01

    Mucopolysaccharidoses (MPS) are due to deficiencies in activities of lysosomal enzymes that degrade glycosaminoglycans. Some attempts at gene therapy for MPS in animal models have involved intravenous injection of vectors derived from an adeno-associated virus (AAV), adenovirus, retrovirus or a plasmid, which primarily results in expression in liver and secretion of the relevant enzyme into blood. Most vectors can correct disease in liver and spleen, although correction in other organs includ...

  18. Gene Therapy Prolongs Survival and Restores Function in Murine and Canine Models of Myotubular Myopathy

    OpenAIRE

    Childers, Martin K.; Joubert, Romain; Poulard, Karine; Moal, Christelle; Grange, Robert W.; Doering, Jonathan A; Lawlor, Michael W.; Rider, Branden E; Jamet, Thibaud; Danièle, Nathalie; Martin, Samia; Rivière, Christel; Soker, Thomas; Hammer, Caroline; Van Wittenberghe, Laetitia

    2014-01-01

    Loss-of-function mutations in the myotubularin gene (MTM1) cause X-linked myotubular myopathy (XLMTM), a fatal, congenital pediatric disease that affects the entire skeletal musculature. Systemic administration of a single dose of a recombinant serotype-8 adeno-associated virus (AAV8) vector expressing murine myotubularin to Mtm1-deficient knockout mice at the onset or at late stages of the disease resulted in robust improvement in motor activity and contractile force, corrected muscle pathol...

  19. Body-wide gene therapy of Duchenne muscular dystrophy in the mdx mouse model

    OpenAIRE

    Denti, Michela Alessandra; Rosa, Alessandro; D’Antona, Giuseppe; Sthandier, Olga; De Angelis, Fernanda Gabriella; Nicoletti, Carmine; Allocca, Mariacarmela; Pansarasa, Orietta; Parente, Valeria; Musarò, Antonio; Auricchio, Alberto; Bottinelli, Roberto; Bozzoni, Irene

    2006-01-01

    Duchenne muscular dystrophy is an X-linked muscle disease characterized by mutations in the dystrophin gene. Many of these can be corrected at the posttranscriptional level by skipping the mutated exon. We have obtained persistent exon skipping in mdx mice by tail vein injection with an adeno-associated viral (AAV) vector expressing antisense sequences as part of the stable cellular U1 small nuclear RNA. Systemic delivery of the AAV construct resulted in effective body-wide colonization, sign...

  20. Single residue AAV capsid mutation improves transduction of photoreceptors in the Abca4-/- mouse and bipolar cells in the rd1 mouse and human retina ex-vivo

    OpenAIRE

    Silva, SR; Charbel Issa, P; Singh, MS; Lipinski, DM; Barnea-Cramer, AO; Walker, NJ; Barnard, AR; Hankins, MW; MacLaren, RE

    2016-01-01

    Gene therapy using adeno-associated viral vectors (AAV) for the treatment of retinal degenerations has shown safety and efficacy in clinical trials. However, very high levels of vector expression may be necessary for the treatment of conditions such as Stargardt disease where a dual vector approach is potentially needed, or in optogenetic strategies for end-stage degeneration in order to achieve maximal light sensitivity. In this study, we assessed two vectors with single ca...

  1. Angiostatin overexpression is associated with an improvement in chronic kidney injury by an anti-inflammatory mechanism

    OpenAIRE

    Mu, Wei; Long, David A.; Ouyang, Xiaosen; Agarwal, Anupam; Cruz, Pedro E; Roncal, Carlos A.; Nakagawa, Takahiko; Yu, Xueqing; Hauswirth, William W.; Johnson, Richard J

    2008-01-01

    Angiostatin, a proteolytic fragment of plasminogen, is a potent anti-angiogenic factor recently shown also to have an inhibitory effect on leukocyte recruitment and macrophage migration. Because both angiogenesis and inflammation play key roles in the progression of chronic kidney disease, we evaluated the effect of angiostatin treatment in the rat remnant kidney model. Rats were pretreated for 4 wk with recombinant adeno-associated viruses expressing either angiostatin or green fluorescence ...

  2. Systemic delivery of rAAV6-microdystrophin preserves muscle function and extends lifespan in a murine model of severe muscular dystrophy

    OpenAIRE

    Gregorevic, Paul; Allen, James M.; Minami, Elina; Blankinship, Michael J; Haraguchi, Miki; Meuse, Leonard; Finn, Eric; Adams, Marvin E.; Froehner, Stanley C.; Murry, Charles E.; Jeffrey S. Chamberlain

    2006-01-01

    Mice carrying mutations in both the dystrophin and utrophin genes die prematurely as a consequence of severe muscular dystrophy. Here, we demonstrate that intravascular administration of recombinant adeno-associated viral (rAAV) vectors carrying a microdystrophin gene restores dystrophin expression in the striated musculature of these animals, considerably reducing skeletal muscle pathology and extending lifespan. These findings suggest rAAV vector-mediated systemic gene transfer may be usefu...

  3. Overexpression of BDNF Increases Excitability of the Lumbar Spinal Network and Leads to Robust Early Locomotor Recovery in Completely Spinalized Rats

    OpenAIRE

    Ewelina Ziemlińska; Sebastian Kügler; Melitta Schachner; Iwona Wewiór; Julita Czarkowska-Bauch; Małgorzata Skup

    2014-01-01

    Strategies to induce recovery from lesions of the spinal cord have not fully resulted in clinical applications. This is a consequence of a number of impediments that axons encounter when trying to regrow beyond the lesion site, and that intraspinal rearrangements are subjected to. In the present study we evaluated (1) the possibility to improve locomotor recovery after complete transection of the spinal cord by means of an adeno-associated (AAV) viral vector expressing the neurotrophin brain-...

  4. Dual AAV therapy ameliorates exercise-induced muscle injury and functional ischemia in murine models of Duchenne muscular dystrophy

    OpenAIRE

    Zhang, Yadong; Yue, Yongping; Li, Liang; Hakim, Chady H.; Zhang, Keqing; Thomas, Gail D.; Duan, Dongsheng

    2013-01-01

    Neuronal nitric oxide synthase (nNOS) membrane delocalization contributes to the pathogenesis of Duchenne muscular dystrophy (DMD) by promoting functional muscle ischemia and exacerbating muscle injury during exercise. We have previously shown that supra-physiological expression of nNOS-binding mini-dystrophin restores normal blood flow regulation and prevents functional ischemia in transgenic mdx mice, a DMD model. A critical next issue is whether systemic dual adeno-associated virus (AAV) g...

  5. Current Challenges and Future Directions in Recombinant AAV-Mediated Gene Therapy of Duchenne Muscular Dystrophy

    OpenAIRE

    Shin'ichi Takeda; Takashi Okada

    2013-01-01

    Various characteristics of adeno-associated virus (AAV)-based vectors with long-term safe expression have made it an exciting transduction tool for clinical gene therapy of Duchenne muscular dystrophy (DMD). Although host immune reactions against the vector as well as transgene products were detected in some instances of the clinical studies, there have been promising observations. Methods of producing AAV vectors for considerable in vivo experimentation and clinical investigations have been ...

  6. Protection from Ebola Virus Mediated by Cytotoxic T Lymphocytes Specific for the Viral Nucleoprotein

    OpenAIRE

    Wilson, Julie A.; Hart, Mary Kate

    2001-01-01

    Cytotoxic T lymphocytes (CTLs) are proposed to be critical for protection from intracellular pathogens such as Ebola virus. However, there have been no demonstrations that protection against Ebola virus is mediated by Ebola virus-specific CTLs. Here, we report that C57BL/6 mice vaccinated with Venezuelan equine encephalitis virus replicons encoding the Ebola virus nucleoprotein (NP) survived lethal challenge with Ebola virus. Vaccination induced both antibodies to the NP and a major histocomp...

  7. 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.

  8. Vaccinia Virus-mediated Therapy of Solid Tumor Xenografts: Intra-tumoral Delivery of Therapeutic Antibodies

    OpenAIRE

    Huang, Ting

    2013-01-01

    Over the past 30 years, much effort and financial support have been invested in the fight against cancer, yet cancer still represents the leading cause of death in the world. Conventional therapies for treatment of cancer are predominantly directed against tumor cells. Recently however, new treatments options have paid more attention to exploiting the advantage of targeting the tumor stroma instead. Vaccinia virus (VACV) has played an important role in human medicine since the 18th century...

  9. Cross-neutralization of influenza A viruses mediated by a single antibody loop.

    Science.gov (United States)

    Ekiert, Damian C; Kashyap, Arun K; Steel, John; Rubrum, Adam; Bhabha, Gira; Khayat, Reza; Lee, Jeong Hyun; Dillon, Michael A; O'Neil, Ryann E; Faynboym, Aleksandr M; Horowitz, Michael; Horowitz, Lawrence; Ward, Andrew B; Palese, Peter; Webby, Richard; Lerner, Richard A; Bhatt, Ramesh R; Wilson, Ian A

    2012-09-27

    Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens. PMID:22982990

  10. Express web application development

    CERN Document Server

    Yaapa, Hage

    2013-01-01

    Express Web Application Development is a practical introduction to learning about Express. Each chapter introduces you to a different area of Express, using screenshots and examples to get you up and running as quickly as possible.If you are looking to use Express to build your next web application, ""Express Web Application Development"" will help you get started and take you right through to Express' advanced features. You will need to have an intermediate knowledge of JavaScript to get the most out of this book.

  11. The effect of rAAV-AS on expression of VEGF and ICAM-1 in HUVECs%腺相关病毒介导血管抑素对血管内皮生长因子和细胞间黏附分子1表达的影响

    Institute of Scientific and Technical Information of China (English)

    徐维; 李伟

    2011-01-01

    目的 探讨腺相关病毒介导血管抑素(rAAV-AS)基因对人脐静脉内皮细胞(HUVECs)血管内皮生长因子(VEGF)和细胞间黏附分子1(ICAM-1)表达的影响,以探讨rAAV-AS抗糖尿病动脉粥样硬化作用.方法 选择体外培养的HUVECs传至第5代,随机分为6组:正常组、高糖组、高糖+rAAV-010(6)v.g./cell组、高糖+rAAV-AS10(4)v.g./cell组、高糖+rAAV-AS10(5)v.g./cell组、高糖+rAAV-AS10(6)v.g./cell组.分别检测rAAV-AS+预后24、48、72h HUVECs凋亡及VEGF、ICAM-1的表达.结果 与正常组比较,高糖组和高糖+rAAV-010(6)v.g./cell组不同时间HUVECs增殖明显,VEGF和ICAM-1表达明显升高(P<0.05);与高糖组比较,高糖+rAAV-AS10(4)v.g./cell组、高糖+rAAV-AS10(5)v.g./cell组、高糖+rAAV-AS10(6)v.g./cell组增殖明显降低,VEGF和ICAM-1表达明显降低,并呈剂量依赖性(P<0.05).结论 rAAV-AS对糖尿病大血管病变具有保护作用,其可能机制为下调内皮细胞中VEGF和ICAM-1的表达,从而减少内皮细胞增殖,抑制炎性反应.%Objective To investigate effects of recombinant adeno-associated virus carrying human angiostatin gene(rAAV-AS) on expression of vascular endothelial growth factor(VEGF) and inter-cellular adhesion moleculel (ICAM-1) in human umbilical vein endothelial cells(HUVECs),and its mechanisms of anti-diabetic atherosclerosis. Methods HUVECs cells were cultured in vitro ,and the fifth passage cells were randomly divided into six groups:normal group, hyperglycemia group, hyperglycemia+rAAV-0106 v.g. group, hyperglycemia+rAAV-AS104 v.g. group,hyperglycemia+rAAV-AS105 v. g. group, and hyperglycemia+rAAV-AS106 v. g. group. The apoptosis of HUVECs and expression of VEGF and ICAM-1 were detected 24,48 and 72 h after rAAV-AS intervention. Results Significant inhibition of proliferation of HUVECs and expression of VEGF and ICAM-1 in a dose-dependent manner was observed in 3 rAAV-AS transfected groups compared with hyperglycemia group. Conclusion rAAV-AS has

  12. 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.

  13. Facial Expression Recognition

    OpenAIRE

    Neeta Sarode; Prof. Shalini Bhatia

    2010-01-01

    Facial expression analysis is rapidly becoming an area of intense interest in computer science and human-computer interaction design communities. The most expressive way humans display emotions is through facial expressions. In this paper a method is implemented using 2D appearance-based local approach for the extraction of intransient facial features and recognition of four facial expressions. The algorithm implements Radial Symmetry Transform and further uses edge projection analysis for fe...

  14. After Effects expressions

    CERN Document Server

    Geduld, Marcus

    2013-01-01

    Put the power of Expressions to work in your animations with controls and efficiencies impossible to achieve with traditional keyframing techniques. No programming skills are required. Foundation concepts and skills orient the new designer and serve as a handy reference to the experienced one. Basics of creating expressions, variables, commands, and expression helpers precede the leap into javascript and math essentials for more advanced expressions that include randomness, physical simularions and 3D. Full color illustrations display the scripts and the resulti

  15. Extrachromosomal inducible expression

    NARCIS (Netherlands)

    Veltman, Douwe M; Van Haastert, Peter J M; Eichinger, L.; Rivero, F.

    2013-01-01

    Inducible expression systems are very convenient for proteins that induce strong side effects such as retardation of growth or development and are essential for the expression of toxic proteins. In this chapter we describe the doxycycline-inducible expression system, optimized for the controlled exp

  16. Effects of rAAV-mediated rhBDNF gene transfection on BDNF gene expression in the retina of a rabbit model of acute high intraocular pressure%rAAV介导hBDNF基因转染对急性高眼压兔眼视网膜BDNF表达的影响

    Institute of Scientific and Technical Information of China (English)

    王建明; 孙乃学; 惠娜; 范雅稚; 冯海晓; 赵世平

    2009-01-01

    Objective To observe the changes in the expression of brain derived neurotrophic factor (BDNF) gene in the retina of rabbits with acute high intraocular pressure (IOP) after injection of recombinant adeno-associated virus (rAAV) vector containing human BDNF gene (rAAV-hBDNF), and investigate the neuroprotective mechanism of rAAV-hBDNF. Methods The unilateral eyes of 24 white rabbits were randomly chosen as the model group with high IOP induced by saline perfusion into the anterior chamber, and the contralateral eyes served as the control group without treatment. In another 24 white rabbits, 10 μl rAAV-BDNF was injected into the vitreous body of one of the eyes 3 days before induction of high IOP. On days 1,3,7, and 14 after perfusion, the bilateral eyes of 6 rabbits were excised for immunohistochemistry for the expression of endogenous BDNF gene in the retina. Results The number of BDNF-positive cells in the retina decreased after induction of high IOP, and injection of rAAV-hBDNF resulted in a significant increase in BDNF-positive cells as compared with the positive cell number in the high IOP model and control groups (P<0.05, P<0.01). Conclusion rAAV-mediated BDNF gene transfection can increase endogenous BDNF expression in the retina of rabbits with acute high IOP. Intravitreous injection is an effective pathway for rAAV-hBDNF gene transfection into the retina.%目的 通过观察视网膜内源性脑源性神经营养因:F(BDNF)表达的变化,探讨玻璃体注射携带人脑源性神经营养因子(hBDNF)的重组腺伴随病毒(rAAV-BDNF)对急性高眼压兔眼神经损害的保护机制.方法 24只健康日本大耳白兔任选一眼作为造模眼(为模型组,共24眼),用生理盐水前房灌注法造成急性高眼压模型,对侧眼不作任何处理作为正常对照组(24眼).另24只健康日本大耳白兔任选一眼作为造模眼(为BDNF组,共24眼),BDNF组在造模前3d玻璃体内注射10μl rAAV-BDNF.于造模后第1、3、7、14d

  17. Holistic facial expression classification

    Science.gov (United States)

    Ghent, John; McDonald, J.

    2005-06-01

    This paper details a procedure for classifying facial expressions. This is a growing and relatively new type of problem within computer vision. One of the fundamental problems when classifying facial expressions in previous approaches is the lack of a consistent method of measuring expression. This paper solves this problem by the computation of the Facial Expression Shape Model (FESM). This statistical model of facial expression is based on an anatomical analysis of facial expression called the Facial Action Coding System (FACS). We use the term Action Unit (AU) to describe a movement of one or more muscles of the face and all expressions can be described using the AU's described by FACS. The shape model is calculated by marking the face with 122 landmark points. We use Principal Component Analysis (PCA) to analyse how the landmark points move with respect to each other and to lower the dimensionality of the problem. Using the FESM in conjunction with Support Vector Machines (SVM) we classify facial expressions. SVMs are a powerful machine learning technique based on optimisation theory. This project is largely concerned with statistical models, machine learning techniques and psychological tools used in the classification of facial expression. This holistic approach to expression classification provides a means for a level of interaction with a computer that is a significant step forward in human-computer interaction.

  18. Memantine Attenuates Alzheimer's Disease-Like Pathology and Cognitive Impairment.

    Directory of Open Access Journals (Sweden)

    Xiaochuan Wang

    Full Text Available Deficiency of protein phosphatase-2A is a key event in Alzheimer's disease. An endogenous inhibitor of protein phosphatase-2A, inhibitor-1, I1PP2A, which inhibits the phosphatase activity by interacting with its catalytic subunit protein phosphatase-2Ac, is known to be upregulated in Alzheimer's disease brain. In the present study, we overexpressed I1PP2A by intracerebroventricular injection with adeno-associated virus vector-1-I1PP2A in Wistar rats. The I1PP2A rats showed a decrease in brain protein phosphatase-2A activity, abnormal hyperphosphorylation of tau, neurodegeneration, an increase in the level of activated glycogen synthase kinase-3beta, enhanced expression of intraneuronal amyloid-beta and spatial reference memory deficit; littermates treated identically but with vector only, i.e., adeno-associated virus vector-1-enhanced GFP, served as a control. Treatment with memantine, a noncompetitive NMDA receptor antagonist which is an approved drug for treatment of Alzheimer's disease, rescued protein phosphatase-2A activity by decreasing its demethylation at Leu309 selectively and attenuated Alzheimer's disease-like pathology and cognitive impairment in adeno-associated virus vector-1-I1PP2A rats. These findings provide new clues into the possible mechanism of the beneficial therapeutic effect of memantine in Alzheimer's disease patients.

