Sample records for adenovirus vector targeting

  1. Recombinant adenovirus vectors with knobless fibers for targeted gene transfer

    van Beusechem, VW; van Rijswijk, ALCT; van Es, HHG; Haisma, HJ; Pinedo, HM; Gerritsen, WR


    Adenoviral vector systems for gene therapy can be much improved by targeting vectors to specific cell types. This requires both the complete ablation of native adenovirus tropism and the introduction of a novel binding affinity in the viral capsid. We reasoned that these requirements could be fulfil

  2. A novel technology to target adenovirus vectors : application in cells involved in atherosclerosis

    Gras, Jan Cornelis Emile


    In this thesis a novel technology is described to target adenovirus vectors. Adenovirus vectors are powerful tools to modulate gene expression. The use of these vectors however, is hampered by the fact that many for gene therapy interesting cell types do not, or only at low levels express the CAR re

  3. Adenovirus Vectors Target Several Cell Subtypes of Mammalian Inner Ear In Vivo

    Li, Wenyan; Shen, Jun


    Mammalian inner ear harbors diverse cell types that are essential for hearing and balance. Adenovirus is one of the major vectors to deliver genes into the inner ear for functional studies and hair cell regeneration. To identify adenovirus vectors that target specific cell subtypes in the inner ear, we studied three adenovirus vectors, carrying a reporter gene encoding green fluorescent protein (GFP) from two vendors or with a genome editing gene Cre recombinase (Cre), by injection into postnatal days 0 (P0) and 4 (P4) mouse cochlea through scala media by cochleostomy in vivo. We found three adenovirus vectors transduced mouse inner ear cells with different specificities and expression levels, depending on the type of adenoviral vectors and the age of mice. The most frequently targeted region was the cochlear sensory epithelium, including auditory hair cells and supporting cells. Adenovirus with GFP transduced utricular supporting cells as well. This study shows that adenovirus vectors are capable of efficiently and specifically transducing different cell types in the mammalian inner ear and provides useful tools to study inner ear gene function and to evaluate gene therapy to treat hearing loss and vestibular dysfunction. PMID:28116172

  4. Dendritic cell based PSMA immunotherapy for prostate cancer using a CD40-targeted adenovirus vector.

    Briana Jill Williams

    Full Text Available Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs with prostate specific membrane antigen (PSMA have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells. To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ. Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.

  5. Dendritic cell based PSMA immunotherapy for prostate cancer using a CD40-targeted adenovirus vector.

    Williams, Briana Jill; Bhatia, Shilpa; Adams, Lisa K; Boling, Susan; Carroll, Jennifer L; Li, Xiao-Lin; Rogers, Donna L; Korokhov, Nikolay; Kovesdi, Imre; Pereboev, Alexander V; Curiel, David T; Mathis, J Michael


    Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.

  6. Development of peritoneal tumor-targeting vector by in vivo screening with a random peptide-displaying adenovirus library.

    Takeshi Nishimoto

    Full Text Available The targeting of gene transfer at the cell-entry level is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success, because the proper targeting ligands on the cells of interest are generally unknown. To overcome this limitation, we have constructed a random peptide library displayed on the adenoviral fiber knob, and have successfully selected targeted vectors by screening the library on cancer cell lines in vitro. The infection of targeted vectors was considered to be mediated by specific receptors on target cells. However, the expression levels and kinds of cell surface receptors may be substantially different between in vitro culture and in vivo tumor tissue. Here, we screened the peptide display-adenovirus library in the peritoneal dissemination model of AsPC-1 pancreatic cancer cells. The vector displaying a selected peptide (PFWSGAV showed higher infectivity in the AsPC-1 peritoneal tumors but not in organs and other peritoneal tumors as compared with a non-targeted vector. Furthermore, the infectivity of the PFWSGAV-displaying vector for AsPC-1 peritoneal tumors was significantly higher than that of a vector displaying a peptide selected by in vitro screening, indicating the usefulness of in vivo screening in exploring the targeting vectors. This vector-screening system can facilitate the development of targeted adenovirus vectors for a variety of applications in medicine.

  7. The relative magnitude of transgene-specific adaptive immune responses induced by human and chimpanzee adenovirus vectors differs between laboratory animals and a target species.

    Dicks, Matthew D J; Guzman, Efrain; Spencer, Alexandra J; Gilbert, Sarah C; Charleston, Bryan; Hill, Adrian V S; Cottingham, Matthew G


    Adenovirus vaccine vectors generated from new viral serotypes are routinely screened in pre-clinical laboratory animal models to identify the most immunogenic and efficacious candidates for further evaluation in clinical human and veterinary settings. Here, we show that studies in a laboratory species do not necessarily predict the hierarchy of vector performance in other mammals. In mice, after intramuscular immunization, HAdV-5 (Human adenovirus C) based vectors elicited cellular and humoral adaptive responses of higher magnitudes compared to the chimpanzee adenovirus vectors ChAdOx1 and AdC68 from species Human adenovirus E. After HAdV-5 vaccination, transgene specific IFN-γ(+) CD8(+) T cell responses reached peak magnitude later than after ChAdOx1 and AdC68 vaccination, and exhibited a slower contraction to a memory phenotype. In cattle, cellular and humoral immune responses were at least equivalent, if not higher, in magnitude after ChAdOx1 vaccination compared to HAdV-5. Though we have not tested protective efficacy in a disease model, these findings have important implications for the selection of candidate vectors for further evaluation. We propose that vaccines based on ChAdOx1 or other Human adenovirus E serotypes could be at least as immunogenic as current licensed bovine vaccines based on HAdV-5.

  8. Increasing the Efficacy of Oncolytic Adenovirus Vectors

    William S. M. Wold


    Full Text Available Oncolytic adenovirus (Ad vectors present a new modality to treat cancer. These vectors attack tumors via replicating in and killing cancer cells. Upon completion of the vector replication cycle, the infected tumor cell lyses and releases progeny virions that are capable of infecting neighboring tumor cells. Repeated cycles of vector replication and cell lysis can destroy the tumor. Numerous Ad vectors have been generated and tested, some of them reaching human clinical trials. In 2005, the first oncolytic Ad was approved for the treatment of head-and-neck cancer by the Chinese FDA. Oncolytic Ads have been proven to be safe, with no serious adverse effects reported even when high doses of the vector were injected intravenously. The vectors demonstrated modest anti-tumor effect when applied as a single agent; their efficacy improved when they were combined with another modality. The efficacy of oncolytic Ads can be improved using various approaches, including vector design, delivery techniques, and ancillary treatment, which will be discussed in this review.

  9. An Adenovirus Vector Incorporating Carbohydrate Binding Domains Utilizes Glycans for Gene Transfer

    Kim, Julius W.; Glasgow, Joel N.; Nakayama, Masaharu; Ak, Ferhat; Ugai, Hideyo; Curiel, David T.


    Background: Vectors based on human adenovirus serotype 5 (HAdV-5) continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene

  10. A new adenovirus based vaccine vector expressing an Eimeria tenella derived TLR agonist improves cellular immune responses to an antigenic target.

    Daniel M Appledorn

    Full Text Available BACKGROUND: Adenoviral based vectors remain promising vaccine platforms for use against numerous pathogens, including HIV. Recent vaccine trials utilizing Adenovirus based vaccines expressing HIV antigens confirmed induction of cellular immune responses, but these responses failed to prevent HIV infections in vaccinees. This illustrates the need to develop vaccine formulations capable of generating more potent T-cell responses to HIV antigens, such as HIV-Gag, since robust immune responses to this antigen correlate with improved outcomes in long-term non-progressor HIV infected individuals. METHODOLOGY/PRINCIPAL FINDINGS: In this study we designed a novel vaccine strategy utilizing an Ad-based vector expressing a potent TLR agonist derived from Eimeria tenella as an adjuvant to improve immune responses from a [E1-]Ad-based HIV-Gag vaccine. Our results confirm that expression of rEA elicits significantly increased TLR mediated innate immune responses as measured by the influx of plasma cytokines and chemokines, and activation of innate immune responding cells. Furthermore, our data show that the quantity and quality of HIV-Gag specific CD8(+ and CD8(- T-cell responses were significantly improved when coupled with rEA expression. These responses also correlated with a significantly increased number of HIV-Gag derived epitopes being recognized by host T cells. Finally, functional assays confirmed that rEA expression significantly improved antigen specific CTL responses, in vivo. Moreover, we show that these improved responses were dependent upon improved TLR pathway interactions. CONCLUSION/SIGNIFICANCE: The data presented in this study illustrate the potential utility of Ad-based vectors expressing TLR agonists to improve clinical outcomes dependent upon induction of robust, antigen specific immune responses.

  11. An Update on Canine Adenovirus Type 2 and Its Vectors

    Eric J. Kremer


    Full Text Available Adenovirus vectors have significant potential for long- or short-term gene transfer. Preclinical and clinical studies using human derived adenoviruses (HAd have demonstrated the feasibility of flexible hybrid vector designs, robust expression and induction of protective immunity. However, clinical use of HAd vectors can, under some conditions, be limited by pre-existing vector immunity. Pre-existing humoral and cellular anti-capsid immunity limits the efficacy and duration of transgene expression and is poorly circumvented by injections of larger doses and immuno-suppressing drugs. This review updates canine adenovirus serotype 2 (CAV-2, also known as CAdV-2 biology and gives an overview of the generation of early region 1 (E1-deleted to helper-dependent (HD CAV-2 vectors. We also summarize the essential characteristics concerning their interaction with the anti-HAd memory immune responses in humans, the preferential transduction of neurons, and its high level of retrograde axonal transport in the central and peripheral nervous system. CAV-2 vectors are particularly interesting tools to study the pathophysiology and potential treatment of neurodegenerative diseases, as anti-tumoral and anti-viral vaccines, tracer of synaptic junctions, oncolytic virus and as a platform to generate chimeric vectors.

  12. Antiviral antibodies target adenovirus to phagolysosomes and amplify the innate immune response.

    Zaiss, Anne K; Vilaysane, Akosua; Cotter, Matthew J; Clark, Sharon A; Meijndert, H Christopher; Colarusso, Pina; Yates, Robin M; Petrilli, Virginie; Tschopp, Jurg; Muruve, Daniel A


    Adenovirus is a nonenveloped dsDNA virus that activates intracellular innate immune pathways. In vivo, adenovirus-immunized mice displayed an enhanced innate immune response and diminished virus-mediated gene delivery following challenge with the adenovirus vector AdLacZ suggesting that antiviral Abs modulate viral interactions with innate immune cells. Under naive serum conditions in vitro, adenovirus binding and internalization in macrophages and the subsequent activation of innate immune mechanisms were inefficient. In contrast to the neutralizing effect observed in nonhematopoietic cells, adenovirus infection in the presence of antiviral Abs significantly increased FcR-dependent viral internalization in macrophages. In direct correlation with the increased viral internalization, antiviral Abs amplified the innate immune response to adenovirus as determined by the expression of NF-kappaB-dependent genes, type I IFNs, and caspase-dependent IL-1beta maturation. Immune serum amplified TLR9-independent type I IFN expression and enhanced NLRP3-dependent IL-1beta maturation in response to adenovirus, confirming that antiviral Abs specifically amplify intracellular innate pathways. In the presence of Abs, confocal microscopy demonstrated increased targeting of adenovirus to LAMP1-positive phagolysosomes in macrophages but not epithelial cells. These data show that antiviral Abs subvert natural viral tropism and target the adenovirus to phagolysosomes and the intracellular innate immune system in macrophages. Furthermore, these results illustrate a cross-talk where the adaptive immune system positively regulates the innate immune system and the antiviral state.

  13. Human adenovirus 52 uses sialic acid-containing glycoproteins and the coxsackie and adenovirus receptor for binding to target cells.

    Annasara Lenman


    Full Text Available Most adenoviruses attach to host cells by means of the protruding fiber protein that binds to host cells via the coxsackievirus and adenovirus receptor (CAR protein. Human adenovirus type 52 (HAdV-52 is one of only three gastroenteritis-causing HAdVs that are equipped with two different fiber proteins, one long and one short. Here we show, by means of virion-cell binding and infection experiments, that HAdV-52 can also attach to host cells via CAR, but most of the binding depends on sialylated glycoproteins. Glycan microarray, flow cytometry, surface plasmon resonance and ELISA analyses reveal that the terminal knob domain of the long fiber (52LFK binds to CAR, and the knob domain of the short fiber (52SFK binds to sialylated glycoproteins. X-ray crystallographic analysis of 52SFK in complex with 2-O-methylated sialic acid combined with functional studies of knob mutants revealed a new sialic acid binding site compared to other, known adenovirus:glycan interactions. Our findings shed light on adenovirus biology and may help to improve targeting of adenovirus-based vectors for gene therapy.

  14. An adenovirus vector incorporating carbohydrate binding domains utilizes glycans for gene transfer.

    Julius W Kim

    Full Text Available BACKGROUND: Vectors based on human adenovirus serotype 5 (HAdV-5 continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting. METHODOLOGY/PRINCIPAL FINDINGS: As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4. This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells. CONCLUSIONS/SIGNIFICANCE: These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers.

  15. Low seroprevalent species D adenovirus vectors as influenza vaccines.

    Eric A Weaver

    Full Text Available Seasonal and pandemic influenza remains a constant threat. While standard influenza vaccines have great utility, the need for improved vaccine technologies have been brought to light by the 2009 swine flu pandemic, highly pathogenic avian influenza infections, and the most recent early and widespread influenza activity. Species C adenoviruses based on serotype 5 (AD5 are potent vehicles for gene-based vaccination. While potent, most humans are already immune to this virus. In this study, low seroprevalent species D adenoviruses Ad26, 28, and 48 were cloned and modified to express the influenza virus A/PR/8/34 hemagglutinin gene for vaccine studies. When studied in vivo, these species D Ad vectors performed quite differently as compared to species C Ad vectors depending on the route of immunization. By intramuscular injection, species D vaccines were markedly weaker than species C vaccines. In contrast, the species D vaccines were equally efficient as species C when delivered mucosally by the intranasal route. Intranasal adenovirus vaccine doses as low as 10(8 virus particles per mouse induced complete protection against a stringent lethal challenge dose of influenza. These data support translation of species D adenoviruses as mucosal vaccines and highlight the fundamental effects of differences in virus tropism on vaccine applications.

  16. Adenovirus-derived vectors for prostate cancer gene therapy.

    de Vrij, Jeroen; Willemsen, Ralph A; Lindholm, Leif; Hoeben, Rob C; Bangma, Chris H; Barber, Chris; Behr, Jean-Paul; Briggs, Simon; Carlisle, Robert; Cheng, Wing-Shing; Dautzenberg, Iris J C; de Ridder, Corrina; Dzojic, Helena; Erbacher, Patrick; Essand, Magnus; Fisher, Kerry; Frazier, April; Georgopoulos, Lindsay J; Jennings, Ian; Kochanek, Stefan; Koppers-Lalic, Daniela; Kraaij, Robert; Kreppel, Florian; Magnusson, Maria; Maitland, Norman; Neuberg, Patrick; Nugent, Regina; Ogris, Manfred; Remy, Jean-Serge; Scaife, Michelle; Schenk-Braat, Ellen; Schooten, Erik; Seymour, Len; Slade, Michael; Szyjanowicz, Pio; Totterman, Thomas; Uil, Taco G; Ulbrich, Karel; van der Weel, Laura; van Weerden, Wytske; Wagner, Ernst; Zuber, Guy


    Prostate cancer is a leading cause of death among men in Western countries. Whereas the survival rate approaches 100% for patients with localized cancer, the results of treatment in patients with metastasized prostate cancer at diagnosis are much less successful. The patients are usually presented with a variety of treatment options, but therapeutic interventions in prostate cancer are associated with frequent adverse side effects. Gene therapy and oncolytic virus therapy may constitute new strategies. Already a wide variety of preclinical studies has demonstrated the therapeutic potential of such approaches, with oncolytic prostate-specific adenoviruses as the most prominent vector. The state of the art and future prospects of gene therapy in prostate cancer are reviewed, with a focus on adenoviral vectors. We summarize advances in adenovirus technology for prostate cancer treatment and highlight areas where further developments are necessary.

  17. Replication-Defective Vector Based on a Chimpanzee Adenovirus

    Farina, Steven F.; Gao, Guang-Ping; Xiang, Z. Q.; Rux, John J.; Burnett, Roger M.; Alvira, Mauricio R.; Marsh, Jonathan; Ertl, Hildegund C.J.; Wilson, James M.


    An adenovirus previously isolated from a mesenteric lymph node from a chimpanzee was fully sequenced and found to be similar in overall structure to human adenoviruses. The genome of this virus, called C68, is 36,521 bp in length and is most similar to subgroup E of human adenovirus, with 90% identity in most adenovirus type 4 open reading frames that have been sequenced. Substantial differences in the hexon hypervariable regions were noted between C68 and other known adenoviruses, including ...

  18. CXCL12 retargeting of an adenovirus vector to cancer cells using a bispecific adapter

    Bhatia S


    Full Text Available Shilpa Bhatia,1 Samia M O’Bryan,1 Angel A Rivera,2 David T Curiel,3 J Michael Mathis1 1Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, 2Departments of Pathology and Surgery, University of Alabama at Birmingham, Birmingham, AL, 3Department of Radiation Oncology, Washington University School of Medicine, St Louis, MO, USA Abstract: Ad vectors are promising delivery vehicles for cancer therapeutic interventions. However, their application is limited by promiscuous tissue tropism and hepatotoxicity. This limitation can be avoided by altering the native tropism of Ads so that they can be redirected to the target cells through alternate cellular receptors. The CXCR4 chemokine receptor belongs to a large superfamily of G-protein-coupled receptors and is known to be upregulated in a wide variety of cancers, including breast cancer and melanoma. These receptors have been associated with cancer cell survival, progression, and metastasis. In the current study, an Ad to cancer cells overexpressing CXCR4 by using a bispecific adapter, sCAR-CXCL12, was retargeted. The sCAR-CXCL12 adapter contained the soluble ectodomain form of the native Ad5 receptor (sCAR, which was fused to a mature human chemokine ligand, CXCL12, through a short peptide linker. A dramatic increase in the infectivity of cancer cells using a targeted Ad vector compared with an untargeted vector was observed. Furthermore, sCAR-CXCL12 attenuated Ad infection of liver ex vivo and in vivo and enhanced Ad vector infection of xenograft tumors implanted in immunodeficient SCID-bg mice. Thus, the sCAR-CXCL12 adapter could be used to retarget Ad vectors to chemokine receptor-positive tumors. Keywords: adapter, adenovirus serotype 5, cancer, hCAR, human coxsackievirus and adenovirus receptor, chemokine receptor, CXCL12, CXCR4, gene therapy, retargeting, viral vector

  19. Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.

    Wickham, T J; Tzeng, E; Shears, L L; Roelvink, P W; Li, Y; Lee, G M; Brough, D E; Lizonova, A; Kovesdi, I


    Alteration of the natural tropism of adenovirus (Ad) will permit gene transfer into specific cell types and thereby greatly broaden the scope of target diseases that can be treated by using Ad. We have constructed two Ad vectors which contain modifications to the Ad fiber coat protein that redirect virus binding to either alpha(v) integrin [AdZ.F(RGD)] or heparan sulfate [AdZ.F(pK7)] cellular receptors. These vectors were constructed by a novel method involving E4 rescue of an E4-deficient Ad with a transfer vector containing both the E4 region and the modified fiber gene. AdZ.F(RGD) increased gene delivery to endothelial and smooth muscle cells expressing alpha(v) integrins. Likewise, AdZ.F(pK7) increased transduction 5- to 500-fold in multiple cell types lacking high levels of Ad fiber receptor, including macrophage, endothelial, smooth muscle, fibroblast, and T cells. In addition, AdZ.F(pK7) significantly increased gene transfer in vivo to vascular smooth muscle cells of the porcine iliac artery following balloon angioplasty. These vectors may therefore be useful in gene therapy for vascular restenosis or for targeting endothelial cells in tumors. Although binding to the fiber receptor still occurs with these vectors, they demonstrate the feasibility of tissue-specific receptor targeting in cells which express low levels of Ad fiber receptor.

  20. Transduction and apoptosis induction in the rat prostate, using adenovirus vectors.

    Kirkman, W; Chen, P; Schroeder, R; Feneley, M R; Rodriguez, R; Wickham, T J; King, C R; Bruder, J T


    Proapoptotic adenovirus vectors offer great promise for the treatment of cancer and nonmalignant conditions. Benign prostate hyperplasia (BPH) is a common nonmalignant enlargement of the prostate that involves epithelial, stromal, and smooth muscle components of the gland. We tested the hypothesis that an adenovirus vector expressing Fas ligand can be used to induce apoptosis in the prostate. We analyzed the efficiency of transduction and apoptosis induction in primary cultures of human prostate cells after adenovirus-mediated gene transfer. Efficient transduction was observed in primary prostate epithelial cells. Stromal and smooth muscle cells were more difficult to transduce, as no coxsackie-adenovirus receptor (CAR) expression was detectable on these cells. However, transduction was achieved in these cells when the multiplicity of infection was increased to 100 focal-forming units per cell, or when the vectors were delivered as calcium phosphate precipitates. Infection of all three primary prostate cell types with an adenovirus vector that expresses Fas ligand (AdFasL/G) resulted in rapid apoptosis. Direct injection of the rat prostate with an adenovirus vector carrying luciferase resulted in substantial luciferase expression. TUNEL analysis demonstrated that AdFasL/G administration induced low-level apoptosis in prostatic epithelial cells throughout the gland. As a first step toward enhancing the efficiency of prostate transduction in vivo, we tested an adenovirus vector that was engineered to have an expanded tropism. This vector, AdZ.F2K(pK7), was 10- to 500-fold more efficient than unmodified vectors in transducing prostate epithelial, smooth muscle, and stromal cells in culture. Moreover, AdZ.F2K(pK7) was more efficient than an unmodified vector at transducing the rat prostate in vivo, although the effect was dose dependent.

  1. Replication-competent human adenovirus 11p vectors can propagate in Vero cells

    Gokumakulapalle, Madhuri; Mei, Ya-Fang, E-mail:


    The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were as capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. - Highlights: • Africa green monkey cell lines were monitored for human adenovirus 11p GFP vector infection. • Human CD46 molecules were detectable in these monkey cell lines. • Adenovirus 11p GFP vector can be propagated in Vero cells increases the safety of Ad11p-based vectors for clinical trials. • To use Vero cells for preparation of Ad11p vector avoids the potential inclusion of oncogenes from tumor cells.

  2. Construction and expression of SET gene and siRNA recombinant adenovirus vectors

    Xu Bo-qun; Lu Pin-hong; Li Ying; Xue Kai; Li Mei; Ma Xiang; Diao Fei-yan; Cui Yu-gui; Liu Jia-yin


    Objective: To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA (SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels.Methods: The cDNA sequence of SET was cloned by reverse transcriptive polymerase chain reaction (RT-PCR) and the SET gene fragment was subcloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-SET. The shuttle plasmid pAdtrack-SET was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant Ad-CMV-SET and the recombinant Ad-CMV-SET was packaged and amplified in the AD293 cells. The expression of SET in AD293 cells was detected by Western blot. In addition, we constructed SET gene SiRNA recombinant adenovirus vector (Ad-H1-SiRNA/SET) and its efficacy of knockdown of SET protein was detected in infected GC-2spd(ts) cells by Western blot. Results: The recombinant adenovirus vectors, both SET gene recombinant adenovirus vector Ad-CMV-SET and SET gene SiRNA recombinant adenovirus vector Ad-H1-SiRNA/SET, were proven to be constructed successfully by the evidence of endonulease digestion and sequencing. AD293 cells infected with either recombinant adenovirus vector of Ad-CMV-SET or Ad-H1-SiRNA/SET were observed to express GFP. The expression of SET protein was up-regulated significantly in AD293 cells infected with SET gene recombinant adenovirus vector. On the contrast, SET protein was significantly down-regulated in the GC-2spd(ts) cells infected with Ad-H1-SiRNA/SET (P<0.05) and the knockdown efficiency was approximately 50%-70%. Conclusion: The recombinant adenovirus vector Ad-CMV-SET and Ad-H1-SiRNA/SET were successfully constructed and effectively expressed in germ cells and somatic cells. It provides an experimental tool for further study of SET gene in the physiological and pathophysiological mechanism of reproduction-related diseases.

  3. Construction and Expression of Human PTEN Tumor Suppressor Gene Recombinant Adenovirus Vector

    CHEN Qingyong; WANG Chunyou; CHEN Daoda; CHEN Jianying; JIANG Chunfang; ZHENG Hai


    The recombinant defective adenovirus vector carrying human PTEN tumor suppres sor gene was constructed by using AdEasy-1 system and its expression was detected in human breast cancer cell line MDA-MB-468. Human PTEN cDNA was cloned into adenovirus shuttle plasmid pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-PTEN, then homologeous recombination was carried out in the E. coli BJ5183 by contransforming linearized shuttle vector with adenovirus backbone plasmid pAdEasy-1. The newly recombined defective adenovirus vector AdPTEN containing green fluorescent protein (GFP) was packaged and propagated in 293 cells. After being purified by cesium chloride gradient centrifugation, the adenovirus was transfected into human breast cancer cell line MDA-MB-468 in vitro. The expression of PTEN mRNA and protein in infected human breast cancer cell line MDA-MB-468 was detected by RT-PCR and Western blot respectively. The recombinant defective adenovirus vector carrying PTEN gene was constructed successfully. The viral titer of purified adenovirus was 2.5×1010 pfu/mL, and about 70 % breast cancer cells were infected with Ad PTEN when multiplicity of infection (MOI) reached 50. The exogenous PTEN mRNA and protein were expressed in MDA-MB-468 cells infected with Ad-PTEN by RT-PCR and Western blot. The recombinant defective adenovirus vector of PTEN gene was constructed successfully using AdEasy-1 system rapidly, which paved a sound foundation for gene study of breast cancer.

  4. Adenovirus as a gene therapy vector for hematopoietic cells.

    Marini, F C; Yu, Q; Wickham, T; Kovesdi, I; Andreeff, M


    Adenovirus (Adv)-mediated gene transfer has recently gained new attention as a means to deliver genes for hematopoietic stem cell (HSC) or progenitor cell gene therapy. In the past, HSCs have been regarded as poor Adv targets, mainly because they lack the specific Adv receptors required for efficient and productive Adv infection. In addition, the nonintegrating nature of Adv has prevented its application to HSC and bone marrow transduction protocols where long-term expression is required. There is even controversy as to whether Adv can infect hematopoietic cells at all. In fact, the ability of Adv to infect epithelium-based targets and its inability to effectively transfect HSCs have been used in the development of eradication schemes that use Adv to preferentially infect and "purge" tumor cell-contaminating HSC grafts. However, there are data supporting the existence of productive Adv infections into HSCs. Such protocols involve the application of cytokine mixtures, high multiplicities of infection, long incubation periods, and more recently, immunological and genetic modifications to Adv itself to enable it to efficiently transfer genes into HSCs. This is a rapidly growing field, both in terms of techniques and applications. This review examines the two sides of the Adv/CD34 controversy as well as the current developments in this field.

  5. Adenovirus tumor targeting and hepatic untargeting by a coxsackie/adenovirus receptor ectodomain anti-carcinoembryonic antigen bispecific adapter.

    Li, Hua-Jung; Everts, Maaike; Pereboeva, Larisa; Komarova, Svetlana; Idan, Anat; Curiel, David T; Herschman, Harvey R


    Adenovirus vectors have a number of advantages for gene therapy. However, because of their lack of tumor tropism and their preference for liver infection following systemic administration, they cannot be used for systemic attack on metastatic disease. Many epithelial tumors (e.g., colon, lung, and breast) express carcinoembryonic antigen (CEA). To block the natural hepatic tropism of adenovirus and to "retarget" the virus to CEA-expressing tumors, we used a bispecific adapter protein (sCAR-MFE), which fuses the ectodomain of the coxsackie/adenovirus receptor (sCAR) with a single-chain anti-CEA antibody (MFE-23). sCAR-MFE untargets adenovirus-directed luciferase transgene expression in the liver by >90% following systemic vector administration. Moreover, sCAR-MFE can "retarget" adenovirus to CEA-positive epithelial tumor cells in cell culture, in s.c. tumor grafts, and in hepatic tumor grafts. The sCAR-MFE bispecific adapter should, therefore, be a powerful agent to retarget adenovirus vectors to epithelial tumor metastases.

  6. Canine adenovirus type 2 vector generation via I-Sce1-mediated intracellular genome release.

    Ibanes, Sandy; Kremer, Eric J


    When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2) vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER) fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.

  7. Canine adenovirus type 2 vector generation via I-Sce1-mediated intracellular genome release.

    Sandy Ibanes

    Full Text Available When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2 vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.

  8. Targeting species D adenoviruses replication to counteract the epidemic keratoconjunctivitis.

    Nikitenko, Natalia A; Speiseder, Thomas; Groitl, Peter; Spirin, Pavel V; Prokofjeva, Maria M; Lebedev, Timofey D; Rubtsov, Petr M; Lam, Elena; Riecken, Kristoffer; Fehse, Boris; Dobner, Thomas; Prassolov, Vladimir S


    Human adenoviruses are non-enveloped DNA viruses causing various infections; their pathogenicity varies dependent on virus species and type. Although acute infections can sometimes take severe courses, they are rarely fatal in immune-competent individuals. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are hyperacute and highly contagious infections of the eye caused by human adenovirus types within species D. Currently there is no causal treatment available to counteract these diseases effectively. The E2B region of the adenovirus genome encodes for the viral DNA polymerase, which is required for adenoviral DNA replication. Here we propose novel model systems to test this viral key factor, DNA polymerase, as a putative target for the development of efficient antiviral therapy based on RNA interference. Using our model cell lines we found that different small interfering RNAs mediate significant suppression (up to 90%) of expression levels of viral DNA polymerase upon transfection. Moreover, permanent expression of short hairpin RNA based on the most effective small interfering RNA led to a highly significant, more than tenfold reduction in replication for different human group D adenoviruses involved in ocular infections.

  9. Novel adenovirus vaccine vectors based on the enteric-tropic serotype 41.

    Lemiale, Franck; Haddada, Hedi; Nabel, Gary J; Brough, Douglas E; King, C Richter; Gall, Jason G D


    Replication-defective adenovirus vectors, primarily developed from serotype 5 (Ad5) viruses, have been widely used for gene transfer and vaccination approaches. Vectors based on other serotypes of adenovirus could be used in conjunction with, or in place of, Ad5 vectors. In this study, Ad41, an enteric adenovirus usually described as 'non-cultivable' or 'fastidious,' has been successfully cloned, rescued and propagated on 293-ORF6 cells. The complementation capabilities of this cell line allow generation of Ad41 vectors at titers comparable to those obtained for Ad5 vectors. Mice immunized with an Ad41 vector containing an HIV envelope (Env) gene mounted anti-Env cellular and humoral immune responses. Ad41-Env vectors appear to be particularly attractive when used in heterologous prime-boost regimens, where they induce significantly higher cellular immune responses to HIV-Env than Ad5-based regimens. Ad41-based constructs are attractive vaccine vectors alone or in combination with Ad5 adenovectors, since each vector type can provide circumvention of pre-existing immunity to the other.

  10. Differential Innate Immune Stimulation Elicited by Adenovirus and Poxvirus Vaccine Vectors

    Teigler, Jeffrey Edward


    Vaccines are one of the most effective advances in medical science and continue to be developed for applications against infectious diseases, cancers, and autoimmunity. A common strategy for vaccine construction is the use of viral vectors derived from various virus families, with Adenoviruses (Ad) and Poxviruses (Pox) being extensively used. Studies utilizing viral vectors have shown a broad variety of vaccine-elicited immune response phenotypes. However, innate immune stimulation elicited b...

  11. Adenovirus Vectors Expressing Hantavirus Proteins Protect Hamsters against Lethal Challenge with Andes Virus ▿


    Hantaviruses infect humans following aerosolization from rodent feces and urine, producing hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Due to the high rates of mortality and lack of therapies, vaccines are urgently needed. Nonreplicating adenovirus (Ad) vectors that express Andes hantavirus (ANDV) nucleocapsid protein (AdN) or glycoproteins (AdGN and AdGC) were constructed. Ad vectors were tested for their ability to protect Syrian hamsters from a lethal ANDV infe...

  12. Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function.

    Stanton, Richard J; McSharry, Brian P; Armstrong, Melanie; Tomasec, Peter; Wilkinson, Gavin W G


    With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector of PCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector Vectors were optimized for high-throughput applications by making them "self-excising" through incorporating the I-SceI homing endonuclease into the vector removing the need to linearize vectors prior to transfection into packaging cells. AdZ vectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.

  13. Toxic activity of the CdtB component of Haemophilus ducreyi cytolethal distending toxin expressed from an adenovirus 5 vector.

    Wising, Catharina; Magnusson, Maria; Ahlman, Karin; Lindholm, Leif; Lagergård, Teresa


    The Haemophilus ducreyi cytolethal distending toxin (HdCDT) catalytic subunit CdtB has DNase-like activity and mediates DNA damage after its delivery into target cells. We constructed a replication-deficient adenovirus type 5 (Ad5) vector expressing CdtB and investigated the toxic properties of this vector on HeLa cells. Ad5CdtB caused loss of cell viability, morphologic changes, and cell cycle arrest, findings similar to HdCDT intoxication. This confirmed that CdtB is responsible for the toxicity of the holotoxin when expressed in cells following transduction by an adenoviral vector, and indicated a possible potential of this novel strategy in studies of activity of intracellular products and in gene therapy of cancer.

  14. Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes.

    Inesi, G; Lewis, D; Sumbilla, C; Nandi, A; Strock, C; Huff, K W; Rogers, T B; Johns, D C; Kessler, P D; Ordahl, C P


    Adenovirus-mediated transfer of cDNA encoding the chicken skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) yielded selective expression in cultured chick embryo cardiac myocytes under control of a segment (-268 base pair) of the cell-specific cardiac troponin T (cTnT) promoter or nonselective expression in myocytes and fibroblasts under control of a constitutive viral [cytomegalovirus (CMV)] promoter. Under optimal conditions nearly all cardiac myocytes in culture were shown to express transgenic SERCA1 ATPase. Expression was targeted to intracellular membranes and was recovered in subcellular fractions with a pattern identical to that of the endogenous SERCA2a ATPase. Relative to control myocytes, transgenic SERCA1 expression increased up to four times the rates of ATP-dependent (and thapsigargin-sensitive) Ca2+ transport activity of cell homogenates. Although the CMV promoter was more active than the cTnT promoter, an upper limit for transgenic expression of functional enzyme was reached under control of either promoter by adjustment of the adenovirus plaque-forming unit titer of infection media. Cytosolic Ca2+ concentration transients and tension development of whole myocytes were also influenced to a similar limit by transgenic expression of SERCA1 under control of either promoter. Our experiments demonstrate that a cell-specific protein promoter in recombinant adenovirus vectors yields highly efficient and selective transgene expression of a membrane-bound and functional enzyme in cardiac myocytes.