  19. Pleiotrophin Expression during Odontogenesis

    OpenAIRE

    Erlandsen, Heidi; Ames, Jennifer E.; Tamkenath, Amena; Mamaeva, Olga; Stidham, Katherine; Wilson, Mary E; Perez-Pinera, Pablo; Deuel, Thomas F.; Macdougall, Mary

    2012-01-01

    Pleiotrophin (PTN) is an extracellular matrix–associated growth factor and chemokine expressed in mesodermal and ectodermal cells. It plays an important role in osteoblast recruitment and differentiation. There is limited information currently available about PTN expression during odontoblast differentiation and tooth formation, and thus the authors aimed to establish the spatiotemporal expression pattern of PTN during mouse odontogenesis. Immortalized mouse dental pulp (MD10-D3, MD10-A11) an...

  20. Regular Expression Pocket Reference

    CERN Document Server

    Stubblebine, Tony

    2007-01-01

    This handy little book offers programmers a complete overview of the syntax and semantics of regular expressions that are at the heart of every text-processing application. Ideal as a quick reference, Regular Expression Pocket Reference covers the regular expression APIs for Perl 5.8, Ruby (including some upcoming 1.9 features), Java, PHP, .NET and C#, Python, vi, JavaScript, and the PCRE regular expression libraries. This concise and easy-to-use reference puts a very powerful tool for manipulating text and data right at your fingertips. Composed of a mixture of symbols and text, regular exp

  1. Regular expressions cookbook

    CERN Document Server

    Goyvaerts, Jan

    2009-01-01

    This cookbook provides more than 100 recipes to help you crunch data and manipulate text with regular expressions. Every programmer can find uses for regular expressions, but their power doesn't come worry-free. Even seasoned users often suffer from poor performance, false positives, false negatives, or perplexing bugs. Regular Expressions Cookbook offers step-by-step instructions for some of the most common tasks involving this tool, with recipes for C#, Java, JavaScript, Perl, PHP, Python, Ruby, and VB.NET. With this book, you will: Understand the basics of regular expressions through a

  2. Regulation of melanopsin expression.

    Science.gov (United States)

    Hannibal, Jens

    2006-01-01

    Circadian rhythms in mammals are adjusted daily to the environmental day/night cycle by photic input via the retinohypothalamic tract (RHT). Retinal ganglion cells (RGCs) of the RHT constitute a separate light-detecting system in the mammalian retina used for irradiance detection and for transmission to the circadian system and other non-imaging forming processes in the brain. The RGCs of the RHT are intrinsically photosensitive due to the expression of melanopsin, an opsin-like photopigment. This notion is based on anatomical and functional data and on studies of mice lacking melanopsin. Furthermore, heterologous expression of melanopsin in non-neuronal mammalian cell lines was found sufficient to render these cells photosensitive. Even though solid evidence regarding the function of melanopsin exists, little is known about the regulation of melanopsin gene expression. Studies in albino Wistar rats showed that the expression of melanopsin is diurnal at both the mRNA and protein levels. The diurnal changes in melanopsin expression seem, however, to be overridden by prolonged exposure to light or darkness. Significant increase in melanopsin expression was observed from the first day in constant darkness and the expression continued to increase during prolonged exposure in constant darkness. Prolonged exposure to constant light, on the other hand, decreased melanopsin expression to an almost undetectable level after 5 days of constant light. The induction of melanopsin by darkness was even more pronounced if darkness was preceded by light suppression for 5 days. These observations show that dual mechanisms regulate melanopsin gene expression and that the intrinsic light-responsive RGCs in the albino Wistar rat adapt their expression of melanopsin to environmental light and darkness.

  3. Duchenne muscular dystrophy gene therapy: Lost in translation?

    Directory of Open Access Journals (Sweden)

    Dongsheng Duan

    2011-03-01

    Full Text Available Dongsheng DuanDepartment of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO, USAAbstract: A milestone of molecular medicine is the identification of dystrophin gene mutation as the cause of Duchenne muscular dystrophy (DMD. Over the last 2 decades, major advances in dystrophin biology and gene delivery technology have created an opportunity to treat DMD with gene therapy. Remarkable success has been achieved in treating dystrophic mice. Several gene therapy strategies, including plasmid transfer, exon skipping, and adeno-associated virus-mediated microdystrophin therapy, have entered clinical trials. However, therapeutic benefit has not been realized in DMD patients. Bridging the gap between mice and humans is no doubt the most pressing issue facing DMD gene therapy now. In contrast to mice, dystrophin-deficient dogs are genetically and phenotypically similar to human patients. Preliminary gene therapy studies in the canine model may offer critical insights that cannot be obtained from murine studies. It is clear that the canine DMD model may represent an important link between mice and humans. Unfortunately, our current knowledge of dystrophic dogs is limited, and the full picture of disease progression remains to be clearly defined. We also lack rigorous outcome measures (such as in situ force measurement to monitor therapeutic efficacy in dystrophic dogs. Undoubtedly, maintaining a dystrophic dog colony is technically demanding, and the cost of dog studies cannot be underestimated. A carefully coordinated effort from the entire DMD community is needed to make the best use of the precious dog resource. Successful DMD gene therapy may depend on valid translational studies in dystrophin-deficient dogs.Keywords: Duchenne muscular dystrophy, gene therapy, dystrophin, adeno-associated virus, exon-skipping, canine model

  4. [Protein expression and purification].

    Science.gov (United States)

    Růčková, E; Müller, P; Vojtěšek, B

    2014-01-01

    Production of recombinant proteins is essential for many applications in both basic research and also in medicine, where recombinant proteins are used as pharmaceuticals. This review summarizes procedures involved in recombinant protein expression and purification, including molecular cloning of target genes into expression vectors, selection of the appropriate expression system, and protein purification techniques. Recombinant DNA technology allows protein engineering to modify protein stability, activity and function or to facilitate protein purification by affinity tag fusions. A wide range of cloning systems enabling fast and effective design of expression vectors is currently available. A first choice of protein expression system is usually the bacteria Escherichia coli. The main advantages of this prokaryotic expression system are low cost and simplicity; on the other hand this system is often unsuitable for production of complex mammalian proteins. Protein expression mediated by eukaryotic cells (yeast, insect and mammalian cells) usually produces properly folded and posttranslationally modified proteins. How-ever, cultivation of insect and, especially, mammalian cells is time consuming and expensive. Affinity tagged recombinant proteins are purified efficiently using affinity chromatography. An affinity tag is a protein or peptide that mediates specific binding to a chromatography column, unbound proteins are removed during a washing step and pure protein is subsequently eluted. PMID:24945544

  5. Expert Oracle application express

    CERN Document Server

    Scott, John Edward

    2011-01-01

    Expert Oracle Application Express brings you groundbreaking insights into developing with Oracle's enterprise-level, rapid-development tool from some of the best practitioners in the field today. Oracle Application Express (APEX) is an entirely web-based development framework that is built into every edition of Oracle Database. The framework rests upon Oracle's powerful PL/SQL language, enabling power users and developers to rapidly develop applications that easily scale to hundreds, even thousands of concurrent users. The 13 authors of Expert Oracle Application Express build their careers aro

  6. Express.js blueprints

    CERN Document Server

    Augarten, Ben; Lin, Eric; Shaikh, Aidha; Soriani, Fabiano Pereira; Tisserand, Geoffrey; Zhang, Chiqing; Zhang, Kan

    2015-01-01

    This book is for beginners to Node.js and also for those who are technically advanced. By the end of this book, every competent developer will have achieved expertise in building web applications with Express.js.

  7. Neuroglobin over expressing mice

    DEFF Research Database (Denmark)

    Raida, Zindy; Hundahl, Christian Ansgar; Nyengaard, Jens R;

    2013-01-01

    thoroughly validated antibodies and oligos, we give a detailed brain anatomical characterization of transgenic mice over expressing Neuroglobin. Moreover, using permanent middle artery occlusion the effect of elevated levels of Neuroglobin on ischemic damage was studied. Lastly, the impact of mouse strain...... genetic background on ischemic damage was investigated. PRINCIPAL FINDINGS: A four to five fold increase in Neuroglobin mRNA and protein expression was seen in the brain of transgenic mice. A β-actin promoter was used to drive Neuroglobin over expression, but immunohistochemistry and in situ hybridization...... infarct volume 24 hours after ischemia. Immunohistochemistry showed no selective sparing of Neuroglobin expressing cells in the ischemic core or penumbra. A significant difference in infarct volume was found between mice of the same strain, but from different colonies. SIGNIFICANCE: In contrast to some...

  8. Gene Expression Omnibus (GEO)

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene Expression Omnibus is a public functional genomics data repository supporting MIAME-compliant submissions of array- and sequence-based data. Tools are provided...

  9. Imaging oncogene expression

    Energy Technology Data Exchange (ETDEWEB)

    Mukherjee, Archana [Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107 (United States)], E-mail: Archana.Mukherjee@jefferson.edu; Wickstrom, Eric [Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233S, 10th street, Philadelphia, PA 19107 (United States)], E-mail: eric@tesla.jci.tju.edu; Thakur, Mathew L. [Department of Radiology, Thomas Jefferson University, Philadelphia, PA 19107 (United States)], E-mail: Mathew.Thakur@jefferson.edu

    2009-05-15

    This review briefly outlines the importance of molecular imaging, particularly imaging of endogenous gene expression for noninvasive genetic analysis of radiographic masses. The concept of antisense imaging agents and the advantages and challenges in the development of hybridization probes for in vivo imaging are described. An overview of the investigations on oncogene expression imaging is given. Finally, the need for further improvement in antisense-based imaging agents and directions to improve oncogene mRNA targeting is stated.

  10. Regular Expression Containment

    DEFF Research Database (Denmark)

    Henglein, Fritz; Nielsen, Lasse

    2011-01-01

    We present a new sound and complete axiomatization of regular expression containment. It consists of the conventional axiomatiza- tion of concatenation, alternation, empty set and (the singleton set containing) the empty string as an idempotent semiring, the fixed- point rule E* = 1 + E × E......* for Kleene-star, and a general coin- duction rule as the only additional rule. Our axiomatization gives rise to a natural computational inter- pretation of regular expressions as simple types that represent parse trees, and of containment proofs as coercions. This gives the axiom- atization a Curry......-Howard-style constructive interpretation: Con- tainment proofs do not only certify a language-theoretic contain- ment, but, under our computational interpretation, constructively transform a membership proof of a string in one regular expres- sion into a membership proof of the same string in another regular expression. We...

  11. In Silico Expression Analysis.

    Science.gov (United States)

    Bolívar, Julio; Hehl, Reinhard; Bülow, Lorenz

    2016-01-01

    Information on the specificity of cis-sequences enables the design of functional synthetic plant promoters that are responsive to specific stresses. Potential cis-sequences may be experimentally tested, however, correlation of genomic sequence with gene expression data enables an in silico expression analysis approach to bioinformatically assess the stress specificity of candidate cis-sequences prior to experimental verification. The present chapter demonstrates an example for the in silico validation of a potential cis-regulatory sequence responsive to cold stress. The described online tool can be applied for the bioinformatic assessment of cis-sequences responsive to most abiotic and biotic stresses of plants. Furthermore, a method is presented based on a reverted in silico expression analysis approach that predicts highly specific potentially functional cis-regulatory elements for a given stress.

  12. In Silico Expression Analysis.

    Science.gov (United States)

    Bolívar, Julio; Hehl, Reinhard; Bülow, Lorenz

    2016-01-01

    Information on the specificity of cis-sequences enables the design of functional synthetic plant promoters that are responsive to specific stresses. Potential cis-sequences may be experimentally tested, however, correlation of genomic sequence with gene expression data enables an in silico expression analysis approach to bioinformatically assess the stress specificity of candidate cis-sequences prior to experimental verification. The present chapter demonstrates an example for the in silico validation of a potential cis-regulatory sequence responsive to cold stress. The described online tool can be applied for the bioinformatic assessment of cis-sequences responsive to most abiotic and biotic stresses of plants. Furthermore, a method is presented based on a reverted in silico expression analysis approach that predicts highly specific potentially functional cis-regulatory elements for a given stress. PMID:27557772

  13. The expressions of emotions

    DEFF Research Database (Denmark)

    Vishnivetz, Berta

    Abstract On the broadness of the vast field called “Expressions of Emotions” this study focuses on the whole bodily emotional expression. The main question posed is: Whether there are movement patterns specific to each emotion?. I carried out a thorough review of the theories of emotion...... and of expressions of emotions and movement notation that provided the sources for a careful research plan for the empirical process of this study. On this basis I chose to record onto video the four previously choreographed movements that I considered to correspond each of the following emotions: joy, fear, sadness......, anger. The selection of these four emotions demanded previously to clear up the problems the above named survey ensued. When researchers want to describe a certain movement in the field of psychology and non-verbal communication, it may result in disagreements and misunderstandings which sometimes lead...

  14. Selectivity of face aftereffects for expressions and anti-expressions

    Directory of Open Access Journals (Sweden)

    Igor eJuricevic

    2012-01-01

    Full Text Available Adapting to a facial expression can alter the perceived expression of subsequently viewed faces. However, it remains unclear whether this adaptation affects each expression independently or transfers from one expression to another, and whether this transfer impedes or enhances responses to a different expression. To test for these interactions, we probed the basic expressions of anger, fear, happiness, sadness, surprise, and disgust, adapting to one expression and then testing on all six. Each expression was varied in strength by morphing it with a common neutral facial expression. Observers determined the threshold level required to correctly identify each expression, before or after adapting to a face with a neutral or intense expression. The adaptation was strongly selective for the adapting category; responses to the adapting expression were reduced, while other categories showed little consistent evidence of either suppression or facilitation. In a second experiment we instead compared adaptation to each expression and its anti-expression. The latter are defined by the physically complementary facial configuration, yet appear much more ambiguous as expressions. In this case, for most expressions the opposing faces produced aftereffects of opposite sign in the perceived expression. These biases suggest that the adaptation acts in part by shifting the perceived neutral point for the facial configuration. This is consistent with the pattern of renormalization suggested for adaptation to other facial attributes, and thus may reflect a generic level of configural coding. However, for most categories aftereffects were stronger for expressions than anti-expressions, pointing to the possible influence of an additional component of the adaptation at sites that explicitly represent facial expressions. At either level our results are consistent with other recent work in suggesting that the six expressions are defined by dimensions that are largely

  15. PCA facial expression recognition

    Science.gov (United States)

    El-Hori, Inas H.; El-Momen, Zahraa K.; Ganoun, Ali

    2013-12-01

    This paper explores and compares techniques for automatically recognizing facial actions in sequences of images. The comparative study of Facial Expression Recognition (FER) techniques namely Principal Component's analysis (PCA) and PCA with Gabor filters (GF) is done. The objective of this research is to show that PCA with Gabor filters is superior to the first technique in terms of recognition rate. To test and evaluates their performance, experiments are performed using real database by both techniques. The universally accepted five principal emotions to be recognized are: Happy, Sad, Disgust and Angry along with Neutral. The recognition rates are obtained on all the facial expressions.

  16. PXI Express specification tutorial

    Institute of Scientific and Technical Information of China (English)

    National Instruments

    2007-01-01

    @@ Introduction The PXI industry standard has quickly gained adoption and grown in prevalence in automated test systems since its release in 1998. PXI is being selected as the platform of choice for thousands of applications, from areas such as military and aerospace, consumer electronics, and communications, to process control and industrial automation. One of the key elements driving the rapid adoption of PXI is its use of PCI in the communication backplane. Now, as the commercial PC industry drastically improves the available bus bandwidth by evolving PCI to PCI Express, PXI has the ability to meet even more application needs by integrating PCI Express into the PXI standard.

  17. Facial expressions recognition with an emotion expressive robotic head

    Science.gov (United States)

    Doroftei, I.; Adascalitei, F.; Lefeber, D.; Vanderborght, B.; Doroftei, I. A.

    2016-08-01

    The purpose of this study is to present the preliminary steps in facial expressions recognition with a new version of an expressive social robotic head. So, in a first phase, our main goal was to reach a minimum level of emotional expressiveness in order to obtain nonverbal communication between the robot and human by building six basic facial expressions. To evaluate the facial expressions, the robot was used in some preliminary user studies, among children and adults.

  18. Collaborating on Referring Expressions

    CERN Document Server

    Heeman, P A; Heeman, Peter A.; Hirst, Graeme

    1995-01-01

    This paper presents a computational model of how conversational participants collaborate in order to make a referring action successful. The model is based on the view of language as goal-directed behavior. We propose that the content of a referring expression can be accounted for by the planning paradigm. Not only does this approach allow the processes of building referring expressions and identifying their referents to be captured by plan construction and plan inference, it also allows us to account for how participants clarify a referring expression by using meta-actions that reason about and manipulate the plan derivation that corresponds to the referring expression. To account for how clarification goals arise and how inferred clarification plans affect the agent, we propose that the agents are in a certain state of mind, and that this state includes an intention to achieve the goal of referring and a plan that the agents are currently considering. It is this mental state that sanctions the adoption of g...

  19. Highly Expressive Hakka Art

    Institute of Scientific and Technical Information of China (English)

    JENNIFER LIM

    1996-01-01

    SOUTHERN Jiangxi Province was the birthplace of the Hakka ethnic group and has since been the native home and main transfer hub for the spread of the nationality. The highly expressive art of the Hakkas, including folk songs in Xingguo, colored lantern performances in Shicheng, ancient

  20. Facial Expression Analysis

    NARCIS (Netherlands)

    Pantic, Maja; Li, S.; Jain, A.