  15. (13) C-metabolic flux analysis of human adenovirus infection: Implications for viral vector production.

    Carinhas, Nuno; Koshkin, Alexey; Pais, Daniel A M; Alves, Paula M; Teixeira, Ana P


    Adenoviruses are human pathogens increasingly used as gene therapy and vaccination vectors. However, their impact on cell metabolism is poorly characterized. We performed carbon labeling experiments with [1,2-(13) C]glucose or [U-(13) C]glutamine to evaluate metabolic alterations in the amniocyte-derived, E1-transformed 1G3 cell line during production of a human adenovirus type 5 vector (AdV5). Nonstationary (13) C-metabolic flux analysis revealed increased fluxes of glycolysis (17%) and markedly PPP (over fourfold) and cytosolic AcCoA formation (nearly twofold) following infection of growing cells. Interestingly, infection of growth-arrested cells increased overall carbon flow even more, including glutamine anaplerosis and TCA cycle activity (both over 1.5-fold), but was unable to stimulate the PPP and was associated with a steep drop in AdV5 replication (almost 80%). Our results underscore the importance of nucleic and fatty acid biosynthesis for adenovirus replication. Overall, we portray a metabolic blueprint of human adenovirus infection, highlighting similarities with other viruses and cancer, and suggest strategies to improve AdV5 production. Biotechnol. Bioeng. 2017;114: 195-207. © 2016 Wiley Periodicals, Inc.

  16. Construction and effect of recombinant adenovirus vector containing siRNA targeting Raf-1 on cardiomyocyte hypertrophy in neonatal rats%Raf-1基因重组腺病毒 siRNA 载体构建及其对大鼠心肌细胞肥大的影响

    郑亚萍; 刘春杰


    Objective To construct an adenovirus vector expressing small interfering RNA ( siRNA) targeting to rat Raf-1 gene and identify its function in cardiomyocytes.Methods The siRNA containing DNA sequence targeting to Raf-1 and its negative control sequence were designed, synthesized, annealed and subcloned into adenoviral shuttle vector pAdTrack-CMV.The recombinant adenovirus vector pAd-siRaf-1 was obtained by homologous recombination with pAdTrack-siRaf-1 linearized by PmeI and pAdeasy-1 in bacteria BJ5183, then transfected into HEK293 cells to package the adenovirus.Cardiomyocytes were infected with the adenovirus pAd-siRaf-1, and the expressions of Raf-1 and NF-κB protein were detected by Western blotting.[ 3 H ]-leu incorporation was evaluated by scintillation.The surface area of cardiomyocytes was measured using a HJ2000 image analysis system.Results The adenovirus vectors were verified by enzyme digestion and DNA sequencing.Compared with the Ang II group, Raf-1 and NF-κB expression, the surface area and [ 3 H]-leu incorporation of cardiomyocytes were significantly decreased in cardiomyocytes infected with the adenovirus PAd-siRaf-1.Conclusions A recombinant adenovirus vector containing rat siRaf-1 gene is successfully constructed.It can effectively reduce Raf-1 and NF-κB expression and cardiomyocyte hypertrophy induced by Ang II.%目的:构建特异性抑制大鼠Raf-1基因的重组腺病毒载体,并将其体外转导大鼠心肌细胞中进行功能鉴定。方法合成针对大鼠Raf-1的靶序列及阴性对照序列,经退火形成的DNA双链定向克隆到穿梭质粒pAdTrackCMV中获得pAdTrack-siRaf-1质粒,PmeI线性化后在BJ5183细菌中与pAdEasy-1骨架质粒进行同源重组获得pAd-siRaf-1质粒,后转染HEK293细胞,包装获得pAd-siRaf-1腺病毒颗粒,继而感染原代培养的心肌细胞,通过Western blot方法检测siRNA对Raf-1、NF-κB基因的抑制效率,液体闪烁计数仪测定3 H-亮氨酸([3 H

  17. Construction and characterization of a recombinant human adenovirus vector expressing bone morphogenetic protein 2

    Zhang, Zheng; WANG, GUOXIAN; Li, Chen; Liu, Danping


    The aim of this study was to construct and characterize a novel recombinant human adenovirus vector expressing bone morphogenetic protein 2 (BMP2) and green fluorescent protein (GFP). The BMP2 gene in the plasmid pcDNA3-BMP2 was sequenced and the restriction enzyme recognition sites were analyzed. Following mutagenesis using polymerase chain reaction (PCR), the gene sequence after the translation termination codon was removed and new restriction sites were added. The mutated BMP2 gene (BMP2+ ...


    Li Jian; Jiang Lei; Xia Yongjing; Li Hongxia; Hu Yajun; Hu Shixue; Xu Hongji


    Objective:To study the role of the most extensively studied tumor suppressor gene, retinoblastoma (Rb) gene,on the growth of lung adenocarcinoma cell line GLC-82 and explore a gene therapy approach for lung adenocarcinoma. Methods: The recombinant Rb gene adenovirus vector was constructed, the control virus which carries LacZ gene was producted by the same method. Infection effects were detected by biochemical staining of β-gal and immunohistochemical analysis of Rb protein. The Rb cDNA of infected cells were determined by PCR. The cell growth rate and cell cycle were observed by cell-counting and flow cytometry. Results: The constructed recombinant adenovirus vector could infect effectively the cells with high level expression of Rb cDNA and Rb protein. The transfection of wild-type Rb gene could suppress GLC-82 cell proliferation and decrease the cellular DNA synthesis. Conclusions: These results showed the possibility of using recombinant Rb gene adenovirus vector in the gene therapy of cancer to inhibit the growth of cancer.

  19. CAR chasing: canine adenovirus vectors-all bite and no bark?

    Kremer, Eric J


    This review deals primarily with canine adenovirus serotype 2 (CAV-2) vectors and gives a simplified overview of how the various domains of virology, cellular and molecular biology, as well as immunology, come into play when trying to understand and ameliorate adenovirus (Ad)-mediated gene transfer. The generation of early region 1 (E1)-deleted (DeltaE1) CAV-2 vectors, the lack of pre-existing humoral immunity, trafficking, the use of the coxsackie B adenovirus receptor (CAR), the surprising neuronal tropism, and the ability to migrate via axons to afferent regions of the central and peripheral nervous system, are described. Due to these intrinsic properties, CAV-2 vectors may be powerful tools for the study of the pathophysiology and potential treatment of neurodegenerative diseases like lysosomal storage disorders, Parkinson's, Alzheimer's, Huntington's, amyotrophic lateral sclerosis, and others. Other potential uses include anti-tumoral and anti-viral vaccines, tracer of synaptic junctions, pain therapy, cancer therapy (e.g. K9 CRAds), and gene transfer to other somatic tissues.

  20. 用RNA干扰腺病毒载体研究Wnt/β-连环蛋白信号途径对人甲状腺细胞增殖的影响%Construction of GSK-3β-targeting RNAi adenovirus vector and the influence of Wnt/β-catenin pathway in proliferation of human thyrocytes

    陈刚; 邹欣; 林丽香; 牟伦盼; 姚瑾; 游婷婷; 沈小燕; 朱香清; 乔玉芳; 林苗; 方晓文


    Objective To construct RNAi recombinant adenoviral expressive vectors specific to glycogen synthase kinase-3β (GSK-3β) and to observe its gene knockdown effect on the expression of GSK-3β, and to explore the effect of Wnt/β-catenin pathway on the proliferation of human thyrocytes using the RNAi adenovirus vector. Methods An adenovirus plasmid that contained the RNAi cassette targeting the GSK-3β gene was constructed by homologous recombination and cloning techniques, transfected into human embryo kidney (HEK) 293A cells to product adenovirus, and then was used to infect the HEK293A cells to amplify the adenoviral stock. Plaque forming assay was used to titer the adenoviral stock . Normal human thyrocytes fart from thyroid adenoma were obtained during resection of adenoma, cultured, and infected by the GSK-3β specific RNAi adenovirus. The GSK-3β gene silencing effect induced by the RNAi adenovirus was detected by Western blotting 0, 24, 48, 72, 120, and 144 hours later. BrdU method was used to detect the cell proliferation. Another HEK293A cells were divided into 3 groups: infected with recombinant adenovirus plasmid Ad-1457, infected with un-recombinant framework plasmid pAd-DEST, and un-infected. 72 hours later Western blotting was used to examine the level of β-catenin. Results The GSK-3β expression of the thyrocytes infected with the recombinant adenovirus plasmid Ad-1457 were significantly lower than those of the thyrocytes infected with Ad-DEST (all P <0.05). The expression of β-catenin of the thyrocytes infected with Ad-DEST was significantly higher than those of the Ad-DEST group and un-infected group (both P<0.05). BrdU assay suggested that the proliferation rates 1,3, 5, and 7 days after infection of the thyrocytes infected with Ad1457 plasmid were significantly higher than those of the thyrocytes infected with the plasmid pAd-DEST (all P<0.05). Conclusion RNAi adenovirus is an important tool inhibiting the expression of target gene efficiently

  1. Gene targeting with retroviral vectors

    Ellis, J.; Bernstein, A. (Toronto Univ., ON (Canada))


    The authors have designed and constructed integration-defective retroviral vectors to explore their potential for gene targeting in mammalian cells. Two nonoverlapping deletion mutants of the bacterial neomycin resistance (neo) gene were used to detect homologous recombination events between viral and chromosomal sequences. Stable neo gene correction events were selected at a frequency of approximately 1 G418/sup r/ cell per 3 x 10/sup 6/ infected cells. Analysis of the functional neo gene in independent targeted cell clones indicated that unintegrated retroviral linear DNA recombined with the target by gene conversion for variable distances into regions of nonhomology. In addition, transient neo gene correction events which were associated with the complete loss of the chromosomal target sequences were observed. These results demonstrated that retroviral vectors can recombine with homologous chromosomal sequences in rodent and human cells.

  2. A targeting ligand enhances infectivity and cytotoxicity of an oncolytic adenovirus in human pancreatic cancer tissues.

    Yamamoto, Yuki; Hiraoka, Nobuyoshi; Goto, Naoko; Rin, Yosei; Miura, Kazuki; Narumi, Kenta; Uchida, Hiroaki; Tagawa, Masatoshi; Aoki, Kazunori


    The addition of a targeting strategy is necessary to enhance oncolysis and secure safety of a conditionally replicative adenovirus (CRAd). We have constructed an adenovirus library displaying random peptides on the fiber, and have successfully identified a pancreatic cancer-targeting ligand (SYENFSA). Here, the usefulness of cancer-targeted CRAd for pancreatic cancer was examined as a preclinical study. First, we constructed a survivin promoter-regulated CRAd expressing enhanced green fluorescent protein gene (EGFP), which displayed the identified targeting ligand (AdSur-SYE). The AdSur-SYE resulted in higher gene transduction efficiency and oncolytic potency than the untargeted CRAd (AdSur) in several pancreatic cancer cell lines. An intratumoral injection of AdSur-SYE significantly suppressed the growth of subcutaneous tumors, in which AdSur-SYE effectively proliferated and spread. An ectopic infection in adjacent tissues and organs of intratumorally injected AdSur-SYE was decreased compared with AdSur. Then, to examine whether the targeting ligand actually enhanced the infectivity of CRAd in human pancreatic cancer tissues, tumor cells prepared from surgical specimens were infected with viruses. The AdSur-SYE increased gene transduction efficiency 6.4-fold higher than did AdSur in single cells derived from human pancreatic cancer, whereas the infectivity of both vectors was almost the same in the pancreas and other cancers. Immunostaining showed that most EGFP(+) cells were cytokeratin-positive in the sliced tissues, indicating that pancreatic cancer cells but not stromal cells were injected with AdSur-SYE. AdSur-SYE resulted in a stronger oncolysis in the primary pancreatic cancer cells co-cultured with mouse embryonic fibroblasts than AdSur did. CRAd in combination with a tumor-targeting ligand is promising as a next-generation of oncolytic virotherapy for pancreatic cancer.

  3. pri-miR-21重组腺病毒载体构建及其对TLR4基因调控的研究%Construction of a recombined adenovirus vector carrying pri-miR-21 gene and research on it's target gene TLR4

    赵婧; 徐广贤; 贾伟; 董辉; 张一琳; 赵志军; 魏军


    目的:构建能高效表达成熟miR-21小分子的腺病毒载体,探讨其对TLR4基因的靶向调控关系.方法:以正常小鼠基因组DNA为模板,PCR扩增pri-miR-21基因,克隆至穿梭载体pAdTrack-CMV中经PCR、酶切及基因测序分析正确无误后,与pAdEasy-1腺病毒骨架质粒进行同源重组,并利用293A细胞,包装成pri-miR-21重组腺病毒感染HeLa细胞,通过Westem blot检测TLR4的蛋白表达水平,验证miR-21与TLR4靶向调控的关系.结果:经PCR、酶切、测序及GFP表达证实,成功构建携带pri-miR-21基因的重组腺病毒载体并制备出高滴度重组病毒.Western blot证实,miR-21可抑制TLR4蛋白的表达.结论:所制备的小鼠pri- miR-21重组腺病毒,可以高效表达成熟miR-21小分子,能够抑制靶基因TLR4的表达.%AIM: To construct the recombined adenovi-rus vector carrying pri-miR-21 gene, which can express mature miR-21 efficiently, and to study the interaction of miR-21 with its target gene TLR4. METHODS: Using healthy mouse's gDNA as template, the primary miR-21 coding sequence was amplified by PCR and cloned into a shuttle vector pAdTrack-CMV. Constructed plasmid was sequenced and linearized for homologous recombination with pAdEasy-1 vector in BJ5183 bacteria. The recombined adenovirus vector carrying pri-miR-21 gene was used to challenge HeLa cell. The candidate target gene of miR-21 was determined by miRNA analysis databases. The expression level of TLR4 protein was detected by western blotting. RESULTS; Through the PCR, restriction enzyme digestion, DNA sequencing and expression of GFP, recombinant adenoviral vector pri-miR-21 gene was constructed successfully. Bioin-formatic analysis suggested a few possible binding sites between miR-21 and TLR4. Results showed that miR-21 down-regulated TLR4 at protein levels. CONCLUSION-. The recombinant adenoviral vector containing pri- miR-21 was successfully constructed. miR-21 gene interfered with the expression of TLR4 target gene.

  4. Peptide targeting of adenoviral vectors to augment tumor gene transfer.

    Ballard, E N; Trinh, V T; Hogg, R T; Gerard, R D


    Adenovirus serotype 5 remains one of the most promising vectors for delivering genetic material to cancer cells for imaging or therapy, but optimization of these agents to selectively promote tumor cell infection is needed to further their clinical development. Peptide sequences that bind to specific cell surface receptors have been inserted into adenoviral capsid proteins to improve tumor targeting, often in the background of mutations designed to ablate normal ligand:receptor interactions and thereby reduce off target effects and toxicities in non-target tissues. Different tumor types also express highly variable complements of cell surface receptors, so a customized targeting strategy using a particular peptide in the context of specific adenoviral mutations may be needed to achieve optimal efficacy. To further investigate peptide targeting strategies in adenoviral vectors, we used a set of peptide motifs originally isolated using phage display technology that evince tumor specificity in vivo. To demonstrate their abilities as targeting motifs, we genetically incorporated these peptides into a surface loop of the fiber capsid protein to construct targeted adenovirus vectors. We then systematically evaluated the ability of these peptide targeted vectors to infect several tumor cell types, both in vitro and in vivo, in a variety of mutational backgrounds designed to reduce CAR and/or HSG-mediated binding. Results from this study support previous observations that peptide insertions in the HI loop of the fiber knob domain are generally ineffective when used in combination with HSG detargeting mutations. The evidence also suggests that this strategy can attenuate other fiber knob interactions, such as CAR-mediated binding, and reduce overall viral infectivity. The insertion of peptides into fiber proved more effective for targeting tumor cell types expressing low levels of CAR receptor, as this strategy can partially compensate for the very low infectivity of wild

  5. Construction of Rat Calcineurin A α cDNA Recombinant Adenovirus Vector and Its Identification


    Rat calcineurin (CaN) A α isoform (Ppp3ca) cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemiareperfusion injury. Total RNA was isolated from the heart of the adult Wistar rat, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EG-FP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment (3.97 kb) obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno-Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect (CPE) appeared.The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results veri fied that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A α (Ppp3ca) cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.

  6. Use of adenovirus vector expressing the mouse full estrogen receptor alpha gene to infect mouse primary neurons

    Xiao HU; Lei Lou; Jun Yuan; Xing Wan; Jianyi Wang; Xinyue Qin


    Estrogen plays important regulatory and protective roles in the central nervous system through estrogen receptor a mediation.Previous studies applied eukaryotic expression and lentiviral vectors carrying estrogen receptor a to clarify the undedying mechanisms,in the present study,an adenovirus vector expressing the mouse full estrogen receptor a gene was constructed to identify biological characteristics of estrogen receptor a recombinant adenovirus infecting nerve cells.Primary cultured mouse nerve cells were first infected with estrogen receptor a recombinant adenovirus at various multiplicities of infection,followed by 100 multiplicity of infection.Results showed overexpression of estrogen receptor a mRNA and protein in the infected nerve cells.Estrogen receptor a recombinant adenovirus at 100 multiplicity of infection successfully infected neurons and upregulated estrogen receptor a mRNA and protein expression.

  7. Modification to the Capsid of the Adenovirus Vector That Enhances Dendritic Cell Infection and Transgene-Specific Cellular Immune Responses

    Worgall, Stefan; Busch, Annette; Rivara, Michael; Bonnyay, David; Leopold, Philip L.; Merritt, Robert; Hackett, Neil R.; Rovelink, Peter W.; Joseph T Bruder; Wickham, Thomas J.; Kovesdi, Imi; Crystal, Ronald G.


    Adenovirus (Ad) gene transfer vectors can be used to transfer and express antigens and function as strong adjuvants and thus are useful platforms for the development of genetic vaccines. Based on the hypothesis that Ad vectors with enhanced infectibility of dendritic cells (DC) may be able to evoke enhanced immune responses against antigens encoded by the vector in vivo, the present study analyzes the vaccine potential of an Ad vector expressing β-galactosidase as a model antigen and genetica...

  8. Adenovirus Vector-Derived VA-RNA-Mediated Innate Immune Responses

    Hiroyuki Mizuguchi


    Full Text Available The major limitation of the clinical use of replication-incompetent adenovirus (Ad vectors is the interference by innate immune responses, including induction of inflammatory cytokines and interferons (IFN, following in vivo application of Ad vectors. Ad vector-induced production of inflammatory cytokines and IFNs also results in severe organ damage and efficient induction of acquired immune responses against Ad proteins and transgene products. Ad vector-induced innate immune responses are triggered by the recognition of Ad components by pattern recognition receptors (PRRs. In order to reduce the side effects by Ad vector-induced innate immune responses and to develop safer Ad vectors, it is crucial to clarify which PRRs and which Ad components are involved in Ad vector-induced innate immune responses. Our group previously demonstrated that myeloid differentiating factor 88 (MyD88 and toll-like receptor 9 (TLR9 play crucial roles in the Ad vector-induced inflammatory cytokine production in mouse bone marrow-derived dendritic cells. Furthermore, our group recently found that virus associated-RNAs (VA-RNAs, which are about 160 nucleotide-long non-coding small RNAs encoded in the Ad genome, are involved in IFN production through the IFN-β promoter stimulator-1 (IPS-1-mediated signaling pathway following Ad vector transduction. The aim of this review is to highlight the Ad vector-induced innate immune responses following transduction, especially VA-RNA-mediated innate immune responses. Our findings on the mechanism of Ad vector-induced innate immune responses should make an important contribution to the development of safer Ad vectors, such as an Ad vector lacking expression of VA-RNAs.

  9. Evaluation of helper-dependent canine adenovirus vectors in a 3D human CNS model.

    Simão, D; Pinto, C; Fernandes, P; Peddie, C J; Piersanti, S; Collinson, L M; Salinas, S; Saggio, I; Schiavo, G; Kremer, E J; Brito, C; Alves, P M


    Gene therapy is a promising approach with enormous potential for treatment of neurodegenerative disorders. Viral vectors derived from canine adenovirus type 2 (CAV-2) present attractive features for gene delivery strategies in the human brain, by preferentially transducing neurons, are capable of efficient axonal transport to afferent brain structures, have a 30-kb cloning capacity and have low innate and induced immunogenicity in preclinical tests. For clinical translation, in-depth preclinical evaluation of efficacy and safety in a human setting is primordial. Stem cell-derived human neural cells have a great potential as complementary tools by bridging the gap between animal models, which often diverge considerably from human phenotype, and clinical trials. Herein, we explore helper-dependent CAV-2 (hd-CAV-2) efficacy and safety for gene delivery in a human stem cell-derived 3D neural in vitro model. Assessment of hd-CAV-2 vector efficacy was performed at different multiplicities of infection, by evaluating transgene expression and impact on cell viability, ultrastructural cellular organization and neuronal gene expression. Under optimized conditions, hd-CAV-2 transduction led to stable long-term transgene expression with minimal toxicity. hd-CAV-2 preferentially transduced neurons, whereas human adenovirus type 5 (HAdV5) showed increased tropism toward glial cells. This work demonstrates, in a physiologically relevant 3D model, that hd-CAV-2 vectors are efficient tools for gene delivery to human neurons, with stable long-term transgene expression and minimal cytotoxicity.

  10. Construction of a recombinant adenovirus Vector of human papillomavirus type 16 L1_E7c


    Human papillomaviruses are closely associated with human cervical cancer, especially HPV types 16 and 18. At present, HPV can not be produced in large quantity; it also has tumorgenicity and these properties of HPV have seriously hampered the development of HPV vaccine. HPV type 16 L1 proteins can assembled into virus-like particles (VLP), which are morphologically identical to the nature virion. In order to develop the recombinant adenovirus vectors of HPV, we constructed a recombinant adenovirus shuttle plasmid pCA14 L1-E7c. Methods: Human papillomavirus type 16 L1 open reading frame without terminator codon (TAA) (5559- 7152) and E7c (682- 855) were amplified using PCR. The L1 and E7c fragments were inserted into pGEM-T easy vectors by T- A strategy, named pTAL1 and pTAE7c. pTAL1 was cut with Hind III and BglII, the pTAE7c with BamHI and ClaI. The L1 DNA fragment, E7c and pBluesscript SK were ligated together using T4 DNA ligase. pBSL1-E7c and pBSL1-E7c was digested with Hind III and Xhol. The L1-E7c fragment was inserted into adenovirus shuttle plasmids pCAl4, named pCAl4L1-E7c. DNA sequence results indicated that The L1-E7c DNA fragment can encode the HPV16L1-E7 fusion protein correctly. Results: The L1 and E7c DNA fragments were amplified by PCR and recombinant plasmid pTAL1, pTAE7c, pBSL1-E7c and pCA14L1-E7c were constructed correctly. The pCAl4L1-E7c can be used in the further research work, cotransfected the 293 cell with the parent adenovirus pBHG10. Conclusion: Our results indicated that we have constructed a HPV16L1-E7 fusion DNA fragments and the adenovirus shuttle plasmids pCALl-E7c for the further research.

  11. Effect of recombinant adenovirus vector mediated human interleukin-24 gene transfection on pancreatic carcinoma growth

    PAN Xin-ting; ZHU Qing-yun; LI De-chun; YANG Ji-cheng; ZHANG Zi-xiang; ZHU Xing-guo; ZHAO Hua


    Background Pancreatic cancer is a highly malignant tumor affecting an ever increasing number of patients with a mean 5-year survival rate below 4%. Therefore, gene therapy for cancer has become a potential novel therapeutic modality. In this study we sought to determine the inhibitory effects of adenovirus-mediated human interleukin-24 (AdhlL-24) on pancreatic cancer.Methods Human interleukin-24 gene was cloned into replication-defective adenovirus specific for patu8988 tumor cells by virus recombination technology. Reverse transcription-polymerase chain reaction and Western blotting analysis were used to determine the expression of human interleukin-24 mRNA in patu8988 cells in vitro. Induction of apoptosis by overexpression of human interleukin-24 in patu8988 cells was determined by flow cytometry. In vivo efficacy of adenoviral delivery of human interleukin-24 was assessed in nude mice (n=10 for each group) bearing patu8988 pancreatic cancer cell lines by determining inhibition of tumor growth, endothelial growth factor and CD34 expression, and intratumoral microvessel density (MVD).Results The recombinant adenovirus vector AdVGFP/IL-24 was constructed with a packaged recombinant retrovirus titer of 1.0x1010 pfu/ml and successfully expressed of both mRNA and protein in patu8988 cells. The AdVGFP/IL-24 induced apoptosis of patu8988 tumor cells in vitro and significantly inhibited tumor growth in vivo (P <0.05). The intratumoral MVD decreased significantly in the treated tumors (P <0.05).Conclusion The recombinant adenovirus AdGFP/IL-24 can effectively express biologically active human interleukin-24, which results in inhibition of pancreatic cancer growth.

  12. Immunogenic comparison of chimeric adenovirus 5/35 vector carrying optimized human immunodeficiency virus clade C genes and various promoters.

    Shoji, Masaki; Yoshizaki, Shinji; Mizuguchi, Hiroyuki; Okuda, Kenji; Shimada, Masaru


    Adenovirus vector-based vaccine is a promising approach to protect HIV infection. However, a recent phase IIb clinical trial using the vector did not show its protective efficacy against HIV infection. To improve the vaccine, we explored the transgene protein expression and its immunogenicity using optimized codon usage, promoters and adaptors. We compared protein expression and immunogenicity of adenovirus vector vaccines carrying native or codon usage-optimized HIV-1 clade C gag and env genes expression cassettes driven by different promoters (CMV, CMVi, and CA promoters) and adapters (IRES and F2A). The adenovirus vector vaccine containing optimized gag gene produced higher Gag protein expression and induced higher immune responses than the vector containing native gag gene in mice. Furthermore, CA promoter generated higher transgene expression and elicited higher immune responses than other two popularly used promoters (CMV and CMVi). The second gene expression using F2A adaptor resulted in higher protein expression and immunity than that of using IRES and direct fusion protein. Taken together, the adenovirus vector containing the expression cassette with CA promoter, optimized HIV-1 clade C gene and an F2A adaptor produced the best protein expression and elicited the highest transgene-specific immune responses. This finding would be promising for vaccine design and gene therapy.

  13. Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles.

    Stefania Piersanti

    Full Text Available Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD human (HAd and canine (CAV-2 adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS. With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways--but in opposite directions--suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer.

  14. Increased gene transfer in acute myeloid leukemic cells by an adenovirus vector containing a modified fiber protein.

    Gonzalez, R; Vereecque, R; Wickham, T J; Vanrumbeke, M; Kovesdi, I; Bauters, F; Fenaux, P; Quesnel, B


    Applications of gene transfer in acute myeloid leukemia (AML) blast cells have still not been developed, mostly due to the lack of an efficient vector. Adenoviruses have many advantages as vectors, but remain poorly efficient in cells lacking fiber receptors. A promising strategy is the retargeting of adenoviruses to other cellular receptors. We report the dramatic enhancement of gene transfer efficiency in AML blasts using AdZ.F(pK7), a modified adenovirus containing a heparin/heparan sulfate binding domain incorporated into the fiber protein of the adenovirus. We transduced 25 AML blast samples with efficiency reaching 100% of the cells in most samples. Optimal results were obtained at 8400 physical particles per cell, corresponding to a multiplicity of infection of 100 plaque forming units per cell. Control AdZ.F adenovirus efficiently transduced leukemic cell lines but gave poor results in AML samples. Both addition of soluble heparin and cell treatment with heparinase inhibited AdZ.F(pK7) gene transfer, showing that heparan sulfates are the major receptors mediating AdZ.F(pK7) transduction of AML blasts. Although adenoviruses can infect nondividing cells, we observed that a combination of growth factors (GM-CSF, IL-3, stem cell factor) was required for efficient transduction in order to maintain AML blast cell viability. This study demonstrates that retargeting the adenovirus fiber protein to heparan sulfates can overcome the low efficiency of adenovirus in AML blast cells and may provide a useful tool for gene therapy approaches in AML.

  15. Gene therapy for allergic rhinitis with recombinant adenovirus vector containing CTLA4Ig in mice

    朱瑾; 吴军; 陈希炜; 易绍萱; 罗高兴; 贺伟峰


    Objective: To examine the role of recombinant adenovirus vector containing CTLA4Ig gene(Ad-CTLA4Ig) in the treatment of induced allergic rhinitis in mice.Methods: Allergic rhinitis was induced by sensitizing and challenging with ovalbumin(OVA).Ad-CTLA4Ig was intraperitoneally injected 30 min before OVA challenge.Adenovirus vector without inserted CTLA4Ig cDNA served as the control.The symptoms and morphological changes of nasal mucosa of each group were observed, and the serum levels of IgE against OVA were detected with ELISA.Results: There were no obvious symptoms and pathological changes in Ad-CTLA4Ig treated group, in which the serum OVA-specific IgE levels were significantly lower than that in control groups(P< 0.05).Conclusion: Ad-CTLA4Ig prevents and treats allergic rhinitis of mice,implying the possibility of the usage of Ad-CTLA4Ig against allergic rhinitis in clinic in future.

  16. Inhibition of HIV-1 replication in alveolar macrophages by adenovirus gene transfer vectors.

    Rice, Joshua; Connor, Ruth; Worgall, Stefan; Moore, John P; Leopold, Philip L; Kaner, Robert J; Crystal, Ronald G


    To assess the hypothesis that infection of alveolar macrophages (AM) with adenovirus (Ad) gene transfer vectors might prevent subsequent human immunodeficiency virus (HIV)-1 replication in AM, AM isolated from normal volunteers were infected with increasing doses of first generation (E1(-)) Ad vectors, followed 72 h later by infection with HIV-1(JRFL), an R5/M-tropic strain that preferentially uses the CCR5 coreceptor. As a measure of HIV-1 replication, p24 Ag was quantified by enzyme-linked imunosorbent assay in supernatants on Days 4 to 14 after HIV-1infection. Pretreatment of the AM with an Ad vector resulted in a dose- and time-dependent suppression of subsequent HIV-1 replication. The Ad vector inhibition of HIV-1 replication was independent of the transgene in the Ad vector expression cassette and E4 genes in the Ad backbone. Moreover, it did not appear to be secondary to a soluble factor released by the AM, nor was it overridden by the concomitant transfer of the CCR5 or CXCR4 receptors to the AM before HIV-1 infection. These observations have implications regarding pulmonary host responses associated with HIV-1 infection, as well as possibly uncovering new therapeutic strategies against HIV-1 infection.

  17. Comparison between Sendai virus and adenovirus vectors to transduce HIV-1 genes into human dendritic cells.

    Hosoya, Noriaki; Miura, Toshiyuki; Kawana-Tachikawa, Ai; Koibuchi, Tomohiko; Shioda, Tatsuo; Odawara, Takashi; Nakamura, Tetsuya; Kitamura, Yoshihiro; Kano, Munehide; Kato, Atsushi; Hasegawa, Mamoru; Nagai, Yoshiyuki; Iwamoto, Aikichi


    Immuno-genetherapy using dendritic cells (DCs) can be applied to human immunodeficiency virus type 1 (HIV-1) infection. Sendai virus (SeV) has unique features such as cytoplasmic replication and high protein expression as a vector for genetic manipulation. In this study, we compared the efficiency of inducing green fluorescent protein (GFP) and HIV-1 gene expression in human monocyte-derived DCs between SeV and adenovirus (AdV). Human monocyte-derived DCs infected with SeV showed the maximum gene expression 24 hr after infection at a multiplicity of infection (MOI) of 2. Although SeV vector showed higher cytopathic effect on DCs than AdV, SeV vector induced maximum gene expression earlier and at much lower MOI. In terms of cell surface phenotype, both SeV and AdV vectors induced DC maturation. DCs infected with SeV as well as AdV elicited HIV-1 specific T-cell responses detected by interferon gamma (IFN-gamma) enzyme-linked immunospot (Elispot). Our data suggest that SeV could be one of the reliable vectors for immuno-genetherapy for HIV-1 infected patients.

  18. An adenoviral vector-based expression and delivery system for the inhibition of wild-type adenovirus replication by artificial microRNAs.

    Ibrišimović, Mirza; Kneidinger, Doris; Lion, Thomas; Klein, Reinhard


    Human adenoviruses are rarely associated with life-threatening infections in healthy individuals. However, immunocompromised patients, and particularly allogeneic hematopoietic stem cell transplant recipients, are at high risk of developing disseminated and potentially fatal disease. The efficacy of commonly used drugs to treat adenovirus infections (i.e., cidofovir in most cases) is limited, and alternative treatment options are needed. Artificial microRNAs (amiRNAs) are a class of synthetic RNAs resembling cellular miRNAs, and, similar to their natural relatives, can mediate the knockdown of endogenous gene expression. This process, termed RNA interference, can be harnessed to target and potentially silence both cellular and viral genes. In this study, we designed amiRNAs directed against adenoviral E1A, DNA polymerase, and preterminal protein (pTP) mRNAs in order to inhibit adenoviral replication in vitro. For the expression of amiRNA-encoding sequences, we utilized replication-deficient adenoviral vectors. In cells transduced with the recombinant vectors and infected with the wild-type (wt) adenovirus, one particular amiRNA that was directed against the pTP mRNA was capable of decreasing the output of infectious wt virus progeny by 2.6 orders of magnitude. This inhibition rate could be achieved by concatemerizing amiRNA-encoding sequences to allow for high intracellular amiRNA concentrations. Because superinfecting wt virus induces the replication and amplification of the recombinant adenoviral vector, amiRNA concentrations were increased in cells infected with wt adenovirus. Furthermore, a combination of amiRNA expression and treatment of infected cells with cidofovir resulted in additive effects that manifested as a total reduction of infectious virus progeny by greater than 3 orders of magnitude.

  19. Evaluation of helper-dependent canine adenovirus vectors in a 3D human CNS model

    Simão, Daniel; Pinto, Catarina; Fernandes, Paulo; Peddie, Christopher J.; Piersanti, Stefania; Collinson, Lucy M.; Salinas, Sara; Saggio, Isabella; Schiavo, Giampietro; Kremer, Eric J.; Brito, Catarina; Alves, Paula M.


    Gene therapy is a promising approach with enormous potential for treatment of neurodegenerative disorders. Viral vectors derived from canine adenovirus type 2 (CAV-2) present attractive features for gene delivery strategies in the human brain, by preferentially transducing neurons, are capable of efficient axonal transport to afferent brain structures, have a 30-kb cloning capacity and have low innate and induced immunogenicity in pre-clinical tests. For clinical translation, in-depth pre-clinical evaluation of efficacy and safety in a human setting is primordial. Stem cell-derived human neural cells have a great potential as complementary tools by bridging the gap between animal models, which often diverge considerably from human phenotype, and clinical trials. Herein, we explore helper-dependent CAV-2 (hd-CAV-2) efficacy and safety for gene delivery in a human stem cell-derived 3D neural in vitro model. Assessment of hd-CAV-2 vector efficacy was performed at different multiplicities of infection, by evaluating transgene expression and impact on cell viability, ultrastructural cellular organization and neuronal gene expression. Under optimized conditions, hd-CAV-2 transduction led to stable long-term transgene expression with minimal toxicity. hd-CAV-2 preferentially transduced neurons, while human adenovirus type 5 (HAdV5) showed increased tropism towards glial cells. This work demonstrates, in a physiologically relevant 3D model, that hd-CAV-2 vectors are efficient tools for gene delivery to human neurons, with stable long-term transgene expression and minimal cytotoxicity. PMID:26181626

  20. De Novo Synthesis of Marburg Virus Antigens from Adenovirus Vectors Induce Potent Humoral and Cellular Immune Responses


    cells and purified by ultra-centrifugation in esium chloride gradients. Briefly, adenoviral lysates from hirty 150-mm plates were banded twice on CsCl...differences of individuals with cystic fibrosis con- sequent to local administration of a normal CFTR cDNA adenovirus gene transfer vector. Hum Gene Ther

  1. Modifications of the fiber in adenovirus vectors increase tropism for malignant glioma models.

    Staba, M J; Wickham, T J; Kovesdi, I; Hallahan, D E


    Recombinant adenovirus (Ad) vectors provide a means of local, therapeutic gene delivery to a wide range of neoplasms. Ad-mediated gene therapy trials in malignant glioma models have been limited by the need for high viral titers and multiple dosages. In an attempt to improve Ad vector gene transfer, we studied human (U87, D54) and rodent (GL261, C6) malignant glioma cell lines transfected with various doses of unmodified Ad vectors (AdZ), Ad vectors that contain an alteration of the fiber-coat protein and that direct virus binding to heparan sulfate receptors (AdZ.F(pK7)), and Ad vectors with modifications of the fiber-coat protein that direct virus binding to alpha1, integrin cellular receptors (AdZ.F(RGD)). AdZ.F(pK7) increased the frequency of cells expressing the reporter gene, beta-galactosidase, and improved transduction by 2- to 20-fold compared with AdZ in U87, D54, and GL261 cells. In U87, D54, GL261, and C6 tumors, AdZ.F(pK7) increased gene transfer by 10- to 100-fold compared with AdZ. AdZ.F(RGD) increased gene expression in C6 xenografts compared with AdZ, but had reduced transduction compared with the C6 xenografts of AdZ in all other glioma tumors. These findings suggest that the increased tropisms resulting from alterations of the Ad vector fiber-coat protein as in AdZ.F(pK7) and AdZ.F(RGD) offer a feasible approach to improving in vitro and in vivo transduction efficiencies in certain malignant glioma cell lines.

  2. In situ tumor vaccination with adenovirus vectors encoding measles virus fusogenic membrane proteins and cytokines

    Dennis Hoffmann; Wibke Bayer; Oliver Wildner


    AIM: To evaluate whether intratumoral expression of measles virus fusogenic membrane glycoproteins H and "F (MV-FMG), encoded by an adenovirus vector Ad.MV-H/ F, alone or in combination with local coexpression of cytokines (IL-2, IL-12, IL-18, IL-21 or GM-CSF), can serve as a platform for inducing tumor-specific immune responses in colon cancer.METHODS: We used confocal laser scanning microscopy and flow cytometry to analyze cell-cell fusion after expression of MV-FMG by dye colocalization. In a syngeneic bilateral subcutaneous MC38 and Colon26 colon cancer model in C57BL/6 and BALB/c mice, we assessed the effect on both the directly vector-treated tumor as well as the contralateral, not directly vector-treated tumor. We assessed the induction of a tumor-specific cytotoxic T lymphocyte (CTL) response with a lactate dehydrogenase (LDH) release assay.RESULTS: We demonstrated in vitro that transduction of MC38 and Colon26 cells with Ad.MV-H/F resulted in dye colocalization, indicative of cell-cell fusion. In addition, in the syngeneic bilateral tumor model we demonstrated a significant regression of the directly vector-inoculated tumor upon intratumoral expression of MV-FMG alone or in combination with the tested cytokines. We observed the highest anti-neoplastic efficacy with MV-FMG and IL-21 coexpression. The degree of tumor regression of the not directly vector-treated tumor correlated with the anti-neoplastic response of the directly vector-treated tumor. This regression was mediated by a tumor-specific CTL response.CONCLUSION: Our data indicate that intratumoral expression of measles virus fusogenic membrane glycoproteins is a promising tool both for direct tumor treatment as well as for tumor vaccination approaches that can be further enhanced by cytokine coexpression.

  3. Qualitative and quantitative analysis of adenovirus type 5 vector-induced memory CD8 T cells

    Steffensen, Maria Abildgaard; Holst, Peter Johannes; Steengaard, Sanne Skovvang


    is followed by sustained expansion of the memory CD8 T-cell population, and the generated memory cells do not appear to have been driven towards exhaustive differentiation. Based on these findings, we suggest that adenovirus based prime-boost regimens (including Ad5 and Ad5-like vectors) represent...... adenoviral boosting and, importantly, the generated secondary memory cells cannot be qualitatively differentiated from those induced by primary infection with replicating virus. Quantitatively, DNA priming prior to Ad-vaccination will lead to even higher numbers of memory cells. In this case, the vaccination...... leads to the generation of a population of memory cells characterized by relatively low CD27 expression and high CD127 and KLRG1 expression. These memory CD8 T cells are capable of proliferating in response to viral challenge, and protect against infection with live virus. Furthermore, viral challenge...

  4. Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication

    Pei, Zheng; Shi, Guoli; Kondo, Saki; Ito, Masahiko; Maekawa, Aya; Suzuki, Mariko; Saito, Izumu; Suzuki, Tetsuro; Kanegae, Yumi


    First-generation adenovirus vectors (FG AdVs) expressing short-hairpin RNA (shRNA) effectively downregulate the expressions of target genes. However, this vector, in fact, expresses not only the transgene product, but also virus-associated RNAs (VA RNAs) that disturb cellular RNAi machinery. We have established a production method for VA-deleted AdVs lacking expression of VA RNAs. Here, we showed that the highest shRNA activity was obtained when the shRNA was inserted not at the popularly used E1 site, but at the E4 site. We then compared the activities of shRNAs against hepatitis C virus (HCV) expressed from VA-deleted AdVs or conventional AdVs. The VA-deleted AdVs inhibited HCV production much more efficiently. Therefore, VA-deleted AdVs were more effective than the currently used AdVs for shRNA downregulation, probably because of the lack of competition between VA RNAs and the shRNAs. These VA-deleted AdVs might enable more effective gene therapies for chronic hepatitis C.

  5. Use of recombinant adenovirus vectored consensus IFN-α to avert severe arenavirus infection.

    Brian B Gowen

    Full Text Available Several arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from 15-30%. Because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the National Institutes of Allergy and Infectious Diseases (NIAID as top priority biodefense Category A pathogens. Recombinant consensus interferon alpha (cIFN-α is a licensed protein with broad clinical appeal. However, while cIFN-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. To address these limitations, we describe the use of DEF201, a replication-deficient adenovirus vector that drives the expression of cIFN-α, for pre- and post-exposure prophylaxis of acute arenaviral infection modeled in hamsters. Intranasal administration of DEF201 24 h prior to challenge with Pichindé virus (PICV was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. A significant protective effect was still observed with a single dosing of DEF201 given two weeks prior to PICV challenge. DEF201 was also efficacious when administered as a treatment 24 to 48 h post-virus exposure. The protective effect of DEF201 was largely attributed to the expression of cIFN-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal PICV challenge. Effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral infections. The DEF201 technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens.

  6. Effect of adenovirus gene transfer vectors on the immunologic functions of mouse dendritic cells.

    Korst, Robert J; Mahtabifard, Ali; Yamada, Reiko; Crystal, Ronald G


    To address the effect of adenovirus (Ad) gene transfer vector transduction on the diverse functions of dendritic cells, we used an Ad vector encoding no transgene (AdNull) to transduce mouse bone-marrow-derived dendritic cells (BMDC). Initial experiments using an Ad vector encoding a marker gene (AdGFP, jellyfish green fluorescent protein) showed that the optimal ratio of infectious Ad particles to each cell was 100, when both transgene expression and resultant BMDC viability were taken into account. Exposure to AdNull resulted in upregulation of both surface activation markers (CD40, MHC class II, B7.1, B7.2, ICAM-1) and IL-12 expression by BMDC. AdNull activation of BMDC was observed in multiple strains of mice. Despite this, AdNull-transduced BMDC displayed only modestly impaired antigen uptake ability, as demonstrated in macropinocytosis and phagocytosis assays, in vitro. However, Ad-modified BMDC migrated to regional lymph nodes five times more efficiently than sham-transduced BMDC in vivo. In addition, Ad transduction significantly enhanced the ability of BMDC to present a model peptide antigen to T-lymphocyte hybridoma cells at low BMDC:T cell ratios. We conclude that Ad modification, in and of itself, induces a state of activation in mouse BMDC. This activation, albeit mild compared with that induced by other stimuli, produces measurable effects of the specific immunological functions of these antigen-presenting cells.

  7. Adenovirus replication-competent vectors (KD1, KD3) complement the cytotoxicity and transgene expression from replication-defective vectors (Ad-GFP, Ad-Luc).

    Habib, Nagy A; Mitry, Ragai; Seth, Prem; Kuppuswamy, Mohan; Doronin, Konstantin; Toth, Karoly; Krajcsi, Peter; Tollefson, Ann E; Wold, William S M


    The successful clinical application of adenovirus (Ad) in cancer control has been of limited success because of the current inability to infect the majority of cancer cells with a large amount of vector. In this study, we show that when human lung tumors growing in immunodeficient nude mice were coinfected with a replication-defective (RD) Ad vector expressing green fluorescent protein and a replication-competent (RC) Ad vector named KD3, KD3 enhanced the expression of green fluorescent protein throughout the tumor. Also, KD3 and another RC vector named KD1 complemented the expression of luciferase from a RD vector in a human liver tumor xenotransplant in nude mice. Altogether, these results suggest that the combination of a RD vector with a RC vector might be a more effective treatment for cancer than either vector alone due to more widespread dissemination of the virus.

  8. TheQ1 Influence of Innate and Pre-Existing Immunity on Adenovirus Therapy

    Zaiss, Anne K.; Machado, Hidevaldo B.; Herschman, Harvey R.


    Recombinant adenovirus serotype 5 (Ad5) vectors have been studied extensively in preclinical gene therapy models and in a range of clinical trials. However, innate immune responses to adenovirus vectors limit effectiveness of Ad5 based therapies. Moreover, extensive pre-existing Ad5 immunity in human populations will likely limit the clinical utility of adenovirus vectors, unless methods to circumvent neutralizing antibodies that bind virus and block target cell transduction can be developed;...

  9. Constructionof the recombinant adenovirus vectors of CALB2 gene and small interfering RNA, and application in testicular Leydig cells

    Luo Jian; Wang Jing; Liu Shan; Sun Xue-ping; Gao Chao; Gao Li; Yang Xiao-yu; Liu Jia-yin; Cui Yu-gui


    Objective:To construct the recombinant adenovirus vectors of calretinin (CALB2) gene and small interfering RNA (siRNA),for over-expression or knock-down of CALB2,as the basis of functional investigation of CALB2 in testicular Leydig cells.Methods:The cDNA sequence of CALB2 was cloned by the reverse transcriptive polymerase chain reaction (RT-PCR).A CALB2 gene fragment was sub-cloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-CALB2.Then it was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant AdCMV-CALB2.The recombinant AdCMV-CALB2 was further packaged and amplificated in AD293 cells.The expression of CALB2 protein in AD293 cells was detected by Western blotting.CALB2 protein was over-expressed in mouse Leydig cell line (MLTC-1 cells) by the constructed AdCMVCALB2.CALB2 gene siRNA recombinant adenovirus vector (Ad-H1-siRNA/CALB2 was also constructed simultaneously.Its efficacy was detected in AD293 cells by Western blotting.Results:The CALB2 gene recombinant adenovirus vector AdCMV-CALB2 and the CALB2 gene siRNA recombinant adenovirus vector Ad-H1-siRNA/CALB2 were constructed successfully by endonulease digestion and sequencing.AD293 cells infected with AdCMV-CALB2 or Ad-H1-SiRNA/CALB2 significantly expressed GFP protein.The expression of CALB2 protein was significantly up-regulated in AD293 cells infected with AdCMV-CALB2 plasmids,while the expression of CALB2 protein was down-regulated by 60% in the CALB2 cells infected with Ad-H1SiRNA/CALB2.MLTC-1 cells did not markedly express CALLB2 protein,while MLTC-1 cells infected with AdCMV-CALB2 expressed CALB2 protein at a high level.Conclusions:The recombinant adenovirus vectors of AdCMV-CALB2 and Ad-H1-SiRNA/CALB2 were successfully constructed.Both vectors effectively expressed in AD293.CALB2 protein was over-expressed in the cultured MLTC-1 cells by AdCMV-CALB2.These vectors of CALB2 gene and Leydig cell line are

  10. Improvements in adenovirus-vector technology : aiming at replication specificity and vector integration

    Rademaker, Hendrik Jan


    Current generation adenoviral vectors (Ads) are not suitable for those gene therapy approaches that require long-term gene expression. This is due to their high immunogenicity and transient gene expression in fast dividing tissue. The development of gutless Ads, also known as helper-dependent Ads, i

  11. Intraductal delivery of adenoviruses targets pancreatic tumors in transgenic Ela-myc mice and orthotopic xenografts.

    José, Anabel; Sobrevals, Luciano; Miguel Camacho-Sánchez, Juan; Huch, Meritxell; Andreu, Núria; Ayuso, Eduard; Navarro, Pilar; Alemany, Ramon; Fillat, Cristina


    Gene-based anticancer therapies delivered by adenoviruses are limited by the poor viral distribution into the tumor. In the current work we have explored the feasibility of targeting pancreatic tumors through a loco-regional route. We have taken advantage of the ductal network in the pancreas to retrogradelly inject adenoviruses through the common bile duct in two different mouse models of pancreatic carcinogenesis: The transgenic Ela-myc mice that develop mixed neoplasms displaying both acinar-like and duct-like neoplastic cells affecting the whole pancreas; and mice bearing PANC-1 and BxPC-3 orthotopic xenografts that constitute a model of localized human neoplastic tumors. We studied tumor targeting and the anticancer effects of newly thymidine kinase-engineered adenoviruses both in vitro and in vivo, and conducted comparative studies between intraductal or intravenous administration. Our data indicate that the intraductal delivery of adenovirus efficiently targets pancreatic tumors in the two mouse models. The in vivo application of AduPARTKT plus ganciclovir (GCV) treatment induced tumor regression in Ela-myc mice. Moreover, the intraductal injection of ICOVIR15-TKT oncolytic adenoviruses significantly improved mean survival of mice bearing PANC-1 and BxPC-3 pancreatic xenografts from 30 to 52 days and from 20 to 68 days respectively (p less than 0.0001) when combined with GCV. Of notice, both AduPARTKT and ICOVIR15-TKT antitumoral responses were stronger by ductal viral application than intravenously, in line with the 38-fold increase in pancreas transduction observed upon ductal administration. In summary our data show that cytotoxic adenoviruses retrogradelly injected to the pancreas can be a feasible approach to treat localized pancreatic tumors.

  12. Bioresorbable microporous stents deliver recombinant adenovirus gene transfer vectors to the arterial wall.

    Ye, Y W; Landau, C; Willard, J E; Rajasubramanian, G; Moskowitz, A; Aziz, S; Meidell, R S; Eberhart, R C


    The use of intravascular stents as an adjunct for percutaneous transluminal revascularization is limited by two principal factors, acute thrombosis and neointimal proliferation, resulting in restenosis. To overcome these limitations, we have investigated the potential of microporous bioresorbable polymer stents formed from poly(L-lactic acid) (PLLA)/poly(epsilon-caprolactone) (PCL) blends to function both to provide mechanical support and as reservoirs for local delivery of therapeutic molecules and particles to the vessel wall. Tubular PLLA/PCL stents were fabricated by the flotation-precipitation method, and helical stents were produced by a casting/winding technique. Hybrid structures in which a tubular sheath is deposited on a helical skeleton were also generated. Using a two-stage solvent swelling technique, polyethylene oxide has been incorporated into these stents to improve hydrophilicity and water uptake, and to facilitate the ability of these devices to function as drug carriers. Stents modified in this manner retain axial and radial mechanical strength sufficient to stabilize the vessel wall against elastic recoil caused by vasoconstrictive and mechanical forces. Because of the potential of direct gene transfer into the vessel wall to ameliorate thrombosis and neointimal proliferation, we have investigated the capacity of these polymer stents to function in the delivery of recombinant adenovirus vectors to the vessel wall. In vitro, virus stock was observed to readily absorb into, and elute from these devices in an infectious form, with suitable kinetics. Successful gene transfer and expression has been demonstrated following implantation of polymer stents impregnated with a recombinant adenovirus carrying a nuclear-localizing betaGal reporter gene into rabbit carotid arteries. These studies suggest that surface-modified polymer stents may ultimately be useful adjunctive devices for both mechanical support and gene transfer during percutaneous

  13. Adenovirus vector expressing mda-7 selectively kills hepatocellular carcinoma cell line Hep3B

    Xin-Bo Xue; Kun Chen; Cong-Jun Wang; Jian-Wei Zheng; Yuan Yu; Zhi-Hai Peng; Zai-De Wu


    BACKGROUND: Melanoma differentiation associated gene-7 (mda-7) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo through activation of various intracellular signaling pathways. In this study, we investigated the potential effect of mda-7 on human hepatocellular carcinoma (HCC) in vitro. METHODS: Cells from the human HCC cell line Hep3B and the human liver cell line L-02 were assigned to three groups. One was cultured in Dulbecco's modiifed Eagle's medium without serum (control). The others were transfected with adenovirus expressing the mda-7 gene (Ad.mda-7) or adenovirus vector serving as negative control (Ad.vec). The expression of MDA-7 and Bcl-2 proteins in Hep3B and L-02 cells was conifrmed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The methyl thiazolyl tetrazolium colorimetric assay and lfow cytometry were used to assess tumor cell proliferation and the cell cycle. Hoechst and Annexin-V/propidium iodide staining were used to study mda-7 gene expression in Hep3B and L-02 cells. The expression of MDA-7, Bcl-2 and Bax proteins were detected by Western blotting. RESULTS: The mda-7 gene was expressed in Hep3B and L-02 cells. The protein concentrations of MDA-7 in supernatants were 790 and 810 pg/ml, respectively. mda-7 induced Hep3B growth suppression and apoptosis, compared with Ad.mda-7 and control (P CONCLUSIONS: mda-7 selectively induces growth inhibi-tion and apoptosis in the HCC cell line Hep3B but not in the normal liver cell line L-02 via downregulating the anti-apoptosis protein Bcl-2. It could be an ideal gene for gene therapy in HCC.

  14. An Adenovirus Vector Containing the Suicide Gene Thymidine Kinase for a Broad Application in Cancer Gene Therapy

    Magalhães GS


    Full Text Available Treatment of cancer using gene therapy is based on adding a property to the cell leading to its elimination. One possibility is the use of suicide genes that code for enzymes that transform a pro-drug into a cytotoxic product. The most extensively used is the herpes simplex virus thymidine kinase (TK gene, followed by administration of the antiviral drug ganciclovir (GCV. The choice of the promoter to drive the transcription of a transgene is one of the determinants of a given transfer vector usefulness, as different promoters show different efficiencies depending on the target cell type. In the experiments presented here, we report the construction of a recombinant adenovirus carrying TK gene (Ad-TK driven by three strong promoters (P CMV IE, SV40 and EN1 and its effectiveness in two cell types. Human HeLa and mouse CCR2 tumor cells were transduced with Ad-TK and efficiently killed after addition of GCV. We could detect two sizes of transcripts of TK gene, one derived from the close together P CMV IE/SV40 promoters and the other from the 1.5 Kb downstream EN1 promoter. The relative amounts of these transcripts were different in each cell type thus indicating a higher flexibility of this system.

  15. Transient detection of E1-containing adenovirus in saliva after the delivery of a first-generation adenoviral vector to human parotid gland†

    Zheng, Changyu; Nikolov, Nikolay P.; Alevizos, Ilias; Cotrim, Ana P.; Liu, Shuying; McCullagh, Linda; Chiorini, John A.; Illei, Gabor G.; Baum, Bruce J.


    Background Radiation-induced salivary hypofunction is a common side-effect of treatment for head and neck cancers. Patients suffer significant morbidity and there is no suitable conventional therapy. We are conducting a Phase I clinical trial, using a first-generation serotype 5 adenoviral (Ad5) vector encoding human aquaporin-1 (AdhAQP1) to treat such patients. One week after the administration of AdhAQP1 to an enrolled, generally healthy patient, E1-containing adenovirus was detected in parotid saliva. Methods The real-time quantitative polymerase chain reactuion (PCR) was used to measure the Ad5 E1 gene and AdhAQP1 in saliva and serum. PCR and sequencing were used to characterize viral/vector DNA extracted from saliva. The presence of infectious adenovirus was assessed by the inoculation of A549 cells with aliquots of saliva. Serum Ad5 neutralizing antibodies were measured by the inhibition of 293-cell transduction with an Ad5 vector encoding luciferase. Multiple clinical evaluations were performed. Results On day 7 after AdhAQP1 delivery, low levels of the Ad5 E1 gene were detected in parotid saliva (82 copies/μl). In addition, significant levels of AdhAQP1 were also detected (1.5 × 103 copies/μl). The patient was asymptomatic and subsequent analysis of parotid saliva samples prior to day 7 and after day 7 until day 42 was negative for both virus and vector. No virus or vector was detected in serum at any time. Detailed PCR analyses of DNA extracted from the day 7 parotid saliva sample suggested the absence of a recombination event, and no infectious virus was found. Conclusions The patient most likely had a latent Ad5 infection in the targeted parotid gland that was activated after gene transfer and was without clinical consequence. Published in 2009 by John Wiley & Sons, Ltd. PMID:19941317

  16. Transduction and oncolytic profile of a potent replication-competent adenovirus 11p vector (RCAd11pGFP in colon carcinoma cells.

    Jim Silver

    Full Text Available Replication-competent adenovirus type 5 (Ad5 vectors promise to be more efficient gene delivery vehicles than their replication-deficient counterparts, and chimeric Ad5 vectors that are capable of targeting CD46 are more effective than Ad5 vectors with native fibers. Although several strategies have been used to improve gene transduction and oncolysis, either by modifying their tropism or enhancing their replication capacity, some tumor cells are still relatively refractory to infection by chimeric Ad5. The oncolytic effects of the vectors are apparent in certain tumors but not in others. Here, we report the biological and oncolytic profiles of a replication-competent adenovirus 11p vector (RCAd11pGFP in colon carcinoma cells. CD46 was abundantly expressed in all cells studied; however, the transduction efficiency of RCAd11pGFP varied. RCAd11pGFP efficiently transduced HT-29, HCT-8, and LS174T cells, but it transduced T84 cells, derived from a colon cancer metastasis in the lung, less efficiently. Interestingly, RCAd11p replicated more rapidly in the T84 cells than in HCT-8 and LS174T cells and as rapidly as in HT-29 cells. Cell toxicity and proliferation assays indicated that RCAd11pGFP had the highest cell-killing activities in HT29 and T84 cells, the latter of which also expressed the highest levels of glycoproteins of the carcinoma embryonic antigen (CEA family. In vivo experiments showed significant growth inhibition of T84 and HT-29 tumors in xenograft mice treated with either RCAd11pGFP or Ad11pwt compared to untreated controls. Thus, RCAd11pGFP has a potent cytotoxic effect on colon carcinoma cells.

  17. Integration profile and safety of an adenovirus hybrid-vector utilizing hyperactive sleeping beauty transposase for somatic integration.

    Wenli Zhang

    Full Text Available We recently developed adenovirus/transposase hybrid-vectors utilizing the previously described hyperactive Sleeping Beauty (SB transposase HSB5 for somatic integration and we could show stabilized transgene expression in mice and a canine model for hemophilia B. However, the safety profile of these hybrid-vectors with respect to vector dose and genotoxicity remains to be investigated. Herein, we evaluated this hybrid-vector system in C57Bl/6 mice with escalating vector dose settings. We found that in all mice which received the hyperactive SB transposase, transgene expression levels were stabilized in a dose-dependent manner and that the highest vector dose was accompanied by fatalities in mice. To analyze potential genotoxic side-effects due to somatic integration into host chromosomes, we performed a genome-wide integration site analysis using linker-mediated PCR (LM-PCR and linear amplification-mediated PCR (LAM-PCR. Analysis of genomic DNA samples obtained from HSB5 treated female and male mice revealed a total of 1327 unique transposition events. Overall the chromosomal distribution pattern was close-to-random and we observed a random integration profile with respect to integration into gene and non-gene areas. Notably, when using the LM-PCR protocol, 27 extra-chromosomal integration events were identified, most likely caused by transposon excision and subsequent transposition into the delivered adenoviral vector genome. In total, this study provides a careful evaluation of the safety profile of adenovirus/Sleeping Beauty transposase hybrid-vectors. The obtained information will be useful when designing future preclinical studies utilizing hybrid-vectors in small and large animal models.

  18. Gene therapy for pathological scar with hepatocyte growth factor mediated by recombinant adenovirus vector

    哈小琴; 苑宾; 李元敏; 劳妙芬; 吴祖泽


    A complementary DNA (cDNA) encoding human hepatocyte growth factor wasintroduced into a replication-defective type 5 adenovirus (lacking E1, E3 domains) vector by homologous recombination of intracellular plasmid DNA, thus a recombinant vector containing HGF (Ad-HGF) was obtained. Ad-HGF and Ad-GFP (adenovirus vector carrying green fluorescence protein gene) were expanded in 293 cells and purified by cesium chloride gradient centrifugation for large-scale preparation, then were infected to the primarily cultured scar fibroblast of rabbit ear to observe the transfer efficiency and expression level of HGF in vitro. To evaluate the effect of Ad-HGF on established scar Ad-HGF solution was injected into excessively formed scar, which bears some clinical and histologic similarities tohuman hypertrophic scars. The results showed that: (i) the transfer efficiency was 36.8%±14.1% on day 3 in primarily cultured scar fibroblasts treated with Ad-GFP and lasted more than 20 d; (ii) high-level expression of HGF protein was detected by means of ELISA in supernatant of scar fibroblasts treated with Ad-HGF,the amount of expression was 76 ng/4.0×105 cells on day 3; (iii) on day 32 after a single intradermal injection of Ad-HGF at different doses (8.6×109 pfu, 8.6×108 pfu, 8.6×107 pfu, 8.6×106 pfu) per scar, most of the scars in the former two dose groups were dramatically flattened, some were even similar to that ofthe normal skin. The value of HI (hypertrophic index) showed that there was a therapeutic effect of Ad-HGF on scars at the dose of 109 pfu and 108 pfu. Whereasno therapeutic effects were seen at lower dose (107 pfu and 106 pfu of Ad-HGF) groups. In addition, clusters of hair were observed to different extent on healed wound treated with Ad-HGF. Histopathologic examination revealed that in most healed wounds of Ad-HGF treated group, the dermal layer was thinner, the amount of fibrous tissue was much fewer, and hair follicles growth and sebaceous glands were observed

  19. Antigen Gene Transfer to Human Plasmacytoid Dendritic Cells Using Recombinant Adenovirus and Vaccinia Virus Vectors

    Hetty J. Bontkes


    Full Text Available Recombinant adenoviruses (RAd and recombinant vaccinia viruses (RVV expressing tumour-associated antigens (TAA are used as anti-tumour vaccines. It is important that these vaccines deliver the TAA to dendritic cells (DC for the induction of a strong immune response. Infection of myeloid DC (MDC with RAd alone is relatively inefficient but CD40 retargeting significantly increases transduction efficiency and DC maturation. Infection with RVV is efficient without DC maturation. Plasmacytoid dendritic cells (PDC play a role in the innate immune response to viral infections through the secretion of IFNα but may also play a role in specific T-cell induction. The aim of our study was to investigate whether PDC are better targets for RAd and RVV based vaccines. RAd alone hardly infected PDC (2% while CD40 retargeting did not improve transduction efficiency, but it did increase PDC maturation (25% CD83 positive cells. Accordingly, specific CTL activation by RAd infected PDC was limited (the number of IFNγ producing CTL was reduced by 75% compared to stimulation with peptide loaded PDC. RVV infected PDC specifically stimulated CTL but PDC were not activated. These Results indicate that PDC are not ideal targets for RAd and RVV based vaccines. However, PDC induced specific CTL activation after pulsing with recombinant protein, indicating that PDC can also cross-present antigens released from surrounding infected cells.

  20. Immunogenicity of the recombinant adenovirus type 5 vector with type 35 fiber containing HIV-1 gag gene



    The immune efficiency of a recombinant adenovirus type 5 with type 35 fiber containing HIV-1 gag gene (rAd5/F35-mod.gag) was investigated in BALB/c mice, in which the rAd5/F35-mod. gag was firstly identified with PCR, then transfected to 293 cells and the in vitro expression level of Gag protein was determined by Western blotting and indirect immuno-fluorescent assay. Mice were immunized with intramuscular injections of rAd5/F35-mod. gag, rAd5-mod. gag or DNA and were boosted after 3 weeks.To test the effect of pre-existing anti-viral immunity on immunization, mice were also injected with Ad5-GFP vector and then immunized 4 and 7 weeks later with Ad5/F35-mod. gag vector. The P24-specific IgG antibody in sera of immunized mice was determined by ELISA and the specific cytotoxic T lymphocyte (CTL) response was assayed by intracellular cytokine staining. It was demonstrated that the rAd5/F35-mod. gag vector could express efficiently the HIV Gag protein in 293 cells in vitro and induce strong HIVspecific immune responses in vivo. The strongest CTL and serum IgG response occurred when mice were immunized twice with injection of rAd5/F35 alone, but the anti-Ad5 antibody after primary infection with adenovirus could inhibit the specific immune responses induced by rAd5/F35 vector. It is concluded that single immunization with recombinant adenovirus rAd5/F35-mod. gag can induce specific CTL and serum IgG antibody responses in mice, but the immunogenicity of rAd5/F35 is comparably weaker than that of rAd5.

  1. Adenovirus vector-mediated RNA interference for the inhibition of human parvovirus B19 replication.

    Brandt, Marius R G; Kirste, Ariane G; Pozzuto, Tanja; Schubert, Steffen; Kandolf, Reinhard; Fechner, Henry; Bock, C-Thomas; Kurreck, Jens


    Human parvovirus B19 (B19V) has been considered to cause acute and chronic myocarditis, which is accompanied by endothelial dysfunction. Currently, no causative treatment option for B19V-infections is available. Since RNA interference (RNAi) has proven to be a highly potent antiviral approach, the aim of the current study was to develop an RNAi-based strategy to inhibit B19V replication. Three B19V-VP2-specific short hairpin RNAs (shRNAs) were designed and tested for their silencing activity in reporter assays and the expression cassette of the best one was introduced into an adenoviral shuttle vector (Ad5). B19V-permissive UT7/Epo-S1 cells were infected with B19V and the RNAi triggers were delivered by the adenoviral vector (Ad5shVP2) 24h thereafter. The shRNA targeting the B19V-VP2 gene significantly suppressed VP2 mRNA levels as determined by quantitative RT-PCR. Additionally, also the expression levels of the non-targeted non-structural B19V-NS1 mRNA were strongly reduced. Our results demonstrate that vector-mediated delivery of shRNA expression cassettes targeting the structural B19-VP2 gene is a suitable approach to inhibit B19V replication.

  2. Combined adenovirus-mediated artificial microRNAs targeting mfgl2, mFas, and mTNFR1 protect against fulminant hepatic failure in mice.

    Dong Xi

    Full Text Available Hepatitis B virus (HBV-related acute-on-chronic liver failure (ACLF has a poor prognosis with high in-hospital mortality. Hepatic and circulating inflammatory cytokines, such as fibrinogen like protein 2 (fgl2, FasL/Fas, and TNFα/TNFR1, play a significant role in the pathophysiology of ACLF. This study aimed to investigate the therapeutic effect of recombinant adenoviral vectors carrying constructed DNA code for non-native microRNA (miRNA targeting mouse fgl2 (mfgl2 or both mFas and mTNFR1 on murine hepatitis virus (MHV-3-induced fulminant hepatitis in BALB/cJ mice. Artificial miRNA eukaryotic expression plasmids against mfgl2, mFas, and mTNFR1 were constructed, and their inhibitory effects on the target genes were confirmed in vitro. pcDNA6.2-mFas-mTNFR1- miRNA,which expresses miRNA against both mFas and mTNFR1 simultaneously,was constructed. To construct a miRNA adenovirus expression vector against mfgl2, pcDNA6.2-mfgl2-miRNA was cloned using Gateway technology. Ad-mFas-mTNFR1- miRNA was also constructed by the same procedure. Adenovirus vectors were delivered by tail-vein injection into MHV-3-infected BALB/cJ mice to evaluate the therapeutic effect. 8 of 18 (44.4% mice recovered from fulminant viral hepatitis in the combined interference group treated with Ad-mfgl2-miRNA and Ad-mFas-mTNFR1-miRNA. But only 4 of 18 (22.2% mice receiving Ad-mfgl2-miRNA and 3 of 18 (16.7% mice receiving Ad-mFas-mTNFR1- miRNA survived. These adenovirus vectors significantly ameliorated inflammatory infiltration, fibrin deposition, hepatocyte necrosis and apoptosis, and prolonged survival time. Our data illustrated that combined interference using adenovirus-mediated artificial miRNAs targeting mfgl2, mFas, and mTNFR1 might have significant therapeutic potential for the treatment of fulminant hepatitis.

  3. Protection of adenovirus from neutralizing antibody by cationic PEG derivative ionically linked to adenovirus

    Sun X; Zhang Z; Gong T; Zhao D.; Han J; Zeng Q


    Qin Zeng, Jianfeng Han, Dong Zhao, Tao Gong, Zhirong Zhang, Xun SunKey Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, People's Republic of ChinaBackground: The generation of anti-adenovirus neutralizing antibody (NAb) in humans severely restricts the utilization of recombinant adenovirus serotype 5 (Ad5) vectors in gene therapy for a wide range of clinical trials. To overcome this limitation, w...