    2009-01-01

    Facial expression recognition is a process performed by humans or computers, which consists of: 1. Locating faces in the scene (e.g., in an image; this step is also referred to as face detection), 2. Extracting facial features from the detected face region (e.g., detecting the shape of facial compon

  1. Encircling Creative Expression

    Science.gov (United States)

    Brown, Susannah

    2007-01-01

    Artworks that are circular in nature are often referred to as mandalas. "Mandala" means center, circle, or circumference. Mandalas are created in many cultures for a variety of reasons, most of which are related to self-expression, ritual, and religion. In this article, the author describes how her students created mandalas. She also provides…

  2. Expressive Costume Portraits

    Science.gov (United States)

    Lott, Debra

    2008-01-01

    This article describes how contemporary costumes, expressive techniques, and mixed media can take "the ordinary" out of figure studies. To pique student interest and create a meaningful figurative study, students are instructed to bring in their latest fashion accessories (hats, shawls, neck warmers, denim jackets, etc.), or shop the local thrift…

  3. New Words and Expressions

    Institute of Scientific and Technical Information of China (English)

    陈福生

    1984-01-01

    @@ To start with, I mention that certain new words and expressions are used relatively often by journalists when they compose reports and articles. Their writing is said to be all right for a newspaper, but that lacks imagination and beauty. Examples:

  4. Expression of Concern

    Science.gov (United States)

    Delvaux, Damien

    2016-08-01

    This is a note of a temporary expression of concern related to the publication titled, "Sapphirine and fluid inclusions in Tel Thanoun mantle xenoliths, Syria" by Ahmad Bilal, which appeared in Journal of African Earth Sciences, 116 (2016) 105-113.

  5. Brandom's Expressive Conception of Logic

    OpenAIRE

    Streed, Adam Joseph

    2015-01-01

    This dissertation is an exploration of the expressive conception of logic, as found in the work of Robert Brandom. I situate the expressive conception against a historical background of thought about logic, investigate the key notion of material inference on which the expressive conception rests, and argue that the expressive conception counts as a form of psychologism about logic which avoids many of the difficulties faced by older forms of psychologism. Finally, I argue that the expressive ...

  6. Boolean Expression Diagrams

    DEFF Research Database (Denmark)

    Andersen, Henrik Reif; Hulgaard, Henrik

    1997-01-01

    This paper presents a new data structure called Boolean Expression Diagrams (BEDs) for representing and manipulating Boolean functions. BEDs are a generalization of Binary Decision Diagrams (BDDs) which can represent any Boolean circuit in linear space and still maintain many of the desirable pro...... standard BDD techniques this problem is infeasible. BEDs are useful in applications where the end-result as a reduced ordered BDD is small, for example for tautology checking...

  7. Overexpression of factor VIII after AAV delivery is transiently associated with cellular stress in hemophilia A mice

    Science.gov (United States)

    Lange, Amy M; Altynova, Ekaterina S; Nguyen, Giang N; Sabatino, Denise E

    2016-01-01

    Factor VIII (FVIII) is a large glycoprotein that is challenging to express both in vitro and in vivo. Several studies suggest that high levels of FVIII expression can lead to cellular stress. After gene transfer, transgene expression is restricted to a subset of cells and the increased FVIII load per cell may impact activation of the unfolded protein response. We sought to determine whether increased FVIII expression in mice after adeno-associated viral liver gene transfer would affect the unfolded protein response and/or immune response to the transgene. The FVIII gene was delivered as B-domain deleted single chain or two chain (light and heavy chains) at a range of doses in hemophilia A mice. A correlation between FVIII expression and anti-FVIII antibody titers was observed. Analysis of key components of the unfolded protein response, binding immunoglobulin protein (BiP), and C/EBP homologous protein (CHOP), showed transient unfolded protein response activation in the single chain treated group expressing >200% of FVIII but not after two chain delivery. These studies suggest that supraphysiological single chain FVIII expression may increase the likelihood of a cellular stress response but does not alter liver function. These data are in agreement with the observed long-term expression of FVIII at therapeutic levels after adeno-associated viral delivery in hemophilia A dogs without evidence of cellular toxicity. PMID:27738645

  8. Freedom of Expression at Yale

    Science.gov (United States)

    AAUP Bulletin, 1975

    1975-01-01

    A report of the Committee on Freedom of Expression at Yale appointed by the president to examine the condition of free expression, peaceful dissent, mutual respect and tolerance at Yale and to draft recommendations for maintenance of those principles. (JT)

  9. Facial Expressions Recognition Using Eigenspaces

    OpenAIRE

    Senthil Ragavan Valayapalayam Kittusamy; Venkatesh Chakrapani

    2012-01-01

    A challenging research topic is to make the Computer Systems to recognize facial expressions from the face image. A method of facial expression recognition, based on Eigenspaces is presented in this study. Here, the authors recognize the userâs facial expressions from the input images, using a method that was customized from eigenface recognition. Evaluation was done for this method in terms of identification correctness using two different Facial Expressions databases, Cohn-Kanade facial exp...

  10. Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice.

    Science.gov (United States)

    Moda, Fabio; Vimercati, Chiara; Campagnani, Ilaria; Ruggerone, Margherita; Giaccone, Giorgio; Morbin, Michela; Zentilin, Lorena; Giacca, Mauro; Zucca, Ileana; Legname, Giuseppe; Tagliavini, Fabrizio

    2012-01-01

    Prion diseases are caused by a conformational modification of the cellular prion protein (PrP (C)) into disease-specific forms, termed PrP (Sc), that have the ability to interact with PrP (C) promoting its conversion to PrP (Sc). In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrP (C) region involved in the interaction with PrP (Sc) thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrP (Sc) in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding. PMID:22842862

  11. Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice.

    Science.gov (United States)

    Moda, Fabio; Vimercati, Chiara; Campagnani, Ilaria; Ruggerone, Margherita; Giaccone, Giorgio; Morbin, Michela; Zentilin, Lorena; Giacca, Mauro; Zucca, Ileana; Legname, Giuseppe; Tagliavini, Fabrizio

    2012-01-01

    Prion diseases are caused by a conformational modification of the cellular prion protein (PrP (C)) into disease-specific forms, termed PrP (Sc), that have the ability to interact with PrP (C) promoting its conversion to PrP (Sc). In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrP (C) region involved in the interaction with PrP (Sc) thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrP (Sc) in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding.

  12. Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

    International Nuclear Information System (INIS)

    We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by ∼ 68% and ∼ 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy

  13. PRELIMINARY STUDY ON TRANSDIFFERENTIATION OF HUMAN AMNIOTIC EPITHELIAL CELLS AND ITS INTRASLENIC TRANSPLANTATION%人羊膜上皮细胞横向分化及脾内移植的初步研究

    Institute of Scientific and Technical Information of China (English)

    罗宏武; 黄湘俊; 黄飞舟; 刘浔阳

    2011-01-01

    Objective The human amniotic epithelial cells (hAECs) are a recently identified new type of stem cells.It has previously been shown that hAECs express hepatocyte-related gene and possess intracellular features and functional properties of hepatocytes.The hAECs may be a candidate seed cell for liver regeneration.To research the survival and migration in vivo of hAECs via adeno-associated virus-mediated the green fluorescent protein gene (AAV-GFP) transfection, and to explore the expression ofhepatocyte-like function.Methods Thirty nude mice (aging 6-8 weeks, halfmales and females, and weighing 20-22 g) were randomly divided into 3 groups (groups A, B, and C, n=10).The mice of groups A and C were made the 2/3 partial hepatectomy model, and the mice of group B underwent open abdominal operation without hepatectomy.The hAECs transfected by AAV-GFP were transplanted into the inferior end of the spleen in groups A and B with a cell density of 5 × 106/mL and a volume of 0.2 mL; the same volume of normal saline was injected in group C.At 4 hours, the nude mice were sacrificed and the samples of liver, spleen, heart, lung, brain, and kidney were harvested and the general observation, histological observation, and immunofluorescence detection were performed for the hAECs survival, migration, and the functional properties of hepatocytes.Results No tumor tissue was found in liver and spleen of 3 groups, and HE staining showed no tumor cells.There were a lot of roundlike and deeply-stained cells with less cytoplasm and large nucleus in the spleen and the liver of group A; no abnormal cells were found in liver and spleen of groups B and C and in kidney, heart, bung, and brain of groups A, B, and C.The GFP+ cells were detected in the spleen and liver of group A with expressing human albumin, but no GFP+ cells was found in liver and spleen of groups B and C and in heart, kidney, lung, and brain of groups A, B, and C.Conclusion AAV-GFP infected hAECs transplanted into SCID nude

  14. Vaccinia virus-mediated melanin production allows MR and optoacoustic deep tissue imaging and laser-induced thermotherapy of cancer

    OpenAIRE

    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.

    2013-01-01

    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...

  15. 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.

  16. Natural Art, False Expressions

    Directory of Open Access Journals (Sweden)

    Oscar Hernando Nossa García

    2011-05-01

    Full Text Available In the documentary My Kid Could Paint That, directed by Bar-Lev, which deals with Marla Olmstead, the child prodigy of painting, several interviews with persons in the art world are conducted, among them an artist who uses a magnifying glass and the thinnest brushes to do his work. This man, although happy for the success of the child’s abstract paintings, saw in the whole spectacle a mockery of art, and stood firmly by her work. The girl’s father, also an artist, was accused of plagiarism. Cameras entered the child’s studio in order to prove that Marla was the real artist. Why should such relevance be given to authorship? What is the cause of the dispute between the expressive and the rational?

  17. Functional correction of neurological and somatic disorders at later stages of disease in MPS IIIA mice by systemic scAAV9-hSGSH gene delivery

    OpenAIRE

    Fu, Haiyan; Cataldi, Marcela P.; Ware, Tierra A.; Zaraspe, Kimberly; Meadows, Aaron S; Murrey, Darren A; McCarty, Douglas M

    2016-01-01

    The reversibility of neuropathic lysosomal storage diseases, including MPS IIIA, is a major goal in therapeutic development, due to typically late diagnoses and a large population of untreated patients. We used self-complementary adeno-associated virus (scAAV) serotype 9 vector expressing human N-sulfoglucosamine sulfohydrolase (SGSH) to test the efficacy of treatment at later stages of the disease. We treated MPS IIIA mice at 1, 2, 3, 6, and 9 months of age with an intravenous injection of s...

  18. Telomerase gene therapy in adult and old mice delays aging and increases longevity without increasing cancer

    OpenAIRE

    Bernardes de Jesus, Bruno; Vera, Elsa; Schneeberger, Kerstin; Tejera, Agueda M.; Ayuso, Eduard; Bosch, Fatima; Blasco, Maria A.

    2012-01-01

    A major goal in aging research is to improve health during aging. In the case of mice, genetic manipulations that shorten or lengthen telomeres result, respectively, in decreased or increased longevity. Based on this, we have tested the effects of a telomerase gene therapy in adult (1 year of age) and old (2 years of age) mice. Treatment of 1- and 2-year old mice with an adeno associated virus (AAV) of wide tropism expressing mouse TERT had remarkable beneficial effects on health and fitness,...

  19. Endogenous leptin signaling in the caudal nucleus tractus solitarius and area postrema is required for energy balance regulation

    OpenAIRE

    Hayes, Matthew R.; Skibicka, Karolina P; Leichner, Theresa M.; Guarnieri, Douglas J; DiLeone, Ralph J; Bence, Kendra K.; Grill, Harvey J.

    2010-01-01

    Medial nucleus tractus solitarius (mNTS) neurons express leptin receptors (LepR) and intramNTS delivery of leptin reduces food intake and body weight. Here, the contribution of endogenous LepR signaling in mNTS neurons to energy balance control was examined. Knockdown of LepR in mNTS and area postrema (AP) neurons of rats (LepRKD) via adeno-associated virus short hairpin RNA-interference (AAV-shRNAi) resulted in significant hyperphagia for chow, high-fat and sucrose diets, yielding increased ...

  20. Development of an intein-mediated split-Cas9 system for gene therapy

    OpenAIRE

    Truong, D.J.J.; Kuehner, K.; Kuehn, R.; Werfel, S.; Engelhardt, S.; WURST, W; Ortiz, O.

    2015-01-01

    Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split–Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 pro...

  1. Effect of Bone Marrow Mesenchymal Stem Cells Transfected with rAAV2-bFGF on Early Angiogenesis of Calvarial Defects in Rats

    Institute of Scientific and Technical Information of China (English)

    陈美玲; 宋珂; 饶念静; 黄梦琪; 黄正江; 曹颖光

    2010-01-01

    The purpose of this study was to evaluate the effect of bone marrow mesenchymal stem cells (MSCs) transfected with the basic fibroblast growth factor (bFGF)-expressing recombinant adeno-associated virus vector (rAAV2-bFGF), on early angiogenesis of calvarial defects in rats. The MSCs were cultured and transfected with rAAV2-bFGF after differential adherence isolation. The transfection efficiency was detected by RT-PCR and Western blotting. The transfected MSCs were compounded with poly-DL-lactide/hydroxyapa...

  2. Remembering Faces with Emotional Expressions

    Directory of Open Access Journals (Sweden)

    Chang Hong eLiu

    2014-12-01

    Full Text Available It is known that happy faces create more robust identity recognition memory than faces with some other expressions. However, this advantage was not verified against all basic expressions. Moreover, no research has assessed whether similar differences also exist among other expressions. To tackle these questions, we compared the effects of six basic emotional expressions on recognition memory using a standard old/new recognition task. The experiment also examined whether exposure to different emotional expressions at training creates variable effects on transfer of the trained faces to a new/neutral expression. Our results suggest that happy faces produced better identity recognition relative to disgusted faces, regardless of whether they were tested in the same image or a new image displaying a neutral expression. None of the other emotional expressions created measurable advantage for recognition memory. Overall, our data lend further support for the happy face advantage for long-term recognition memory. However, our detailed analyses also show that the advantage of happy expression on identity recognition may not be equally discernible from all other emotional expressions.

  3. Targeted disruption of LDLR causes hypercholesterolemia and atherosclerosis in Yucatan miniature pigs.

    Directory of Open Access Journals (Sweden)

    Bryan T Davis

    Full Text Available Recent progress in engineering the genomes of large animals has spurred increased interest in developing better animal models for diseases where current options are inadequate. Here, we report the creation of Yucatan miniature pigs with targeted disruptions of the low-density lipoprotein receptor (LDLR gene in an effort to provide an improved large animal model of familial hypercholesterolemia and atherosclerosis. Yucatan miniature pigs are well established as translational research models because of similarities to humans in physiology, anatomy, genetics, and size. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, male and female LDLR+/- pigs were generated. Subsequent breeding of heterozygotes produced LDLR-/- pigs. When fed a standard swine diet (low fat, no cholesterol, LDLR+/- pigs exhibited a moderate, but consistent increase in total and LDL cholesterol, while LDLR-/- pigs had considerably elevated levels. This severe hypercholesterolemia in homozygote animals resulted in atherosclerotic lesions in the coronary arteries and abdominal aorta that resemble human atherosclerosis. These phenotypes were more severe and developed over a shorter time when fed a diet containing natural sources of fat and cholesterol. LDLR-targeted Yucatan miniature pigs offer several advantages over existing large animal models including size, consistency, availability, and versatility. This new model of cardiovascular disease could be an important resource for developing and testing novel detection and treatment strategies for coronary and aortic atherosclerosis and its complications.

  4. Restoration of synaptic function in sight for degenerative retinal disease.

    Science.gov (United States)

    Schubert, Timm; Wissinger, Bernd

    2015-07-01

    Synaptic disorganization is a prominent feature of many neurological diseases of the CNS, including Parkinson's disease, intellectual development disorders, and autism. Although synaptic plasticity is critical for learning and memory, it is unclear whether this innate property helps restore synaptic function in disease once the primary cause of disease is abrogated. An answer to this question may come from a recent investigation in X-linked retinoschisis, a currently untreatable retinopathy. In this issue of the JCI, Ou, Vijayasarathy, and colleagues showed progressive disorganization of key functional elements of the synapse between photoreceptors and ON-bipolar cells in a retinoschisin-deficient mouse model. Moreover, they demonstrated that adeno-associated virus-mediated (AAV-mediated) delivery of the retinoschisin gene restores structure and function to the photoreceptor to ON-bipolar cell synapse in mouse models, even in adults at advanced stages of the disease. The results of this study hold promise that AAV-based supplemental gene therapy will benefit patients with X-linked retinoschisis in a forthcoming clinical trial. PMID:26098210

  5. Techniques in Facial Expression Recognition

    OpenAIRE

    Avinash Prakash Pandhare; Umesh Balkrishna Chavan

    2016-01-01

    Facial expression recognition is gaining widespread importance as the applications related to Human – Computer interactions are increasing. This paper mentions various techniques and approaches that have been used in the field of facial expression recognition. Facial expression recognition takes place in various stages and these stages have been implemented by various approaches. Viola and Jones for face detection, Gabor filters for feature extraction, SVM classifiers for classifi...

  6. Oracle Application Express 4 Recipes

    CERN Document Server

    Zehoo, Edmund

    2011-01-01

    Oracle Application Express 4 Recipes provides an example-based approach to learning Application Express - the ground-breaking, rapid application development platform included with every Oracle Database license. The recipes format is ideal for the quick-study who just wants a good example or two to kick start their thinking and get pointed in the right direction. The recipes cover the gamut of Application Express development. Author and Application Express expert Edmund Zehoo shows how to create data entry screens, visualize data in the form of reports and charts, implement validation and back-

  7. Generational Differences of Emotional Expression

    Institute of Scientific and Technical Information of China (English)

    李学勇

    2014-01-01

    As a kind of subjective psychological activity, emotion can only be known and perceived by a certain expressive form. Varies as the different main bodies, difference of emotional expression can be reflected not only among individuals but between generations. The old conceals their emotions inside, the young express their emotions boldly, and the middle-aged are rational and deep in their expressions. Facing and understanding such differences is the premise and foundation of the con-struction of a harmonious relationship between different generations.

  8. Expression modeling for expression-invariant face recognition

    NARCIS (Netherlands)

    Haar, F.B. Ter; Veltkamp, R.C.

    2010-01-01

    Morphable face models have proven to be an effective tool for 3D face modeling and face recognition, but the extension to 3D face scans with expressions is still a challenge. The two main difficulties are (1) how to build a new morphable face model that deals with expressions, and (2) how to fit thi

  9. Tumor-specific gene expression patterns with gene expression profiles

    Institute of Scientific and Technical Information of China (English)

    RUAN Xiaogang; LI Yingxin; LI Jiangeng; GONG Daoxiong; WANG Jinlian

    2006-01-01

    Gene expression profiles of 14 common tumors and their counterpart normal tissues were analyzed with machine learning methods to address the problem of selection of tumor-specific genes and analysis of their differential expressions in tumor tissues. First, a variation of the Relief algorithm, "RFE_Relief algorithm" was proposed to learn the relations between genes and tissue types. Then, a support vector machine was employed to find the gene subset with the best classification performance for distinguishing cancerous tissues and their counterparts. After tissue-specific genes were removed, cross validation experiments were employed to demonstrate the common deregulated expressions of the selected gene in tumor tissues. The results indicate the existence of a specific expression fingerprint of these genes that is shared in different tumor tissues, and the hallmarks of the expression patterns of these genes in cancerous tissues are summarized at the end of this paper.

  10. Human papillomavirus gene expression

    International Nuclear Information System (INIS)

    To determine the role of tissue differentiation on expression of each of the papillomavirus mRNA species identified by electron microscopy, the authors prepared exon-specific RNA probes that could distinguish the alternatively spliced mRNA species. Radioactively labeled single-stranded RNA probes were generated from a dual promoter vector system and individually hybridized to adjacent serial sections of formalin-fixed, paraffin-embedded biopsies of condylomata. Autoradiography showed that each of the message species had a characteristic tissue distribution and relative abundance. The authors have characterized a portion of the regulatory network of the HPVs by showing that the E2 ORF encodes a trans-acting enhancer-stimulating protein, as it does in BPV-1 (Spalholz et al. 1985). The HPV-11 enhancer was mapped to a 150-bp tract near the 3' end of the URR. Portions of this region are duplicated in some aggressive strains of HPV-6 (Boshart and zur Hausen 1986; Rando et al. 1986). To test the possible biological relevance of these duplications, they cloned tandem arrays of the enhancer and demonstrated, using a chloramphenicol acetyltransferase (CAT) assay, that they led to dramatically increased transcription proportional to copy number. Using the CAT assays, the authors found that the E2 proteins of several papillomavirus types can cross-stimulate the enhancers of most other types. This suggests that prior infection of a tissue with one papillomavirus type may provide a helper effect for superinfection and might account fo the HPV-6/HPV-16 coinfections in condylomata that they have observed

  11. Analysis of Animal Metaphorical Expression

    Institute of Scientific and Technical Information of China (English)

    姜晴川

    2016-01-01

    Animal metaphor, as a kind of metaphor, refers to a cognitive process in which some aspects of human beings are understood or experienced through the aspects of animals. The meanings of animal metaphor are based on people's experience, cultural background, custom and the ways of thinking. Animal metaphorical expression is an important part of human's language expressions and communication.

  12. Emotional Expression in Reality TV

    DEFF Research Database (Denmark)

    Rasmussen, Tove Arendt

    , that information ‘given off’ in lying behavior will must often be found in non-verbal mikro expressions and mikro gestures. I will end by discussing the strategic emotional expressions as production of floating identities in a broader framework on the basis of among others Gergen, Lipovetsky and Baumann...

  13. Precise Analysis of String Expressions

    DEFF Research Database (Denmark)

    Christensen, Aske Simon; Møller, Anders; Schwartzbach, Michael Ignatieff

    2003-01-01

    , including statically checking the syntax of dynamically generated expressions, such as SQL queries. Our analysis constructs flow graphs from class files and generates a context-free grammar with a nonterminal for each string expression. The language of this grammar is then widened into a regular language...

  14. Creating an Expressive Performance Mindset

    Science.gov (United States)

    Broomhead, Paul; Skidmore, Jon B.

    2014-01-01

    Students in performance situations sometimes experience physiological symptoms that inhibit their ability to perform as expressively as they otherwise might possess the understanding and ability to do. As students set out to perform with an expressive mindset, the brain's limbic system may detect some perceived danger in the situation and…

  15. Expression in E. coli systems

    DEFF Research Database (Denmark)

    Krogsdam, Anne-M; Kristiansen, Karsten; Nøhr, Jane

    2003-01-01

    Owing to cost advantage, speed of production, and often high product yield (up to 50% of total cell protein), expression in Escherichia coli is generally the first choice when attempting to express a recombinant protein. Expression systems exist to produce recombinant protein intracellularly...... (soluble or in inclusion bodies), secreted to the periplasm, or to the surrounding medium. When deciding on a genetic design strategy, it is important to consider the nature of the recombinant protein. The mildest and thus the obvious first-choice expression strategy is to attempt to express the protein...... intracellularly in soluble form. In E. coli, proteins containing disulfide bonds are best produced by secretion because the disulfide forming foldases reside in the periplasm. Likewise, a correct N-terminus is more likely to be obtained upon secretion. Moreover, potentially toxic proteins are more likely...

  16. Spontaneous Emotional Facial Expression Detection

    Directory of Open Access Journals (Sweden)

    Zhihong Zeng

    2006-08-01

    Full Text Available Change in a speaker’s emotion is a fundamental component in human communication. Automatic recognition of spontaneous emotion would significantly impact human-computer interaction and emotion-related studies in education, psychology and psychiatry. In this paper, we explore methods for detecting emotional facial expressions occurring in a realistic human conversation setting—the Adult Attachment Interview (AAI. Because non-emotional facial expressions have no distinct description and are expensive to model, we treat emotional facial expression detection as a one- class classification problem, which is to describe target objects (i.e., emotional facial expressions and distinguish them from outliers (i.e., non-emotional ones. Our preliminary experiments on AAI data suggest that one-class classification methods can reach a good balance between cost (labeling and computing and recognition performance by avoiding non-emotional expression labeling and modeling.

  17. Techniques in Facial Expression Recognition

    Directory of Open Access Journals (Sweden)

    Avinash Prakash Pandhare

    2016-05-01

    Full Text Available Facial expression recognition is gaining widespread importance as the applications related to Human – Computer interactions are increasing. This paper mentions various techniques and approaches that have been used in the field of facial expression recognition. Facial expression recognition takes place in various stages and these stages have been implemented by various approaches. Viola and Jones for face detection, Gabor filters for feature extraction, SVM classifiers for classification, L1 minimization for sparse representation, facial expression recognition, geometric deformation model, multiple gabor filters for robust feature extraction, parallel implementation of Viola and Jones for face detection and parallel implementation of SVM classifier for classification of expressions are discussed in this paper.

  18. Expression and communication of doubt/uncertainty through facial expression

    Directory of Open Access Journals (Sweden)

    Pio E. Ricci Bitti

    2014-04-01

    Full Text Available There is a wide debate on the mental state of doubt/uncertainty; one wonders whether it is a predominantly cognitive or emotional state of mind and whether typical facial expressions communicate doubt/uncertainty. To this purpose,through a role playing procedure, a large sample of expressions were collected and afterwards evaluated through a combination of encoding and decoding procedures,including also FACS (Facial Action Coding System analysis. The results have partially confirmed our hypothesis, identifying two typical facial expressions of doubt/uncertainty, which share the same facial actions in the inferior part of the face and show differential facial actions in the upper face.

  19. Construction and expression of recombined human AFP eukaryotic expression vector

    Institute of Scientific and Technical Information of China (English)

    Li-Wang Zhang; Yang-Lin Pan; Stephen M Festein; Jun Ren; Liang Zhang; Hong-Mei Zhang; Bin Jin; Bo-Rong Pan; Xiao-Ming Si; Yan-Jun Zhang; Zhong-Hua Wang

    2003-01-01

    AIM: To construct a recombined human AFP eukaryotic expression vector for the purpose of gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: The full length AFP-cDNA of prokaryotic vector was digested, and subcloned to the multi-clony sites of the eukaryotic vector. The constructed vector was confirmed by enzymes digestion and electrophoresis, and the product expressed was detected by electrochemiluminescence and immunofluorescence methods.RESULTS: The full length AFP-cDNA successfully cloned to the eukaryotic vector through electrophoresis, 0.9723 IU/ml AFP antigen was detected in the supernatant of AFPCHO by electrochemiluminescence method. Compared with the control groups, the differences were significant (P<0.05).AFP antigen molecule was observed in the plasma of AFPCHO by immunofluorescence staining.CONCLUSION: The recombined human AFP eukaryotic expression vector can express in CHO cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma.

  20. TempoExpress: An Expressivity-Preserving Musical Tempo Transformation

    OpenAIRE

    Grachten, Maarten; Arcos, Josep Ll.; Lopez De Mantaras, Ramon

    2006-01-01

    The research described in this paper focuses on global tempo transformations of monophonic audio recordings of saxophone jazz performances. More concretely, we have investigated the problem of how a performance played at a particular tempo can be automatically rendered at another tempo while preserving its expressivity. To do so we have developed a case-based reasoning system called TempoExpress. The results we have obtained have been extensively compared against a sta...

  1. Shuffling Yeast Gene Expression Data

    CERN Document Server

    Bilke, S

    2000-01-01

    A new method to sort gene expression patterns into functional groups is presented. The method is based on a sorting algorithm using a non-local similarity score, which takes all other patterns in the dataset into account. The method is therefore very robust with respect to noise. Using the expression data for yeast, we extract information about functional groups. Without prior knowledge of parameters the cell cycle regulated genes in yeast can be identified. Furthermore a second, independent cell clock is identified. The capability of the algorithm to extract information about signal flow in the regulatory network underlying the expression patterns is demonstrated.

  2. A Tattoo Is Expression, Too.

    Science.gov (United States)

    Dowling-Sendor, Benjamin

    1997-01-01

    In "Stephenson v. Davenport Community School District," the U.S. Eighth Circuit Court of Appeals ruled that schools cannot adopt unduly vague policies to regulate student expression, in this case, a cross-shaped tattoo. (LMI)

  3. "Fractal Expression" in Chinese Calligraphy

    OpenAIRE

    Li, Yuelin

    2008-01-01

    We show that from historical record and mathematic analysis, "fractal expressions" may have been a conscious pursuit at least one thousand years ago as an element of beauty in ancient Chinese calligraphy.

  4. Homeobox gene expression in Brachiopoda

    DEFF Research Database (Denmark)

    Altenburger, Andreas; Martinez, Pedro; Wanninger, Andreas

    2011-01-01

    The molecular control that underlies brachiopod ontogeny is largely unknown. In order to contribute to this issue we analyzed the expression pattern of two homeobox containing genes, Not and Cdx, during development of the rhynchonelliform (i.e., articulate) brachiopod Terebratalia transversa....... Not is a homeobox containing gene that regulates the formation of the notochord in chordates, while Cdx (caudal) is a ParaHox gene involved in the formation of posterior tissues of various animal phyla. The T. transversa homolog, TtrNot, is expressed in the ectoderm from the beginning of gastrulation until...... formation. TtrNot expression is absent in unfertilized eggs, in embryos prior to gastrulation, and in settled individuals during and after metamorphosis. Comparison with the expression patterns of Not genes in other metazoan phyla suggests an ancestral role for this gene in gastrulation and germ layer...

  5. Emotion Classification Using Facial Expression

    Directory of Open Access Journals (Sweden)

    Devi Arumugam

    2011-08-01

    Full Text Available Human emotional facial expressions play an important role in interpersonal relations. This is because humans demonstrate and convey a lot of evident information visually rather than verbally. Although humans recognize facial expressions virtually without effort or delay, reliable expression recognition by machine remains a challenge as of today. To automate recognition of emotional state, machines must be taught to understand facial gestures. In this paper we developed an algorithm which is used to identify the person’s emotional state through facial expression such as angry, disgust, happy. This can be done with different age group of people with different situation. We Used a Radial Basis Function network (RBFN for classification and Fisher’s Linear Discriminant (FLD, Singular Value Decomposition (SVD for feature selection.

  6. Noninvasive tracking of gene transcript and neuroprotection after gene therapy.

    Science.gov (United States)

    Ren, J; Chen, Y I; Liu, C H; Chen, P-C; Prentice, H; Wu, J-Y; Liu, P K

    2016-01-01

    Gene therapy holds exceptional potential for translational medicine by improving the products of defective genes in diseases and/or providing necessary biologics from endogenous sources during recovery processes. However, validating methods for the delivery, distribution and expression of the exogenous genes from such therapy can generally not be applicable to monitor effects over the long term because they are invasive. We report here that human granulocyte colony-stimulating factor (hG-CSF) complimentary DNA (cDNA) encoded in self-complementary adeno-associated virus-type 2 adeno-associated virus, as delivered through eye drops at multiple time points after cerebral ischemia using bilateral carotid occlusion for 60 min (BCAO-60) led to significant reduction in mortality rates, cerebral atrophy and neurological deficits in C57black6 mice. Most importantly, we validated hG-CSF cDNA expression using translatable magnetic resonance imaging (MRI) in living brains. This noninvasive approach for monitoring exogenous gene expression in the brains has potential for great impact in the area of experimental gene therapy in animal models of heart attack, stroke, Alzheimer's dementia, Parkinson's disorder and amyotrophic lateral sclerosis, and the translation of such techniques to emergency medicine. PMID:26207935

  7. Antiangiogenic therapy enhances the efficacy of transcatheter arterial embolization for hepatocellular carcinomas.

    Science.gov (United States)

    Jiang, Hongchi; Meng, Qinghui; Tan, Hongtao; Pan, Shangha; Sun, Bei; Xu, Ruian; Sun, Xueying

    2007-07-15

    Transcatheter arterial embolization (TAE) is a well-established technique for unresectable hepatic malignancies. However, TAE can temporally halt the growth of hepatic tumors by blocking their arterial supply, but often tumors rapidly develop collateral blood vessels via angiogenesis. We have previously demonstrated that intraportal delivery of adeno-associated viral particles expressing angiostatin leads to long-term expression of angiostatin capable of inhibiting angiogenesis and suppressing the outgrowth of tumors in the liver. Thus, we hypothesize that adeno-associated virus (AAV)-mediated antiangiogenic therapy could enhance the efficacy of TAE to combat liver cancers. To achieve this objective, we engineered a recombinant AAV vector encoding rat angiostatin. Intraportal delivery of this vector led to long term and stable transgenic expression of angiostatin locally in rat hepatocytes and suppressed the growth of CBRH7919 hepatocellular carcinomas established in rat livers by inhibiting formation of neovessels. Although TAE therapy led to necrosis of liver tumors and suppressed their growth, the neovessels of tumors were rapidly reformed 3 weeks after TAE. However, intraportal injection of AAV-angiostatin inhibited the angiogenesis stimulated by TAE, synergized with TAE in suppressing growth of tumors established in livers and prolonged the survival of rats. In conclusion, these encouraging results warrant future investigation of the use of antiangiogenic therapy for enhancing the therapeutic efficacy of TAE to treat unresectable liver cancers. PMID:17330237

  8. “Persistente secrezione di Exendin-4, un agonista recettoriale del Glucagon-like peptide-1, mediante terapia genica mediata da Adeno-associated Viruses, su ghiandole salivari di roditori in due differenti modelli di obesità/diabete tipo 2”

    OpenAIRE

    Ilaria Dicembrini

    2013-01-01

    Exendin-4 (Ex-4)è un agonista recettoriale del Glucagon-like peptide 1 (GLP-1)attualmente approvato per il trattamento del Diabete Mellito tipo 2 e prevede una somministrazione per via iniettiva sottocutanea due volte al giorno. Nei pazienti affetti da Diabete tipo 2, la somministrazione di GLP-1 riduce significativamente la glicemia ed i livelli di HbA1c con una modesta, ma significativa riduzione del peso corporeo. In questa tesi di dottorato si è valutata la persistenza di espressione d...

  9. 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.

  10. Expressive alignment language and implementation

    OpenAIRE

    Euzenat, Jérôme; Scharffe, François; Zimmermann, Antoine

    2007-01-01

    euzenat2007e This deliverable provides the description of an alignment language which is both expressive and independent from ontology languages. It defines the language through its abstract syntax and semantics depending on ontology language semantics. It then describes two concrete syntax: an exchange syntax in RDF/XML and a surface syntax for human consumption. Finally, it presents the current implementation of this expressive language within the Alignment API taking advantage of the OM...

  11. Shuffling Yeast Gene Expression Data

    OpenAIRE

    Bilke, Sven

    2000-01-01

    A new method to sort gene expression patterns into functional groups is presented. The method is based on a sorting algorithm using a non-local similarity score, which takes all other patterns in the dataset into account. The method is therefore very robust with respect to noise. Using the expression data for yeast, we extract information about functional groups. Without prior knowledge of parameters the cell cycle regulated genes in yeast can be identified. Furthermore a second, independent ...

  12. Measuring facial expression of emotion

    OpenAIRE

    Wolf, Karsten

    2015-01-01

    Research into emotions has increased in recent decades, especially on the subject of recognition of emotions. However, studies of the facial expressions of emotion were compromised by technical problems with visible video analysis and electromyography in experimental settings. These have only recently been overcome. There have been new developments in the field of automated computerized facial recognition; allowing real-time identification of facial expression in social environments. This rev...