  4. Adenovirus 5-vectored P. falciparum vaccine expressing CSP and AMA1. Part A: safety and immunogenicity in seronegative adults.

    Martha Sedegah

    Full Text Available BACKGROUND: Models of immunity to malaria indicate the importance of CD8+ T cell responses for targeting intrahepatic stages and antibodies for targeting sporozoite and blood stages. We designed a multistage adenovirus 5 (Ad5-vectored Plasmodium falciparum malaria vaccine, aiming to induce both types of responses in humans, that was tested for safety and immunogenicity in a Phase 1 dose escalation trial in Ad5-seronegative volunteers. METHODOLOGY/PRINCIPAL FINDINGS: The NMRC-M3V-Ad-PfCA vaccine combines two adenovectors encoding circumsporozoite protein (CSP and apical membrane antigen-1 (AMA1. Group 1 (n = 6 healthy volunteers received one intramuscular injection of 2×10∧10 particle units (1×10∧10 each construct and Group 2 (n = 6 a five-fold higher dose. Transient, mild to moderate adverse events were more pronounced with the higher dose. ELISpot responses to CSP and AMA1 peaked at 1 month, were higher in the low dose (geomean CSP = 422, AMA1 = 862 spot forming cells/million than in the high dose (CSP = 154, p = 0.049, AMA1 = 423, p = 0.045 group and were still positive at 12 months in a number of volunteers. ELISpot depletion assays identified dependence on CD4+ or on both CD4+ and CD8+ T cells, with few responses dependent only on CD8+ T cells. Intracellular cytokine staining detected stronger CD8+ than CD4+ T cell IFN-γ responses (CSP p = 0.0001, AMA1 p = 0.003, but similar frequencies of multifunctional CD4+ and CD8+ T cells secreting two or more of IFN-γ, TNF-α or IL-2. Median fluorescence intensities were 7-10 fold higher in triple than single secreting cells. Antibody responses were low but trended higher in the high dose group and did not inhibit growth of cultured P. falciparum blood stage parasites. SIGNIFICANCE: As found in other trials, adenovectored vaccines appeared safe and well-tolerated at doses up to 1×10∧11 particle units. This is the first demonstration in humans of a

  5. Experimental Study of Adenovirus Vector Mediated-hVEGF165 Gene on Prevention of Restenosis after Angioplasty

    刘启功; 陆再英; 岳远坤; 林立; 张卫东; 颜进


    This study evaluated the effects of adenovirus vector mediated human vascular endothelial growth factor-165 (hVEGF165) gene on prevention of restenosis after angioplasty. Rabbit models of bilateral carotid artery injury were established by balloon denudation. The recombinant adenoviruses containing hVEGF165 cDNA was directly injected into left side of the injured carotid arteries.On day 3 and week 3 after transfection the expression of VEGF was observed by RT-PCR and immunohistochemistry. The thrombokinesis, reendothelialization (rET) and intimal hyperplasia in carotid arteries were evaluated by computerized image analysis system 3 weeks after gene transfer.The changes in the VEGF gene-treated side were compared with the control side. Our results showed that 3 days and 3 weeks after hVEGF165 gene transfer the VEGF mRNA and antigen expression were detected in vivo. 3 weeks after the transfer, the carotid artery rET was markedly better in the VEGF gene-treated group compared with the control. The thrombokinesis, intima area/media area (I/M), maximal intimal and medial thicknesses (ITmax and MTmax) demonstrated a statistically significant decrease in arteries treated with VEGF gene as compared with the control group. It is concluded that VEGF gene transfer could be achieved by intra-arterial injection of recombinant adenoviruses. It might accelerate the restoration of endothelial integrity, inhibit thrombokinesis and attenuate intimal hyperplasia in the injured arteries after VEGF gene transfer. This procedure could be useful in preventing restenosis after angioplasty.

  6. Multiple efficacy studies of an adenovirus-vectored foot-and-mouth disease virus serotype A24 subunit vaccine in cattle using direct homologous challenge

    The safety and efficacy of an experimental, replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was examined in eight independent cattle studies. AdtA24 non-adjuvanted vaccine was administered intramuscularl...

  7. Adenovirus-vectored foot-and-mouth disease vaccine confers early and full protection against FMDV O1 Manisa in swine

    A human adenovirus (Ad5) vectored foot-and-mouth disease virus (FMDV) sero-type O1-Manisa subunit vaccine (Ad5-O1Man) was engineered to deliver FMDV O1-Manisa empty capsids. Swine inoculated with Ad5-O1Man developed an FMDV-specific neutralizing antibody response as compared to animals inoculated wi...

  8. Evaluation of a Fiber-Modified Adenovirus Vector Vaccine against Foot-and-Mouth Disease in Cattle.

    Medina, Gisselle N; Montiel, Nestor; Diaz-San Segundo, Fayna; Sturza, Diego; Ramirez-Medina, Elizabeth; Grubman, Marvin J; de los Santos, Teresa


    Novel vaccination approaches against foot-and-mouth disease (FMD) include the use of replication-defective human adenovirus type 5 (Ad5) vectors that contain the capsid-encoding regions of FMD virus (FMDV). Ad5 containing serotype A24 capsid sequences (Ad5.A24) has proved to be effective as a vaccine against FMD in livestock species. However, Ad5-vectored FMDV serotype O1 Campos vaccine (Ad5.O1C.2B) provides only partial protection of cattle against homologous challenge. It has been reported that a fiber-modified Ad5 vector expressing Arg-Gly-Asp (RGD) enhances transduction of antigen-presenting cells (APC) in mice. In the current study, we assessed the efficacy of a fiber-modified Ad5 (Adt.O1C.2B.RGD) in cattle. Expression of FMDV capsid proteins was superior in cultured cells infected with the RGD-modified vector. Furthermore, transgene expression of Adt.O1C.2B.RGD was enhanced in cell lines that constitutively express integrin αvβ6, a known receptor for FMDV. In contrast, capsid expression in cattle-derived enriched APC populations was not enhanced by infection with this vector. Our data showed that vaccination with the two vectors yielded similar levels of protection against FMD in cattle. Although none of the vaccinated animals had detectable viremia, FMDV RNA was detected in serum samples from animals with clinical signs. Interestingly, CD4(+) and CD8(+) gamma interferon (IFN-γ)(+) cell responses were detected at significantly higher levels in animals vaccinated with Adt.O1C.2B.RGD than in animals vaccinated with Ad5.O1C.2B. Our results suggest that inclusion of an RGD motif in the fiber of Ad5-vectored FMD vaccine improves transgene delivery and cell-mediated immunity but does not significantly enhance vaccine performance in cattle.

  9. Targeting an adenoviral gene vector to cytokine-activated vascular endothelium via E-selectin.

    Harari, O A; Wickham, T J; Stocker, C J; Kovesdi, I; Segal, D M; Huehns, T Y; Sarraf, C; Haskard, D O


    We have aimed at selective gene delivery to vascular endothelial cells (EC) at sites of inflammation, by targeting E-selectin, a surface adhesion molecule that is only expressed by activated EC. An anti-E-selectin mAb, 1.2B6, was complexed with the adenovirus vector AdZ.FLAG (expressing the FLAG peptide) by conjugating it to an anti-FLAG mAb. Gene transduction of cultured EC was increased 20-fold compared with AdZ.FLAG complexed with a control bsAb providing EC were activated by cytokines. The anti-E-selectin-complexed vector transduced 29 +/- 9% of intimal EC in segments of pig aorta cultured with cytokines ex vivo, compared with less than 0.1% transduced with the control construct (P < 0.05). This strategy could be developed to target endothelium in inflammation with genes capable of modifying the inflammatory response.

  10. The nucleotide sequence and a first generation gene transfer vector of species B human adenovirus serotype 3.

    Sirena, Dominique; Ruzsics, Zsolt; Schaffner, Walter; Greber, Urs F; Hemmi, Silvio


    Human adenovirus (Ad) serotype 3 causes respiratory infections. It is considered highly virulent, accounting for about 13% of all Ad isolates. We report here the complete Ad3 DNA sequence of 35,343 base pairs (GenBank accession DQ086466). Ad3 shares 96.43% nucleotide identity with Ad7, another virulent subspecies B1 serotype, and 82.56 and 62.75% identity with the less virulent species B2 Ad11 and species C Ad5, respectively. The genomic organization of Ad3 is similar to the other human Ads comprising five early transcription units, E1A, E1B, E2, E3, and E4, two delayed early units IX and IVa2, and the major late unit, in total 39 putative and 7 hypothetical open reading frames. A recombinant E1-deleted Ad3 was generated on a bacterial artificial chromosome. This prototypic virus efficiently transduced CD46-positive rodent and human cells. Our results will help in clarifying the biology and pathology of adenoviruses and enhance therapeutic applications of viral vectors in clinical settings.

  11. Adenovirus DNA Replication

    Hoeben, Rob C.; Uil, Taco G.


    Adenoviruses have attracted much attention as probes to study biological processes such as DNA replication, transcription, splicing, and cellular transformation. More recently these viruses have been used as gene-transfer vectors and oncolytic agents. On the other hand, adenoviruses are notorious pathogens in people with compromised immune functions. This article will briefly summarize the basic replication strategy of adenoviruses and the key proteins involved and will deal with the new deve...

  12. Protective Efficacy in Sheep of Adenovirus-Vectored Vaccines against Bluetongue Virus Is Associated with Specific T Cell Responses

    Martín, Verónica; Pascual, Elena; Avia, Miguel; Peña, Lourdes; Valcárcel, Félix; Sevilla, Noemí


    Bluetongue virus (BTV) is an economically important Orbivirus of the Reoviridae family that causes a hemorrhagic disease in ruminants. Its control has been achieved by inactivated-vaccines that have proven to protect against homologous BTV challenge although unable to induce long-term immunity. Therefore, a more efficient control strategy needs to be developed. Recombinant adenovirus vectors are lead vaccine candidates for protection of several diseases, mainly because of their potency to induce potent T cell immunity. Here we report the induction of humoral and T-cell mediated responses able to protect animals against BTV challenge by recombinant replication-defective human adenovirus serotype 5 (Ad5) expressing either VP7, VP2 or NS3 BTV proteins. First we used the IFNAR(-/-) mouse model system to establish a proof of principle, and afterwards we assayed the protective efficacy in sheep, the natural host of BTV. Mice were completely protected against BTV challenge, developing humoral and BTV-specific CD8+- and CD4+-T cell responses by vaccination with the different rAd5. Sheep vaccinated with Ad5-BTV-VP2 and Ad5-BTV-VP7 or only with Ad5-BTV-VP7 and challenged with BTV showed mild disease symptoms and reduced viremia. This partial protection was achieved in the absence of neutralizing antibodies but strong BTV-specific CD8+ T cell responses in those sheep vaccinated with Ad5-BTV-VP7. These data indicate that rAd5 is a suitable vaccine vector to induce T cell immunity during BTV vaccination and provide new data regarding the relevance of T cell responses in protection during BTV infection. PMID:26619062

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

    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


    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.

  14. HCCS1-armed, quadruple-regulated oncolytic adenovirus specific for liver cancer as a cancer targeting gene-viro-therapy strategy

    Xu Hai-Neng


    Full Text Available Abstract Background In previously published studies, oncolytic adenovirus-mediated gene therapy has produced good results in targeting cancer cells. However, safety and efficacy, the two most important aspects in cancer therapy, remain serious challenges. The specific expression or deletion of replication related genes in an adenovirus has been frequently utilized to regulate the cancer cell specificity of a virus. Accordingly, in this study, we deleted 24 bp in E1A (bp924-bp947 and the entirety of E1B, including those genes encoding E1B 55kDa and E1B19kDa. We used the survivin promoter (SP to control E1A in order to construct a new adenovirus vector named Ad.SP.E1A(Δ24.ΔE1B (briefly Ad.SPDD. HCCS1 (hepatocellular carcinoma suppressor 1 is a novel tumor suppressor gene that is able to specifically induce apoptosis in cancer cells. The expression cassette AFP-HCCS1-WPRE-SV40 was inserted into Ad.SPDD to form Ad.SPDD-HCCS1, enabling us to improve the safety and efficacy of oncolytic-mediated gene therapy for liver cancer. Results Ad.SPDD showed a decreased viral yield and less toxicity in normal cells but enhanced toxicity in liver cancer cells, compared with the cancer-specific adenovirus ZD55 (E1B55K deletion. Ad.SPDD-HCCS1 exhibited a potent anti-liver-cancer ability and decreased toxicity in vitro. Ad.SPDD-HCCS1 also showed a measurable capacity to inhibit Huh-7 xenograft tumor growth on nude mice. The underlying mechanism of Ad.SPDD-HCCS1-induced liver cancer cell death was found to be via the mitochondrial apoptosis pathway. Conclusions These results demonstrate that Ad.SPDD-HCCS1 was able to elicit reduced toxicity and enhanced efficacy both in vitro and in vivo compared to a previously constructed oncolytic adenovirus. Ad.SPDD-HCCS1 could be a promising candidate for liver cancer therapy.

  15. Construction of human BMP2-IRES-HIF1αmu adenovirus expression vector and its expression in mesenchymal stem cells.

    Liu, Danping; Hu, Liang; Zhang, Zheng; Li, Quan Ying; Wang, Guoxian


    The present study aimed to construct a novel recombinant adenovirus expression vector Ad-BMP2-IRES-HIF1αmu that expresses human bone morphogenetic protein (BMP2) and mutant hypoxia-inducible factor 1α, and investigated its effects in promoting neogenesis of bone and angiogenesis. The recombinant adenovirus BMP2, HIF1αmu and pIRES2-EGFP expression vectors were constructed and transfected into HEK293A cells. The groups were divided into group A, transfection with Ad-BMP2-IRES-HIF1αmu; group B, transfection with Ad-HIF1αmu-IRES-hrGFP-1; group C, transfection with Ad-BMP2-IRES-hrGFP-1; group D, transfection with Ad-IRES-hrGFP-1; group E, not transfected. Adenovirus liquid was transferred into rabbit mesenchymal stem cells (MSCs) pretreated with dexamethasone at the best multiplicity of infection (MOI). The mRNA and protein expression of BMP2 and HIF1α were detected by RT-PCR and western blot analysis. Adenovirus was successfully packaged. The expression level of HIF1α mRNA in group A and B was markedly higher than that in groups C, D and E, showing a significant difference (PBMP2 mRNA between group A and C (PBMP2 in group A and C was markedly higher than that in groups B, D and E (PBMP2-IRES-HIF1αmu adenovirus expression vector was successfully constructed and the experimental groups formed bone and blood vessels prior to the positive and negative control groups.

  16. Modification to the capsid of the adenovirus vector that enhances dendritic cell infection and transgene-specific cellular immune responses.

    Worgall, Stefan; Busch, Annette; Rivara, Michael; Bonnyay, David; Leopold, Philip L; Merritt, Robert; Hackett, Neil R; Rovelink, Peter W; Bruder, Joseph T; Wickham, Thomas J; Kovesdi, Imi; Crystal, Ronald G


    Adenovirus (Ad) gene transfer vectors can be used to transfer and express antigens and function as strong adjuvants and thus are useful platforms for the development of genetic vaccines. Based on the hypothesis that Ad vectors with enhanced infectibility of dendritic cells (DC) may be able to evoke enhanced immune responses against antigens encoded by the vector in vivo, the present study analyzes the vaccine potential of an Ad vector expressing beta-galactosidase as a model antigen and genetically modified with RGD on the fiber knob [AdZ.F(RGD)] to more selectively infect DC and consequently enhance immunity against the beta-galactosidase antigen. Infection of murine DC in vitro with AdZ.F(RGD) showed an eightfold-increased transgene expression following infection compared to AdZ (also expressing beta-galactosidase, but with a wild-type capsid). Binding, cellular uptake, and trafficking in DC were also increased with AdZ.F(RGD) compared to AdZ. To determine whether AdZ.F(RGD) could evoke enhanced immune responses to beta-galactosidase in vivo, C57BL/6 mice were immunized with AdZ.F(RGD) or AdZ subcutaneously via the footpad. Humoral responses with both vectors were comparable, with similar anti-beta-galactosidase antibody levels following vector administration. However, cellular responses to beta-galactosidase were significantly enhanced, with the frequency of CD4(+) as well as the CD8(+) beta-galactosidase-specific gamma interferon response in cells isolated from the draining lymph nodes increased following immunization with AdZ.F(RGD) compared to Ad.Z (P AdZ.F(RGD) vector was sufficient to evoke enhanced inhibition of the growth of preexisting tumors expressing beta-galactosidase: BALB/c mice implanted with the CT26 syngeneic beta-galactosidase-expressing colon carcinoma cell line and subsequently immunized with AdZ.F(RGD) showed decreased tumor growth and improved survival compared to mice immunized with AdZ. These data demonstrate that addition of an RGD motif

  17. Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector


    AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gen...

  18. Efficient induction of cross-presentating human B cell by transduction with human adenovirus type 7 vector.

    Peng, Ying; Lai, Meimei; Lou, Yunyan; Liu, Yanqing; Wang, Huiyan; Zheng, Xiaoqun


    Although human autologous B cells represent a promising alternative to dendritic cells (DCs) for easy large-scale preparation, the naive human B cells are always poor at antigen presentation. The safe and effective usage record of human adenovirus type 7 (HAdV7) live vaccines makes it attractive as a promising vaccine vector candidate. To investigate whether HAdV7 vector could be used to induce the human B cells cross-presentation, in the present study, we constructed the E3-defective recombinant HAdV7 vector encoding green fluorescent protein (GFP) and carcinoembryonic antigen (CEA). We demonstrated that naive human B cells can efficiently be transduced, and that the MAPKs/NF-κB pathway can be activated by recombinant HAdV7. We proved that cytokine TNF-α, IL-6 and IL-10, surface molecule MHC class I and the CD86, antigen-processing machinery (APM) compounds ERp57, TAP-1, and TAP-2. were upregulated in HAdV7 transduced human B cells. We also found that CEA-specific IFNγ expression, degranulation, and in vitro and ex vivo cytotoxicities are induced in autologous CD8(+) T cells presensitized by HAd7CEA modified human B cells. Meanwhile, our evidences clearly show that Toll-like receptors 9 (TLR9) antagonist IRS 869 significantly eliminated most of the HAdV7 initiated B cell activation and CD8(+) T cells response, supporting the role and contribution of TLR9 signaling in HAdV7 induced human B cell cross-presentation. Besides a better understanding of the interactions between recombinant HAdV7 and human naive B cells, to our knowledge, the present study provides the first evidence to support the use of HAdV7-modified B cells as a vehicle for vaccines and immunotherapy.

  19. Genetic stability of a recombinant adenovirus vaccine vector seed library expressing human papillomavirus type 16 E6 and E7 proteins



    The aim of the present study was to understand the genetic stability of a master seed bank (MSB) and a working seed bank (WSB) of an adenovirus vector vaccine expressing the human papillomavirus (HPV) type 16 E6 and E7 fusion proteins (Ad-HPV16E6E7). Microscopic examination and viral infectious efficacy were used to measure the infectious titers of the Ad-HPV16E6E7 MSB and WSB. Polymerase chain reaction was used to analyze the stability of the Ad-HPV16E6E7 target gene insertion, while western blot analysis and immunofluorescence were used to assess the expression levels of the Ad-HPV16E6E7 target protein. A C57BL/6 mouse TC-1 tumor cell growth inhibition model was used to evaluate the biological effect of Ad-HPV16E6E7 administration. The infectious titers of the Ad-HPV16E6E7 MSB and WSB were 6.31×109 IU/ml and 3.0×109 IU/ml, respectively. In addition, the expression levels of the inserted target genes and target proteins were found to be stable. In the mouse TC-1 tumor inhibition analysis, when the virus titers of the Ad-HPV16E6E7 MSB and WSB were 109 IU/ml, the tumor inhibition rate was 100%, which was significantly different when compared with the control group (χ2MSB=20.00 and χ2WSB=20.00; P<0.01). Therefore, the Ad-HPV16E6E7 vaccine seed bank is genetically stable and meets the requirements for vaccine development. PMID:25780403

  20. Adenovirus structure.

    Rux, John J; Burnett, Roger M


    Structural studies continue to play an essential role as the focus of adenovirus research shifts in emphasis from basic biology to adenovirus-based vector technologies. A crucial step in developing novel therapeutics for gene replacement, cancer, and vaccines is often to modify the virion. Such engineered changes are designed to retarget the virus, or to reduce the immunological responses to infection. These efforts are far more effective when they are based on detailed structural knowledge. This minireview provides a brief summary of the wealth of information that has been obtained from the combined application of X-ray crystallography and electron microscopy. This knowledge now includes a good working model for the architectural organization of the virion, and atomic resolution molecular structures for all the major capsid proteins, hexon, penton, and fiber. We highlight new developments, which include the structure of the penton base and the discovery that adenovirus has several relatives. We sketch how the structural information can be used to engineer novel virions and conclude with the prospects for future progress.

  1. Protection against P. aeruginosa with an adenovirus vector containing an OprF epitope in the capsid.

    Worgall, Stefan; Krause, Anja; Rivara, Michael; Hee, Kyung-Kim; Vintayen, Enrico V; Hackett, Neil R; Roelvink, Peter W; Bruder, Joseph T; Wickham, Thomas J; Kovesdi, Imre; Crystal, Ronald G


    Pseudomonas aeruginosa is an important opportunistic pathogen that can cause chronic and often life-threatening infections of the respiratory tract, particularly in individuals with cystic fibrosis (CF). Because infections with P. aeruginosa remain the major cause of the high morbidity and mortality of CF, a vaccine against P. aeruginosa would be very useful for preventing this disorder. The outer membrane protein F (OprF) of P. aeruginosa is a promising vaccine candidate and various B cell epitopes within OprF have been identified. Given that adenovirus (Ad) vectors have strong immunogenic potential and can function as adjuvants for genetic vaccines, the present study evaluates the immunogenic and protective properties of a novel replication-deficient Ad vector in which the Ad hexon protein was modified to include a 14-amino acid epitope of P. aeruginosa OprF (Epi8) in loop 1 of the hypervariable region 5 of the hexon (AdZ.Epi8). Immunization of C57BL/6 mice with AdZ.Epi8 resulted in detectable serum anti-P. aeruginosa and anti-OprF humoral responses. These responses were haplotype dependent, with higher serum anti-OprF titers in CBA mice than in BALB/c or C57BL/6 mice. AdZ.Epi8 induced Epi8-specific IFN-gamma-positive CD4 and CD8 T cell responses and resulted in protection against a lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeated administration of AdZ.Epi8 resulted in boosting of the anti-OprF humoral and anti-Epi8 cellular response, whereas no boosting effect was present in the response against the transgene beta-galactosidase. These observations suggest that Ad vectors expressing pathogen epitopes in their capsid will protect against an extracellular pathogen and will allow boosting of the epitope-specific humoral response with repeated administration, a strategy that should prove useful in developing Ad vectors as vaccines where humoral immunity will be protective.

  2. Intramuscular delivery of adenovirus serotype 5 vector expressing humanized protective antigen induces rapid protection against anthrax that may bypass intranasally originated preexisting adenovirus immunity.

    Wu, Shipo; Zhang, Zhe; Yu, Rui; Zhang, Jun; Liu, Ying; Song, Xiaohong; Yi, Shaoqiong; Liu, Ju; Chen, Jianqin; Yin, Ying; Xu, Junjie; Hou, Lihua; Chen, Wei


    Developing an effective anthrax vaccine that can induce a rapid and sustained immune response is a priority for the prevention of bioterrorism-associated anthrax infection. Here, we developed a recombinant replication-deficient adenovirus serotype 5-based vaccine expressing the humanized protective antigen (Ad5-PAopt). A single intramuscular injection of Ad5-PAopt resulted in rapid and robust humoral and cellular immune responses in Fisher 344 rats. Animals intramuscularly inoculated with a single dose of 10⁸ infectious units of Ad5-PAopt achieved 100% protection from challenge with 10 times the 50% lethal dose (LD₅₀) of anthrax lethal toxin 7 days after vaccination. Although preexisting intranasally induced immunity to Ad5 slightly weakened the humoral and cellular immune responses to Ad5-PAopt via intramuscular inoculation, 100% protection was achieved 15 days after vaccination in Fisher 344 rats. The protective efficacy conferred by intramuscular vaccination in the presence of preexisting intranasally induced immunity was significantly better than that of intranasal delivery of Ad5-PAopt and intramuscular injection with recombinant PA and aluminum adjuvant without preexisting immunity. As natural Ad5 infection often occurs via the mucosal route, the work here largely illuminates that intramuscular inoculation with Ad5-PAopt can overcome the negative effects of immunity induced by prior adenovirus infection and represents an efficient approach for protecting against emerging anthrax.

  3. Construction of recombinant adenovirus vector containing AFP and generation of adenovirus-mediated AFP gene modified dendritic cells vaccine%含人AFP基因重组腺病毒载体的构建及其转染树突状细胞瘤苗的制备

    杨静悦; 曹大勇; 刘文超; 斯小明


    Objective:To construct recombinant adenovirus vectors containing human AFP genes,and infect dendritic cell. Methods: Full length AFP cDNAs were subcloned into pIND vector,followed by being cloned into shuttle2 vector.The AFP gene fragments resulted from the shuttle2-AFP digested with PI-Sce and I-Ceu were linked to the linear adeno-X virus DNA.After packaged with HEK293 cells,the adenovirus expression vector was obtained.The plasmid pAdeno-AFP was identified by endonuclease and PCR.After dendritic cells were infected pAdeno-AFP,the surface molecules of pAdeno-AFP/DC were analysed by flow cytometry.AFP levels in culture supernatant of pAdeno-AFP/DC were measured by ELISA. Results: AFP gene in the inserted DNA of adeno-AFP was confirmed by PCR,and predictive fragments proved by restriction enzyme digestion analysis were exhibited.All the above results indicated that human AFP gene had been connected with pAdeno-X vectors correctly.The recombinant adenovirus vector of human AFP gene packaged in HEK293 cells,it will be used to introduce the target gene into dendritic cell.pAdeno-AFP/DC were able to upregulate CD1a,CD11c,CD80,CD86 and HLA-DR.And pAdeno-AFP/DC could secrete high level of AFP in vitro. Conclusion: The recombinant adenovirus vector of human AFP gene have been constructed successfully.The established AFP -DC vaccine may be a tool of the hepatocellular carcinoma immunotherapy,and it will be the foundation of future clinical use of DC vaccine.%目的:构建含人AFP基因的腺病毒载体,体外转染树突状细胞,制备树突状细胞肝癌瘤苗.方法: 将AFP基因亚克隆到pIND 载体和Shuttle2载体中,构建穿梭载体Shuttle2-AFP.用PI-Sce Ⅰ和I-CeuⅠ双酶切后将所获AFP基因片段再与线性化的腺病毒载体pAdeno-X连接,构成pAdeno-AFP重组腺病毒载体.其后,用重组腺病毒载体转染HEK293细胞,包装腺病毒表达载体.通过酶切、PCR对腺病毒载体进行鉴定.包装好的重组病毒载体pAdeno-AFP体外

  4. Pharmacological Interventions for Improving Adenovirus Usage in Gene Therapy

    Haisma, Hidde J.; Bellu, Anna Rita


    Gene therapy may be an innovative and promising new treatment strategy for cancer but is limited due to a low efficiency and specificity of gene delivery to the target cells. Adenovirus is the preferred gene therapy vector for systemic delivery because of its unparalleled in vivo transduction effici

  5. Enhanced immunity against classical swine fever in pigs induced by prime-boost immunization using an alphavirus replicon-vectored DNA vaccine and a recombinant adenovirus.

    Sun, Yuan; Li, Na; Li, Hong-Yu; Li, Miao; Qiu, Hua-Ji


    Classical swine fever (CSF) - caused by the classical swine fever virus (CSFV) - is a fatal disease of pigs that is responsible for extensive losses to the swine industry worldwide. We had demonstrated previously that a prime-boost vaccination strategy using an alphavirus (Semliki Forest virus, SFV) replicon-vectored DNA vaccine (pSFV1CS-E2) and a recombinant adenovirus (rAdV-E2) expressing the E2 glycoprotein of CSFV induced enhanced immune responses in a mouse model. In this study, we evaluated further the efficacy of the heterologous prime-boost immunization approach in pigs, the natural host of CSFV. The results showed that the pigs (n=5) receiving pSFV1CS-E2/rAdV-E2 heterologous prime-boost immunization developed significantly higher titers of CSFV-specific neutralizing antibodies and comparable CD4(+) and CD8(+) T-cell proliferation, compared to the pigs receiving double immunizations with rAdV-E2 alone. When challenged with virulent CSFV Shimen strain, the pigs of the heterologous prime-boost group did not show clinical symptoms or viremia, which were observed in one of the 5 pigs immunized with rAdV-E2 alone and all the 5 control pigs immunized with an empty adenovirus. The results demonstrate that the heterologous DNA prime and recombinant adenovirus boost strategy can induce solid protective immunity.

  6. Expanding specificity of class I restricted CD8+ T cells for viral epitopes following multiple inoculations of swine with a human adenovirus vectored foot-and-mouth disease virus (FMDV) vaccine

    Pedersen, Lasse E.; Patch, Jared R; Kenney, Mary


    The immune response to the highly acute foot-and-mouth disease virus (FMDV) is routinely reported as a measure of serum antibody. However, a critical effector function of immune responses combating viral infection of mammals is the cytotoxic T lymphocyte (CTL) response mediated by virus specific CD......8 expressing T cells. This immune mechanism arrests viral spread by killing virus infected cells before new, mature virus can develop. We have previously shown that infection of swine by FMDV results in a measurable CTL response and have correlated CTL killing of virus-infected cells with specific...... class I major histocompatibility complex (MHC) tetramer staining. We also showed that a modified replication defective human adenovirus 5 vector expressing the FMDV structural proteins (Ad5-FMDV-T vaccine) targets the induction of a CD8(+) CTL response with a minimal humoral response. In this report, we...

  7. Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

    Andrew H. Baker


    Full Text Available Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX, which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs. These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon, pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies, can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX, or alternatively, through the use of polymer

  8. Support vector regression model for complex target RCS predicting

    Wang Gu; Chen Weishi; Miao Jungang


    The electromagnetic scattering computation has developed rapidly for many years; some computing problems for complex and coated targets cannot be solved by using the existing theory and computing models. A computing model based on data is established for making up the insufficiency of theoretic models. Based on the "support vector regression method", which is formulated on the principle of minimizing a structural risk, a data model to predicate the unknown radar cross section of some appointed targets is given. Comparison between the actual data and the results of this predicting model based on support vector regression method proved that the support vector regression method is workable and with a comparative precision.

  9. Targeting of adenoviral vectors through a bispecific single-chain antibody

    Haisma, HJ; Grill, J; Curiel, DT; Hoogeland, S; van Beusechem, VW; Pinedo, HM; Gerritsen, WR


    Recombinant adenoviral vectors are attractive in the context of cancer gene therapy because they are capable of delivering genes to a wide variety of tissues. The utility of adenoviruses is limited by their lack of specificity and by the absence of the receptor(s) for these viruses on many tumor cel

  10. Adenovirus serotype 5 vaccine vectors trigger IL-27-dependent inhibitory CD4+ T cell responses that impair CD8+ T cell function

    Larocca, Rafael A.; Provine, Nicholas M.; Aid, Malika; Iampietro, M. Justin; Borducchi, Erica N.; Badamchi-Zadeh, Alexander; Abbink, Peter; Ng’ang’a, David; Bricault, Christine A.; Blass, Eryn; Penaloza-MacMaster, Pablo; Stephenson, Kathryn E.; Barouch, Dan H.


    Adenovirus serotype 5 (Ad5) vaccine vectors elicit robust CD8+ T cell responses, but these responses typically exhibit a partially exhausted phenotype. However, the immunologic mechanism by which Ad5 vectors induce dysfunctional CD8+ T cells has not previously been elucidated. Here we demonstrate that, following immunization of B6 mice, Ad5 vectors elicit antigen-specific IL-10+CD4+ T cells with a distinct transcriptional profile in a dose-dependent fashion. In rhesus monkeys, we similarly observed upregulated expression of IL-10 and PD-1 by CD4+ T cells following Ad5 vaccination. These cells markedly suppressed vaccine-elicited CD8+ T cell responses in vivo and IL-10 blockade increased the frequency and functionality of antigen-specific CD8+ T cells as well as improved protective efficacy against challenge with recombinant Listeria monocytogenes. Moreover, induction of these inhibitory IL-10+CD4+ T cells correlated with IL-27 expression and IL-27 blockade substantially improved CD4+ T cell functionality. These data highlight a role for IL-27 in the induction of inhibitory IL-10+CD4+ T cells, which suppress CD8+ T cell magnitude and function following Ad5 vector immunization. A deeper understanding of the cytokine networks and transcriptional profiles induced by vaccine vectors should lead to strategies to improve the immunogenicity and protective efficacy of viral vector-based vaccines.

  11. 腺病毒介导的HSV-tk 体外抗喉癌作用%Anti-laryngeal carcinoma with adenovirus vector mediated HSV-tk gene

    陈立; 李凡; 易世红; 黄红兰; 赵春燕; 魏强


    Objective To observe killing action of HSV-tk/GCV system onlaryngeal cancer cells. Methods Hep-2 cells were transfected by adenovirus vector with LacZ gene, then stained with X-gal for measurement of the adenovirus transfected efficiency. The recombinant adenovirus carrying HSV-tk was constructed by homologous recombination techniques. Hep-2 cells survival and by stander effect were observed after adding 10 mg/L GCV to transfected cells. Results The more the multiplicity of infection (MOI), the more the adenovirus transfected efficiency to Hep-2 cells. 100% transfected efficiency needs MOI 200. The killing action of HSV-tk/GCV on Hep-2 cells depends on the amount of AdCMVtk. There is a weak by stander effect. Conclusion Adenovirus mediated HSV-tk/GCV can kill Hep-2 cells.%目的 观察HSV-tk/GCV系统对喉癌细胞的杀伤作用。方法 将带有报告基因LacZ的5型缺陷性腺病毒载体AdCMVLacZ感染喉癌细胞Hep-2,X-gal染色,测定腺病毒的转染率。利用同源重组技术构建含HSV-tk基因的重组腺病毒载体AdCMVtk。AdCMVtk感染Hep-2细胞后,加入10mg/LGCV,观察瘤细胞存活率及旁观者效应。结果 随着腺病毒感染复数量(multiplicityofinfection,MOI)增加,对Hep-2细胞转染率亦增加,100%转染所需的MOI为200。AdCMVtk/GCV对Hep-2细胞的杀伤强度与AdCMVtk量呈正向关系,即有浓度依赖性。此杀伤作用存在旁观者效应,但较弱。结论 腺病毒介导的HSV-tk/GCV具有杀伤喉癌细胞作用。

  12. The α2 helix in the DNA ligase IV BRCT-1 domain is required for targeted degradation of ligase IV during adenovirus infection.