  13. Spontaneous Emotional Facial Expression Detection

    OpenAIRE

    Zhihong Zeng; Yun Fu; Roisman, Glenn I.; Zhen Wen; Yuxiao Hu; Thomas S. Huang

    2006-01-01

    Change in a speaker’s emotion is a fundamental component in human communication. Automatic recognition of spontaneous emotion would significantly impact human-computer interaction and emotion-related studies in education, psychology and psychiatry. In this paper, we explore methods for detecting emotional facial expressions occurring in a realistic human conversation setting—the Adult Attachment Interview (AAI). Because non-emotional facial expressions have no distinct description and are exp...

  14. On Fuzzy Expressions in English

    Institute of Scientific and Technical Information of China (English)

    张桂君

    2005-01-01

    In this paper Ⅰ attempt to explain my general understanding of fuzzy expressions in language, and I take English as an example.Fuzziness is one of the basic characteristics of language.It differs from generality, ambiguity and vagueness.There are many examples of fuzzy expressions in English.I supply examples of color words, some adjectives and linguistic hedges.Finally I attempt to explain fuzziness from pragmatic point of view.

  15. Vascular gene expression: a hypothesis

    OpenAIRE

    Martínez-Navarro, Angélica C.; Galván-Gordillo, Santiago V.; Xoconostle-Cázares, Beatriz; Ruiz-Medrano, Roberto

    2013-01-01

    The phloem is the conduit through which photoassimilates are distributed from autotrophic to heterotrophic tissues and is involved in the distribution of signaling molecules that coordinate plant growth and responses to the environment. Phloem function depends on the coordinate expression of a large array of genes. We have previously identified conserved motifs in upstream regions of the Arabidopsis genes, encoding the homologs of pumpkin phloem sap mRNAs, displaying expression in vascular ti...

  16. Compound facial expressions of emotion.

    Science.gov (United States)

    Du, Shichuan; Tao, Yong; Martinez, Aleix M

    2014-04-15

    Understanding the different categories of facial expressions of emotion regularly used by us is essential to gain insights into human cognition and affect as well as for the design of computational models and perceptual interfaces. Past research on facial expressions of emotion has focused on the study of six basic categories--happiness, surprise, anger, sadness, fear, and disgust. However, many more facial expressions of emotion exist and are used regularly by humans. This paper describes an important group of expressions, which we call compound emotion categories. Compound emotions are those that can be constructed by combining basic component categories to create new ones. For instance, happily surprised and angrily surprised are two distinct compound emotion categories. The present work defines 21 distinct emotion categories. Sample images of their facial expressions were collected from 230 human subjects. A Facial Action Coding System analysis shows the production of these 21 categories is different but consistent with the subordinate categories they represent (e.g., a happily surprised expression combines muscle movements observed in happiness and surprised). We show that these differences are sufficient to distinguish between the 21 defined categories. We then use a computational model of face perception to demonstrate that most of these categories are also visually discriminable from one another. PMID:24706770

  17. Compound facial expressions of emotion.

    Science.gov (United States)

    Du, Shichuan; Tao, Yong; Martinez, Aleix M

    2014-04-15

    Understanding the different categories of facial expressions of emotion regularly used by us is essential to gain insights into human cognition and affect as well as for the design of computational models and perceptual interfaces. Past research on facial expressions of emotion has focused on the study of six basic categories--happiness, surprise, anger, sadness, fear, and disgust. However, many more facial expressions of emotion exist and are used regularly by humans. This paper describes an important group of expressions, which we call compound emotion categories. Compound emotions are those that can be constructed by combining basic component categories to create new ones. For instance, happily surprised and angrily surprised are two distinct compound emotion categories. The present work defines 21 distinct emotion categories. Sample images of their facial expressions were collected from 230 human subjects. A Facial Action Coding System analysis shows the production of these 21 categories is different but consistent with the subordinate categories they represent (e.g., a happily surprised expression combines muscle movements observed in happiness and surprised). We show that these differences are sufficient to distinguish between the 21 defined categories. We then use a computational model of face perception to demonstrate that most of these categories are also visually discriminable from one another.

  18. Referring Expressions: A Unified Approach

    Institute of Scientific and Technical Information of China (English)

    K.M. Jaszczolt

    2001-01-01

    @@ 0. Introduction Expressions used by speakers to refer are commonly divided into two categories: that of directly referring expressions and that of expressions whose referring function is secured by the context of utterance. Directly referring expressions are normally said to include proper names, some pronouns including demonstrative, and demonstrative phrases. The other category comprises mainly definite and indefinite descriptions, the first being their most acclaimed representative. Definite descriptions are widely acknowledged to have referential uses. They are not referring expressions, so to speak, by default,but rather as a result of a contextually determined interpretation. As a category, they are frequently said to belong with quantifiers (Neale 1990; Recanati 1993). However, the arguments for classifying them with referring expressions are ample (Bach 1987a; Larson & Segal 1995; Brown 1995; Jaszczolt 1997a,1997b, 1999b). It is argued in Part I of this paper that although definite descriptions exhibit an ambiguity of use between the referential and the attributive reading, they also exhibit the property of having an unmarked, salient interpretation which makes them akin to directly referential terms. This salient reading is the referential interpretation, arrived at with the help of the hearer′s presumption of the presence of strong referential intention that supports the speaker′s utterance.

  19. Extracting expression modules from perturbational gene expression compendia

    Directory of Open Access Journals (Sweden)

    Van Dijck Patrick

    2008-04-01

    Full Text Available Abstract Background Compendia of gene expression profiles under chemical and genetic perturbations constitute an invaluable resource from a systems biology perspective. However, the perturbational nature of such data imposes specific challenges on the computational methods used to analyze them. In particular, traditional clustering algorithms have difficulties in handling one of the prominent features of perturbational compendia, namely partial coexpression relationships between genes. Biclustering methods on the other hand are specifically designed to capture such partial coexpression patterns, but they show a variety of other drawbacks. For instance, some biclustering methods are less suited to identify overlapping biclusters, while others generate highly redundant biclusters. Also, none of the existing biclustering tools takes advantage of the staple of perturbational expression data analysis: the identification of differentially expressed genes. Results We introduce a novel method, called ENIGMA, that addresses some of these issues. ENIGMA leverages differential expression analysis results to extract expression modules from perturbational gene expression data. The core parameters of the ENIGMA clustering procedure are automatically optimized to reduce the redundancy between modules. In contrast to the biclusters produced by most other methods, ENIGMA modules may show internal substructure, i.e. subsets of genes with distinct but significantly related expression patterns. The grouping of these (often functionally related patterns in one module greatly aids in the biological interpretation of the data. We show that ENIGMA outperforms other methods on artificial datasets, using a quality criterion that, unlike other criteria, can be used for algorithms that generate overlapping clusters and that can be modified to take redundancy between clusters into account. Finally, we apply ENIGMA to the Rosetta compendium of expression profiles for

  20. The motivation to express prejudice.

    Science.gov (United States)

    Forscher, Patrick S; Cox, William T L; Graetz, Nicholas; Devine, Patricia G

    2015-11-01

    Contemporary prejudice research focuses primarily on people who are motivated to respond without prejudice and the ways in which unintentional bias can cause these people to act in a manner inconsistent with this motivation. However, some real-world phenomena (e.g., hate speech, hate crimes) and experimental findings (e.g., Plant & Devine, 2001, 2009) suggest that some prejudice is intentional. These phenomena and findings are difficult to explain solely from the motivations to respond without prejudice. We argue that some people are motivated to express prejudice, and we develop the Motivation to Express Prejudice Scale (MP) to measure this motivation. In 7 studies involving more than 6,000 participants, we demonstrate that, across scale versions targeted at Black people and gay men, the MP has good reliability and convergent, discriminant, and predictive validity. In normative climates that prohibit prejudice, the internal and external motivations to express prejudice are functionally nonindependent, but they become more independent when normative climates permit more prejudice toward a target group. People high in the motivation to express prejudice are relatively likely to resist pressure to support programs promoting intergroup contact and to vote for political candidates who support oppressive policies. The motivation to express prejudice predicted these outcomes even when controlling for attitudes and the motivations to respond without prejudice. This work encourages contemporary prejudice researchers to give greater consideration to the intentional aspects of negative intergroup behavior and to broaden the range of phenomena, target groups, and samples that they study.

  1. The motivation to express prejudice.

    Science.gov (United States)

    Forscher, Patrick S; Cox, William T L; Graetz, Nicholas; Devine, Patricia G

    2015-11-01

    Contemporary prejudice research focuses primarily on people who are motivated to respond without prejudice and the ways in which unintentional bias can cause these people to act in a manner inconsistent with this motivation. However, some real-world phenomena (e.g., hate speech, hate crimes) and experimental findings (e.g., Plant & Devine, 2001, 2009) suggest that some prejudice is intentional. These phenomena and findings are difficult to explain solely from the motivations to respond without prejudice. We argue that some people are motivated to express prejudice, and we develop the Motivation to Express Prejudice Scale (MP) to measure this motivation. In 7 studies involving more than 6,000 participants, we demonstrate that, across scale versions targeted at Black people and gay men, the MP has good reliability and convergent, discriminant, and predictive validity. In normative climates that prohibit prejudice, the internal and external motivations to express prejudice are functionally nonindependent, but they become more independent when normative climates permit more prejudice toward a target group. People high in the motivation to express prejudice are relatively likely to resist pressure to support programs promoting intergroup contact and to vote for political candidates who support oppressive policies. The motivation to express prejudice predicted these outcomes even when controlling for attitudes and the motivations to respond without prejudice. This work encourages contemporary prejudice researchers to give greater consideration to the intentional aspects of negative intergroup behavior and to broaden the range of phenomena, target groups, and samples that they study. PMID:26479365

  2. Metallothionein expression in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Geng-Wen Huang; Lian-Yue Yang

    2002-01-01

    AIM: To investigate the expression of metallothioneins(MTs), which were recently thought to have closerelationship with tumors, in human hepatocellularcarcinoma.METHODS: Histological specimens of 35 cases of primaryhuman hepatocellular carcinoma with para-neoplasticliver tissue and 5 cases of normal liver were stainedfor MTs with monoclonal mouse anti-MTs serum (Eg)by the immunohistochemical ABC technique. RESULTS: MTs were stained in the 35 cases of HCC,including 6 cases negative (17.1 %), 23 weaklypositive (65.7 %), and 6 strongly positive(17.1%).But MTs were stained strongly positive in all the fivecases of normal liver and 35 cases of para-neoplasticliver tissue. The differences of MTs expression betweenHCC and normal liver tissue or para-neoplastic livertissue were highly significant (P<0.01). The rate ofMTs expression in HCC grade I was 100 percent,higher than that in grade Ⅱ(81%) and grade Ⅲand Ⅳ (78 %). But the differences were notsignificant (P>0.05). No obvious correlationsbetween MTs expression in HCC and tumor size,clinical stage or serum alpha fetoproteinconcentration were found (P>0.05).CONCLUSION: Decrease of MTs expression in HCC mayplay a role in carcinogenesis of HCC. MTs are stainedheterogenously in HCC. We can choose the anticanceragents according to the MTs concentraiton in HCC, whichmay improve the results of chemotherapy for HCC.

  3. Facial Asymmetry and Emotional Expression

    CERN Document Server

    Pickin, Andrew

    2011-01-01

    This report is about facial asymmetry, its connection to emotional expression, and methods of measuring facial asymmetry in videos of faces. The research was motivated by two factors: firstly, there was a real opportunity to develop a novel measure of asymmetry that required minimal human involvement and that improved on earlier measures in the literature; and secondly, the study of the relationship between facial asymmetry and emotional expression is both interesting in its own right, and important because it can inform neuropsychological theory and answer open questions concerning emotional processing in the brain. The two aims of the research were: first, to develop an automatic frame-by-frame measure of facial asymmetry in videos of faces that improved on previous measures; and second, to use the measure to analyse the relationship between facial asymmetry and emotional expression, and connect our findings with previous research of the relationship.

  4. An Expressive Extension of TLC

    DEFF Research Database (Denmark)

    Henriksen, Jesper Gulmann

    2002-01-01

    A temporal logic of causality (TLC) was introduced by Alur, Penczek and Peled in [1]. It is basically a linear time temporal logic interpreted over Mazurkiewicz traces which allows quantification over causal chains. Through this device one can directly formulate causality properties of distributed...... systems. In this paper we consider an extension of TLC by strengthening the chain quantification operators. We show that our logic TLC* adds to the expressive power of TLC. We do so by defining an Ehrenfeucht-Fraïssé game to capture the expressive power of TLC. We then exhibit a property and by means...... of this game prove that the chosen property is not definable in TLC. We then show that the same property is definable in TLC*. We prove in fact the stronger result that TLC* is expressively stronger than TLC exactly when the dependency relation associated with the underlying trace alphabet is not transitive....

  5. Gankyrin expression during mouse embryogenesis

    Institute of Scientific and Technical Information of China (English)

    秦建民; 刘淑琴; 曾锦章; 李慎菁; 付晓勇; 邱秀华; 吴孟超; 王红阳

    2004-01-01

    Objective: To observe the gene expression of Gankyrin during mouse embryogenesis and reveal the gene biological significance during organs and tissues formation. Methods: The expressions of Gankyrin mRNA in various organs and tissues were detected by in situ hybridization at indicated times during embryogenesis. Results: The expression of Gankyrin mRNA in mouse day 12.5 embryo was mainly in midbrain, interbrain and endbrain; in mouse day 14.5 embryo mainly in midbrain, aorta, liver, gonad, cranium and rib; in mouse day 16.5 embryo mainly in cranium, rib and vertebra;and in mouse day 18.5 embryo mainly in cranium, rib and intestinal mucosa. Conclusion: Gankyrin gene probably participates in the development of the neural tissues (such as midbrain, interbrain and endbrain etc. ), aorta, liver and gonad, intestinal mucosa and bone tissues, which may be closely associated with the function of the organs and tissues.

  6. Extracting expression modules from perturbational gene expression compendia

    OpenAIRE

    Van Dijck Patrick; Maere Steven; Kuiper Martin

    2008-01-01

    Abstract Background Compendia of gene expression profiles under chemical and genetic perturbations constitute an invaluable resource from a systems biology perspective. However, the perturbational nature of such data imposes specific challenges on the computational methods used to analyze them. In particular, traditional clustering algorithms have difficulties in handling one of the prominent features of perturbational compendia, namely partial coexpression relationships between genes. Biclus...

  7. Advanced express web application development

    CERN Document Server

    Keig, Andrew

    2013-01-01

    A practical book, guiding the reader through the development of a single page application using a feature-driven approach.If you are an experienced JavaScript developer who wants to build highly scalable, real-world applications using Express, this book is ideal for you. This book is an advanced title and assumes that the reader has some experience with node, Javascript MVC web development frameworks, and has heard of Express before, or is familiar with it. You should also have a basic understanding of Redis and MongoDB. This book is not a tutorial on Node, but aims to explore some of the more

  8. Zipf's Law in Gene Expression

    CERN Document Server

    Furusawa, C; Furusawa, Chikara; Kaneko, Kunihiko

    2002-01-01

    Using data from gene expression databases on various organisms and tissues, including yeast, nematodes, human normal and cancer tissues, and embryonic stem cells, we found that the abundances of expressed genes exhibit a power-law distribution with an exponent close to -1, i.e., they obey Zipf's law. Furthermore, by simulations of a simple model with an intra-cellular reaction network, we found that Zipf's law of chemical abundance is a universal feature of cells where such a network optimizes the efficiency and faithfulness of self-reproduction. These findings provide novel insights into the nature of the organization of reaction dynamics in living cells.

  9. Correction of gene expression data

    DEFF Research Database (Denmark)

    Darbani Shirvanehdeh, Behrooz; Stewart, C. Neal, Jr.; Noeparvar, Shahin;

    2014-01-01

    This report investigates for the first time the potential inter-treatment bias source of cell number for gene expression studies. Cell-number bias can affect gene expression analysis when comparing samples with unequal total cellular RNA content or with different RNA extraction efficiencies....... For maximal reliability of analysis, therefore, comparisons should be performed at the cellular level. This could be accomplished using an appropriate correction method that can detect and remove the inter-treatment bias for cell-number. Based on inter-treatment variations of reference genes, we introduce...

  10. Reconhecimento Expressões Faciais

    OpenAIRE

    Costa, Fernando Gil Abreu Mesquita da

    2010-01-01

    Dissertação de Mestrado em Engenharia Electrotécnica de Computadores A expressão facial é uma forma de comunicação não-verbal que permite a partilha de sentimentos e emoções, numa linguagem subentendida entre seres humanos. A detecção facial e reconhecimento automático de expressões baseia-se no processamento de imagem e reconhecimento de padrões. O desenvolvimento nestas áreas deve-se a avanços multidisciplinares recentes, desde a aquisição e processamento digital de imagem, caracteriz...

  11. Microsoft Expression Web for dummies

    CERN Document Server

    Hefferman, Linda

    2013-01-01

    Expression Web is Microsoft's newest tool for creating and maintaining dynamic Web sites. This FrontPage replacement offers all the simple ""what-you-see-is-what-you-get"" tools for creating a Web site along with some pumped up new features for working with Cascading Style Sheets and other design options. Microsoft Expression Web For Dummies arrives in time for early adopters to get a feel for how to build an attractive Web site. Author Linda Hefferman teams up with longtime FrontPage For Dummies author Asha Dornfest to show the easy way for first-time Web designers, FrontPage ve

  12. The Change of Expression Configuration Affects Identity-Dependent Expression Aftereffect but Not Identity-Independent Expression Aftereffect.

    Science.gov (United States)

    Song, Miao; Shinomori, Keizo; Qian, Qian; Yin, Jun; Zeng, Weiming

    2015-01-01

    The present study examined the influence of expression configuration on cross-identity expression aftereffect. The expression configuration refers to the spatial arrangement of facial features in a face for conveying an emotion, e.g., an open-mouth smile vs. a closed-mouth smile. In the first of two experiments, the expression aftereffect is measured using a cross-identity/cross-expression configuration factorial design. The facial identities of test faces were the same or different from the adaptor, while orthogonally, the expression configurations of those facial identities were also the same or different. The results show that the change of expression configuration impaired the expression aftereffect when the facial identities of adaptor and tests were the same; however, the impairment effect disappears when facial identities were different, indicating the identity-independent expression representation is more robust to the change of the expression configuration in comparison with the identity-dependent expression representation. In the second experiment, we used schematic line faces as adaptors and real faces as tests to minimize the similarity between the adaptor and tests, which is expected to exclude the contribution from the identity-dependent expression representation to expression aftereffect. The second experiment yields a similar result as the identity-independent expression aftereffect observed in Experiment 1. The findings indicate the different neural sensitivities to expression configuration for identity-dependent and identity-independent expression systems.

  13. Mapping and Manipulating Facial Expression

    Science.gov (United States)

    Theobald, Barry-John; Matthews, Iain; Mangini, Michael; Spies, Jeffrey R.; Brick, Timothy R.; Cohn, Jeffrey F.; Boker, Steven M.

    2009-01-01

    Nonverbal visual cues accompany speech to supplement the meaning of spoken words, signify emotional state, indicate position in discourse, and provide back-channel feedback. This visual information includes head movements, facial expressions and body gestures. In this article we describe techniques for manipulating both verbal and nonverbal facial…

  14. Freedom of Expression? It's Academic.

    Science.gov (United States)

    Hayes, Louis

    1999-01-01

    Examines the educational environment in Japan since World War II. Assesses both elementary and secondary levels of education, and evaluates the psychology of dependence as a factor in the lack of freedom of expression in academic settings. Delves into the external and internal structure of Japanese cultural society. (CCM)

  15. Ascidian gene-expression profiles

    OpenAIRE

    William R Jeffery

    2002-01-01

    With the advent of gene-expression profiling, a large number of genes can now be investigated simultaneously during critical stages of development. This approach will be particularly informative in studies of ascidians, basal chordates whose genomes and embryology are uniquely suited for mapping developmental gene networks.

  16. Academic Freedom and Artistic Expression.

    Science.gov (United States)

    Academe, 1990

    1990-01-01

    The concluding statement by participants in the 1990 Wolf Trap Conference on Academic Freedom and Artistic Expression (Virginia, April 29-May 1) proposes policies to assist institutions in responding to issues of accountability, audience, and public funding arising from presentation of artistic works to the public in a manner that preserves…

  17. Expressing Sentiments in Game Reviews

    NARCIS (Netherlands)

    Secui, Ana; Sirbu, Maria; Dascalu, Mihai; Crossley, Scott; Ruseti, Stefan; Trausan-Matu, Stefan

    2016-01-01

    Opinion mining and sentiment analysis are important research areas of Natural Language Processing (NLP) tools and have become viable alternatives for automatically extracting the affective information found in texts. Our aim is to build an NLP model to analyze gamers’ sentiments and opinions express

  18. DNA expressions - A formal notation for DNA

    NARCIS (Netherlands)

    Vliet, Rudy van

    2015-01-01

    We describe a formal notation for DNA molecules that may contain nicks and gaps. The resulting DNA expressions denote formal DNA molecules. Different DNA expressions may denote the same molecule. Such DNA expressions are called equivalent. We examine which DNA expressions are minimal, which

  19. Facial and vocal expressions of emotion.

    Science.gov (United States)

    Russell, James A; Bachorowski, Jo-Anne; Fernandez-Dols, Jose-Miguel

    2003-01-01

    A flurry of theoretical and empirical work concerning the production of and response to facial and vocal expressions has occurred in the past decade. That emotional expressions express emotions is a tautology but may not be a fact. Debates have centered on universality, the nature of emotion, and the link between emotions and expressions. Modern evolutionary theory is informing more models, emphasizing that expressions are directed at a receiver, that the interests of sender and receiver can conflict, that there are many determinants of sending an expression in addition to emotion, that expressions influence the receiver in a variety of ways, and that the receiver's response is more than simply decoding a message.

  20. Vascular Gene Expression: A Hypothesis

    Directory of Open Access Journals (Sweden)

    Angélica Concepción eMartínez-Navarro

    2013-07-01

    Full Text Available The phloem is the conduit through which photoassimilates are distributed from autotrophic to heterotrophic tissues and is involved in the distribution of signaling molecules that coordinate plant growth and responses to the environment. Phloem function depends on the coordinate expression of a large array of genes. We have previously identified conserved motifs in upstream regions of the Arabidopsis genes, encoding the homologs of pumpkin phloem sap mRNAs, displaying expression in vascular tissues. This tissue-specific expression in Arabidopsis is predicted by the overrepresentation of GA/CT-rich motifs in gene promoters. In this work we have searched for common motifs in upstream regions of the homologous genes from plants considered to possess a primitive vascular tissue (a lycophyte, as well as from others that lack a true vascular tissue (a bryophyte, and finally from chlorophytes. Both lycophyte and bryophyte display motifs similar to those found in Arabidopsis with a significantly low E-value, while the chlorophytes showed either a different conserved motif or no conserved motif at all. These results suggest that these same genes are expressed coordinately in non- vascular plants; this coordinate expression may have been one of the prerequisites for the development of conducting tissues in plants. We have also analyzed the phylogeny of conserved proteins that may be involved in phloem function and development. The presence of CmPP16, APL, FT and YDA in chlorophytes suggests the recruitment of ancient regulatory networks for the development of the vascular tissue during evolution while OPS is a novel protein specific to vascular plants.

  1. Positively regulated bacterial expression systems.

    Science.gov (United States)

    Brautaset, Trygve; Lale, Rahmi; Valla, Svein

    2009-01-01

    Regulated promoters are useful tools for many aspects related to recombinant gene expression in bacteria, including for high-level expression of heterologous proteins and for expression at physiological levels in metabolic engineering applications. In general, it is common to express the genes of interest from an inducible promoter controlled either by a positive regulator or by a repressor protein. In this review, we discuss established and potentially useful positively regulated bacterial promoter systems, with a particular emphasis on those that are controlled by the AraC-XylS family of transcriptional activators. The systems function in a wide range of microorganisms, including enterobacteria, soil bacteria, lactic bacteria and streptomycetes. The available systems that have been applied to express heterologous genes are regulated either by sugars (L-arabinose, L-rhamnose, xylose and sucrose), substituted benzenes, cyclohexanone-related compounds, ε-caprolactam, propionate, thiostrepton, alkanes or peptides. It is of applied interest that some of the inducers require the presence of transport systems, some are more prone than others to become metabolized by the host and some have been applied mainly in one or a limited number of species. Based on bioinformatics analyses, the AraC-XylS family of regulators contains a large number of different members (currently over 300), but only a small fraction of these, the XylS/Pm, AraC/P(BAD), RhaR-RhaS/rhaBAD, NitR/PnitA and ChnR/Pb regulator/promoter systems, have so far been explored for biotechnological applications.

  2. Facial Expressions with Some Mixed Expressions Recognition Using Neural Networks

    Directory of Open Access Journals (Sweden)

    Dr.R.Parthasarathi

    2011-01-01

    Full Text Available Facial feature extraction is the essential step of facial expression recognition. The automatic facial impression evaluation applies for wide area use. The important facial feature vectors for expressionanalysis are analyzed. The extracted feature vector loads all known feature vectors and trains the NN using as input training vectors while PCA is used for dimensionality reduction. The method is effective for both dimension reduction and good recognition performance in comparison with other proposed methods as shown in experiment results.

  3. Facial Expressions with Some Mixed Expressions Recognition Using Neural Networks

    OpenAIRE

    Dr.R.Parthasarathi; V.Lokeswar Reddy,; K.Vishnuthej,; G.Vishnu Vandan

    2011-01-01

    Facial feature extraction is the essential step of facial expression recognition. The automatic facial impression evaluation applies for wide area use. The important facial feature vectors for expressionanalysis are analyzed. The extracted feature vector loads all known feature vectors and trains the NN using as input training vectors while PCA is used for dimensionality reduction. The method is effective for both dimension reduction and good recognition performance in comparison with other p...

  4. Gene expression in colorectal cancer

    DEFF Research Database (Denmark)

    Birkenkamp-Demtroder, Karin; Christensen, Lise Lotte; Olesen, Sanne Harder;

    2002-01-01

    Understanding molecular alterations in colorectal cancer (CRC) is needed to define new biomarkers and treatment targets. We used oligonucleotide microarrays to monitor gene expression of about 6,800 known genes and 35,000 expressed sequence tags (ESTs) on five pools (four to six samples in each...... pool) of total RNA from left-sided sporadic colorectal carcinomas. We compared normal tissue to carcinoma tissue from Dukes' stages A-D (noninvasive to distant metastasis) and identified 908 known genes and 4,155 ESTs that changed remarkably from normal to tumor tissue. Based on intensive filtering 226...... known genes and 157 ESTs were found to be highly relevant for CRC. The alteration of known genes was confirmed in >70% of the cases by array analysis of 25 single samples. Two-way hierarchical average linkage cluster analysis clustered normal tissue together with Dukes' A, clustered Dukes' B with Dukes...

  5. Derivatives of Parsing Expression Grammars

    OpenAIRE

    Moss, Aaron

    2014-01-01

    This paper introduces a new memoized derivative parsing algorithm for recognition of parsing expression grammars. The algorithm runs in worst case quartic time and cubic space. However, existing research suggests that due to the limited amount of backtracking and recursion in real-world grammars and input, practical performance may be closer to linear time and constant space; experimental validation of this conjecture is in progress.

  6. Professional expression of technology teacher's

    OpenAIRE

    Jacevič, Jolanta

    2005-01-01

    Professional expression of technology teacher’s is important in nowadays society and educational system, especially when new educational paradigm is becoming stronger. The teacher of technology has to help pupils to understand the value of present time life: to set pupils’ sights on country’s production and use policy, individual usage culture, healthy living, ability to develop creatively the national ethnic cultural traditions and their integration into work and housekeeping, and also to...

  7. Summer Oral Expression English Course

    CERN Multimedia

    HR Department

    2011-01-01

    An English Oral Expression course will take place between 15 August and 30 September 2011. Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enrol here. Or contact: Kerstin FUHRMEISTER (70896) Tessa OSBORNE (72957)  

  8. Parsing Expression Grammars Made Practical

    OpenAIRE

    Laurent, Nicolas; Mens, Kim

    2015-01-01

    Parsing Expression Grammars (PEGs) define languages by specifying recursive-descent parser that recognises them. The PEG formalism exhibits desirable properties, such as closure under composition, built-in disambiguation, unification of syntactic and lexical concerns, and closely matching programmer intuition. Unfortunately, state of the art PEG parsers struggle with left-recursive grammar rules, which are not supported by the original definition of the formalism and can lead to infinite recu...

  9. Transgenic Arabidopsis Gene Expression System

    Science.gov (United States)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  10. The Expression of a Need

    OpenAIRE

    Alfort, Esben

    2013-01-01

    This thesis provides a framework for information retrieval based on a set of models which together illustrate how users of search engines come to express their needs in a particular way. With such insights, we may be able to improve systems’ capabilities of understanding users’ requests and through that eventually the ability to satisfy their needs. Developing the framework necessitates discussion of context, relevance, need development, and the cybernetics of search, all of which...

  11. Expressive Sound Synthesis for Animation

    OpenAIRE

    Picard-Limpens, Cécile

    2009-01-01

    The main objective of this thesis is to provide tools for an expressive and real-time synthesis of sounds resulting from physical interactions of various objects in a 3D virtual environment. Indeed, these sounds, such as collisions sounds or sounds from continuous interaction between surfaces, are difficult to create in a pre-production process since they are highly dynamic and vary drastically depending on the interaction and objects. To achieve this goal, two approaches are proposed; the fi...

  12. Vénus version Express

    Science.gov (United States)

    Nazé, Yaël

    2010-04-01

    En avril 2006, Vénus a "capturé" un objet d'un genre particulier: une sonde robotique européenne, baptisée Venus Express et destinée à scruter cette planète sous tous les angles. Bilan de cette mission 5 ans après le lancement de la sonde, dont 4 d'observations vénusiennes.

  13. Misrecognition of facial expressions in delinquents

    Directory of Open Access Journals (Sweden)

    Matsuura Naomi

    2009-09-01

    Full Text Available Abstract Background Previous reports have suggested impairment in facial expression recognition in delinquents, but controversy remains with respect to how such recognition is impaired. To address this issue, we investigated facial expression recognition in delinquents in detail. Methods We tested 24 male adolescent/young adult delinquents incarcerated in correctional facilities. We compared their performances with those of 24 age- and gender-matched control participants. Using standard photographs of facial expressions illustrating six basic emotions, participants matched each emotional facial expression with an appropriate verbal label. Results Delinquents were less accurate in the recognition of facial expressions that conveyed disgust than were control participants. The delinquents misrecognized the facial expressions of disgust as anger more frequently than did controls. Conclusion These results suggest that one of the underpinnings of delinquency might be impaired recognition of emotional facial expressions, with a specific bias toward interpreting disgusted expressions as hostile angry expressions.

  14. Emotional context influences micro-expression recognition.

    Directory of Open Access Journals (Sweden)

    Ming Zhang

    Full Text Available Micro-expressions are often embedded in a flow of expressions including both neutral and other facial expressions. However, it remains unclear whether the types of facial expressions appearing before and after the micro-expression, i.e., the emotional context, influence micro-expression recognition. To address this question, the present study used a modified METT (Micro-Expression Training Tool paradigm that required participants to recognize the target micro-expressions presented briefly between two identical emotional faces. The results of Experiments 1 and 2 showed that negative context impaired the recognition of micro-expressions regardless of the duration of the target micro-expression. Stimulus-difference between the context and target micro-expression was accounted for in Experiment 3. Results showed that a context effect on micro-expression recognition persists even when the stimulus similarity between the context and target micro-expressions was controlled. Therefore, our results not only provided evidence for the context effect on micro-expression recognition but also suggested that the context effect might result from both the stimulus and valence differences.

  15. Gene expression profile of pulpitis.

    Science.gov (United States)

    Galicia, J C; Henson, B R; Parker, J S; Khan, A A

    2016-06-01

    The cost, prevalence and pain associated with endodontic disease necessitate an understanding of the fundamental molecular aspects of its pathogenesis. This study was aimed to identify the genetic contributors to pulpal pain and inflammation. Inflamed pulps were collected from patients diagnosed with irreversible pulpitis (n=20). Normal pulps from teeth extracted for various reasons served as controls (n=20). Pain level was assessed using a visual analog scale (VAS). Genome-wide microarray analysis was performed using Affymetrix GeneTitan Multichannel Instrument. The difference in gene expression levels were determined by the significance analysis of microarray program using a false discovery rate (q-value) of 5%. Genes involved in immune response, cytokine-cytokine receptor interaction and signaling, integrin cell surface interactions, and others were expressed at relatively higher levels in the pulpitis group. Moreover, several genes known to modulate pain and inflammation showed differential expression in asymptomatic and mild pain patients (⩾30 mm on VAS) compared with those with moderate to severe pain. This exploratory study provides a molecular basis for the clinical diagnosis of pulpitis. With an enhanced understanding of pulpal inflammation, future studies on treatment and management of pulpitis and on pain associated with it can have a biological reference to bridge treatment strategies with pulpal biology. PMID:27052691

  16. The Gene Expression Omnibus Database.

    Science.gov (United States)

    Clough, Emily; Barrett, Tanya

    2016-01-01

    The Gene Expression Omnibus (GEO) database is an international public repository that archives and freely distributes high-throughput gene expression and other functional genomics data sets. Created in 2000 as a worldwide resource for gene expression studies, GEO has evolved with rapidly changing technologies and now accepts high-throughput data for many other data applications, including those that examine genome methylation, chromatin structure, and genome-protein interactions. GEO supports community-derived reporting standards that specify provision of several critical study elements including raw data, processed data, and descriptive metadata. The database not only provides access to data for tens of thousands of studies, but also offers various Web-based tools and strategies that enable users to locate data relevant to their specific interests, as well as to visualize and analyze the data. This chapter includes detailed descriptions of methods to query and download GEO data and use the analysis and visualization tools. The GEO homepage is at http://www.ncbi.nlm.nih.gov/geo/. PMID:27008011

  17. Gene Expression in Trypanosomatid Parasites

    Directory of Open Access Journals (Sweden)

    Santiago Martínez-Calvillo

    2010-01-01

    Full Text Available The parasites Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are the trypanosomatid protozoa that cause the deadly human diseases leishmaniasis, African sleeping sickness, and Chagas disease, respectively. These organisms possess unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes and trans-splicing. Little is known about either the DNA sequences or the proteins that are involved in the initiation and termination of transcription in trypanosomatids. In silico analyses of the genome databases of these parasites led to the identification of a small number of proteins involved in gene expression. However, functional studies have revealed that trypanosomatids have more general transcription factors than originally estimated. Many posttranslational histone modifications, histone variants, and chromatin modifying enzymes have been identified in trypanosomatids, and recent genome-wide studies showed that epigenetic regulation might play a very important role in gene expression in this group of parasites. Here, we review and comment on the most recent findings related to transcription initiation and termination in trypanosomatid protozoa.