    Gilson, Timra; Greer, Amy E; Vindigni, Alessandro; Ketner, Gary; Hanakahi, Leslyn A


    In adenovirus E4 mutant infections, viral DNAs form concatemers through a process that requires host Non-homologous End Joining (NHEJ) proteins including DNA Ligase IV (LigIV). Adenovirus proteins E4 34k and E1b 55k form the substrate-selection component of an E3 ubiquitin ligase and prevent concatenation by targeting LigIV for proteasomal degradation. The mechanisms and sites involved in targeting this and other E3 ligase substrates generally are poorly-understood. Through genetic analysis, we identified the α2 helix of one LigIV BRCT domain (BRCT-1) as essential for adenovirus-mediated degradation. Replacement of the BRCT domain of DNA ligase III (LigIII), which is resistant to degradation, with LigIV BRCT-1 does not promote degradation. A humanized mouse LigIV that possesses a BRCT-1 α2 helix identical to the human protein, like its parent, is also resistant to adenovirus-mediated degradation. Thus, both the BRCT-1 α2 helix and an element outside BRCT-1 are required for adenovirus-mediated degradation of LigIV.

  13. Bovine adenoviral vector-based H5N1 influenza vaccine overcomes exceptionally high levels of pre-existing immunity against human adenovirus.

    Singh, Neetu; Pandey, Aseem; Jayashankar, Lakshmi; Mittal, Suresh K


    Because of the high prevalence of adenovirus (Ad) infections in humans, it is believed that pre-existing Ad-neutralizing antibodies (vector immunity) may negatively impact the immune response to vaccine antigens when delivered by human Ad (HAd) vectors. In order to evaluate whether bovine Ad subtype 3 (BAd3), a non-HAd vector, can effectively elude high levels of pre-existing vector immunity, naïve and HAd serotype 5 (HAd)-primed mice were immunized with BAd-H5HA [BAd3 vector expressing the hemagglutinin (HA) gene from H5N1 influenza virus]. Even in the presence of very high levels of HAd-specific neutralizing antibody, no significant reductions in HA-specific humoral and cell-mediated immune (CMI) responses were observed in HAd-primed mice immunized with BAd-H5HA. In naïve mice immunized with HAd-H5HA (HAd5 vector expressing H5N1 HA) and boosted with BAd-H5HA, the humoral responses elicited were significantly higher (P BAd-H5HA alone, while the CMI responses were comparable in the groups. This finding underlines the importance of a heterologous prime-boost approach for achieving an enhanced immune response. The immunization of naïve or HAd-primed mice with BAd-H5HA bestowed full protection from morbidity and mortality following a potentially lethal challenge with A/Hong Kong/483/97. These results demonstrate the importance of BAd vectors as an alternate or supplement to HAd vectors for influenza pandemic preparedness.

  14. Increasing the potential for malaria elimination by targeting zoophilic vectors

    Waite, Jessica L.; Swain, Sunita; Lynch, Penelope A.; Sharma, S. K.; Haque, Mohammed Asrarul; Montgomery, Jacqui; Thomas, Matthew B.


    Countries in the Asia Pacific region aim to eliminate malaria by 2030. A cornerstone of malaria elimination is the effective management of Anopheles mosquito vectors. Current control tools such as insecticide treated nets or indoor residual sprays target mosquitoes in human dwellings. We find in a high transmission region in India, malaria vector populations show a high propensity to feed on livestock (cattle) and rest in outdoor structures such as cattle shelters. We also find evidence for a shift in vector species complex towards increased zoophilic behavior in recent years. Using a malaria transmission model we demonstrate that in such regions dominated by zoophilic vectors, existing vector control tactics will be insufficient to achieve elimination, even if maximized. However, by increasing mortality in the zoophilic cycle, the elimination threshold can be reached. Current national vector control policy in India restricts use of residual insecticide sprays to domestic dwellings. Our study suggests substantial benefits of extending the approach to treatment of cattle sheds, or deploying other tactics that target zoophilic behavior. Optimizing use of existing tools will be essential to achieving the ambitious 2030 elimination target. PMID:28091570

  15. Construction of recombinant adenovirus vector of mouse keratinocyte growth factor receptor%小鼠角质化生长因子受体(KGFR)重组腺病毒载体的构建

    陈力; 曹春蕾; 李蓓; 周璇; 李银平; 黎檀实


    Objective To construct a recombinant adenovirus vector expressing keratinocyte growth factor receptor (KGFR) gene of mouse. Methods KGFR gene was amplified by polymerase chain reaction (PCR) from mouse cDNA which had been reverse transcripted from RNA harvested from C57BL/6 mouse, and was then inserted into the plasmid pShuttle-CMV to construct the shuttle plasmid pShuttle-CMV-KGFR (pKGFR). After linearized by restriction enzyme, the plasmid was transformed into Escherichia. coli BJ5183 containing adenovirus backbone. The homologous recombinant pAdeasy-1-pShuttle-CMV-KGFR(pAd-KGFR) was identified, linearized, and then transfected into HEK293 cells using lipofectamineTM 2000 to package the adenovirus, Adeasy-1-pShuttle-CMV-KGFR (Ad-KGFR), followed by further amplification, caesium chloride density gradient centrifugation purification and measurement of virus titer. Results Firstly, the shuttle plasmid pKGFR was proved to be successfully constructed by gene sequencing and restriction enzyme, as well as the recombinant adenovirus plasmid. Then, the cytopathic effects of HEK293 cells ohserved under the microscope suggested the successful duplication of the virus. Finally, the plague titration of on HEK293 cells showed virus titers were 2.25 × 1010 PFU/ml, which was enough concentration for future test in vivo or in vitro.Conclusion The recombinant adenovirus vector of pAd-KGFR is constructed successfully to be used in the future test to investigate the effects of KGFR in pulmonary diseases, and provides attractive therapeutic target in the pulmonary disease.%目的 构建表达小鼠KGFR基因的重组腺病毒.方法 提取小鼠肺组织RNA,RT-PCR获得cDNA后,PCR扩增目的 基因片段.将获得的KGFR基因插入载体质粒pShuttle-CMV,构建穿梭质粒pShuttle-CMV-KGFR(pKGFR).线性化pKGFR后,转化入含Adeasy-1病毒骨架的BJ5183细菌中.筛选正确的同源重组质粒pAdeasy-1-pShuttle-CMV-KGFR(pAd-KGFR),转染HEK293细胞,产生病毒颗粒Adeasy-1

  16. Canine Recombinant Adenovirus Vector Induces an Immunogenicity-Related Gene Expression Profile in Skin-Migrated CD11b+ -Type DCs

    Jouneau, Luc; Bourge, Mickael; Bouet-Cararo, Coraline; Bonneau, Michel; Zientara, Stephan; Klonjkowski, Bernard; Schwartz-Cornil, Isabelle


    Gene expression profiling of the blood cell response induced early after vaccination has previously been demonstrated to predict the immunogenicity of vaccines. In this study, we evaluated whether the analysis of the gene expression profile of skin-migrated dendritic cells (DCs) could be informative for the in vitro prediction of immunogenicity of vaccine, using canine adenovirus serotype 2 (CAV2) as vaccine vector. CAV2 has been shown to induce immunity to transgenes in several species including sheep and is an interesting alternative to human adenovirus-based vectors, based on the safety records of the parental strain in dogs and the lack of pre-existing immunity in non-host species. Skin-migrated DCs were collected from pseudo-afferent lymph in sheep. Both the CD11b+ -type and CD103+ -type skin-migrated DCs were transduced by CAV2. An analysis of the global gene response to CAV2 in the two skin DC subsets showed that the gene response in CD11b+ -type DCs was far higher and broader than in the CD103+ -type DCs. A newly released integrative analytic tool from Ingenuity systems revealed that the CAV2-modulated genes in the CD11b+ -type DCs clustered in several activated immunogenicity-related functions, such as immune response, immune cell trafficking and inflammation. Thus gene profiling in skin-migrated DC in vitro indicates that the CD11b+ DC type is more responsive to CAV2 than the CD103+ DC type, and provides valuable information to help in evaluating and possibly improving viral vector vaccine effectiveness. PMID:23300693

  17. Canine recombinant adenovirus vector induces an immunogenicity-related gene expression profile in skin-migrated CD11b⁺ -type DCs.

    Vanessa Contreras

    Full Text Available Gene expression profiling of the blood cell response induced early after vaccination has previously been demonstrated to predict the immunogenicity of vaccines. In this study, we evaluated whether the analysis of the gene expression profile of skin-migrated dendritic cells (DCs could be informative for the in vitro prediction of immunogenicity of vaccine, using canine adenovirus serotype 2 (CAV2 as vaccine vector. CAV2 has been shown to induce immunity to transgenes in several species including sheep and is an interesting alternative to human adenovirus-based vectors, based on the safety records of the parental strain in dogs and the lack of pre-existing immunity in non-host species. Skin-migrated DCs were collected from pseudo-afferent lymph in sheep. Both the CD11b(+ -type and CD103(+ -type skin-migrated DCs were transduced by CAV2. An analysis of the global gene response to CAV2 in the two skin DC subsets showed that the gene response in CD11b(+ -type DCs was far higher and broader than in the CD103(+ -type DCs. A newly released integrative analytic tool from Ingenuity systems revealed that the CAV2-modulated genes in the CD11b(+ -type DCs clustered in several activated immunogenicity-related functions, such as immune response, immune cell trafficking and inflammation. Thus gene profiling in skin-migrated DC in vitro indicates that the CD11b(+ DC type is more responsive to CAV2 than the CD103(+ DC type, and provides valuable information to help in evaluating and possibly improving viral vector vaccine effectiveness.

  18. Construction and Expression of Recombinant Adenovirus Vector Ad5-hTRX-EGFP%重组腺病毒载体Ad5-hTRX-EGFP的构建及其表达

    扈江伟; 王军; 徐曼; 苏永锋; 孔维霞; 盛红霞; 张斌; 陈虎


    -defective recombinant adenovirus pAd-hTRX-EGFP was co-transfected in HEK293 cells, purified by CsCl gradient centrifugation, counted for virus particles and determined for tiler. The recombinant adenovirus was identified by PCR. The HEK293 cells were then transfected with adenoviruses and assayed by flow cytometry. The expression of hTRX was confirmed by Western blot. The results showed that according to PCR and restriction endonuclease assay, the target gene was inserted into recombinant adenovirus vector successfully. The sequence of fusion gene was the same as that of designed fragments. The titer of the purified recombinant adenovirus pAd-hTRX-EGFP was 5. 558 × 1010pfu/mL A transfection efficiency of 92. 25% could be achieved at MOI = 100. Western blot further confirmed that hTRX was efficiently expressed in HEK293 cells. It is concluded that recombinant adenovirus vector containing hTRX has been constructed successfully and obtained highly efficient virus that can express efficiently in HEK293 cells, which laid a foundation for further investigation.

  19. Adenovirus delivered short hairpin RNA targeting a conserved site in the 5' non-translated region inhibits all four serotypes of dengue viruses.

    Anil Babu Korrapati

    Full Text Available BACKGROUND: Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs. This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi to attenuate DENV replication may offer one approach to dengue therapy. METHODOLOGY/PRINCIPAL FINDINGS: We screened the non-translated regions (NTRs of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5' NTR that maps to the 5' upstream AUG region, a highly conserved cis-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5 vector to deliver a short-hairpin RNA (shRNA targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes. CONCLUSION/SIGNIFICANCE: The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection.

  20. 利用腺病毒载体介导的RNA干扰技术在悬浮细胞中获得基因沉默效应%Adenovirus expression vector mediated gene silencing in suspension cells

    王淳; 方文刚; 李波


    目的 利用重组腺病毒载体携带短发夹RNA (shRNA),使难于转染的悬浮细胞达到较高的目的基因沉默效率.方法 分别将含有人胎盘生长因子(PLGF)的干扰序列及阴性对照序列(scramble)连入pRNAT-H1.1/Adeno载体,获得重组穿梭质粒;将线性化的重组穿梭质粒与腺病毒骨架重组;将重组子转染HEK 293细胞,经过3轮病毒包装,收获病毒颗粒并测定滴度.病毒颗粒感染悬浮细胞——人小细胞肺癌细胞(NCI-H 250和NCI-H 209),通过实时定量PCR方法鉴定靶基因沉默效应,并利用肿瘤细胞重建基质膜侵袭实验进行功能鉴定.结果 成功构建带有靶基因干扰序列及scramble、空载体的3种腺病毒重组子(AD-shuttle-shPLGF、AD-shuttlescramble和AD-shuttle).经过HEK 293细胞包装,最终获得3株病毒颗粒,病毒滴度分别为1.5×1011、1.6×1011和1.6×1011.NCI-H 250和NCI-H 209细胞感染腺病毒颗粒48 h后,大部分细胞表达GFP,感染率接近100%;且两株细胞感染AD-shuttle-shPLGF病毒48 h后,PLGF mRNA表达水平降低(P<0.01),肿瘤细胞侵袭能力明显下降(P<0.01).结论 携带shRNA的腺病毒表达载体可以代替电转、脂质体介导等方法,使难于转染细胞的目的基因达到有效沉默.%Objective Using recombinant adenovirus to make gene silencing in suspension cell. Methods Placen-tal growth factor (PLGF) silencing target sequence and scramble sequence were inserted into shuttle plasmid pR-NAT-H1. 1/Adenb respectively to gain recombinant shuttle vectors. Then the two recombinant shuttle vectors and empty vector were co-transformed with pAdeasy-1 through homologous recombination. These recombinant adenovi-ruses were packaged and amplified in HEK 293 cells. Human small cell lung cancer cells NCI-H 250 and NCI-H 209, were infected by recombinant adenovirus. And the expression of target gene was detected by real - time PCRand cancer cells' invation function was analyzed by Transwell cancer cell

  1. Adenovirus-5-vectored P. falciparum vaccine expressing CSP and AMA1. Part B: safety, immunogenicity and protective efficacy of the CSP component.

    Cindy Tamminga

    Full Text Available BACKGROUND: A protective malaria vaccine will likely need to elicit both cell-mediated and antibody responses. As adenovirus vaccine vectors induce both these responses in humans, a Phase 1/2a clinical trial was conducted to evaluate the efficacy of an adenovirus serotype 5-vectored malaria vaccine against sporozoite challenge. METHODOLOGY/PRINCIPAL FINDINGS: NMRC-MV-Ad-PfC is an adenovirus vector encoding the Plasmodium falciparum 3D7 circumsporozoite protein (CSP. It is one component of a two-component vaccine NMRC-M3V-Ad-PfCA consisting of one adenovector encoding CSP and one encoding apical membrane antigen-1 (AMA1 that was evaluated for safety and immunogenicity in an earlier study (see companion paper, Sedegah et al. Fourteen Ad5 seropositive or negative adults received two doses of NMRC-MV-Ad-PfC sixteen weeks apart, at 1 x 1010 particle units per dose. The vaccine was safe and well tolerated. All volunteers developed positive ELISpot responses by 28 days after the first immunization (geometric mean 272 spot forming cells/million[sfc/m] that declined during the following 16 weeks and increased after the second dose to levels that in most cases were less than the initial peak (geometric mean 119 sfc/m. CD8+ predominated over CD4+ responses, as in the first clinical trial. Antibody responses were poor and like ELISpot responses increased after the second immunization but did not exceed the initial peak. Pre-existing neutralizing antibodies (NAb to Ad5 did not affect the immunogenicity of the first dose, but the fold increase in NAb induced by the first dose was significantly associated with poorer antibody responses after the second dose, while ELISpot responses remained unaffected. When challenged by the bite of P. falciparum-infected mosquitoes, two of 11 volunteers showed a delay in the time to patency compared to infectivity controls, but no volunteers were sterilely protected. SIGNIFICANCE: The NMRC-MV-Ad-PfC vaccine expressing CSP was

  2. Co-expression of the C-terminal domain of Yersinia enterocolitica invasin enhances the efficacy of classical swine-fever-vectored vaccine based on human adenovirus

    Helin Li; Pengbo Ning; Zhi Lin; Wulong Liang; Kai Kang; Lei He; Yanming Zhang


    The use of adenovirus vector-based vaccines is a promising approach for generating antigen-specific immune responses. Improving vaccine potency is necessary in other approaches to address their inadequate protection for the majority of infectious diseases. This study is the first to reconstruct a recombinant replication-defective human adenovirus co-expressing E2 and invasin C-terminal (InvC) glycoproteins (rAd-E2-InvC). rAd-E2-InvC with 2×106 TCID50 was intramuscularly administered two times to CSFV-free pigs at 14 day intervals. No adverse clinical reactions were observed in any of the pigs after the vaccination. The CSFV E2-specific antibody titer was significantly higher in the rAd-E2-InvC group than that in the rAdV-E2 group as measured by NPLA and blocking ELISA. Pigs immunized with rAd-E2-InvC were completely protected against lethal challenge. Neither CSFV RNA nor pathological changes were detected in the tissues after CSFV challenge. These results demonstrate that rAd-E2-InvC could be an alternative to the existing CSF vaccine. Moreover, InvC that acts as an adjuvant could enhance the immunogenicity of rAdV-E2 and induce high CSFV E2-specific antibody titer and protection level.

  3. Construction of gene targeting vectors from lambda KOS genomic libraries.

    Wattler, S; Kelly, M; Nehls, M


    We describe a highly redundant murine genomic library in a new lambda phage, lambda knockout shuttle (lambda KOS) that facilitates the very rapid construction of replacement-type gene targeting vectors. The library consists of 94 individually amplified subpools, each containing an average of 40,000 independent genomic clones. The subpools are arrayed into a 96-well format that allows a PCR-based efficient recovery of independent genomic clones. The lambda KOS vector backbone permits the CRE-mediated conversion into high-copy number pKOS plasmids, wherein the genomic inserts are automatically flanked by negative-selection cassettes. The lambda KOS vector system exploits the yeast homologous recombination machinery to simplify the construction of replacement-type gene targeting vectors independent of restriction sites within the genomic insert. We outline procedures that allow the generation of simple and more sophisticated conditional gene targeting vectors within 3-4 weeks, beginning with the screening of the lambda KOS genomic library.

  4. Construction of RNAi lentiviral vector targeting mouse Islet-1 gene

    Shen-shen ZHI


    Full Text Available Objective To construct and select RNAi lentiviral vectors that can silence mouse Islet-1 gene effectively.Methods Three groups of RNAi-target of mouse Islet-1 gene were designed,and corresponding shRNA oligo(sh1,sh2 and sh3 were synthesized,and then they were respectively inserted to the PLVTHM vector that had been digested by endonuclease.Agarose gel electrophoresis and sequencing were used to select and indentify the positive clones.The positive clones were extracted and then mixed with E.coli to amplify positive clones.The amplified clones were then infected into 293T along with the other 3 helper plasmids to produce lentiviral vector.After the construction of the lentiviral vector,plaque formation test was performed to determine the titer of lentiviral vector.The lentiviral vectors were then infected into C3H10T1/2 cells.The transfect efficiency of the lentiviral vectors was determined with flow cytometry with detection of green fluorescent protein(GFP.Q-PCR was employed to detect the RNAi efficiency of the lentiviral vectors.Results Agarose gel electrophoresis analysis showed that the clones with right gene at the target size were successfully established;gene sequencing showed that the right DNA fragments had been inserted;plaque formation test showed that the titer of the virus solution was 3.87×108TU/ml;the transfect efficiency of the lentiviral vector infected into C3H10T1/2 cells was 90.36%.All the 3 groups of shRNA targets(sh1,sh2 and sh3 showed an inhibitory effect on Islet-1 gene,and the sh1 showed the highest inhibitory effect(76.8%,as compared with that of normal cells(P < 0.05.Conclusion The RNAi lentiviral vector that can effectively silence the mouse Islet-1 gene has been constructed successfully,which may lay a foundation for further investigation of Islet-1 gene.

  5. Targeted cancer immunotherapy with oncolytic adenovirus coding for a fully human monoclonal antibody specific for CTLA-4.

    Dias, J D; Hemminki, O; Diaconu, I; Hirvinen, M; Bonetti, A; Guse, K; Escutenaire, S; Kanerva, A; Pesonen, S; Löskog, A; Cerullo, V; Hemminki, A


    Promising clinical results have been achieved with monoclonal antibodies (mAbs) such as ipilimumab and tremelimumab that block cytotoxic T lymphocyte-associated antigen-4 (CTLA-4, CD152). However, systemic administration of these agents also has the potential for severe immune-related adverse events. Thus, local production might allow higher concentrations at the target while reducing systemic side effects. We generated a transductionally and transcriptionally targeted oncolytic adenovirus Ad5/3-Δ24aCTLA4 expressing complete human mAb specific for CTLA-4 and tested it in vitro, in vivo and in peripheral blood mononuclear cells (PBMCs) of normal donors and patients with advanced solid tumors. mAb expression was confirmed by western blotting and immunohistochemistry. Biological functionality was determined in a T-cell line and in PBMCs from cancer patients. T cells of patients, but not those of healthy donors, were activated by an anti-CTLA4mAb produced by Ad5/3-Δ24aCTLA4. In addition to immunological effects, a direct anti-CTLA-4-mediated pro-apoptotic effect was observed in vitro and in vivo. Local production resulted in 43-fold higher (P<0.05) tumor versus plasma anti-CTLA4mAb concentration. Plasma levels in mice remained below what has been reported safe in humans. Replication-competent Ad5/3-Δ24aCTLA4 resulted in 81-fold higher (P<0.05) tumor mAb levels as compared with a replication-deficient control. This is the first report of an oncolytic adenovirus producing a full-length human mAb. High mAb concentrations were seen at tumors with lower systemic levels. Stimulation of T cells of cancer patients by Ad5/3-Δ24aCTLA4 suggests feasibility of testing the approach in clinical trials.

  6. 11R-P53 and GM-CSF Expressing Oncolytic Adenovirus Target Cancer Stem Cells with Enhanced Synergistic Activity

    Lv, Sai-qun; Ye, Zhen-long; Liu, Pin-yi; Huang, Yao; Li, Lin-fang; Liu, Hui; Zhu, Hai-li; Jin, Hua-jun; Qian, Qi-jun


    Targeting cancer stem cells with oncolytic virus (OV) holds great potential for thorough elimination of cancer cells. Based on our previous studies, we here established 11R-P53 and mGM-CSF carrying oncolytic adenovirus (OAV) SG655-mGMP and investigated its therapeutic effect on hepatocellular carcinoma stem cells Hep3B-C and teratoma stem cells ECCG5. Firstly, the augmenting effect of 11R in our construct was tested and confirmed by examining the expression of EGFP with Fluorescence and FCM assays after transfecting Hep3B-C and ECCG5 cells with OVA SG7605-EGFP and SG7605-11R-EGFP. Secondly, the expressions of 11R-P53 and GM-CSF in Hep3B-C and ECCG5 cells after transfection with OAV SG655-mGMP were detected by Western blot and Elisa assays, respectively. Thirdly, the enhanced growth inhibitory and augmented apoptosis inducing effects of OAV SG655-mGMP on Hep3B-C and ECCG5 cells were tested with FCM assays by comparing with the control, wild type 5 adenovirus, 11R-P53 carrying OVA in vitro. Lastly, the in vivo therapeutic effect of OAV SG655-mGMP toward ECCG5 cell-formed xenografts was studied by measuring tumor volumes post different treatments with PBS, OAV SG655-11R-P53, OAV SG655-mGM-CSF and OAV SG655-mGMP. Treatment with OAV SG655-mGMP induced significant xenograft growth inhibition, inflammation factor AIF1 expression and immune cells infiltration. Therefore, our OAV SG655-mGMP provides a novel platform to arm OVs to target cancer stem cells.

  7. Qualitative and quantitative analysis of adenovirus type 5 vector-induced memory CD8 T cells: not as bad as their reputation.

    Steffensen, Maria Abildgaard; Holst, Peter Johannes; Steengaard, Sanne Skovvang; Jensen, Benjamin Anderschou Holbech; Bartholdy, Christina; Stryhn, Anette; Christensen, Jan Pravsgaard; Thomsen, Allan Randrup


    It has been reported that adenovirus (Ad)-primed CD8 T cells may display a distinct and partially exhausted phenotype. Given the practical implications of this claim, we decided to analyze in detail the quality of Ad-primed CD8 T cells by directly comparing these cells to CD8 T cells induced through infection with lymphocytic choriomeningitis virus (LCMV). We found that localized immunization with intermediate doses of Ad vector induces a moderate number of functional CD8 T cells which qualitatively match those found in LCMV-infected mice. The numbers of these cells may be efficiently increased by additional adenoviral boosting, and, importantly, the generated secondary memory cells cannot be qualitatively differentiated from those induced by primary infection with replicating virus. Quantitatively, DNA priming prior to Ad vaccination led to even higher numbers of memory cells. In this case, the vaccination led to the generation of a population of memory cells characterized by relatively low CD27 expression and high CD127 and killer cell lectin-like receptor subfamily G member 1 (KLRG1) expression. These memory CD8 T cells were capable of proliferating in response to viral challenge and protecting against infection with live virus. Furthermore, viral challenge was followed by sustained expansion of the memory CD8 T-cell population, and the generated memory cells did not appear to have been driven toward exhaustive differentiation. Based on these findings, we suggest that adenovirus-based prime-boost regimens (including Ad serotype 5 [Ad5] and Ad5-like vectors) represent an effective means to induce a substantially expanded, long-lived population of high-quality transgene-specific memory CD8 T cells.

  8. Novel viral vectors utilizing intron splice-switching to activate genome rescue, expression and replication in targeted cells

    El Andaloussi Samir


    Full Text Available Abstract Background The outcome of virus infection depends from the precise coordination of viral gene expression and genome replication. The ability to control and regulate these processes is therefore important for analysis of infection process. Viruses are also useful tools in bio- and gene technology; they can efficiently kill cancer cells and trigger immune responses to tumors. However, the methods for constructing tissue- or cell-type specific viruses typically suffer from low target-cell specificity and a high risk of reversion. Therefore novel and universal methods of regulation of viral infection are also important for therapeutic application of virus-based systems. Methods Aberrantly spliced introns were introduced into crucial gene-expression units of adenovirus vector and alphavirus DNA/RNA layered vectors and their effects on the viral gene expression, replication and/or the release of infectious genomes were studied in cell culture. Transfection of the cells with splice-switching oligonucleotides was used to correct the introduced functional defect(s. Results It was demonstrated that viral gene expression, replication and/or the release of infectious genomes can be blocked by the introduction of aberrantly spliced introns. The insertion of such an intron into an adenovirus vector reduced the expression of the targeted gene more than fifty-fold. A similar insertion into an alphavirus DNA/RNA layered vector had a less dramatic effect; here, only the release of the infectious transcript was suppressed but not the subsequent replication and spread of the virus. However the insertion of two aberrantly spliced introns resulted in an over one hundred-fold reduction in the infectivity of the DNA/RNA layered vector. Furthermore, in both systems the observed effects could be reverted by the delivery of splice-switching oligonucleotide(s, which corrected the splicing defects. Conclusions Splice-switch technology, originally developed for

  9. Construction and in vitro analysis of recombinant adenovirus vector carrying red fluorescent protein reporter gene%携带红色荧光蛋白报告基因的重组腺病毒载体的构建及其体外试验研究

    卢建溪; 阳莉; 钱师宇; 王强


    construct recombinant vector pShuttle - TurboRFP - N. I - Ceu I/PI - Sce I were used to double digest recombinant vector pShuttle -TurboRFP - N and backbone of vector pH5 040. pkGFP - II. The target fragment was collected and ligated, and recombinant adenovirus vector AdH5'. 040. CMV. RFP - N was obtained. After linearization, the vector was transfected into AD293 cells by liposome for virus packaging. The efficiency of virus packaging and RFP expression level in AD293 cells were examined using fluorescent microscope. In addition, the biological activity and titer of the virus were tested. Human lung cancer cell line A549 and breast cancer cell line MDA - MB - 231 were infected with recombinant adenovirus vector AdH5 .040. CMV. RFP - N and control adenovirus vector AdH5. CMV. EGFP respectively. The infection efficiencies of the 2 vectors to different cell lines were compared by evaluating the expression levels of RFP and enhanced green fluorescent protein ( EGFP ). RESULTS: The recombinant adenovirus vector AdH5 '. 040. CMV. RFP - N was correctly constructedand confirmed by enzyme digestion. The virus was packaged by the vector in AD293 cells and had the ability to infect the target cells. The target gene in eukaryotic cells was also expressed. The number of recombinant adenoviruses and the titer of the virus after amplification and purification were 3. 6 x 10 vp/L and 1 x 10 pfu/L, respectively. The infection efficiencies of recombinant adenovirus vector Ad5 . 040. CMV. RFP - N to human lung cancer cell line A549 and breast cancer cell line MDA - MB - 231 were higher than those in control adenovirus vector AdH5. CMV. EGFP ( P < 0. 05 ). CONCLUSION: We have constructed recombinant virus vector carrying RFP reporter gene and provide an important tool for gene therapy and gene vaccine research. The reporter gene can be highly expressed in AD293 cells and has high infection efficiency to cancer cells. RFP is a good substitution and supplement to green fluorescent protein.

  10. Protection of adenovirus from neutralizing antibody by cationic PEG derivative ionically linked to adenovirus

    Sun X


    Full Text Available Qin Zeng, Jianfeng Han, Dong Zhao, Tao Gong, Zhirong Zhang, Xun SunKey Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, People's Republic of ChinaBackground: The generation of anti-adenovirus neutralizing antibody (NAb in humans severely restricts the utilization of recombinant adenovirus serotype 5 (Ad5 vectors in gene therapy for a wide range of clinical trials. To overcome this limitation, we ionically complexed Ad5 with a newly synthesized copolymer, which we called APC, making an adenovirus shielded from NAb.Methods: APC, a cationic polyethylene glycol derivative, was synthesized via two steps of ring-opening copolymerization of ethylene oxide and allyl glycidyl ether, followed by the addition of 2-mercaptoethylamine. The copolymer or the control PEI-2k was ionically complexed to anionic Ad5 in 5% glucose, and in vitro transduction assays were carried out in coxsackievirus and adenovirus receptor-positive cells (A549 and coxsackievirus and adenovirus receptor-negative cells (B16 and SKOV3. The physical properties and morphology of adenovirus alone or the complexes were investigated respectively by zeta potential, size distribution, and transmission electron microscopy image. Then cytotoxicity of APC was examined using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assays. Finally, the ability of APC to protect adenovirus from NAb was evaluated by transfection assays after a neutralizing effect.Results: APC was successfully synthesized and showed a low cytotoxicity. Positively charged Ad5/APC exhibited slightly increased diameter (130.2 ± 0.60 nm than naked Ad5 (115.6 ± 5.46 nm while Ad5/PEI-2k showed severe aggregation (1382 ± 79.9 nm. Ad5/APC achieved a gene transfection level as high as Ad5/PEI-2k in A549 or B16 cells, and significantly higher than Ad5/PEI-2k in SKOV3 cells. Most importantly, after the exposure to the neutralizing

  11. Isolated limb perfusion for local gene delivery: efficient and targeted adenovirus-mediated gene transfer into soft tissue sarcomas

    W.K. de Roos; J.H.W. de Wilt (Johannes); M.E. van der Kaaden; E.R. Manusama (Eric); M.W. de Vries; A. Bout; T.L.M. ten Hagen (Timo); D. Valerio (Dinko); A.M.M. Eggermont (Alexander)


    textabstractOBJECTIVE: To evaluate the potential of isolated limb perfusion (ILP) for efficient and tumor-specific adenovirus-mediated gene transfer in sarcoma-bearing rats. SUMMARY BACKGROUND DATA: A major concern in adenovirus-mediated gene therapy in cancer is the transfer of ge

  12. Single dose adenovirus vectored vaccine induces a potent and long-lasting immune response against rabbit hemorrhagic disease virus after parenteral or mucosal administration.

    Fernández, Erlinda; Toledo, Jorge R; Chiong, Maylin; Parra, Francisco; Rodríguez, Elsa; Montero, Carlos; Méndez, Lídice; Capucci, Lorenzo; Farnós, Omar


    Rabbit hemorrhagic disease virus (RHDV) is the etiological agent of a lethal and contagious disease of rabbits that remains as a serious problem worldwide. As this virus does not replicate in cell culture systems, the capsid protein gene has been expressed in heterologous hosts or inserted in replication-competent viruses in order to obtain non-conventional RHDV vaccines. However, due to technological or safety issues, current RHDV vaccines are still prepared from organs of infected rabbits. In this work, two human type 5 derived replication-defective adenoviruses encoding the rabbit hemorrhagic disease virus VP60 capsid protein were constructed. The recombinant protein was expressed as a multimer in mouse and rabbit cell lines at levels that ranged from approximately 120 to 160 mg/L of culture. Mice intravenously or subcutaneously inoculated with a single 10(8) gene transfer units (GTU) dose of the AdVP60 vector (designed for VP60 intracellular expression) seroconverted at days 7 and 14 post-immunization, respectively. This vector generated a stronger response than that obtained with a second vector (AdVP60sec) designed for VP60 secretion. Rabbits were then immunized by parenteral or mucosal routes with a single 10(9)GTU dose of the AdVP60 and the antibody response was evaluated using a competition ELISA specific for RHDV or RHDVa. Protective hemagglutination inhibition (HI) titers were also promptly detected and IgG antibodies corresponding with inhibition percentages over 85% persisted up to one year in all rabbits, independently of the immunization route employed. These levels were similar to those elicited with inactivated RHDV or with VP60 obtained from yeast or insect cells. IgA specific antibodies were only found in saliva of rabbits immunized by intranasal instillation. The feasibility of VP60 production and vaccination of rabbits with replication-defective adenoviral vectors was demonstrated.

  13. Comparative Immunization in BALB/c Mice with Recombinant Replication-Defective Adenovirus Vector and DNA Plasmid Expressing a SARS-CoV Nucleocapsid Protein Gene

    Chunling Ma; Kun Yao; Feng Zhou; Minsheng Zhu


    In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus (SARS-CoV)-N gene recombinant replication-defective adenoviral vector, rAd-N, was generated and immunized BALB/c mice in a pcDNA3.1-N prime-rAd-N boost regimen. After humoral and cellular immune response detection, different levels of SARS-CoV N protein specific antibodies and interferon-γ (IFN-γ) secretion are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. There is a significant difference between heterogeneous and homologous vaccinations. The heterogeneous combinations were all higher than those of the homologous combinations in the induction of anti-N antibody response. Among the three heterogeneous combinations, pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N induced the strongest antibody response. In the induction of IFN-γ production, the homologous combination of rAd-N/rAd-N/rAd-N/rAd- N was significantly stronger than that of pcDNA3.1-N/pcDNA3. 1-N/pcDNA3.1-N/pcDNA3.1-N, but was relatively weaker than the heterogeneous combination of pcDAN3.1-N/pcDAN3.1-N/pcDAN3.1-N/rAd-N. This combination was a most efficient immunization regimen in induction of SARS-CoV-N-specific (IFN-γ) secretion just as the antibody response. These results suggest that DNA immunization followed by recombinant adenovirus boosting could be used as a potential SARS-CoV vaccine.

  14. Targeting male mosquito swarms to control malaria vector density

    Sawadogo, Simon Peguedwinde; Niang, Abdoulaye; Bilgo, Etienne; Millogo, Azize; Maïga, Hamidou; Dabire, Roch K.; Tripet, Frederic; Diabaté, Abdoulaye


    Malaria control programs are being jeopardized by the spread of insecticide resistance in mosquito vector populations. It has been estimated that the spread of resistance could lead to an additional 120000 deaths per year, and interfere with the prospects for sustained control or the feasibility of achieving malaria elimination. Another complication for the development of resistance management strategies is that, in addition to insecticide resistance, mosquito behavior evolves in a manner that diminishes the impact of LLINs and IRS. Mosquitoes may circumvent LLIN and IRS control through preferential feeding and resting outside human houses and/or being active earlier in the evening before people go to sleep. Recent developments in our understanding of mosquito swarming suggest that new tools targeting mosquito swarms can be designed to cut down the high reproductive rate of malaria vectors. Targeting swarms of major malaria vectors may provide an effective control method to counteract behavioral resistance developed by mosquitoes. Here, we evaluated the impact of systematic spraying of swarms of Anopheles gambiae s.l. using a mixed carbamate and pyrethroid aerosol. The impact of this intervention on vector density, female insemination rates and the age structure of males was measured. We showed that the resulting mass killing of swarming males and some mate-seeking females resulted in a dramatic 80% decrease in population size compared to a control population. A significant decrease in female insemination rate and a significant shift in the age structure of the male population towards younger males incapable of mating were observed. This paradigm-shift study therefore demonstrates that targeting primarily males rather than females, can have a drastic impact on mosquito population. PMID:28278212

  15. The targeted transduction of MMP-overexpressing tumor cells by ACPP-HPMA copolymer-coated adenovirus conjugates.

    Shuhua Li

    Full Text Available We have designed and tested a new way to selectively deliver HPMA polymer-coated adenovirus type 5 (Ad5 particles into matrix metalloproteinase (MMP-overexpressing tumor cells. An activatable cell penetrating peptide (ACPP was designed and attached to the reactive 4-nitrophenoxy groups of HPMA polymers by the C-terminal amino acid (asparagine, N. ACPPs are activatable cell penetrating peptides (CPPs with a linker between polycationic and polyanionic domains, and MMP-mediated cleavage releases the CPP portion and its attached cargo to enable cell entry. Our data indicate that the transport of these HPMA polymer conjugates by a single ACPP molecule to the cytoplasm occurs via a nonendocytotic and concentration-independent process. The uptake was observed to finish within 20 minutes by inverted fluorescence microscopy. In contrast, HPMA polymer-coated Ad5 without ACPPs was internalized solely by endocytosis. The optimal formulation was not affected by the presence of Ad5 neutralizing antibodies during transduction, and ACPP/polymer-coated Ad5 also retained high targeting capability to several MMP-overexpressing tumor cell types. For the first time, ACPP-mediated cytoplasmic delivery of polymer-bound Ad5 to MMP-overexpressing tumor cells was demonstrated. These findings are significant, as they demonstrate the use of a polymer-based system for the targeted delivery into MMP-overexpressing solid tumors and highlight how to overcome major cellular obstacles to achieve intracellular macromolecular delivery.

  16. Library screening and receptor-directed targeting of gammaretroviral vectors.

    Mazari, Peter M; Roth, Monica J


    Gene- and cell-based therapies hold great potential for the advancement of the personalized medicine movement. Gene therapy vectors have made dramatic leaps forward since their inception. Retroviral-based vectors were the first to gain clinical attention and still offer the best hope for the long-term correction of many disorders. The fear of nonspecific transduction makes targeting a necessary feature for most clinical applications. However, this remains a difficult feature to optimize, with specificity often coming at the expense of efficiency. The aim of this article is to discuss the various methods employed to retarget retroviral entry. Our focus will lie on the modification of gammaretroviral envelope proteins with an in-depth discussion of the creation and screening of envelope libraries.

  17. Avian CD154 enhances humoral and cellular immune responses induced by an adenovirus vector-based vaccine in chickens.

    Sánchez Ramos, Oliberto; González Pose, Alain; Gómez-Puerta, Silvia; Noda Gomez, Julia; Vega Redondo, Armando; Águila Benites, Julio César; Suárez Amarán, Lester; Parra, Natalie C; Toledo Alonso, Jorge R


    Recombinant adenoviral vectors have emerged as an attractive system for veterinary vaccines development. However, for poultry vaccination a very important criterion for an ideal vaccine is its low cost. The objective of this study was to test the ability of chicken CD154 to enhance the immunogenicity of an adenoviral vector-based vaccine against avian influenza virus in order to reduce the amount of antigen required to induce an effective immune response in avian. Chickens were vaccinated with three different doses of adenoviral vectors encoding either HA (AdHA), or HA fused to extracellular domain chicken's CD154 (AdHACD). Hemagglutination inhibition (HI) assay and relative quantification of IFN-γ showed that the adenoviral vector encoding for the chimeric antigen is able to elicit an improved humoral and cellular immune response, which demonstrated that CD154 can be used as a molecular adjuvant allowing to reduce in about 50-fold the amount of adenoviral vector vaccine required to induce an effective immune response.

  18. Cancer targeting Gene-Viro-Therapy specific for liver cancer by α-fetoprotein-controlled oncolytic adenovirus expression of SOCS3 and IL-24

    Xin Cao; Ruicheng Wei; Xinran Liu; Yan Zeng; Hongling Huang; Miao Ding; Kangjian Zhang; Xin-Yuan Liu


    The combination of gene therapy and virotherapy for cancer treatment has received close attention and has become a trend in the field of cancer biotherapy.A strategy called 'Cancer Targeting Gene-Viro-Therapy' (CTGVT) or 'Gene Armed Oncolytic Viral Therapy'(GAOVT) has been proposed,in which an antitumor gene is inserted into an oncolytic viral vector.In our previous study,a dual-regulated oncolytic adenovirus with enhanced safety for normal cells and strict liver cancertargeting ability,designated Ad·enAFP· E1A· E1 B (A55)(briefly Ad·enAFP·D55),was successfully constructed. In the current work,interleukin-24 (IL-24) and suppressor of cytokine signaling 3 (SOCS3) genes were packaged into Ad·enAFP·D55.The new constructs,Ad·enAFP·D55-(IL-24) and Ad·enAFP·D55-(SOCS3),showed improved tumoricidal activity in hepatoma cell lines compared with the oncolytic viral vector Ad·enAFP·D55.The coadministrationofAd · enAFP· D55-(IL-24)and Ad·enAFP·D55-(SOCS3) showed much better antitumor effect than Ad·enAFP·D55-(IL-24) or Ad·enAFP·D55-(SOCS3) alone both in vitro and in a nude mouse xenograft model.Moreover,our results also showed that blockade of the Jak/Stat3 pathway by Ad·enAFP·D55-(SOCS3) infection in HuH-7 cells could down-regulate some anti-apoptosis proteins,such as XIAP,Bcl-xL,and survivin,whichmightsensitizethecellsto Ad·enAFP·D55-(IL-24)-induced apoptosis.These results indicate that co-administration of Ad·enAFP·D55-(IL-24) and Ad·enAFP·D55-(SOCS3) may serve as a candidate therapeutic approach for the treatment of liver cancer.

  19. The Early Activation of CD8+ T Cells Is Dependent on Type I IFN Signaling following Intramuscular Vaccination of Adenovirus Vector

    Masahisa Hemmi


    Full Text Available Few of the vaccines in current use can induce antigen- (Ag- specific immunity in both mucosal and systemic compartments. Hence, the development of vaccines that realize both mucosal and systemic protection against various pathogens is a high priority in global health. Recently, it has been reported that intramuscular (i.m. vaccination of an adenovirus vector (Adv can induce Ag-specific cytotoxic T lymphocytes (CTLs in both systemic and gut mucosal compartments. We previously revealed that type I IFN signaling is required for the induction of gut mucosal CTLs, not systemic CTLs. However, the molecular mechanism via type I IFN signaling is largely unknown. Here, we report that type I IFN signaling following i.m. Adv vaccination is required for the expression of type I IFN in the inguinal lymph nodes (iLNs, which are the draining lymph nodes of the administration site. We also showed that the type I IFN signaling is indispensable for the early activation of CTLs in iLNs. These data suggested that type I IFN signaling has an important role in the translation of systemic innate immune response into mucosal adaptive immunity by amplifying the innate immune signaling and activating CTLs in the iLN.

  20. Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

    Frandsen, Rasmus John Normand; Andersson, Jens A.; Kristensen, Matilde Bylov;


    technique that allows single step cloning of the two required homologous recombination sequences into different sites of a recipient vector. The advantages are: A simple experimental design, free choice of target sequence, few procedures and user convenience. The vectors are intented for Agrobacterium...... with an average efficiency of 84% for gene replacement and 80% for targeted overexpression. Conclusion: The new vectors designed for USER Friendly cloning provided a fast reliable method to construct vectors for targeted gene manipulations in fungi....

  1. Mucosal vaccination with heterologous viral vectored vaccine targeting subdominant SIV accessory antigens strongly inhibits early viral replication

    Xu, Huanbin; Andersson, Anne-Marie; Ragonnaud, Emeline


    Conventional HIV T cell vaccine strategies have not been successful in containing acute peak viremia, nor in providing long-term control. We immunized rhesus macaques intramuscularly and rectally using a heterologous adenovirus vectored SIV vaccine regimen encoding normally weakly immunogenic tat...

  2. Breadth of T cell responses after immunization with adenovirus vectors encoding ancestral antigens or polyvalent papillomavirus antigens

    Ragonnaud, Emeline; Pedersen, Anders Gorm; Holst, Peter Johannes


    to the other PV proteins. The PV sequences were fused to a T cell adjuvant, the murine invariant chain and encoded in a recombinant adenoviral vector which was administered to naïve outbred mice. By measuring T cell responses induced by these different vaccines and towards peptide pools representing 3...

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

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


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

  4. Protection of Nonhuman Primates Against Two Species of Ebola Virus Infection With a Single Complex Adenovirus Vector


    Zaire Ebola virus . Virology 346:394–401. 21. Leffel, E. K., and D. S. Reed. 2004. Marburg and Ebola viruses as aerosol threats. Biosecur. Bioterror. 2...the event of a natural virus outbreak or biological threat. The filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), cause outbreaks of severe...vaccine efficacy are justifiable. While the combination of the two CAdVax-based vectors for Ebola virus (EBO7) and Marburg virus (M8) GPs constitute the

  5. Adenovirus-mediated siRNA targeting TNF-α and overexpression of bone morphogenetic protein-2 promotes early osteoblast differentiation on a cell model of Ti particle-induced inflammatory response in vitro

    Guo, H.H.; Yu, C.C.; Sun, S.X. [Affiliated Hospital of Ningxia Medical University, Department of Orthopedic Surgery, Yinchuan (China); Ma, X.J. [Ningxia Medical Autonomous Region of the First People' s Hospital, Department of Orthopedic Surgery, Yinchuan (China); Yang, X.C.; Sun, K.N.; Jin, Q.H. [Affiliated Hospital of Ningxia Medical University, Department of Orthopedic Surgery, Yinchuan (China)


    Wear particles are phagocytosed by macrophages and other inflammatory cells, resulting in cellular activation and release of proinflammatory factors, which cause periprosthetic osteolysis and subsequent aseptic loosening, the most common causes of total joint arthroplasty failure. During this pathological process, tumor necrosis factor-alpha (TNF-α) plays an important role in wear-particle-induced osteolysis. In this study, recombination adenovirus (Ad) vectors carrying both target genes [TNF-α small interfering RNA (TNF-α-siRNA) and bone morphogenetic protein 2 (BMP-2)] were synthesized and transfected into RAW264.7 macrophages and pro-osteoblastic MC3T3-E1 cells, respectively. The target gene BMP-2, expressed on pro-osteoblastic MC3T3-E1 cells and silenced by the TNF-α gene on cells, was treated with titanium (Ti) particles that were assessed by real-time PCR and Western blot. We showed that recombinant adenovirus (Ad-siTNFα-BMP-2) can induce osteoblast differentiation when treated with conditioned medium (CM) containing RAW264.7 macrophages challenged with a combination of Ti particles and Ad-siTNFα-BMP-2 (Ti-ad CM) assessed by alkaline phosphatase activity. The receptor activator of nuclear factor-κB ligand was downregulated in pro-osteoblastic MC3T3-E1 cells treated with Ti-ad CM in comparison with conditioned medium of RAW264.7 macrophages challenged with Ti particles (Ti CM). We suggest that Ad-siTNFα-BMP-2 induced osteoblast differentiation and inhibited osteoclastogenesis on a cell model of a Ti particle-induced inflammatory response, which may provide a novel approach for the treatment of periprosthetic osteolysis.

  6. Adenovirus-mediated siRNA targeting TNF-α and overexpression of bone morphogenetic protein-2 promotes early osteoblast differentiation on a cell model of Ti particle-induced inflammatory response in vitro.

    Guo, H H; Yu, C C; Sun, S X; Ma, X J; Yang, X C; Sun, K N; Jin, Q H


    Wear particles are phagocytosed by macrophages and other inflammatory cells, resulting in cellular activation and release of proinflammatory factors, which cause periprosthetic osteolysis and subsequent aseptic loosening, the most common causes of total joint arthroplasty failure. During this pathological process, tumor necrosis factor-alpha (TNF-α) plays an important role in wear-particle-induced osteolysis. In this study, recombination adenovirus (Ad) vectors carrying both target genes [TNF-α small interfering RNA (TNF-α-siRNA) and bone morphogenetic protein 2 (BMP-2)] were synthesized and transfected into RAW264.7 macrophages and pro-osteoblastic MC3T3-E1 cells, respectively. The target gene BMP-2, expressed on pro-osteoblastic MC3T3-E1 cells and silenced by the TNF-α gene on cells, was treated with titanium (Ti) particles that were assessed by real-time PCR and Western blot. We showed that recombinant adenovirus (Ad-siTNFα-BMP-2) can induce osteoblast differentiation when treated with conditioned medium (CM) containing RAW264.7 macrophages challenged with a combination of Ti particles and Ad-siTNFα-BMP-2 (Ti-ad CM) assessed by alkaline phosphatase activity. The receptor activator of nuclear factor-κB ligand was downregulated in pro-osteoblastic MC3T3-E1 cells treated with Ti-ad CM in comparison with conditioned medium of RAW264.7 macrophages challenged with Ti particles (Ti CM). We suggest that Ad-siTNFα-BMP-2 induced osteoblast differentiation and inhibited osteoclastogenesis on a cell model of a Ti particle-induced inflammatory response, which may provide a novel approach for the treatment of periprosthetic osteolysis.

  7. Portfolio Optimization with Target-Shortfall-Probability Vector

    Leo Schubert


    Full Text Available Traditional portfolio optimization uses the standard deviation of the returns as a measure of risk. In recent years, the Target-Shortfall-Probability (TSP was discussed as an alternative measure. From the utility-theoretical point of view, the TSP is not perfect. Furthermore it is criticized due to the insufficient description of the risk. The advantages of the TSP are the usage independent of the distribution and the intuitive understanding by the investor. The use of a TSP-vector reduces an utility-theoretical disadvantage of a single TSP and offers an sufficient description of risk. The developed Mean-TSP-vector model is a mixed-integer linear program. The CPU-Time of the program to get a solution demonstrates that the model is suitable for practical applications. A test of the performance shows, that the average return of the model when used in bear markets is equal to the results of the traditional portfolio optimization but - due to skewness - in bullish markets can achieve better returns.

  8. Development of a novel adenovirus-alphavirus hybrid vector with RNA replicon features for malignant hematopoietic cell transduction.

    Yang, Y; Xiao, F; Lu, Z; Li, Z; Zuo, H; Zhang, Q; Li, Q; Wang, H; Wang, L-S


    To improve the expression levels of transgenes in malignant hematopoietic cells, we developed a novel adenoviral-alphavirus hybrid vector Ad5/F11p-SFV-GFP that contains a Semliki Forest Virus (SFV) replicon and chimeric fibers of Ad5 and Ad11p. Ad5/F11p-SFV-GFP infected >95% of K562, U937 or Jurkat cells and 23.65% of HL-60 cells, and led to moderate Enhanced Green Fluorescent Protein (EGFP) transgene expression intensity. The infection efficiency of Ad5/F11p-SFV-GFP in primary human leukemia cells ranged from 9.34-89.63% (median, 28.58%) at a multiplicity of infection (MOI) of 100, compared with only 3.37-44.54% (median, 10.42%) in cells infected by Ad5/F11p-GFP. Importantly, Ad5/F11p-SFV-GFP led to a significantly higher transgene expression level in primary leukemia cells, as indicated by the relative fluorescence intensity, compared to cells infected with Ad5/F11p-GFP. The increased expression of EGFP in Ad5/F11p-SFV-GFP-infected cells was associated with the accumulation of abundant subgenomic mRNA. Additionally, infection of K562, U937 or Jurkat cells by Ad5/F11p-SFV-GFP was significantly inhibited by blocking CD46 receptor; however, other factors may affect the gene-transfer efficiency of Ad5/F11p-SFV-GFP in primary leukemia cells. In conclusion, we successfully developed a novel adenoviral-alphavirus hybrid vector with RNA replicon features, which represents a promising vector for gene modifications during the production of cell-based vaccines for leukemia patients.

  9. Threat Assessment of Targets Based on Support Vector Machine

    CAI Huai-ping; LIU Jing-xu; CHEN Ying-wu


    In the context of cooperative engagement of armored vehicles, the threat factors of offensive targets are analyzed, and a threat assessment (TA) model is built based on a support v.ector machine (SVM) method. The SVM-based model has some advantages over the traditional method-based models: the complex factors of threat are considered in the cooperative engagement; the shortcomings of neural networks, such as local minimum and "over fitting", are overcome to improve the generalization ability; its operation speed is high and meets the needs of real time C2 of cooperative engagement; the assessment results could be more reasonable because of its self-learning capability. The analysis and simulation indicate that the SVM method is an effective method to resolve the TA problems.

  10. HBsAg及B7-2抗原重组腺病毒载体感染免疫研究%Humoral immunization and cell-mediated immunization evoked by HBsAg and B7-2 Ag coexpression recombinant adenovirus vector

    周智; 张定凤; 任红


    目的为激发机体对HBsAg的CTL反应,寻求对慢性乙型肝炎更有效的治疗方法。方法构建了共表达HBsAg及B7-2抗原的E1区插入重组腺病毒载体,用脂质体法转染293细胞,经空斑筛选表达目的抗原的重组腺病毒,用ELISA法及Western blotting法分别检测HBsAg及B7-2抗原表达。在293细胞中扩增,再纯化、浓缩后,体内感染C57小鼠,检测体液免疫反应和细胞免疫反应。结果重组腺病毒在体外培养的293细胞及HepG2细胞中高效表达HBsAg及B7-2抗原。感染小鼠体液免疫较弱,但可通过注射乙型肝炎疫苗而加强;重组腺病毒载体能诱导强而有效的细胞免疫反应;未观察到明显毒副作用。结论 HBsAg及B7-2抗原重组腺病毒载体体外感染293、HepG2细胞后高效表达目的抗原,感染免疫后诱导有效的细胞免疫反应。初步结果显示腺病毒载体是一种高效、安全的载体系统。%Objective To evoke cytotoxic T lymphocytes (CTL) response and seek for a more effective method to treat chronic hepatitis B. Methods The adenovirus vector was constructed with the foreign genes inserted in the early region 1(E1), which directed coexpression of HBV-S and B7-2 antigens by means of an internal ribosomal entry site placed between the two coding sequences. The vector was transfected into 293 cell lines by liposome and the adenovirus expressing the target antigens was obtained by plaque select. The HBsAg and B7-2 antigen expression in in vitro cell culture was measured by ELISA and Western blotting, respectively. The immune responses were measured by ELISA for antibody response and a LDH release assay for CTL activity after immunization with the recombinant adenovirus vector in C57 mice. Results HBsAg and B7-2 antigens were highly expressed after infecting the 293 and HepG2 cell lines in vitro. The humoral response to hepatitis B surface antigen was mildly induced and could be enhanced by reinjecting a

  11. A novel bicistronic high-capacity gutless adenovirus vector that drives constitutive expression of herpes simplex virus type 1 thymidine kinase and tet-inducible expression of Flt3L for glioma therapeutics.

    Puntel, Mariana; Muhammad, A K M G; Candolfi, Marianela; Salem, Alireza; Yagiz, Kader; Farrokhi, Catherine; Kroeger, Kurt M; Xiong, Weidong; Curtin, James F; Liu, Chunyan; Bondale, Niyati S; Lerner, Jonathan; Pechnick, Robert N; Palmer, Donna; Ng, Philip; Lowenstein, Pedro R; Castro, Maria G


    Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Conditional cytotoxic/immune-stimulatory gene therapy (Ad-TK and Ad-Flt3L) elicits tumor regression and immunological memory in rodent GBM models. Since the majority of patients enrolled in clinical trials would exhibit adenovirus immunity, which could curtail transgene expression and therapeutic efficacy, we used high-capacity adenovirus vectors (HC-Ads) as a gene delivery platform. Herein, we describe for the first time a novel bicistronic HC-Ad driving constitutive expression of herpes simplex virus type 1 thymidine kinase (HSV1-TK) and inducible Tet-mediated expression of Flt3L within a single-vector platform. We achieved anti-GBM therapeutic efficacy with no overt toxicities using this bicistronic HC-Ad even in the presence of systemic Ad immunity. The bicistronic HC-Ad-TK/TetOn-Flt3L was delivered into intracranial gliomas in rats. Survival, vector biodistribution, neuropathology, systemic toxicity, and neurobehavioral deficits were assessed for up to 1 year posttreatment. Therapeutic efficacy was also assessed in animals preimmunized against Ads. We demonstrate therapeutic efficacy, with vector genomes being restricted to the brain injection site and an absence of overt toxicities. Importantly, antiadenoviral immunity did not inhibit therapeutic efficacy. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L genes. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.

  12. Construction and expression of recombinant adenovirus vector encoding BDNF%重组大鼠脑源性神经营养因子腺病毒的构建及体外表达分析

    郎洪刚; 曹文斌; 牛道立; 何芬


    Objective To construct adenovirus vector encoding rat brain derived neurotrophic factor (BDNF) gene and identify the expression in vitro.Methods The specific BDNF sequence was cloned into the plasmid of pAdTrack-CMV to construct the BDNF expression plasmid pAd-BDNF.The recombinant plasmids were identified hy DNA sequencing and restriction digestion.The homologous recomhination between the recombinant vector and the adenovirus hone vector pAdeasy-1 in the Ecoli BJ5183 resulted to the formation of recombinant adenovirus vector Ad-BDNF.The infecting recomhinant virus particles Ad-BDNF was produced after the recombinant adenovirus vector had been transfected to the human embry kidney 293 cells.The recombinant adenovirus vector infected the hela cell, the expression of BDNF was detected by RT-PCR, Western blot and immunocytochemistry.Results The sequence results of pAd-BDNF were consistent with expectancy.After homologous recombination and packaging with 293 cells , the recombinant Ad-BDNF adenovirus was obtained.RT-PCR, Western blot and immunocytochemistry suggested that the BDNF was expressed.Conclusions The Ad-BDNF was constructed successfully.It is confirmed that the interest proteins were expressed in the infected cells , which lays the basis for its application in the treatment of the neurodamaged diseases.%目的 构建含有大鼠脑源性神经营养因子(BDNF)基因的重组腺病毒(AD)载体,并分析其体外表达情况.方法 将采用RT-PCR技术获取的大鼠BDNF的cDNA基因定向克隆入穿梭质粒pAdTrack-CMV中.通过与腺病毒骨架载体pAdeasy-1在细菌内同源重组形成重组腺病毒质粒pAd-BDNF,转染人胚肾293细胞后包装成有感染能力的重组腺病毒颗粒(Ad-BDNF).重组病毒感染体外培养的Hela细胞后,用RT-PCR、Western blot及免疫细胞化学检测细胞BDNF基因及蛋白表达情况.结果 pAd-BDNF测序结果和预期一致,同源重组并经293细胞包装后形成重组腺病毒Ad-BDNF,重组病毒

  13. Adenovirus-mediated gene transfer to tumor cells.

    Cascalló, Manel; Alemany, Ramon


    Cell transduction in vitro is only the first step toward proving that a genetherapy vector can be useful to treat tumors. However, tumor targeting in vivo is now the milestone for gene therapy to succeed against disseminated cancer. Therefore, most valuable information is obtained from studies of vector biodistribution. Owing to the hepatotropism of adenoviral vectors, a particularly important parameter is the tumor/liver ratio. This ratio can be given at the level of gene expression if the amount of transgene expression is measured. To optimize the targeting, however, the levels of viral particles that reach the tumor compared to other organs must be studied. Most of this chapter deals with methods to quantify the virus fate in tumor-bearing animals. We present a radioactive labeling method that can be used to study biodistribution. After a small section dealing with tumor models, we describe methods to quantify different parameters related to adenovirus-mediated tumor targeting.

  14. Clustered Integrin Ligands as a Novel Approach for the Targeting of Non-Viral Vectors

    Ng, Quinn Kwan Tai

    Gene transfer or gene delivery is described as the process in which foreign DNA is introduced into cells. Over the years, gene delivery has gained the attention of many researchers and has been developed as powerful tools for use in biotechnology and medicine. With the completion of the Human Genome Project, such advances in technology allowed for the identification of diseases ranging from hereditary disorders to acquired ones (cancer) which were thought to be incurable. Gene therapy provides the means necessary to treat or eliminate genetic diseases from its origin, unlike traditional medicine which only treat symptoms. With ongoing clinical trials for gene therapy increasing, the greatest difficulty still lies in developing safe systems which can target cells of interest to provide efficient delivery. Nature, over millions of years of evolution, has provided an example of one of the most efficient delivery systems: viruses. Although the use of viruses for gene delivery has been well studied, the safety issues involving immunogenicity, insertional mutagenesis, high cost, and poor reproducibility has provided problems for their clinical application. From understanding viruses, we gain insight to designing new systems for non-viral gene delivery. One of these techniques utilized by adenoviruses is the clustering of ligands on its surface through the use of a protein called a penton base. Through the use of nanotechnology we can mimic this basic concept in non-viral gene delivery systems. This dissertation research is focused on developing and applying a novel system for displaying the integrin binding ligand (RGD) in a constrained manner to form a clustered integrin ligand binding platform to be used to enhance the targeting and efficiency of non-viral gene delivery vectors. Peptide mixed monolayer protected gold nanoparticles provides a suitable surface for ligand clustering. A relationship between the peptide ratios in the reaction solution used to form these

  15. Therapeutic induction of angiogenesis by direct myocardial administration of an adenovirus vector encoding human hepatocyte growth factor gene and its safety

    WU Danli; ZHANG Yourong; LAO Miaofen; YUAN Lizhen; WANG Lan; HA Xiaoqin; WU Zuze(WU Cutse)


    After the study in vitro and in rats, we assessed further the effects and safety of local angiogen therapy using intramyocardial delivery of an adenovirus carrying hepatocyte growth factor gene (Ad-HGF) in a canine ischemia model. The angiogenic activity of Ad-HGF was evaluated from three aspects. First, the augmentation of collateral vessel development was assessed by angiography 30 d after surgery. The results showed that the density of collateral vessels in treated group was higher than that of control group. Secondly, infarct size was evaluated by TTC staining and image analysis. The results showed that the infarct size of treated group was smaller than that of control group. Thirdly, the myocardial regional blood flow was determined by the method of colored microspheres. The results showed that the blood flow recovered to the level before ligation in treated group, but that of the control group was lower than normal level. In addition, during the study of chronic toxicity, we tested the anti-adenovirus antibodies by neutralization method. The antibodies yielded after the fourth injection decreased slowly from peak level and disappeared 12 weeks after drug withdrawal. Overall, Ad-HGF can promote angiogenesis in ischemic myocardium and reduce infarct size.So this method may be considered as a therapeutic angiogenesis induction strategy for ischemic disease including myocardial infarction and peripheral artery disease. At the same time, Ad-HGF could induce the yield of anti-adenovirus antibodies to neutralize adenovirus, which may be the mechanism of adenovirus clearance.

  16. Mucosal vaccination by adenoviruses displaying reovirus sigma 1

    Weaver, Eric A. [Department of Internal Medicine, Division of Infectious Diseases, Translational Immunovirology and Biodefense Program, Mayo Clinic, Rochester, MN 55902 (United States); Camacho, Zenaido T. [Department of Cell Biology, Department of Natural Sciences, Western New Mexico University, Silver City, NM 88062 (United States); Hillestad, Matthew L. [Nephrology Training Program, Mayo Clinic, Rochester, MN 55902 (United States); Crosby, Catherine M.; Turner, Mallory A.; Guenzel, Adam J.; Fadel, Hind J. [Virology and Gene Therapy Graduate Program, Mayo Clinic, Rochester, MN 55902 (United States); Mercier, George T. [Department of Physics, University of Houston, Houston, TX 77004 (United States); Barry, Michael A., E-mail: [Department of Internal Medicine, Division of Infectious Diseases, Translational Immunovirology and Biodefense Program, Mayo Clinic, Rochester, MN 55902 (United States); Department of Immunology and Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55902 (United States)


    We developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but has 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generates stronger T cell responses than Ad5 when used for mucosal immunization. In this work, new Ad5-fiber-sigma vectors were generated by varying the number of fiber β-spiral shaft repeats (R) between the fiber tail and sigma. Increasing chimera length led to decreasing insertion of these proteinsAd5 virions. Ad-R3 and R14 vectors effectively targeted JAM-1 in vitro while R20 did not. When wereused to immunize mice by the intranasal route, Ad5-R3-sigma produced higher serum and vaginal antibody responses than Ad5. These data suggest optimized Ad-sigma vectors may be useful vectors for mucosal vaccination. - Highlights: • Constructed adenoviruses (Ads) displaying different reovirus sigma 1 fusion proteins. • Progressively longer chimeras were more poorly encapsidated onto Ad virions. • Ad5-R3-sigma mediated better systemic and mucosal immune responses than Ad5.

  17. Nucleic acid sequences encoding D1 and D1/D2 domains of human coxsackievirus and adenovirus receptor (CAR)

    Freimuth, Paul I.


    The invention provides recombinant human CAR (coxsackievirus and adenovirus receptor) polypeptides which bind adenovirus. Specifically, polypeptides corresponding to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2 are provided. In another aspect, the invention provides nucleic acid sequences encoding these domains and expression vectors for producing the domains and bacterial cells containing such vectors. The invention also includes an isolated fusion protein comprised of the D1 polypeptide fused to a polypeptide which facilitates folding of D1 when expressed in bacteria. The functional D1 domain finds application in a therapeutic method for treating a patient infected with a CAR D1-binding virus, and also in a method for identifying an antiviral compound which interferes with viral attachment. The invention also provides a method for specifically targeting a cell for infection by a virus which binds to D1.

  18. A phase I double blind, placebo-controlled, randomized study of a multigenic HIV-1 adenovirus subtype 35 vector vaccine in healthy uninfected adults.

    Michael C Keefer

    Full Text Available BACKGROUND: We conducted a phase I, randomized, double-blind, placebo-controlled trial to assess the safety and immunogenicity of escalating doses of two recombinant replication defective adenovirus serotype 35 (Ad35 vectors containing gag, reverse transcriptase, integrase and nef (Ad35-GRIN and env (Ad35-ENV, both derived from HIV-1 subtype A isolates. The trial enrolled 56 healthy HIV-uninfected adults. METHODS: Ad35-GRIN/ENV (Ad35-GRIN and Ad35-ENV mixed in the same vial in equal proportions or Ad35-GRIN was administered intramuscularly at 0 and 6 months. Participants were randomized to receive either vaccine or placebo (10/4 per group, respectively within one of four dosage groups: Ad35-GRIN/ENV 2×10(9 (A, 2×10(10 (B, 2×10(11 (C, or Ad35-GRIN 1×10(10 (D viral particles. RESULTS: No vaccine-related serious adverse event was reported. Reactogenicity events reported were dose-dependent, mostly mild or moderate, some severe in Group C volunteers, all transient and resolving spontaneously. IFN-γ ELISPOT responses to any vaccine antigen were detected in 50, 56, 70 and 90% after the first vaccination, and in 75, 100, 88 and 86% of Groups A-D vaccine recipients after the second vaccination, respectively. The median spot forming cells (SFC per 10(6 PBMC to any antigen was 78-139 across Groups A-C and 158-174 in Group D, after each of the vaccinations with a maximum of 2991 SFC. Four to five HIV proteins were commonly recognized across all the groups and over multiple timepoints. CD4+ and CD8+ T-cell responses were polyfunctional. Env antibodies were detected in all Group A-C vaccinees and Gag antibodies in most vaccinees after the second immunization. Ad35 neutralizing titers remained low after the second vaccination. CONCLUSION/SIGNIFICANCE: Ad35-GRIN/ENV reactogenicity was dose-related. HIV-specific cellular and humoral responses were seen in the majority of volunteers immunized with Ad35-GRIN/ENV or Ad35-GRIN and increased after the second

  19. Direct demonstration of Ca2+ binding defects in sarco-endoplasmic reticulum Ca2+ ATPase mutants overexpressed in COS-1 cells transfected with adenovirus vectors.