  18. The Gene Expression Omnibus database

    Science.gov (United States)

    Clough, Emily; Barrett, Tanya

    2016-01-01

    The Gene Expression Omnibus (GEO) database is an international public repository that archives and freely distributes high-throughput gene expression and other functional genomics data sets. Created in 2000 as a worldwide resource for gene expression studies, GEO has evolved with rapidly changing technologies and now accepts high-throughput data for many other data applications, including those that examine genome methylation, chromatin structure, and genome–protein interactions. GEO supports community-derived reporting standards that specify provision of several critical study elements including raw data, processed data, and descriptive metadata. The database not only provides access to data for tens of thousands of studies, but also offers various Web-based tools and strategies that enable users to locate data relevant to their specific interests, as well as to visualize and analyze the data. This chapter includes detailed descriptions of methods to query and download GEO data and use the analysis and visualization tools. The GEO homepage is at http://www.ncbi.nlm.nih.gov/geo/. PMID:27008011

  19. Optimal Expression Condition of Recombinant RAP

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jie; ZHANG Hong; BI Hao; LIU Zhiguo; GUO Jianli; QU Shen

    2007-01-01

    In order to construct the expression recombinant of human receptor associated protein (RAP), optimize its expression condition and obtain the recombinant protein after expression with high efficiency, two prokaryotic expression vectors-pT7-PL and pET-28a(+) were used to construct the expression recombinant containing RAP cDNA, and the expression efficiency of two kinds of expression E. coli of BL21 strains was compared. The effect of different induction conditions on the expression of recombinant RAP was observed. After recombinant protein was purified with Ni+-nitrilotriacetic acid (Ni+-NTA) affinity chromatogram, its binding ability with microphage was observed. The results showed that two recombinant plasmids both obtained high expression of RAP. The expression levels of RAP in plasmid pT7-PL-RAP in BL21 (DE3, plysS) strain were significantly higher than in BL21 (DE3) strain. The expression of pT7-PL-RAP in the presence of chloramphenicol was higher than in the absence of chloramphenicol, and most of the inducible expressed RAP was soluble. The RAP which was purified by Ni+-NTA resin could strongly bind with the RAW264.7 cells rich in low density lipoprotein receptor (LDLR) family receptors. It was concluded that the expression condition of recombinant RAP was optimized and functional RAP was obtained, which offered a good foundation for the further production of RAP as research tool.

  20. Global expression differences and tissue specific expression differences in rice evolution result in two contrasting types of differentially expressed genes

    KAUST Repository

    Horiuchi, Youko

    2015-12-23

    Background Since the development of transcriptome analysis systems, many expression evolution studies characterized evolutionary forces acting on gene expression, without explicit discrimination between global expression differences and tissue specific expression differences. However, different types of gene expression alteration should have different effects on an organism, the evolutionary forces that act on them might be different, and different types of genes might show different types of differential expression between species. To confirm this, we studied differentially expressed (DE) genes among closely related groups that have extensive gene expression atlases, and clarified characteristics of different types of DE genes including the identification of regulating loci for differential expression using expression quantitative loci (eQTL) analysis data. Results We detected differentially expressed (DE) genes between rice subspecies in five homologous tissues that were verified using japonica and indica transcriptome atlases in public databases. Using the transcriptome atlases, we classified DE genes into two types, global DE genes and changed-tissues DE genes. Global type DE genes were not expressed in any tissues in the atlas of one subspecies, however changed-tissues type DE genes were expressed in both subspecies with different tissue specificity. For the five tissues in the two japonica-indica combinations, 4.6 ± 0.8 and 5.9 ± 1.5 % of highly expressed genes were global and changed-tissues DE genes, respectively. Changed-tissues DE genes varied in number between tissues, increasing linearly with the abundance of tissue specifically expressed genes in the tissue. Molecular evolution of global DE genes was rapid, unlike that of changed-tissues DE genes. Based on gene ontology, global and changed-tissues DE genes were different, having no common GO terms. Expression differences of most global DE genes were regulated by cis-eQTLs. Expression

  1. The College Student's Freedom of Expression

    Science.gov (United States)

    Gibbs, Annette

    1974-01-01

    Discussion of means to ensure freedom of expression by college students. Areas of expression noted are student newspapers, lectures by off-campus speakers, freedom to assemble peaceably and freedom to associate. (EK)

  2. Process expression of bounded Petri nets

    Institute of Scientific and Technical Information of China (English)

    吴哲辉

    1996-01-01

    The concept of process expression of bounded Petri nets is presented.Moreover,an algorithm to find the process expression for a bounded Petri net is given.A process expression of a bounded Petri net is a regular expression whose every alphabet symbol represents a basic subprocess of the net.The regular set expressed by the regular expression is the set of all surjective processes of a bounded Petri net.A surjective process of a bounded Petri net is a process of this net in which every s-cut corresponds to a reachable marking of the net.Therefore,all surjective processes of a bounded Petri net can be obtained as long as its process expression and the basic subprocess represented by the alphabet symbols of the process expression are given.

  3. Expressiveness considerations of XML signatures

    DEFF Research Database (Denmark)

    Jensen, Meiko; Meyer, Christopher

    2011-01-01

    more and more challenging. In this paper, we investigate this issue, describing how an attacker can still interfere with Web Services communication even in the presence of XML Signatures. Additionally, we discuss the interrelation of XML Signatures and XML Encryption, focussing on their security......XML Signatures are used to protect XML-based Web Service communication against a broad range of attacks related to man-in-the-middle scenarios. However, due to the complexity of the Web Services specification landscape, the task of applying XML Signatures in a robust and reliable manner becomes...... properties and expressiveness in different application scenarios. © 2011 IEEE....

  4. Summer Oral Expression English course

    CERN Multimedia

    2013-01-01

    An English Oral Expression course will take place this summer at some time between August 19 and October 4.   Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enroll through this link. Please be sure to indicate your planned absences in the comments field so we can schedule the course. If you need more information please send a message to English.training@cern.ch.

  5. Summer Oral Expression English course

    CERN Multimedia

    2012-01-01

    An English Oral Expression course will take place this summer at some time between 25 June and 28 September. The exact dates will be decided according to the preferences of the students.   Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enroll through this link. Please be sure to indicate your planned absences in the comments field so we can schedule the course. If you need more information please send a message to English.training@cern.ch

  6. Summer Oral Expression English course

    CERN Multimedia

    2012-01-01

    An English Oral Expression course will take place this summer from 20 August to 29 September.   Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enroll through this link. Please be sure to indicate your planned absences in the comments field so we can schedule the course. If you need more information please send a message to English.training@cern.ch

  7. Summer Oral Expression English Course

    CERN Multimedia

    HR Department

    2011-01-01

    An English Oral Expression course will take place between 15 August and 30 September 2011. Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enrol through the following link https://cta.cern.ch/cta2/f?p=110:9:1576796470009589::::X_STATUS,XS_COURSE_NAME,XS_PROGRAMME,XS_SUBCATEGORY,X_COURSE_ID,XS_LANGUAGE,XS_SESSION:D,,1,,4368,B, Or contact: Kerstin FUHRMEISTER (70896) Tessa OSBORNE (72957)  

  8. Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales.

    Science.gov (United States)

    Margres, Mark J; Wray, Kenneth P; Seavy, Margaret; McGivern, James J; Herrera, Nathanael D; Rokyta, Darin R

    2016-01-01

    Protein expression level is one of the strongest predictors of protein sequence evolutionary rate, with high-expression protein sequences evolving at slower rates than low-expression protein sequences largely because of constraints on protein folding and function. Expression evolutionary rates also have been shown to be negatively correlated with expression level across human and mouse orthologs over relatively long divergence times (i.e., ∼100 million years). Long-term evolutionary patterns, however, often cannot be extrapolated to microevolutionary processes (and vice versa), and whether this relationship holds for traits evolving under directional selection within a single species over ecological timescales (i.e., protein is predicted to be a tradeoff between the benefit of its function and the costs of its expression. Selection should drive the expression level of all proteins close to values that maximize fitness, particularly for high-expression proteins because of the increased energetic cost of production. Therefore, stabilizing selection may reduce the amount of standing expression variation for high-expression proteins, and in combination with physiological constraints that may place an upper bound on the range of beneficial expression variation, these constraints could severely limit the availability of beneficial expression variants. To determine whether rapid-expression evolution was restricted to low-expression proteins owing to these constraints on highly expressed proteins over ecological timescales, we compared venom protein expression levels across mainland and island populations for three species of pit vipers. We detected significant differentiation in protein expression levels in two of the three species and found that rapid-expression differentiation was restricted to low-expression proteins. Our results suggest that various constraints on high-expression proteins reduce the availability of beneficial expression variants relative to low-expression

  9. Kivy and Langer on expressiveness in music

    OpenAIRE

    van der Schoot Albert

    2013-01-01

    From 1980 onwards, Peter Kivy has put forward that music does not so much express emotions but rather is expressive of emotions. The character of the music does not represent the character or mood of the composer, but reflects his knowledge of emotional life. Unfortunately, Kivy fails to give credit to Susanne Langer, who brought these views to the fore as early as 1942, claiming that the vitality of music lies in expressiveness, not in expression.

  10. Kivy and Langer on expressiveness in music

    Directory of Open Access Journals (Sweden)

    van der Schoot Albert

    2013-01-01

    Full Text Available From 1980 onwards, Peter Kivy has put forward that music does not so much express emotions but rather is expressive of emotions. The character of the music does not represent the character or mood of the composer, but reflects his knowledge of emotional life. Unfortunately, Kivy fails to give credit to Susanne Langer, who brought these views to the fore as early as 1942, claiming that the vitality of music lies in expressiveness, not in expression.

  11. Survey on Human Face Expression Recognition

    OpenAIRE

    Atul Chinchamalatpure; Prof. Anurag Jain

    2014-01-01

    Facial expression recognition plays an important and effective role in the interaction in Human. There are seven basic facial expressions namely fear, surprise, happy, sad, disgust, Neutral and anger. Facial Expression Recognition is process performed by computers which consist of detect the face in the image and pre-process the face region, extracting facial expression features from image by analyzing the motion of facial features or change in the appearance of facial features and classifyin...

  12. Computer-enhanced emotion in facial expressions.

    OpenAIRE

    Calder, A J; Young, A W; Rowland, D.; Perrett, D.I

    1997-01-01

    Benson & Perrett's (1991 b) computer-based caricature procedure was used to alter the positions of anatomical landmarks in photographs of emotional facial expressions with respect to their locations in a reference norm face (e.g. a neutral expression). Exaggerating the differences between an expression and its norm produces caricatured images, whereas reducing the differences produces 'anti-caricatures'. Experiment 1 showed that caricatured (+50% different from neutral) expressions were recog...

  13. Reconnaissance de l'expression du visage

    OpenAIRE

    Zhao, Xi

    2010-01-01

    This Ph.D thesis work is dedicated to automatic facial analysis in 3D, including facial landmarking and facial expression recognition. Indeed, facial expression plays an important role both in verbal and non verbal communication, and in expressing emotions. Thus, automatic facial expression recognition has various purposes and applications and particularly is at the heart of "intelligent" human-centered human/computer(robot) interfaces. Meanwhile, automatic landmarking provides aprior knowled...

  14. Technology Corner: A Regular Expression Training App

    Directory of Open Access Journals (Sweden)

    Nick Flor

    2012-12-01

    Full Text Available Regular expressions enable digital forensic analysts to find information in files. The best way for an analyst to become proficient in writing regular expressions is to practice. This paper presents the code for an app that allows an analyst to practice writing regular expressions.

  15. Facial Expressivity in Infants of Depressed Mothers.

    Science.gov (United States)

    Pickens, Jeffrey; Field, Tiffany

    1993-01-01

    Facial expressions were examined in 84 3-month-old infants of mothers classified as depressed, nondepressed, or low scoring on the Beck Depression Inventory. Infants of both depressed and low-scoring mothers showed significantly more sadness and anger expressions and fewer interest expressions than infants of nondepressed mothers. (Author/MDM)

  16. 7 CFR 27.99 - Values; expression.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Values; expression. 27.99 Section 27.99 Agriculture... CLASSIFICATION UNDER COTTON FUTURES LEGISLATION Regulations Price Quotations and Differences § 27.99 Values; expression. For the purpose of this subpart values shall be expressed in terms of cents and hundredths of...

  17. Mitochondrial RNA granules: Compartmentalizing mitochondrial gene expression.

    Science.gov (United States)

    Jourdain, Alexis A; Boehm, Erik; Maundrell, Kinsey; Martinou, Jean-Claude

    2016-03-14

    In mitochondria, DNA replication, gene expression, and RNA degradation machineries coexist within a common nondelimited space, raising the question of how functional compartmentalization of gene expression is achieved. Here, we discuss the recently characterized "mitochondrial RNA granules," mitochondrial subdomains with an emerging role in the regulation of gene expression. PMID:26953349

  18. A constructive approach to gene expression dynamics

    International Nuclear Information System (INIS)

    Recently, experiments on mRNA abundance (gene expression) have revealed that gene expression shows a stationary organization described by a scale-free distribution. Here we propose a constructive approach to gene expression dynamics which restores the scale-free exponent and describes the intermediate state dynamics. This approach requires only one assumption: Markov property

  19. Expressiveness modulo Bisimilarity of Regular Expressions with Parallel Composition (Extended Abstract)

    CERN Document Server

    Baeten, Jos C M; Muller, Tim; van Tilburg, Paul; 10.4204/EPTCS.41.1

    2010-01-01

    The languages accepted by finite automata are precisely the languages denoted by regular expressions. In contrast, finite automata may exhibit behaviours that cannot be described by regular expressions up to bisimilarity. In this paper, we consider extensions of the theory of regular expressions with various forms of parallel composition and study the effect on expressiveness. First we prove that adding pure interleaving to the theory of regular expressions strictly increases its expressiveness up to bisimilarity. Then, we prove that replacing the operation for pure interleaving by ACP-style parallel composition gives a further increase in expressiveness. Finally, we prove that the theory of regular expressions with ACP-style parallel composition and encapsulation is expressive enough to express all finite automata up to bisimilarity. Our results extend the expressiveness results obtained by Bergstra, Bethke and Ponse for process algebras with (the binary variant of) Kleene's star operation.

  20. Expressiveness modulo Bisimilarity of Regular Expressions with Parallel Composition (Extended Abstract

    Directory of Open Access Journals (Sweden)

    Jos C. M. Baeten

    2010-11-01

    Full Text Available The languages accepted by finite automata are precisely the languages denoted by regular expressions. In contrast, finite automata may exhibit behaviours that cannot be described by regular expressions up to bisimilarity. In this paper, we consider extensions of the theory of regular expressions with various forms of parallel composition and study the effect on expressiveness. First we prove that adding pure interleaving to the theory of regular expressions strictly increases its expressiveness up to bisimilarity. Then, we prove that replacing the operation for pure interleaving by ACP-style parallel composition gives a further increase in expressiveness. Finally, we prove that the theory of regular expressions with ACP-style parallel composition and encapsulation is expressive enough to express all finite automata up to bisimilarity. Our results extend the expressiveness results obtained by Bergstra, Bethke and Ponse for process algebras with (the binary variant of Kleene's star operation.

  1. 病毒序列促转基因在斑马鱼胚胎发育过程中的高效表达%Highly Efficient Expression of Transgenic Genes in Early Embryo Development of Zebrafish

    Institute of Scientific and Technical Information of China (English)

    柳明洙; 张学武; 高圭永; 金善姬; 张琚

    2006-01-01

    目的 探讨转基因在斑马鱼胚胎发育过程中高效表达的有效途径和手段.方法 以增强处理的绿色荧光蛋白(GFP)作为报告基因,在表达质粒的两翼连接构建腺关联病毒(adeno-associated virus, AAV)来源的病毒性反转末端重复序列(inverted terminal repeats, ITRs),以显微注射方式注入斑马鱼受精卵的单一细胞和双细胞时相.结果 病毒序列中的ITRs可使转基因表达效率达到33.0%,远远高于未与病毒序列结合的质粒.结论 源于病毒的ITRs可显著促进转基因在斑马鱼胚胎发育过程中的表达.

  2. Moxibustion upregulates hippocampal progranulin expression

    Directory of Open Access Journals (Sweden)

    Tao Yi

    2016-01-01

    Full Text Available In China, moxibustion is reported to be useful and has few side effects for chronic fatigue syndrome, but its mechanisms are largely unknown. More recently, the focus has been on the wealth of information supporting stress as a factor in chronic fatigue syndrome, and largely concerns dysregulation in the stress-related hypothalamic-pituitary-adrenal axis. In the present study, we aimed to determine the effect of moxibustion on behavioral symptoms in chronic fatigue syndrome rats and examine possible mechanisms. Rats were subjected to a combination of chronic restraint stress and forced swimming to induce chronic fatigue syndrome. The acupoints Guanyuan (CV4 and Zusanli (ST36, bilateral were simultaneously administered moxibustion. Untreated chronic fatigue syndrome rats and normal rats were used as controls. Results from the forced swimming test, open field test, tail suspension test, real-time PCR, enzyme-linked immunosorbent assay, and western blot assay showed that moxibustion treatment decreased mRNA expression of corticotropin-releasing hormone in the hypothalamus, and adrenocorticotropic hormone and corticosterone levels in plasma, and markedly increased progranulin mRNA and protein expression in the hippocampus. These findings suggest that moxibustion may relieve the behavioral symptoms of chronic fatigue syndrome, at least in part, by modulating the hypothalamic-pituitary-adrenal axis and upregulating hippocampal progranulin.

  3. Hepcidin expression in psoriasis patients

    Directory of Open Access Journals (Sweden)

    Nursel Dilek

    2014-01-01

    Full Text Available Background: Iron is an essential nutrient for mammals. Accelerated loss of nutrients through hyperproliferation and desquamation from the skin in psoriasis is known. Hepcidin is an important and recently discovered regulator of iron homeostasis. Aims and Objectives: The present study was undertaken to investigate the hepcidin expression in psoriasis patients. Materials and Methods: We examined peripheral blood cell counts, serum Fe, ferritin, interleukin-6 (IL-6 and hepcidin levels using respectively automated hematology analyzer, Iron assay on the AEROSET system, chemiluminescent microparticle immunoassay with automated analyzer, and enzyme-linked immunosorbent assay. Results: The independent comparison of Fe, ferritin, IL-6 and hepcidin levels in psoriasis patients and control group (healthy volunteers revealed lower Fe and higher IL-6, hepcidin levels in psoriasis patients. No significant difference was seen in the ferritin level between the psoriasis and the control group. Conclusions: We think that studies on hepcidin expression in psoriatic plaques will contribute to our understanding the role of iron and hepcidin in the pathogenesis of psoriasis.