    Strock, C; Cavagna, M; Peiffer, W E; Sumbilla, C; Lewis, D; Inesi, G


    Single mutations of specific amino acids within the membrane-bound region of the sarco-endoplasmic reticulum Ca2+ (SERCA)-1 ATPase interfere with Ca2+ inhibition of ATPase phosphorylation by Pi (1), suggesting that these residues may be involved in complexation of two Ca2+ that are known to bind to the enzyme. However, direct measurements of Ca2+ binding in the absence of ATP have been limited by the low quantities of available mutant protein. We have improved the transfection efficiency by means of recombinant adenovirus vectors, yielding sufficient expression of wild type and mutant SERCA-1 ATPase for measurements of Ca2+ binding to the microsomal fraction of the transfected cells. We find that in the presence of 20 microM Ca2+ and in the absence of ATP, the Glu771 --> Gln, Thr799 --> Ala, Asp800 --> Asn, and Glu908 --> Ala mutants exhibit negligible binding, indicating that the oxygen functions of Glu771, Thr799, Asp800, and Glu908 are involved in interactions whose single disruption causes major changes in the highly cooperative "duplex" binding. Total loss of Ca2+ binding is accompanied by loss of Ca2+ inhibition of the Pi reaction. We also find that, at pH 7.0, the Glu309 --> Gln and the Asn796 --> Ala mutants bind approximately half as much Ca2+ as the wild type ATPase and do not interfere with Ca2+ inhibition of the Pi reaction. At pH 6.2, the Glu309 --> Gln mutant does not bind any Ca2+, and its phosphorylation by Pi is not inhibited by Ca2+. On the contrary, the Asn796 --> Ala mutant retains the behavior displayed at pH 7.0. This suggests that in the Glu309 --> Gln mutant, ionization of acidic functions in other amino acids (e.g. Glu771 and Asp800) occurs as the pH is shifted, thereby rendering Ca2+ binding possible. In the Asn796 --> Ala mutant, on the other hand, the Glu309 carboxylic function allows binding of inhibitory Ca2+ even at pH 6.2. In all cases mutational interference with the inhibition of the Pi reaction by Ca2+ can be overcome by raising

  20. Gene therapy for colorectal cancer by adenovirus-mediated siRNA targeting CD147 based on loss of the IGF2 imprinting system.

    Pan, Yuqin; He, Bangshun; Chen, Jie; Sun, Huiling; Deng, Qiwen; Wang, Feng; Ying, Houqun; Liu, Xian; Lin, Kang; Peng, Hongxin; Xie, Hongguang; Wang, Shukui


    Colorectal cancer (CRC) is one of the most common malignant tumors worldwide. Loss of imprinting (LOI) of the insulin-like growth factor 2 (IGF2) gene is an epigenetic abnormality phenomenon in CRC. Recently observed association of CRC with cluster of differentiation 147 (CD147) could provide a novel approach for gene therapy. In the present study, we investigated the feasibility of using adenovirus‑mediated siRNA targeting CD147 based on the IGF2 LOI system for targeted gene therapy of CRC. A novel adenovirus-mediated siRNA targeting CD147, rAd-H19-CD147mirsh, which was driven by the IGF2 imprinting system, was constructed. The results showed that the EGFP expression was detected only in the IGF2 LOI cell lines (HT-29 and HCT-8), but that no EGFP was produced in cell lines with maintenance of imprinting (MOI) (HCT116). Moreover, rAd-H19-CD147mirsh significantly inhibited the expression of CD147, decreased cell viability and invasive ability, and increased sensitivity to chemotherapeutic drugs only in the LOI cell lines in vitro. Furthermore, mice bearing HT-29 xenografted tumors, which received intratumoral administration of the rAd-H19-CD147mirsh, showed significantly reduced tumor growth and enhanced survival. We conclude that recombinant adenovirus-mediated siRNA targeting CD147 based on the IGF2 LOI system inhibited the growth of the LOI cells in vitro and in vivo, which would provide a novel approach for targeted CRC gene therapy.

  1. Construction of recombinant adenovirus co-expression vector carrying the human transforming growth factor-β1 and vascular endothelial growth factor genes and its effect on anterior cruciate ligament fibroblasts

    WEI Xue-lei; LIN Lin; HOU Yu; FU Xin; ZHANG Ji-ying; MAO Ze-bin; YU Chang-long


    Background Remodeling of the anterior cruciate ligament (ACL) graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal ribosome entry site (IRES) sequence was used to construct a new recombinant adenovirus which permits co-expression of transforming growth factor-β1 (TGFβ1) and vascular endothelial growth factor 165 (VEGF165) genes (named Ad-VEGF165-1RES-TGFβ1). We investigated the effects of the new adenovirus on the migration of and matrix synthesis by ACL fibroblasts.Methods Adenoviral vector containing TGFβ1 and VEGF165 genes was constructed. ACL fibroblasts were obtained from New Zealand white rabbits. After ACL fibroblasts were exposed to Ad-VEGF165-1RES-TGFβ1, the expression of VEGF165 and TGFβ1 proteins were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. Bioassay of VEGF165 and TGFβ1 proteins were assessed by Western blotting analysis. Proliferation and migration of ACL fibroblasts were assessed by in vitro wound closure assay. Gene expression of collagen type I, collagen type Ⅲ, and fibronectin mRNA among matrix markers were assessed by real-time PCR.Results The results showed the successful construction of a recombinant co-expression adenovirus vector containing TGFβI and VEGF165 genes. Co-expression of TGFβ1 and VEGF165 can induce relatively rapid and continuous proliferation of ACL fibroblasts and high gene expression of collagen type Ⅰ, collagen typeⅢ, and fibronectin mRNA among matrix markers.Conclusion Co-expression of TGFβ1 and VEGF165 genes has more powerful and efficient effects on the migration of and matrix synthesis by ACL fibroblasts.

  2. Intranuclear targeting and nuclear export of the adenovirus E1B-55K protein are regulated by SUMO1 conjugation

    Kindsmüller, Kathrin; Groitl, Peter; Härtl, Barbara; Blanchette, Paola; Hauber, Joachim; Dobner, Thomas


    We have investigated the requirements for CRM1-mediated nuclear export and SUMO1 conjugation of the adenovirus E1B-55K protein during productive infection. Our data show that CRM1 is the major export receptor for E1B-55K in infected cells. Functional inactivation of the E1B-55K CRM1-dependent nuclear export signal (NES) or leptomycin B treatment causes an almost complete redistribution of the viral protein from the cytoplasm to the nucleus and its accumulation at the periphery of the viral re...

  3. 大鼠乙醛脱氢酶2基因调控腺病毒载体构建方法及意义%Methodology on construction of rat aldehyde dehydrogenase 2 gene regulation recombinant adenovirus vectors

    李鸿博; 郎小娥


    目的:观察持续活化突变体腺病毒转染大鼠心肌细胞的转染效果及对乙醛脱氢酶2(ALDH2)表达的影响。方法分别将扩增得到的 ALDH2持续活化突变体基因及合成 ALDH2-siRNA 序列颈环状 DNA,连接相应载体后,得到重组穿梭质粒;对2种穿梭质粒分别进行扩增和酶切鉴定,并导入 pAdeno 腺病毒载体,转染293细胞进行扩增与纯化。将1日龄雄性 SD 大鼠心肌细胞进行培养,将2种重组腺病毒及对照空载体分别感染细胞,随后检测 ALDH2表达量。结果2种载体构建正确,纯化后两者滴度分别为2×1010,1.6×1010 PFU・ mL-1。实验组 ALDH2表达量与对照组相比差异具有统计学意义(P <0.01)。结论成功构建大鼠 ALDH2基因双向调控腺病毒载体,可以有效调控离体大鼠心肌细胞 ALDH2表达。%Objective To construct adenovirus specific for rat aldehyde dehydrogenase 2 (ALDH2) gene interference and consistent activation and transfect the viruses into rat cardiomyocytes to observe transfection effect and its influence on ALDH2 expression.Methods Consistently active ALDH2 (CA -ALDH2) mutant gene was amplified and linked to shuttle vector, thus recombinant shuttle plasmid was subsequently con -structed.Stem -loop DNA for ALDH2 silencing RNA ( ALDH2 -siRNA) sequence was synthesized and loaded it into vector thus recombi -nant shuttle plasmid was constructed .Both kinds of plasmid were imple -mented amplification and enzyme identification.Verified plasmids were loaded into pAdeno adenovirus vectors.The viruses were then transfected into 293 cell linage to replicate and be purified.Treat cultured cardio-myocytes from 1 -day -old neonatal male Sprague Dawley (SD) rat with empty adenovirus vector control and both kinds of recombinant adenovirus vector, and perform subsequent assay for ALDH2 expression.Results Both vectors are identified by endonuclease with titre of 2 ×10 10 , 1.6 ×10 10 PFU ・ mL-1

  4. Vectores


    Documento que contiene la explicación sobre las temáticas de Sistemas coordenados, Cantidades vectoriales y escalares, Algunas propiedades de los vectores, Componentes de un vector y vectores unitarios

  5. 羊传染性脓疱病毒B2L基因片段重组腺病毒载体的构建%Construction of Recombinant Adenovirus Vector Containing CEVB2L Gene

    邵洪泽; 毛文智; 宋阳; 李琳; 程荣华; 孙健; 孙强


    [目的]利用腺病毒载体系统构建羊传染性脓疱病毒B2L基因重组腺病毒载体.[方法]以从羊传染性脓疱病毒株JLSY04中提取的基因组DNA为模板,PCR扩增获得B2L目的基因片段;然后将B2L目的基因克隆至PDNR-CMV载体,筛选阳性克隆获得质粒CTC572-6;再将质粒CTC572-6与腺病毒载体进行同源重组,筛选阳性克隆,并进行菌液PCR、酶切、测序等鉴定.[结果]经酶切和基因测序等鉴定,成功构建了携带羊传染性脓疱病毒B2L基因的重组腺病毒载体CTC572Ade-30.[结论]为羊传染性脓疱基因工程疫苗的进一步研究奠定基础.%[Objective] Sheep contagious ecthyma virus B2L gene recombinant adenovirus was built by adenovirus vector system.[Method] Genome DNA extracted from sheep contagious ecthyma virus strain JLSY04 as a template,Gene fragments obtained from B2L by PCR amplification; B2L gene cloning was cloned into PDNR-CMD vector,screening positive clones and plasmid CTC572-6 was obtained; CTC572-6 plasmid for homologous was recombined with the adenoviral vector.Screening positive clones and bacilli PCR,digestion and sequencing and so on were identified.[Result] After identified by enzyme digestion and gene sequencing,recombinant adenovirus vector CTC572Ade-30 of carrying sheep contagious ecthyma virus B2L gene was constructed successfully.[Conclusion] Which laid the foundation for sheep contagious ecthyma genetically engineered vaccine.

  6. Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

    Frandsen, Rasmus John Normand; Andersson, Jens A.; Kristensen, Matilde Bylov;


    of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene. Results: Here, we present a USER Friendly cloning based...... tumefaciens and protoplast based transformation technologies. The system has been tested by the construction of vectors for targeted replacement of 17 genes and overexpression of 12 genes in Fusarium graminearum. The results show that four fragment vectors can be constructed in a single cloning step...... with an average efficiency of 84% for gene replacement and 80% for targeted overexpression. Conclusion: The new vectors designed for USER Friendly cloning provided a fast reliable method to construct vectors for targeted gene manipulations in fungi....

  7. A Tetravalent Dengue Vaccine Based on a Complex Adenovirus Vector Provides Significant Protection in Rhesus Monkeys against All Four Serotypes of Dengue Virus▿

    Raviprakash, Kanakatte; Wang, Danher; Ewing, Dan; Holman, David H.; Block, Karla; Woraratanadharm, Jan; Chen, Lan; Hayes, Curtis; Dong, John Y.; Porter, Kevin


    Nearly a third of the human population is at risk of infection with the four serotypes of dengue viruses, and it is estimated that more than 100 million infections occur each year. A licensed vaccine for dengue viruses has become a global health priority. A major challenge to developing a dengue vaccine is the necessity to produce fairly uniform protective immune responses to all four dengue virus serotypes. We have developed two bivalent dengue virus vaccines, using a complex adenovirus vect...

  8. Oncolytic Adenoviruses in Cancer Treatment

    Ramon Alemany


    Full Text Available The therapeutic use of viruses against cancer has been revived during the last two decades. Oncolytic viruses replicate and spread inside tumors, amplifying their cytotoxicity and simultaneously reversing the tumor immune suppression. Among different viruses, recombinant adenoviruses designed to replicate selectively in tumor cells have been clinically tested by intratumoral or systemic administration. Limited efficacy has been associated to poor tumor targeting, intratumoral spread, and virocentric immune responses. A deeper understanding of these three barriers will be required to design more effective oncolytic adenoviruses that, alone or combined with chemotherapy or immunotherapy, may become tools for oncologists.

  9. CBF mediates adenovirus Ela trans-activation by interaction at the C-terminal promoter targeting domain of conserved region 3.

    Agoff, S N; Wu, B


    Genetic and biochemical evidence suggest that conserved region 3 (CR3) of the adenovirus Ela polypeptide can provide two distinct and separable functions: an N-terminal transcriptional activation region and a C-terminal promoter targeting region. It is thought that the promoter targeting region of Ela CR3 interacts with promoter-specific transcription factors, thereby bringing the activation region of Ela CR3 in proximity of the promoter. Here we report that CBF, a CCAAT-box-binding factor that regulates hsp70 gene expression and mediates Ela trans-activation in vivo, interacts with the promoter targeting region of Ela CR3 in vitro. Point mutations in Ela CR3 that are defective in stimulating transcription from the hsp70 promoter are also defective in stimulating transcription directed by a synthetic activator, GAL-CBF, composed of the DNA-binding domain of yeast GAL4 fused to CBF. These mutations fall into two classes with respect to their abilities to interact with CBF in vitro. Mutations in the transcriptional activation region of Ela CR3 do not affect binding to CBF, but mutation of the promoter targeting region of Ela CR3 prevents association with CBF in vitro.

  10. Pre-existing vector immunity does not prevent replication deficient adenovirus from inducing efficient CD8 T-cell memory and recall responses

    Steffensen, Maria Abildgaard; Jensen, Benjamin Anderschou Holbech; Holst, Peter Johannes


    in a large part of the human population. We have recently developed an improved adenoviral vaccine vector system in which the vector expresses the transgene tethered to the MHC class II associated invariant chain (Ii). To further evaluate the potential of this system, the concept of pre-existing inhibitory......Adenoviral vectors have shown a great potential for vaccine development due to their inherent ability to induce potent and protective CD8 T-cell responses. However, a critical issue regarding the use of these vectors is the existence of inhibitory immunity against the most commonly used Ad5 vector...... immunity to adenoviral vectors was revisited to investigate whether the inhibition previously seen with the Ad5 vector also applied to the optimized vector system. We found this to be the case, and antibodies dominated as the mechanism underlying inhibitory vector immunity. However, presence of CD8 T cells...

  11. Multiple targets vector miss distance measurement accuracy based on 2-D assignment algorithms


    An extension of 2-D assignment approach is proposed for measurement-to-target association for improving multiple targets vector miss distance measurement accuracy.When the multiple targets move so closely,the measurements can not be fully resolved due to finite resolution.The proposed method adopts an auction algorithm to compute the feasible measurement-to-target assignment with unresolved measurements for solving this 2-D assignment problem.Computer simulation results demonstrate the effectiveness and feasibility of this method.

  12. A Tumor-stroma Targeted Oncolytic Adenovirus Replicated in Human Ovary Cancer Samples and Inhibited Growth of Disseminated Solid Tumors in Mice

    Lopez, M Veronica; Rivera, Angel A; Viale, Diego L; Benedetti, Lorena; Cuneo, Nicasio; Kimball, Kristopher J; Wang, Minghui; Douglas, Joanne T; Zhu, Zeng B; Bravo, Alicia I; Gidekel, Manuel; Alvarez, Ronald D; Curiel, David T; Podhajcer, Osvaldo L


    Targeting the tumor stroma in addition to the malignant cell compartment is of paramount importance to achieve complete tumor regression. In this work, we modified a previously designed tumor stroma-targeted conditionally replicative adenovirus (CRAd) based on the SPARC promoter by introducing a mutated E1A unable to bind pRB and pseudotyped with a chimeric Ad5/3 fiber (Ad F512v1), and assessed its replication/lytic capacity in ovary cancer in vitro and in vivo. AdF512v1 was able to replicate in fresh samples obtained from patients: (i) with primary human ovary cancer; (ii) that underwent neoadjuvant treatment; (iii) with metastatic disease. In addition, we show that four intraperitoneal (i.p.) injections of 5 × 1010 v.p. eliminated 50% of xenografted human ovary tumors disseminated in nude mice. Moreover, AdF512v1 replication in tumor models was enhanced 15–40-fold when the tumor contained a mix of malignant and SPARC-expressing stromal cells (fibroblasts and endothelial cells). Contrary to the wild-type virus, AdF512v1 was unable to replicate in normal human ovary samples while the wild-type virus can replicate. This study provides evidence on the lytic capacity of this CRAd and highlights the importance of targeting the stromal tissue in addition to the malignant cell compartment to achieve tumor regression. PMID:22948673

  13. 大鼠SOCS3基因重组腺病毒载体的构建及转染血管平滑肌细胞后的表达%Construction of Rat SOCS3 Recombinant Adenovirus Vector and Its Expression in Rat Primary Vascular Smooth Muscle Cells

    向水; 董念国; 刘金平; 史嘉玮; 肖雅琼; 王玉


    目的 构建携带大鼠SOCS3基因的重组腺病毒载体并转染大鼠血管平滑肌细胞,评价其转染效果,为血管增殖性疾病转基因治疗的实验研究奠定基础.方法 用BamH Ⅰ和EcoR Ⅰ将目的基因pUC57-Simple-rSOCS3及腺病毒穿梭载体pYr-adshuttle- 4(带GFP标记)行双酶切.回收酶切质粒的目的片段进行连接,产物转化至DH5α感受态.提取质粒酶切鉴定正确后测序.通过LR体外同源重组将rSOCS3表达框构建至pAd/PL-DEST腺病毒表达载体上.将腺病毒线性化后转染HEK293细胞包装腺病毒,PCR鉴定此腺病毒中是否含有rSOCS3基因.放大培养并收集病毒,TCD法测定病毒滴度.将此病毒感染大鼠血管平滑肌细胞,荧光显微镜观察感染效率,real-time PCR及免疫印迹检测SOCS3 mRNA及蛋白表达.结果 rSOCS3腺病毒穿梭质粒酶切鉴定正确,测序与rSOCS3基因库中的序列完全相符.PCR鉴定pAd/PL-DEST腺病毒表达载体成功携带目的基因rSOCS3.TCD法测定的最终滴度为2×1010pfu/mL.荧光显微镜观察此病毒感染血管平滑肌细胞的效率在80%以上,real-time PCR及免疫印迹检测结果显示血管平滑肌细胞中SOCS3 mRNA及蛋白表达明显上调.结论 成功构建携带大鼠SOCS3基因的腺病毒载体,并在血管平滑肌细胞中高表达,为血管增殖性疾病转基因治疗的实验研究奠定了基础,具有一定的应用价值.%Objective To construct the recombinant adenovirus vector carrying rat SOCS3 gene and assess its transfection efficiency in vascular smooth muscle cells(VSMCs). Methods The rat SOCS3 plasmid(pUC57-Simple-rSOCS3)and advenovir-us shuttle plasmid(pYr-adshuttle-4)containing green fluorescent protein(GFP)reporter gene were cleaved by restriction endonu-clease BamW I and EcoR I . The target gene fragments were connected together to generate a recombinant plasmid pYr-ad-shuttle-4-rSOCS3 that was transfected into E. coli DH5a. The plasmid was confirmed to be constructed as

  14. 小鼠CD200基因重组腺病毒载体的构建及鉴定%Construction and identification of recombinant adenovirus vector containing mouse CD200 gene

    王琼; 陈冬梅; 王立斌; 魏军; 李玉奎


    Objective:To construct the recombinant adenovirus vector containing mouse CD200 gene and to detect its expression in mice pancreas endothelial cells(mile sven 1,MS1).Methods:mCD200 gene was subcloned into the shuttle plasmid pYr-adshuttle-4 and recombinant plasmid pYr-ads-4-mCD200 was acquired.After that mCD200 mRNA expression frame was transfered to pAd/PLDEST adenovirus vector via LR(attL × attR) in vitro homologous recombination from pYr-ads-4-mCD200.Recombinant adenovirus plasmid was digested by Pac Ⅰ and transfected into HEK 293 cells and packaged out recombinant adenovirus rAd-4-mCD200,which was identified by digestion and PCR analysis.Virus titer was measured by the tissue cell infectious dosage 50(TCID50) method.Meanwhile,adenovirus was amplified in HEK 293 cells and used to infect MS1 cells.Expression of mCD200 gene was measured by fluorescence microscope and real-time PCR.Results:PCR identification demonstrated the construction of recombinant adenovirus carrying rAd-4-mCD200 was successful and its titer was about 109-1010 pfu/ml after amplification.Through the fluorescence microscope and real-time PCR,recombinant adenoviral vector of mCD200 gene was constructed successfully in the MS1 cells.Conclusions:MS1 cells infected by constructed adenovirus vector rAd-4-mCD200 could express mCD200 successfully and may provide the foundation for the next step to carry out the CD200 gene immunosuppression regulation and other related study.%目的:构建携带小鼠CD200基因的重组腺病毒载体,并鉴定其在小鼠胰腺内皮细胞(mile sven 1,MS1)中mCD200基因的表达情况.方法:mCD200基因亚克隆至腺病毒穿梭质粒pYr-adshuttle-4,获得重组质粒pYr-ads-4-mCD200.从该重组质粒上利用LR(attL×attR)特异性同源重组的方法,将mCD200表达框构建至腺病毒骨架质粒pAd/PL-DEST上,获得的重组腺病毒质粒经PacⅠ酶切,转染HEK293细胞,包装成重组腺病毒rAd-4-mCD200,并用酶切和PCR等鉴定.采用TCID 50

  15. Vectors

    Boeriis, Morten; van Leeuwen, Theo


    This article revisits the concept of vectors, which, in Kress and van Leeuwen’s Reading Images (2006), plays a crucial role in distinguishing between ‘narrative’, action-oriented processes and ‘conceptual’, state-oriented processes. The use of this concept in image analysis has usually focused...... on the most salient vectors, and this works well, but many images contain a plethora of vectors, which makes their structure quite different from the linguistic transitivity structures with which Kress and van Leeuwen have compared ‘narrative’ images. It can also be asked whether facial expression vectors...... should be taken into account in discussing ‘reactions’, which Kress and van Leeuwen link only to eyeline vectors. Finally, the question can be raised as to whether actions are always realized by vectors. Drawing on a re-reading of Rudolf Arnheim’s account of vectors, these issues are outlined...

  16. Analysis of the role of homology arms in gene-targeting vectors in human cells.

    Ayako Ishii

    Full Text Available Random integration of targeting vectors into the genome is the primary obstacle in human somatic cell gene targeting. Non-homologous end-joining (NHEJ, a major pathway for repairing DNA double-strand breaks, is thought to be responsible for most random integration events; however, absence of DNA ligase IV (LIG4, the critical NHEJ ligase, does not significantly reduce random integration frequency of targeting vector in human cells, indicating robust integration events occurring via a LIG4-independent mechanism. To gain insights into the mechanism and robustness of LIG4-independent random integration, we employed various types of targeting vectors to examine their integration frequencies in LIG4-proficient and deficient human cell lines. We find that the integration frequency of targeting vector correlates well with the length of homology arms and with the amount of repetitive DNA sequences, especially SINEs, present in the arms. This correlation was prominent in LIG4-deficient cells, but was also seen in LIG4-proficient cells, thus providing evidence that LIG4-independent random integration occurs frequently even when NHEJ is functionally normal. Our results collectively suggest that random integration frequency of conventional targeting vectors is substantially influenced by homology arms, which typically harbor repetitive DNA sequences that serve to facilitate LIG4-independent random integration in human cells, regardless of the presence or absence of functional NHEJ.

  17. 构建含人胰岛素样生长因子基因腺病毒载体及在兔骨髓间充质干细胞的表达%Construction of adenovirus vectors containing human insulin-like growth factor-1 gene and its expression in rabbit mesenchymal stem cells

    丁幸坡; 金先庆; 罗小辑; 邱林; 刘伟


    transfect them to mesenchymal stem cells (MSCs)and detect the expression of target gene hlGF-I at gene and protein levels.DESIGN: Repetitive measurement wail.SETTING: The Institute of Pediatric Research, Chongqing University of Medical Science.METHODS: The study was performed at the Institute of Pediatric Research, Chongqing University of Medical Science from November 2004 to March 2005. After the amplification of truncated hlGF-1 gene from pcDNA3.l-hlGF-I by polymerase chain reaction (PCR), the gene fragment was inserted into the shuttle plasmid pAdtrack-CMV for homologous recombination with backbone plasmid pAdeasy-I in bacteria BJ5183 to get adenovirus.Ad-hlGF-1. The high titer adenovirus supernatant was obtained by repeated transducing of HEK 293 cells by adenovirus harvested after confirmation of the adenovirus structure. As target cells,MSCs were infected with adenovirus earned target gene, hIGF-1, to determine the expression of hlGF-1 gene.MAIN OUTCOME MEASURES: ① The construction of recombinant adenovirus vector;② the expression of target gene hIGF-1 in HEK 293 cells and the proper multiplicity of infection (MOI); ③ hIGF-1 gene expression in MSCs.RESULTS: The adenovirus vector based on adeasy system was constructed successfully and the Ad-hlGF transducing was successfully or efficiently expressed in MSCs cells. The ideal expression of harvested recombinant adenovirus in MSCs was detected by fluorescence microscope, RT-PCR, immunocytochemistry, and Western Blot.CONCLUSION: Adenovirus vector is an effective vector tools for gene expression and wansfection of MSCs. MSCs transduced with Ad-hIGF-1 maybe another option to gene-modified seed cells for articular cartilage tissue engineering.

  18. Engineering fusogenic molecules to achieve targeted transduction of enveloped lentiviral vectors

    Wang Pin


    Full Text Available Abstract Background Lentiviral vectors with broad tropism are one of the most promising gene delivery systems capable of efficiently delivering genes of interest into both dividing and non-dividing cells while maintaining long-term transgene expression. However, there are needs for developing lentiviral vectors with the capability to deliver genes to specific cell types, thus reducing the "off-target" effect of gene therapy. In the present study, we investigated the possibility of engineering the fusion-active domain of a fusogenic molecule (FM with the aim to improve targeted transduction of lentiviral vectors co-displaying an anti-CD20 antibody (αCD20 and a FM. Results Specific mutations were introduced into the fusion domain of a binding-deficient Sindbis virus glycoprotein to generate several mutant FMs. Lentiviral vectors incorporated with αCD20 and one of the engineered FMs were successfully produced and demonstrated to be able to preferentially deliver genes to CD-20-expressing cells. Lentiviral vectors bearing engineered FMs exhibited 8 to 17-fold enhanced transduction towards target cells as compared to the parental FM. Different levels of enhancement were observed for the different engineered FMs. A pH-dependent study of vector transduction showed that the broader pH range of the engineered FM is a possible mechanism for the resulted increase in transduction efficiency. Conclusion The fusion domain of Sindbis virus glycoprotein is amenable for engineering and the engineered proteins provide elevated capacity to mediate lentiviral vectors for targeted transduction. Our data suggests that application of such an engineering strategy can optimize the two-molecular targeting method of lentiviral vectors for gene delivery to predetermined cells.

  19. Construction of adenovirus vector expressing SDF-1/RUNX1 and detection of viral titre%SDF-1/RUNX1融合蛋白腺病毒载体的构建及滴度测定

    罗红春; 张红宾; 秦波; 汪嘉莉; 刘倩


    目的 构建SDF-1/RUNX1融合蛋白腺病毒表达载体,并测定其滴度,为研究SDF-1/RUNX1融合蛋白介导的间充质干细胞对造血干细胞定向募集的作用打下基础.方法 全基因合成SDF-1-(GlySer)3-RUNX1片段,并在其两端添加限制性酶切位点XhoI及EcoRI,经测序验证基因序列是否正确.采用分子克隆技术构建pAD-SDF-1-(GlySer)3-RUNX1-IRES-GFP腺病毒载体,转染入293A细胞中进行包装,采用免疫法测定腺病毒滴度.结果 全基因合成SDF-1-(GlySer)3-RUNX1片段经测序验证基因序列正确,长约3 000 bp.将其连接至目的 载体pIRES2-EGFP,构建出含有SDF-1-(GlySer)3-RUNX1基因序列的GFP共表达质粒编号为B.以B质粒为模板,扩增出带attB1和attB2位点的SDF-1-(GlySer)3-RUNX1-IRES-EGFP片段,长约4 300 bp.采用BP重组系统将上述目的 片段重组到载体pDONR221,构建出BP重组质粒C.再采用LR重组系统将目的 序列重组到腺病毒载体pAD/CMV/v5-DEST上,构建出pAD-SDF-1-(GlySer)3-RUNX1-IRES-GFP腺病毒载体.经293细胞包装后,采用免疫法测定腺病毒滴度为1.03×1011 ifu/mL.结论 成功构建出SDF-11/RUNX1融合蛋白腺病毒表达载体,且腺病毒滴度较高,有利于后续试验.%To construct SDF1/RUNX1 fusion protein adenovirus vector, and to assay its titre, in order to lay the groundwork for researching the oriented recruitment of hematopoietic stem cells influenced by mesenchymal stem cells mediated by SDF1/RUNX1 fusion protein. Methods We synthesized SDF-l-(GlySer) 3-RUNX1 gene fragment with restriction enzyme cutting sites of Xhol and EcoRI at both ends, and sequenced the SDF-l-(GlySer) 3-RUNX1 gene fragment. We constructed pAD-SDF-1-(GlySer)3-RUNX1-IRKS-GFP adenovirus vector by molecular cloning,transfected this adenovirus vector into 293A cell strain for packing,and assayed virus titre using immunization. Results We synthesized SDF-l-(GlySer)3-RUNXl gene fragment successfully,and it was about 3000bp. This right gene

  20. Pre-existing vector immunity does not prevent replication deficient adenovirus from inducing efficient CD8 T-cell memory and recall responses.

    Steffensen, Maria Abildgaard; Jensen, Benjamin Anderschou Holbech; Holst, Peter Johannes; Bassi, Maria Rosaria; Christensen, Jan Pravsgaard; Thomsen, Allan Randrup


    Adenoviral vectors have shown a great potential for vaccine development due to their inherent ability to induce potent and protective CD8 T-cell responses. However, a critical issue regarding the use of these vectors is the existence of inhibitory immunity against the most commonly used Ad5 vector in a large part of the human population. We have recently developed an improved adenoviral vaccine vector system in which the vector expresses the transgene tethered to the MHC class II associated invariant chain (Ii). To further evaluate the potential of this system, the concept of pre-existing inhibitory immunity to adenoviral vectors was revisited to investigate whether the inhibition previously seen with the Ad5 vector also applied to the optimized vector system. We found this to be the case, and antibodies dominated as the mechanism underlying inhibitory vector immunity. However, presence of CD8 T cells directed against epitopes in the adenoviral vector seemed to correlate with repression of the induced response in re-vaccinated B-cell deficient mice. More importantly, despite a repressed primary effector CD8 T-cell response in Ad5-immune animals subjected to vaccination, memory T cells were generated that provided the foundation for an efficient recall response and protection upon subsequent viral challenge. Furthermore, the transgene specific response could be efficiently boosted by homologous re-immunization. Taken together, these studies indicate that adenoviral vectors can be used to induce efficient CD8 T-cell memory even in individuals with pre-existing vector immunity.

  1. Pre-existing vector immunity does not prevent replication deficient adenovirus from inducing efficient CD8 T-cell memory and recall responses.

    Maria Abildgaard Steffensen

    Full Text Available Adenoviral vectors have shown a great potential for vaccine development due to their inherent ability to induce potent and protective CD8 T-cell responses. However, a critical issue regarding the use of these vectors is the existence of inhibitory immunity against the most commonly used Ad5 vector in a large part of the human population. We have recently developed an improved adenoviral vaccine vector system in which the vector expresses the transgene tethered to the MHC class II associated invariant chain (Ii. To further evaluate the potential of this system, the concept of pre-existing inhibitory immunity to adenoviral vectors was revisited to investigate whether the inhibition previously seen with the Ad5 vector also applied to the optimized vector system. We found this to be the case, and antibodies dominated as the mechanism underlying inhibitory vector immunity. However, presence of CD8 T cells directed against epitopes in the adenoviral vector seemed to correlate with repression of the induced response in re-vaccinated B-cell deficient mice. More importantly, despite a repressed primary effector CD8 T-cell response in Ad5-immune animals subjected to vaccination, memory T cells were generated that provided the foundation for an efficient recall response and protection upon subsequent viral challenge. Furthermore, the transgene specific response could be efficiently boosted by homologous re-immunization. Taken together, these studies indicate that adenoviral vectors can be used to induce efficient CD8 T-cell memory even in individuals with pre-existing vector immunity.

  2. Survivin promoter-regulated oncolytic adenovirus with Hsp70 gene exerts effective antitumor efficacy in gastric cancer immunotherapy

    Wang, Weiguo; Ji, Weidan; Hu, Huanzhang; Ma, Juming; Li, Xiaoya; Mei, Weiqun; Xu, Yang; Hu, Huizhen; Yan, Yan; Song, Qizhe; Li, Zhigang; Su, Changqing


    Gene therapy is a promising adjuvant therapeutic strategy for cancer treatment. To overcome the limitations of current gene therapy, such as poor transfection efficiency of vectors, low levels of transgene expression and lack of tumor targeting, the Survivin promoter was used to regulate the selective replication of oncolytic adenovirus in tumor cells, and the heat shock protein 70 (Hsp70) gene was loaded as the anticancer transgene to generate an AdSurp-Hsp70 viral therapy system. The effica...