  4. Moxibustion upregulates hippocampal progranulin expression

    Institute of Scientific and Technical Information of China (English)

    Tao Yi; Li Qi; Ji Li; Jing-jing Le; Lei Shao; Xin Du; Jing-cheng Dong

    2016-01-01

    In China, moxibustion is reported to be useful and has few side effects for chronic fatigue syndrome, but its mechanisms are largely un-known. More recently, the focus has been on the wealth of information supporting stress as a factor in chronic fatigue syndrome, and largely concerns dysregulation in the stress-related hypothalamic-pituitary-adrenal axis. In the present study, we aimed to determine the effect of moxibustion on behavioral symptoms in chronic fatigue syndrome rats and examine possible mechanisms. Rats were subjected to a combination of chronic restraint stress and forced swimming to induce chronic fatigue syndrome. The acupointsGuanyuan (CV4) and Zusanli (ST36, bilateral) were simultaneously administered moxibustion. Untreated chronic fatigue syndrome rats and normal rats were used as controls. Results from the forced swimming test, open ifeld test, tail suspension test, real-time PCR, enzyme-linked immunosor-bent assay, and western blot assay showed that moxibustion treatment decreased mRNA expression of corticotropin-releasing hormone in the hypothalamus, and adrenocorticotropic hormone and corticosterone levels in plasma, and markedly increased progranulin mRNA and protein expression in the hippocampus. These ifndings suggest that moxibustion may relieve the behavioral symptoms of chronic fatigue syndrome, at least in part, by modulating the hypothalamic-pituitary-adrenal axis and upregulating hippocampal progranulin.

  5. Expressing Model Constraints Visually with VMQL

    DEFF Research Database (Denmark)

    Störrle, Harald

    2011-01-01

    OCL is the de facto standard language for expressing constraints and queries on UML models. However, OCL expressions are very difficult to create, understand, and maintain, even with the sophisticated tool support now available. In this paper, we propose to use the Visual Model Query Language (VMQL......) for specifying constraints on UML models. We examine VMQL's usability by controlled experiments and its expressiveness by a representative sample. We conclude that VMQL is less expressive than OCL, although expressive enough for most of the constraints in the sample. In terms of usability, however, VMQL...

  6. Analysis of Gene Expression Patterns Using Biclustering.

    Science.gov (United States)

    Roy, Swarup; Bhattacharyya, Dhruba K; Kalita, Jugal K

    2016-01-01

    Mining microarray data to unearth interesting expression profile patterns for discovery of in silico biological knowledge is an emerging area of research in computational biology. A group of functionally related genes may have similar expression patterns under a set of conditions or at some time points. Biclustering is an important data mining tool that has been successfully used to analyze gene expression data for biologically significant cluster discovery. The purpose of this chapter is to introduce interesting patterns that may be observed in expression data and discuss the role of biclustering techniques in detecting interesting functional gene groups with similar expression patterns. PMID:26350227

  7. Effective Clustering Algorithms for Gene Expression Data

    CERN Document Server

    Chandrasekhar, T; Elayaraja, E

    2012-01-01

    Microarrays are made it possible to simultaneously monitor the expression profiles of thousands of genes under various experimental conditions. Identification of co-expressed genes and coherent patterns is the central goal in microarray or gene expression data analysis and is an important task in Bioinformatics research. In this paper, K-Means algorithm hybridised with Cluster Centre Initialization Algorithm (CCIA) is proposed Gene Expression Data. The proposed algorithm overcomes the drawbacks of specifying the number of clusters in the K-Means methods. Experimental analysis shows that the proposed method performs well on gene Expression Data when compare with the traditional K- Means clustering and Silhouette Coefficients cluster measure.

  8. Increased fibroblast telomerase expression precedes myofibroblast α-smooth muscle actin expression in idiopathic pulmonary fibrosis

    Directory of Open Access Journals (Sweden)

    Daniel Reis Waisberg

    2012-09-01

    Full Text Available OBJECTIVE: This study sought to identify the relationship between fibroblast telomerase expression, myofibroblasts, and telomerase-mediated regulatory signals in idiopathic pulmonary fibrosis. METHODS: Thirty-four surgical lung biopsies, which had been obtained from patients with idiopathic pulmonary fibrosis and histologically classified as usual interstitial pneumonia, were examined. Immunohistochemistry was used to evaluate fibroblast telomerase expression, myofibroblast α-smooth muscle actin expression and the tissue expression of inter leu kin-4, transforming growth factor-β, and basic fibroblast growth factor. The point-counting technique was used to quantify the expression of these markers in unaffected, collapsed, mural fibrosis, and honeycombing areas. The results were correlated to patient survival. RESULTS: Fibroblast telomerase expression and basic fibroblast growth factor tissue expression were higher in collapsed areas, whereas myofibroblast expression and interleukine-4 tissue expression were higher in areas of mural fibrosis. Transforming growth factor-β expression was higher in collapsed, mural fibrosis and honeycombing areas in comparison to unaffected areas. Positive correlations were found between basic fibroblast growth factor tissue expression and fibroblast telomerase expression and between interleukin-4 tissue expression and myofibroblast α-smooth muscle actin expression. Negative correlations were observed between interleukin-4 expression and basic fibroblast growth factor tissue expression in areas of mural fibrosis. Myofibroblast α-smooth muscle actin expression and interleukin-4 tissue expression in areas of mural fibrosis were negatively associated with patient survival. CONCLUSION: Fibroblast telomerase expression is higher in areas of early remodeling in lung tissues demonstrating typical interstitial pneumonia, whereas myofibroblast α-smooth muscle actin expression predominates in areas of late remodeling

  9. Cyclooxygenase-2 expression in the normal human eye and its expression pattern in selected eye tumours

    DEFF Research Database (Denmark)

    Wang, Jinmei; Wu, Yazhen; Heegaard, Steffen;

    2011-01-01

    and retina. The COX-2 expression was less in tumours deriving from the ciliary epithelium and also in retinoblastoma. Conclusion: Cyclooxygenase-2 is constitutively expressed in normal human eyes. The expression of COX-2 is much lower in selected eye tumours involving COX-2 expressing cells....

  10. Regular Expression Matching and Operational Semantics

    CERN Document Server

    Rathnayake, Asiri; 10.4204/EPTCS.62.3

    2011-01-01

    Many programming languages and tools, ranging from grep to the Java String library, contain regular expression matchers. Rather than first translating a regular expression into a deterministic finite automaton, such implementations typically match the regular expression on the fly. Thus they can be seen as virtual machines interpreting the regular expression much as if it were a program with some non-deterministic constructs such as the Kleene star. We formalize this implementation technique for regular expression matching using operational semantics. Specifically, we derive a series of abstract machines, moving from the abstract definition of matching to increasingly realistic machines. First a continuation is added to the operational semantics to describe what remains to be matched after the current expression. Next, we represent the expression as a data structure using pointers, which enables redundant searches to be eliminated via testing for pointer equality. From there, we arrive both at Thompson's lock...

  11. Automatic Facial Expression Analysis A Survey

    Directory of Open Access Journals (Sweden)

    C.P. Sumathi

    2013-01-01

    Full Text Available The Automatic Facial Expression Recognition has been one of the latest research topic since1990’s.There have been recent advances in detecting face, facial expression recognition andclassification. There are multiple methods devised for facial feature extraction which helps in identifyingface and facial expressions. This paper surveys some of the published work since 2003 till date. Variousmethods are analysed to identify the Facial expression. The Paper also discusses about the facialparameterization using Facial Action Coding System(FACS action units and the methods whichrecognizes the action units parameters using facial expression data that are extracted. Various kinds offacial expressions are present in human face which can be identified based on their geometric features,appearance features and hybrid features . The two basic concepts of extracting features are based onfacial deformation and facial motion. This article also identifies the techniques based on thecharacteristics of expressions and classifies the suitable methods that can be implemented.

  12. Definition, Detection and Generation of Iyashi Expressions

    Science.gov (United States)

    Kitaoka, Tetsuko; Diago, Luis A.; Hagiwara, Ichiro; Kitazaki, Satoshi; Yamane, Shigeru

    This paper concerns the engineering analysis of “Iyashi”, a peculiar concept to the Japanese, which affect person's heart and may change their expression and behavior. We have integrated the advocator's view of “Iyashi”, analyzed the social background of “Iyashi” and have defined Iyashi and also the Iyashi expression. As the facial expression is the special and important stimulus for both observers and people who show expressions, we want to prove the existence of expressions that change the observer's emotion with Iyashi. We have developed the system to clarify the combination of facial features important for Iyashi through the psychological experiments and the analysis by Holographic Neural Networks (HNN). HNN analysis gave the structure of the Iyashi expression, that is the important combination of the physical facial parameters contributing to the high degree of Iyashi. Based on the structure of Iyashi we are able to generate the Iyashi expression appropriate for each person.

  13. Cyclooxygenase-2 expression in the normal human eye and its expression pattern in selected eye tumours

    DEFF Research Database (Denmark)

    Wang, Jinmei; Wu, Yazhen; Heegaard, Steffen;

    2011-01-01

    Purpose: Cyclooxygenase-2 (COX-2) is an enzyme involved in neoplastic processes. The purpose of the present study is to investigate COX-2 expression in the normal human eye and the expression pattern in selected eye tumours involving COX-2 expressing cells. Methods: Immunohistochemical staining...... using antibodies against COX-2 was performed on paraffin sections of normal human eyes and selected eye tumours arising from cells expressing COX-2. Results: Cyclooxygenase-2 expression was found in various structures of the normal eye. Abundant expression was seen in the cornea, iris, ciliary body...... and retina. The COX-2 expression was less in tumours deriving from the ciliary epithelium and also in retinoblastoma. Conclusion: Cyclooxygenase-2 is constitutively expressed in normal human eyes. The expression of COX-2 is much lower in selected eye tumours involving COX-2 expressing cells....

  14. Abstract Expression Grammar Symbolic Regression

    Science.gov (United States)

    Korns, Michael F.

    This chapter examines the use of Abstract Expression Grammars to perform the entire Symbolic Regression process without the use of Genetic Programming per se. The techniques explored produce a symbolic regression engine which has absolutely no bloat, which allows total user control of the search space and output formulas, which is faster, and more accurate than the engines produced in our previous papers using Genetic Programming. The genome is an all vector structure with four chromosomes plus additional epigenetic and constraint vectors, allowing total user control of the search space and the final output formulas. A combination of specialized compiler techniques, genetic algorithms, particle swarm, aged layered populations, plus discrete and continuous differential evolution are used to produce an improved symbolic regression sytem. Nine base test cases, from the literature, are used to test the improvement in speed and accuracy. The improved results indicate that these techniques move us a big step closer toward future industrial strength symbolic regression systems.

  15. E-mail: Outlook Express

    Directory of Open Access Journals (Sweden)

    Zainul Bakri

    2012-10-01

    Full Text Available Salah satu layanan Internet yang sangat penting adalah electronic mail atau sering hanya disebut sebagai e-mail. Untuk menggunakan e-mail, diperlukan piranti lunak khusus supaya pengguna dapat mengirim dan menerima e-mail. Jenis piranti lunak e-mail diantaranya adalah Outlook Express yang merupakan satu paket yang didistribusikan bersama Internet Explorer versi 4. Piranti lunak ini dijalankan pada PC yang mempunyai sistem operasi Windows 95 atau 98. Jenis piranti lunak e-mail yang lain adalah Eudora, Pegasus dan sebagainya. Bahkan ada yang diintegrasikan dengan Web Browser (alat untuk menelusuri situs Web misalnya IE,dan Netscape.Sebagai layaknya pelayanan pos, maka setiap pengguna e-mail mempunyai alamat tertentu yang tidak mungkin dipunyai oleh pengguna lainnya diseluruh dunia. Untuk keperluan pendistribusian, maka e-mail mempunyai semacam kantor pos yang ditempatkan dalam sebuah komputer server (mail server atau sering disebut sebagai host. 

  16. Process Expression of Security Automaton

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Security is an essential aspect for mobile systems. Usually, mobile system modeling and its security policies specification are realized in different techniques. So when constructed a mobile system using formal methods it is difficult to verify if the system comply with any given security policies. A method was introduced to express security automata which specifying enforceable security policies as processes in an extended π-calculus. In this extended π-calculus, an exception termination process was introduced, called bad. Any input which violating a security automaton will correspond to a step of transformation of the process that specifying the security automaton to exception termination process. Our method shows that any security automata which specifying enforceable security policies would decide a process in the extended π-calculus.

  17. Visualizing structures of speech expressiveness

    DEFF Research Database (Denmark)

    Herbelin, Bruno; Jensen, Karl Kristoffer; Graugaard, Lars

    2008-01-01

    Speech is both beautiful and informative. In this work, a conceptual study of the speech, through investigation of the tower of Babel, the archetypal phonemes, and a study of the reasons of uses of language is undertaken in order to create an artistic work investigating the nature of speech....... The Babel myth speaks about distance created when aspiring to the heaven as the reason for language division. Meanwhile, Locquin states through thorough investigations that only a few phonemes are present throughout history. Our interpretation is that a system able to recognize archetypal phonemes through...... vowels and consonants, and which converts the speech energy into visual particles that form complex visual structures, provides us with a mean to present the expressiveness of speech into a visual mode. This system is presented in an artwork whose scenario is inspired from the reasons of language...

  18. Anti-viral state segregates two molecular phenotypes of pancreatic adenocarcinoma: potential relevance for adenoviral gene therapy

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    Chiorini Jay A

    2010-01-01

    Full Text Available Abstract Background Pancreatic ductal adenocarcinoma (PDAC remains a leading cause of cancer mortality for which novel gene therapy approaches relying on tumor-tropic adenoviruses are being tested. Methods We obtained the global transcriptional profiling of primary PDAC using RNA from eight xenografted primary PDAC, three primary PDAC bulk tissues, three chronic pancreatitis and three normal pancreatic tissues. The Affymetrix GeneChip HG-U133A was used. The results of the expression profiles were validated applying immunohistochemical and western blot analysis on a set of 34 primary PDAC and 10 established PDAC cell lines. Permissivity to viral vectors used for gene therapy, Adenovirus 5 and Adeno-Associated Viruses 5 and 6, was assessed on PDAC cell lines. Results The analysis of the expression profiles allowed the identification of two clearly distinguishable phenotypes according to the expression of interferon-stimulated genes. The two phenotypes could be readily recognized by immunohistochemical detection of the Myxovirus-resistance A protein, whose expression reflects the activation of interferon dependent pathways. The two molecular phenotypes discovered in primary carcinomas were also observed among established pancreatic adenocarcinoma cell lines, suggesting that these phenotypes are an intrinsic characteristic of cancer cells independent of their interaction with the host's microenvironment. The two pancreatic cancer phenotypes are characterized by different permissivity to viral vectors used for gene therapy, as cell lines expressing interferon stimulated genes resisted to Adenovirus 5 mediated lysis in vitro. Similar results were observed when cells were transduced with Adeno-Associated Viruses 5 and 6. Conclusion Our study identified two molecular phenotypes of pancreatic cancer, characterized by a differential expression of interferon-stimulated genes and easily recognized by the expression of the Myxovirus-resistance A protein. We

  19. Gene expression profile of sprinter's muscle.

    Science.gov (United States)

    Yoshioka, M; Tanaka, H; Shono, N; Shindo, M; St-Amand, J

    2007-12-01

    We have characterized the global gene expression profile in left vastus lateralis muscles of sprinters and sedentary men. The gene expression profile was analyzed by using serial analysis of gene expression (SAGE) method. The abundantly expressed transcripts in the sprinter's muscle were mainly involved in contraction and energy metabolism, whereas six transcripts were corresponding to potentially novel transcripts. Thirty-eight transcripts were differentially expressed between the sprinter and sedentary individuals. Moreover, sprinters showed higher expressions of both uncharacterized and potentially novel transcripts. Sprinters also highly expressed seven transcripts, such as glycine-rich protein, myosin heavy polypeptide (MYH) 2, expressed sequence tag similar to (EST) fructose-bisphosphate aldolase 1 isoform A (ALDOA), glyceraldehyde-3-phosphate dehydrogenase and ATP synthase F0 subunit 6. On the other hand, 20 transcripts such as MYH1, tropomyosin 2 and 3, troponin C slow, C2 fast, I slow, T1 slow and T3 fast, myoglobin, creatine kinase, ALDOA, glycogen phosphorylase, cytochrome c oxidase II and III, and NADH dehydrogenase 1 and 2 showed lower expression levels in the sprinters than the sedentary controls. The current study has characterized the global gene expressions in sprinters and identified a number of transcripts that can be subjected to further mechanistic analysis.

  20. Anticausatives are weak scalar expressions, not reflexive expressions

    Directory of Open Access Journals (Sweden)

    Florian Schäfer

    2016-07-01

    Full Text Available We discuss conceptual and empirical arguments from Germanic, Romance and Slavic languages against an analysis treating anticausative verbs as derived from their lexical causative counterparts under reflexivization. Instead, we defend the standard account to the semantics of the causative alternation according to which anticausatives in general, and anticausatives marked with reflexive morphology in particular, denote simple one-place inchoative events that are logically entailed by their lexical causative counterparts. Under such an account, anticausative verbs are weak scalar expressions that stand in a semantico-pragmatic opposition to their strong lexical causative counterparts. Due to this scalar relation, the use of an anticausative can trigger the implicature that the use of its lexical causative counterpart is too strong. As usual with implicatures, they can be ‘metalinguistically’ denied, cancelled, or reinforced and we argue that these mechanisms explain all central empirical facts brought up in the literature in favor of a treatment of anticausatives as semantically reflexive predicates. Our results reinforce the view that the reflexive morphemes used in many (Indo-European languages to mark anticausatives do not necessarily trigger reflexive semantics. However, we also show that a string involving a reflexively marked (anti-causative verb can be forced into a semantically reflexive construal under particular conceptual or grammatical circumstances.