  3. Construction of conditionally replicative adenovirus vector mediated by dual specific promoters%双特异性启动子调控条件复制腺病毒载体的构建

    李玮; 谭建


    Objective To construct and identify the conditionally replicative adenovirus vectors which may induce the human sodium iodide symporter (hNIS) expression in the early region 1A (E1A) gene of the human telomerase reverse transcriptase (hTERT) and the glial fibrillary acidic protein (GFAP)promoter regions.Methods Immunoblotting assay was employed to detect the variation in GFAP and telomerase protein expression prior to and following viral infection of the cerebral stellar glioblastoma cells (U87),neuroglioma cells (U251) and human embryonic lung fibroblasts (MRC-5) cells.The hTERT and GFAP promoters and the hNIS genes were amplified by polymerase chain reaction for synthesis of adenoviral E1A genes.The recombinant plasmids containing hTERT and GFAP gene promoters were adopted for transfection into MRC-5,U251 and U87,which entailed assessment of the hTERT and GFAP promoter activity via fluorescent analysis after 24 h.This was followed by ligation with the E1A and hNIS genes and subsequent cloning into the plasmid pDC311 for construction of the recombinant plasmid pDC311-Tp-E1A-Gp-NIS that was identified by double enzyme digestion (EcoR Ⅰ and Sal Ⅰ) and gene sequencing.This recombinant plasmid was co-transfected with adenoviral genomic plasmid pBHGlox△E1-3Cre into the human embryonic kidney 293 cells forming the conditionally replicative recombinant adenovirus Ad-Tp-E1A-Gp-NIS.This Ad-Tp-E1A-Gp-NIS was employed to transfect the 293,MRC-5,U87 and U251 cells,whose conditional replicability was measured by plaque forming assay.The recombinant virus Ad-Tp-E1A-Gp-NIS and Ad-CMV-EGFP controls were used to transfect the U251,U87 and MRC-5 cells for detection of the capacity of 125I uptake by using a γ-ray counter.Results The expression of the 120 000 telomerase and 49 000 GFAP protein could be found in U87 and U251 cells,but not in MRC-5 cells.Glioma target gene expression could be induced by the GFAP and hTERT promoters,the efficiency of which was (62.10±6.26)

  4. Efficient gene targeting mediated by a lentiviral vector-associated meganuclease.

    Izmiryan, Araksya; Basmaciogullari, Stéphane; Henry, Adrien; Paques, Frédéric; Danos, Olivier


    Gene targeting can be achieved with lentiviral vectors delivering donor sequences along with a nuclease that creates a locus-specific double-strand break (DSB). Therapeutic applications of this system would require an appropriate control of the amount of endonuclease delivered to the target cells, and potentially toxic sustained expression must be avoided. Here, we show that the nuclease can be transferred into cells as a protein associated with a lentiviral vector particle. I-SceI, a prototypic meganuclease from yeast, was incorporated into the virions as a fusion with Vpr, an HIV accessory protein. Integration-deficient lentiviral vectors containing the donor sequences and the I-SceI fusion protein were tested in reporter cells in which targeting events were scored by the repair of a puromycin resistance gene. Molecular analysis of the targeted locus indicated a 2-fold higher frequency of the expected recombination event when the nuclease was delivered as a protein rather than encoded by a separate vector. In both systems, a proportion of clones displayed multiple integrated copies of the donor sequences, either as tandems at the targeted locus or at unrelated loci. These integration patterns were dependent upon the mode of meganuclease delivery, suggesting distinct recombination processes.

  5. Enhanced structural stability of adenovirus nanocapsule

    Ding Weng; Ziyue Karen Jiang; Jing Jin; Lily Wu; Yunfeng Lu


    Application of viral vector in gene therapy and vaccination is still limited by their structural stability, which significantly increased avoidable cost in storage and transportation. Herein a non-covalent conjugated low-pH degradable nanocapsule has been adopted to stabilize viral vectors. By utilizing a luciferase expressing adenovirus, AdCMVLuc, we succeeded in a raise of over 11 folds in AdCMVLuc's structural stability after 12 days storage at 4 1C.

  6. The Research and Application of Visual Saliency and Adaptive Support Vector Machine in Target Tracking Field

    Yuantao Chen


    Full Text Available The efficient target tracking algorithm researches have become current research focus of intelligent robots. The main problems of target tracking process in mobile robot face environmental uncertainty. They are very difficult to estimate the target states, illumination change, target shape changes, complex backgrounds, and other factors and all affect the occlusion in tracking robustness. To further improve the target tracking’s accuracy and reliability, we present a novel target tracking algorithm to use visual saliency and adaptive support vector machine (ASVM. Furthermore, the paper’s algorithm has been based on the mixture saliency of image features. These features include color, brightness, and sport feature. The execution process used visual saliency features and those common characteristics have been expressed as the target’s saliency. Numerous experiments demonstrate the effectiveness and timeliness of the proposed target tracking algorithm in video sequences where the target objects undergo large changes in pose, scale, and illumination.

  7. Evaluation of spatially targeted strategies to control non-domiciliated Triatoma dimidiata vector of Chagas disease.

    Corentin Barbu


    Full Text Available BACKGROUND: Chagas disease is a major neglected tropical disease with deep socio-economical effects throughout Central and South America. Vector control programs have consistently reduced domestic populations of triatomine vectors, but non-domiciliated vectors still have to be controlled efficiently. Designing control strategies targeting these vectors is challenging, as it requires a quantitative description of the spatio-temporal dynamics of village infestation, which can only be gained from combinations of extensive field studies and spatial population dynamic modelling. METHODOLOGY/PRINCIPAL FINDINGS: A spatially explicit population dynamic model was combined with a two-year field study of T. dimidiata infestation dynamics in the village of Teya, Mexico. The parameterized model fitted and predicted accurately both intra-annual variation and the spatial gradient in vector abundance. Five different control strategies were then applied in concentric rings to mimic spatial design targeting the periphery of the village, where vectors were most abundant. Indoor insecticide spraying and insect screens reduced vector abundance by up to 80% (when applied to the whole village, and half of this effect was obtained when control was applied only to the 33% of households closest to the village periphery. Peri-domicile cleaning was able to eliminate up to 60% of the vectors, but at the periphery of the village it has a low effect, as it is ineffective against sylvatic insects. The use of lethal traps and the management of house attractiveness provided similar levels of control. However this required either house attractiveness to be null, or ≥ 5 lethal traps, at least as attractive as houses, to be installed in each household. CONCLUSION/SIGNIFICANCE: Insecticide and insect screens used in houses at the periphery of the village can contribute to reduce house infestation in more central untreated zones. However, this beneficial effect remains insufficient

  8. Targeting the central nervous system with herpes simplex virus / Sleeping Beauty hybrid amplicon vectors.

    de Silva, Suresh; Bowers, William J


    The pursuits of sustainable treatments for diseases and disorders that afflict the central nervous system (CNS) have proven challenging for the field of viral vector-based gene therapy. However, recent advances in viral vector technology coupled with efficient delivery methods have opened up new avenues that show promise at the preclinical testing stage. The development of the Herpes Simplex Virus/Sleeping Beauty (HSV/SB) hybrid vector represents such an advance for devising treatments targeting the CNS with its potential for stably integrating large transgenomic segments of DNA within the genomes of transduced cells. In utero administration of this hybrid vector into the embryonic mouse brain has revealed the capacity for widespread transgene dissemination due to the targeting of a neuronal precursor cell population. This unique feature has provided the means to stably express a transgene throughout the brain for prolonged periods, which is a prerequisite for the treatment of progressive CNS disorders. In this review we provide a comprehensive breakdown of the characteristics of the HSV/SB vector system and how it can be efficiently employed in the derivation of CNS-targeted gene therapeutic strategies.

  9. Inhibitory effect of adenovirus-mediated short hairpin RNA targeting P85 and Akt1 on growth of human gastric adenocarcinoma cell%腺病毒介导的靶向P85和Akt1短发夹RNA对人胃腺癌细胞生长抑制作用的研究

    张靖; 付彦超; 康春生; 张庆瑜; 王涛; 张洁


    目的 构建靶向P85和蛋白激酶B1(PKB1/Akt1)的短发夹RNA(shRNA)腺病毒载体,研究其对人胃腺癌细胞SGC-7901生长的抑制效果.方法 构建腺病毒载体rAd5-P+A,体外转染SGC-7901细胞后,以实时定量PCR和Western blot分别检测P85和Akt1的mRNA和蛋白质的表达.以噻唑蓝比色分析法(MTT法)和流式细胞法评价转染后胃癌细胞的增殖活性.构建裸鼠皮下荷瘤模型进一步观察rAd5-P+A对SGC-7901细胞生长的抑制效果,并应用原位末端标记技术(TUNEL法)检测肿瘤细胞的凋亡情况.结果 成功构建的rAd5-P+A重组腺病毒载体转染SGC-7901细胞后可显著抑制p85和Akt1的mRNA表达,而P85和Aktl蛋白表达量在转染48 h、72 h后分别下调57.5%、63.7%和67.8%、75.6%,与空白对照组和通用腺病毒对照(rAd5-HK)组相比,差异具有统计学意义(P=0.005,P=0.003).与空白对照组和rAd5-HK组相比,SGC-7901细胞的增殖活性在rAd5-P+A转染后第2天明显下降(P<0.001),且rAd5-P+A转染组进入S期的细胞数减少了5.9%~7.1%,而进入G0/G1期的细胞增加了12.1%~13.7%.裸鼠皮下荷瘤模型治疗实验也显示,rAd5-P+A可抑制胃癌细胞的生长,诱导细胞的凋亡.结论 腺病毒介导的靶向P85和Akt1的shRNA可抑制人胃腺癌细胞的生长,这可能为胃腺癌靶向性联合基因治疗提供新的策略.%Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting P85 and protein kinase B1 (PKB1/Akt1) and study its effects on the growth of SGC-7901 human gastric adenocareinoma cells. Methods P85 and Aktl shRNA expression frames were subcloned to pGSadeno adenovirus vector with homologous recombination technology to construct pGSadeno-P85 + Akt1 (rAd5-P + A) vector. After screening and amplification, the recombinant adenovirus vector was digested with PacI and transfected into SGC-7901 cells and then its titer and transfection efficiency were detected with fluorescent microscope. P85 and Akt1 mRNA protein

  10. Predicted structure of two adenovirus tumor antigens.

    Perricaudet, M; Le Moullec, J M; Pettersson, U


    Early adenovirus type 2(Ad2) mRNA sequences have been cloned by using the pBR322 plasmid as a vector. Two clones that include sequences from region E1B were identified and their DNAs were characterized by hybridization, restriction enzyme cleavage, and DNA sequence analysis. The results showed that the clones were derived from two different spliced mRNAs. By combining our results with the established DNA sequence for region E1B of the closely related adenovirus type 5[Maat, J., van Beveren, C...

  11. A comparative study on the immunotherapeutic efficacy of recombinant Semliki Forest virus and adenovirus vector systems in a murine model for cervical cancer

    Riezebos-Brilman, A.; Walczak, M.; Regts, J.; Rots, Mg; Kamps, G.; Dontje, B.; Haisma, Hy; Wilschut, J.; Daemen, T.


    Currently, various therapeutic strategies are being explored as a potential means to immunize against metastatic malignant cells or even primary tumours. Using recombinant viral vectors systems or protein-based immunization approaches, we are developing immunotherapeutic strategies against cervical

  12. Safety and tolerability of conserved region vaccines vectored by plasmid DNA, simian adenovirus and modified vaccinia virus ankara administered to human immunodeficiency virus type 1-uninfected adults in a randomized, single-blind phase I trial.

    Emma-Jo Hayton

    Full Text Available TRIAL DESIGN: HIV-1 vaccine development has advanced slowly due to viral antigenic diversity, poor immunogenicity and recently, safety concerns associated with human adenovirus serotype-5 vectors. To tackle HIV-1 variation, we designed a unique T-cell immunogen HIVconsv from functionally conserved regions of the HIV-1 proteome, which were presented to the immune system using a heterologous prime-boost combination of plasmid DNA, a non-replicating simian (chimpanzee adenovirus ChAdV-63 and a non-replicating poxvirus, modified vaccinia virus Ankara. A block-randomized, single-blind, placebo-controlled phase I trial HIV-CORE 002 administered for the first time candidate HIV-1- vaccines or placebo to 32 healthy HIV-1/2-uninfected adults in Oxford, UK and elicited high frequencies of HIV-1-specific T cells capable of inhibiting HIV-1 replication in vitro. Here, detail safety and tolerability of these vaccines are reported. METHODS: Local and systemic reactogenicity data were collected using structured interviews and study-specific diary cards. Data on all other adverse events were collected using open questions. Serum neutralizing antibody titres to ChAdV-63 were determined before and after vaccination. RESULTS: Two volunteers withdrew for vaccine-unrelated reasons. No vaccine-related serious adverse events or reactions occurred during 190 person-months of follow-up. Local and systemic events after vaccination occurred in 27/32 individuals and most were mild (severity grade 1 and predominantly transient (<48 hours. Myalgia and flu-like symptoms were more strongly associated with MVA than ChAdV63 or DNA vectors and more common in vaccine recipients than in placebo. There were no intercurrent HIV-1 infections during follow-up. 2/24 volunteers had low ChAdV-63-neutralizing titres at baseline and 7 increased their titres to over 200 with a median (range of 633 (231-1533 post-vaccination, which is of no safety concern. CONCLUSIONS: These data demonstrate

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

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


    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...

  14. MicroRNA-mediated suppression of oncolytic adenovirus replication in human liver.

    Ylösmäki, Erkko; Lavilla-Alonso, Sergio; Jäämaa, Sari; Vähä-Koskela, Markus; af Hällström, Taija; Hemminki, Akseli; Arola, Johanna; Mäkisalo, Heikki; Saksela, Kalle


    MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3' untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5) in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

  15. MicroRNA-mediated suppression of oncolytic adenovirus replication in human liver.

    Erkko Ylösmäki

    Full Text Available MicroRNAs (miRNAs are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3' untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5 in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

  16. The antisaccade task: Vector inversion contributes to a statistical summary representation of target eccentricities.

    Heath, Matthew; Gillen, Caitlin; Weiler, Jeffrey


    Antisaccades require the top-down suppression of a stimulus-driven prosaccade (i.e., response suppression) and the inversion of a target's spatial location to mirror-symmetrical space (i.e., vector inversion). Moreover, recent work has shown that antisaccade amplitudes are characterized by a statistical summary representation (SSR) of the target eccentricities included in a stimulus-set--a result suggesting that antisaccades are supported via the same relative visual information as perceptions. The present investigation sought to determine whether response suppression and the disruption of real-time control or vector inversion contribute to a SSR in oculomotor control. Participants completed pro- and antisaccades (target eccentricities of 10.5°, 15.5°, and 20.5°) in blocks of trials that differed with regard to the frequency that individual target eccentricities were presented. The manipulation of target eccentricity frequency was used to determine whether the most frequently presented target within a stimulus-set (i.e., the SSR) influences saccade amplitudes. Moreover, we disrupted the real-time control of prosaccades by requiring participants to suppress their response for a brief visual delay (i.e., 2000 ms: so-called delay prosaccade). As expected, antisaccades and delay prosaccades produced equivalent reaction times. In turn, amplitudes for delay prosaccades were refractory to the manipulation of target eccentricity frequency, whereas antisaccades were biased in the direction of the most frequently presented target within a stimulus-set. Accordingly, we propose that vector inversion contributes to the mediation of target eccentricities via a SSR and that such a phenomenon provides convergent evidence that a relative visual percept mediates antisaccades.

  17. Retinal degeneration progression changes lentiviral vector cell targeting in the retina.

    Maritza Calame

    Full Text Available In normal mice, the lentiviral vector (LV is very efficient to target the RPE cells, but transduces retinal neurons well only during development. In the present study, the tropism of LV has been investigated in the degenerating retina of mice, knowing that the retina structure changes during degeneration. We postulated that the viral transduction would be increased by the alteration of the outer limiting membrane (OLM. Two different LV pseudotypes were tested using the VSVG and the Mokola envelopes, as well as two animal models of retinal degeneration: light-damaged Balb-C and Rhodopsin knockout (Rho-/- mice. After light damage, the OLM is altered and no significant increase of the number of transduced photoreceptors can be obtained with a LV-VSVG-Rhop-GFP vector. In the Rho-/- mice, an alteration of the OLM was also observed, but the possibility of transducing photoreceptors was decreased, probably by ongoing gliosis. The use of a ubiquitous promoter allows better photoreceptor transduction, suggesting that photoreceptor-specific promoter activity changes during late stages of photoreceptor degeneration. However, the number of targeted photoreceptors remains low. In contrast, LV pseudotyped with the Mokola envelope allows a wide dispersion of the vector into the retina (corresponding to the injection bleb with preferential targeting of Müller cells, a situation which does not occur in the wild-type retina. Mokola-pseudotyped lentiviral vectors may serve to engineer these glial cells to deliver secreted therapeutic factors to a diseased area of the retina.

  18. Construction and immunogenicity of Aβ42 and GM-CSF coexpression adenovirus vector%Aβ42及GM-CSF双基因重组腺病毒的构建和免疫原性

    何芬; 靳晓宇; 邹俊涛; 牛道立; 杨俊华; 魏旋; 李建锋; 姚志彬


    目的 构建含有β-淀粉样蛋白( Aβ42)及核糖体进入位点(IRES)引导的粒细胞-巨噬细胞集落刺激因子(GM-CSF)双表达重组腺病毒疫苗.方法 PCR法扩增带有IgGK轻链信号肽序列的Aβ42基因、IRES基因和小鼠GM-CSF基因,定向克隆于穿梭质粒pAdTrack-CMV中.通过与腺病毒骨架载体pAdeasy-1在细菌内同源重组形成重组腺病毒质粒Ad-Aβ42-IRES-GMCSF,转染人胚肾293细胞后包装成有感染能力的重组腺病毒颗粒(AD-Aβ42).重组腺病毒经氯化铯密度梯度离心纯化后感染293细胞及Hela细胞,用免疫细胞化学和ELISA方法分别检测Aβ42及GM-CSF在细胞内和培养液中的分泌表达.免疫动物鉴定其免疫原性.结果 pAdTrack-Aβ42-GMCSF测序结果和预期一致,纯化的AD-Aβ42感染Hela细胞后,免疫细胞化学证实Aβ42基因在Hela细胞内表达.AD-Aβ42感染293细胞后,ELISA检测培养上清中Aβ42含量为(159.87±33.26)ng/mL,GM-CSF含量为(205.45±38.20)ng/mL.免疫动物能诱导机体产生高滴度的抗Aβ抗体.结论 成功制备了AD-Aβ42,核糖体进入位点(IRES)成功引导的GM-CSF的表达,感染细胞证实其呈分泌表达,接种动物能诱导高滴度抗Aβ抗体.为老年性痴呆(AD)的基因治疗做好铺垫.%Objective To construct coexpression adenovirus vaccine containing Aβ42 and GM-CSF guided by the internal ri-bosome entry site sequence (IRES) to prevent and therapy the Alzheimer' s Disease (AD). Methods The IgG K light chain fused Aβ42 sequence, IRES and the mouse GM-CSF gene were generated by PCR. And then they were cloned to the shuttle plasmid pAd-Track-CMV. The homologous recombination between the recombinant vector and the adenovirus bone vector pAdeasy-1 in the Ecoli BJ5183 resulted to the formation of recombinant adenovirus vector Ad-Aβ42-IRES-GMCSF. The infecting recombinant virus particles AD-Ap42 was produced after the recombinant adenovirus vector had been transfected to the human embry kidney 293

  19. Automatic target recognition of moving target based on empirical mode decomposition and genetic algorithm support vector machine

    张军; 欧建平; 占荣辉


    In order to improve measurement accuracy of moving target signals, an automatic target recognition model of moving target signals was established based on empirical mode decomposition (EMD) and support vector machine (SVM). Automatic target recognition process on the nonlinear and non-stationary of Doppler signals of military target by using automatic target recognition model can be expressed as follows. Firstly, the nonlinearity and non-stationary of Doppler signals were decomposed into a set of intrinsic mode functions (IMFs) using EMD. After the Hilbert transform of IMF, the energy ratio of each IMF to the total IMFs can be extracted as the features of military target. Then, the SVM was trained through using the energy ratio to classify the military targets, and genetic algorithm (GA) was used to optimize SVM parameters in the solution space. The experimental results show that this algorithm can achieve the recognition accuracies of 86.15%, 87.93%, and 82.28%for tank, vehicle and soldier, respectively.

  20. Safety and High Level Efficacy of the Combination Malaria Vaccine Regimen of RTS,S/AS01B With Chimpanzee Adenovirus 63 and Modified Vaccinia Ankara Vectored Vaccines Expressing ME-TRAP

    Rampling, Tommy; Ewer, Katie J.; Bowyer, Georgina; Bliss, Carly M.; Edwards, Nick J.; Wright, Danny; Payne, Ruth O.; Venkatraman, Navin; de Barra, Eoghan; Snudden, Claudia M.; Poulton, Ian D.; de Graaf, Hans; Sukhtankar, Priya; Roberts, Rachel; Ivinson, Karen; Weltzin, Rich; Rajkumar, Bebi-Yassin; Wille-Reece, Ulrike; Lee, Cynthia K.; Ockenhouse, Christian F.; Sinden, Robert E.; Gerry, Stephen; Lawrie, Alison M.; Vekemans, Johan; Morelle, Danielle; Lievens, Marc; Ballou, Ripley W.; Cooke, Graham S.; Faust, Saul N.; Gilbert, Sarah; Hill, Adrian V. S.


    Background. The need for a highly efficacious vaccine against Plasmodium falciparum remains pressing. In this controlled human malaria infection (CHMI) study, we assessed the safety, efficacy and immunogenicity of a schedule combining 2 distinct vaccine types in a staggered immunization regimen: one inducing high-titer antibodies to circumsporozoite protein (RTS,S/AS01B) and the other inducing potent T-cell responses to thrombospondin-related adhesion protein (TRAP) by using a viral vector. Method. Thirty-seven healthy malaria-naive adults were vaccinated with either a chimpanzee adenovirus 63 and modified vaccinia virus Ankara–vectored vaccine expressing a multiepitope string fused to TRAP and 3 doses of RTS,S/AS01B (group 1; n = 20) or 3 doses of RTS,S/AS01B alone (group 2; n = 17). CHMI was delivered by mosquito bites to 33 vaccinated subjects at week 12 after the first vaccination and to 6 unvaccinated controls. Results. No suspected unexpected serious adverse reactions or severe adverse events related to vaccination were reported. Protective vaccine efficacy was observed in 14 of 17 subjects (82.4%) in group 1 and 12 of 16 subjects (75%) in group 2. All control subjects received a diagnosis of blood-stage malaria parasite infection. Both vaccination regimens were immunogenic. Fourteen protected subjects underwent repeat CHMI 6 months after initial CHMI; 7 of 8 (87.5%) in group 1 and 5 of 6 (83.3%) in group 2 remained protected. Conclusions. The high level of sterile efficacy observed in this trial is encouraging for further evaluation of combination approaches using these vaccine types. Clinical Trials Registration. NCT01883609. PMID:27307573

  1. Construction of recombinant adenovirus carrying HSV-TK controlled by hMAM enhancer and promoter and its targeted killing effect on human breast cancer cells%hMAM-EP调控HSV-TK腺病毒载体的构建及其对乳腺癌细胞的靶向杀伤

    于建刚; 吴金香; 庞春秀; 林德馨


    目的:分别构建人乳腺珠蛋白(human mammaglobin,hMAM)基因增强子和启动子(enhancer and promoter of hMAM,hMAM-EP)调控的增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因和单纯疱疹病毒胸苷激酶(herpes simple virus thymidine kinase,HSV-TK)自杀基因两种重组腺病毒载体,探讨hMAM-EP调控的HSV-TK在乳腺癌细胞特异性表达及其对乳腺癌的靶向治疗作用.方法:构建hMAM-EP-EGFP和hMAM-EP-TK重组质粒载体,将重组质粒目的基因转移到腺病毒骨架黏粒载体pAxcwit2,并转染HEK 293细胞获得重组腺病毒载体Ad-EP-EGFP和Ad-EP-TK.将Ad-EP-EGFP感染乳腺癌细胞T-47D、ZR-75-30和鼻咽癌细胞5-8F,荧光显微镜观察EGFP的表达.将Ad-EP-TK感染T47D细胞,给予1、10、20、50 μg/ml前药更昔洛韦(ganciclovir,GCV),观察TK基因对乳腺癌细胞的特异杀伤作用.结果:成功构建hMAM-EP调控的重组腺病毒载体Ad-EP-EGFP和Ad-EP-TK.Ad-EP-EGFP感染后,乳腺癌T-47D细胞可见EGFP表达,但ZR-75-30细胞和5-SF细胞无表达.与未感染组或感染Ad-EP-EGFP组相比,Ad-EP-TK重组腺病毒联合GCV(50 μg/ml)组T-47D细胞存活率显著降低[(35.69±0.07)%vs (91.74±0.02)%,(87.69 ±0.11)%;P <0.05],且随GCV质量浓度的增加,T-47D细胞存活率逐渐下降,在MOI=100、GCV质量浓度分别为1、10、20、50 μg/ml条件下,细胞存活率分别为(94.34 ±0.04)%、(86.26 ±0.02)%、(66.51±0.09)%、(35.69±0.07)%.结论:hMAM-EP调控的HSV-TK自杀基因在乳腺癌T-47D细胞中特异性表达,Ad-EP-TK联合GCV可靶向杀伤乳腺癌T-47D细胞.%Objective:To construct two recombinant adenovirus vectors carrying reporter gene enhanced green fluorescent protein (EGFP) or suicide gene herpes simple virus thymidine kinase (HSV-TK) at the downstream of enhancer and promoter of human mammaglobin (hMAM-EP). To explore breast-cancer-cell-specific regulation effect of hMAM-EP and new methods of targeted therapy for

  2. Application of conditionally replicating adenoviruses in tumor early diagnosis technology, gene-radiation therapy and chemotherapy.

    Li, Shun; Ou, Mengting; Wang, Guixue; Tang, Liling


    Conditionally replicating adenoviruses (CRAds), or known as replication-selective adenoviruses, were discovered as oncolytic gene vectors several years ago. They have a strong ability of scavenging tumor and lesser toxicity to normal tissue. CRAds not only have a tumor-killing ability but also can combine with gene therapy, radiotherapy, and chemotherapy to induce tumor cell apoptosis. In this paper, we review the structure of CRAds and CRAd vectors and summarize the current application of CRAds in tumor detection as well as in radiotherapy and suicide gene-mediating chemotherapy. We also propose further research strategies that can improve the application value of CRAds, including enhancing tumor destruction effect, further reducing toxic effect, reducing immunogenicity, constructing CRAds that can target tumor stem cells, and trying to use mesenchymal stem cells (MSCs) as the carriers for oncolytic adenoviruses. As their importance to cancer diagnosis, gene-radiation, and chemotherapy, CRAds may play a considerable role in clinical diagnosis and various cancer treatments in the future.

  3. Investigation of gene therapy of adenovirus in immune suppression

    Xi XIA; Beibei WANG; Li CAO; Gang CHEN; Peng WU; Yunping LU; Jianfeng ZHOU; Ding MA


    The aim of this paper is to investigate the safety of reconstructed adenovirus in immunosuppressive ther-apeutics and to explore the role of ciclosporin A in ant-agonizing the elimination of the vector. Several rats were given retroperitoneal injection of purified ADV-TK in order to obtain models. After 14 days' treatment of ciclos-porin A, samples of different periods were obtained, then stained with hematoxylin-eosin (HE) to detect inflam-mation reactions. Immunohistochemistry was used to examine the expression of adenovirus in organs. The results are as follows: (1) In HE stained sections of the organs, some transitory and reversible inflammation was detected. (2) In immunohistochemistry assay, recon-structed adenovirus decreased gradually as time went by in the control group, while it did not happen in the experi-mental group in which the adenovirus showed a relative increase compared with their counterparts (P<0.05). (3) The distributions of adenovirus in the liver, spleen and lung were higher than those in the other organs detected. Reconstructed adenovirus as a vector is definitely safe in immunosuppressive therapeutics, and ciclosporin A, to some extent, is able to consequently inhibit the immune response of the rats and prolong the existing period of adenovirus.

  4. Comparison of lentivirus and adenovirus vector mediated gene transfer into cultured spiral ganglion cells%慢病毒载体和腺病毒载体转染原代培养的螺旋神经节细胞的比较

    王国鹏; 罗凌惠; 谢静; 陈沛; 付勇; 钟刚; 龚树生


    Objective:We aimed to compare the characteristics between lentivirus and adenovirus vector mediated gene transfer into cultured spiral ganglion cells (SG Cs). Method:SGCs from newborn rats were cultured and exposed to lentivirus-GFP and adenovirus-GFP vectors. GFP expression and the cell morphology were evaluated under epi-fluorescence microscope at 3 days and 7 days after exposure. Survival number of SGCs was counted, and the average percentage of SGCs with GFP expression was calculated, and axon length was measured by ImageJ software. Result:Cultured SGCs were transfected by either adenovirus or lentivirus vector successfully. The adenovirus vector presented an instant and efficient transfection. However, the expression of GFP went down after 7 days. In lentivirus-GFP group, GFP expression was detected at 7 days after exposure, and the number of cells with GFP expression increased gradually in the following days. Statistical analysis revealed that there were no differences in survival number of SGCs and average axon length among lentivirus-GFP group, adenovirus-GFP group and control group. Conclusion: Cultured SGCs can be transfected by either lentivirus vector or adenovirus vector safely and efficiently. SGCs are more susceptible to adenovirus vector, but GFP persists for a longer period after the lentivirus-mediated gene transfer.%目的:比较慢病毒载体和腺病毒载体转染新生大鼠耳蜗螺旋神经节细胞(SGCs)的不同转染特性.方法:体外培养新生大鼠SGCs,适当滴度的慢病毒载体和腺病毒载体转染SGCs,于病毒转染后3 d(培养后6 d)和转染后7 d(培养后10 d),荧光显微镜下观察绿色荧光蛋白报告基因(GFP)表达情况和SGCs的细胞形态.行SGCs计数,计算转染效率,并通过ImageJ软件测量各组SGCs轴突长度.结果:慢病毒载体和腺病毒载体均可有效转染体外培养的原代SGCs;腺病毒载体转染效率高,GFP表达快,而1周后GFP表达开始下降;慢病毒载体转染7 d

  5. Effective gene-viral therapy for telomerase-positive cancers by selective replicative-competent adenovirus combining with endostatin gene

    Zhang Q; Liu C; Jiang M; Fang G; Liu X; Wu M; Qian Q; Nie M; Sham J; Su C; Xue H; Chua D; Wang W; Cui Z; Liu Y


    Gene-viral therapy, which uses replication-selective transgene-expressing viruses to manage tumors, can exploit the virtues of gene therapy and virotherapy and overcome the limitations of conventional gene therapy. Using a human telomerase reverse transcriptase-targeted replicative adenovirus as an antiangiogenic gene transfer vector to target new angiogenesis and making use of its unrestrained proliferation are completely new concepts in tumor management. CNHK300-mE is a selective replication transgene-expressing adenovirus constructed to carry mouse endostatin gene therapeutically. Infection with CNHK300-mE was associated with selective replication of the adenovirus and production of mouse endostatin in telomerase-positive cancer cells. Endostatin secreted from a human gastric cell line, SGC-7901, infected with CNHK300-mE was significantly higher than that infected with nonreplicative adenovirus Ad-mE in vitro (800±94.7 ng/ml versus 132.9±9.9 ng/ml) and in vivo (610±42 ng/ml versus 126 +/- 13 ng/ml). Embryonic chorioallantoic membrane assay showed that the mouse endostatin secreted by CNHK300-mE inhibited angiogenesis efficiently and also induced distortion of pre-existing vasculature. CNHK300-mE exhibited a superior suppression of xenografts in nude mice compared with CNHK300 and Ad-mE. In summary, we provided a more efficient gene-viral therapy strategy by combining oncolysis with antiangiogenesis.

  6. Survivin promoter-regulated oncolytic adenovirus with Hsp70 gene exerts effective antitumor efficacy in gastric cancer immunotherapy.

    Wang, Weiguo; Ji, Weidan; Hu, Huanzhang; Ma, Juming; Li, Xiaoya; Mei, Weiqun; Xu, Yang; Hu, Huizhen; Yan, Yan; Song, Qizhe; Li, Zhigang; Su, Changqing


    Gene therapy is a promising adjuvant therapeutic strategy for cancer treatment. To overcome the limitations of current gene therapy, such as poor transfection efficiency of vectors, low levels of transgene expression and lack of tumor targeting, the Survivin promoter was used to regulate the selective replication of oncolytic adenovirus in tumor cells, and the heat shock protein 70 (Hsp70) gene was loaded as the anticancer transgene to generate an AdSurp-Hsp70 viral therapy system. The efficacy of this targeted immunotherapy was examined in gastric cancer. The experiments showed that the oncolytic adenovirus can selectively replicate in and lyse the Survivin-positive gastric cancer cells, without significant toxicity to normal cells. AdSurp-Hsp70 reduced viability of cancer cells and inhibited tumor growth of gastric cancer xenografts in immuno-deficient and immuno-reconstruction mouse models. AdSurp-Hsp70 produced dual antitumor effects due to viral replication and high Hsp70 expression. This therapeutic system used the Survivin promoter-regulated oncolytic adenovirus vector to mediate targeted expression of the Hsp70 gene and ensure safety and efficacy for subsequent gene therapy programs against a variety of cancers.

  7. A Transient Three-plasmid Expression System for the Production of Hepatocytes Targeting Retroviral Vectors

    Peng QI; Jinxiang HAN; Yanqin LU; Chuanxi WANG; Bo ZHU


    Targeting of retroviral vectors to specific cells was attempted through modifying the surface protein of the murine leukemia viruses (MLVs), but in many cases the protein function was affected, and it is difficult to achieve the targeted delivery. In this study, we have tried to engineer ecotropic Moloney murine leukemia viruses (MoMLV)-based retroviral vectors to transduce hepatocytes. A chimeric envelope (Env)expression plasmid was constructed containing the hepatitis B virus PreS2 peptide fused to aa +1 at the Nterminus of Env. Following simultaneous transfection of pgag-pol, pLEGFP and chimeric env plasmids into 293T cells, helper-free retrovirus stocks with the titer of approximately 104 infectious units/ml were achieved at 48 h post-transfection. These pseudotype vectors showed the normal host range of retrovirus, infecting host NIH 3T3 cells, although the efficiency was reduced compared with that of virions carrying wild-type ecotropic MoMLV envelope. In addition, the resultant pseudotype viruses could transduce human hepatoma cells mediated by polymerized human serum albumin with relatively high titers in comparison with those transductions without polymerized human serum albumin. This approach can be used to target hepatocytes selectively.

  8. Silencing E1A mRNA by RNA interference inhibits adenovirus replication.

    Chung, Y-S; Kim, M-K; Lee, W-J; Kang, C


    The adenovirus family contains 51 human serotypes, and most human adenoviruses cause widespread respiratory tract infections. Adenovirus infections can result in severe complications in some cases, such as in adenovirus type 11 infection in immunocompromised patients. However, effective treatment methods for adenovirus infections are currently unavailable. This prompted the search for antiviral agents effective against adenovirus infections. In the present study, adenovirus E1A was targeted by RNA interference (RNAi) using synthetic small interfering RNAs (siRNAs) in an attempt to inhibit viral replication, since adenovirus E1A proteins are known to be involved in the transcriptional activation of the viral and cellular genes necessary for controlling the cell cycle and viral replication. The results indicated that the siRNAs effectively reduced the amount of adenovirus E1A mRNA and the levels of replicative intermediates. Additionally, siRNA-mediated gene silencing inhibited adenovirus replication by suppressing the E1A mRNA. These results suggest that the RNAi-mediated targeting of adenovirus E1A may have a potentially therapeutic effect in controlling adenovirus infections.

  9. Application of support vector machine and quantum genetic algorithm in infrared target recognition

    Wang, Hongliang; Huang, Yangwen; Ding, Haifei


    In this paper, a kind of classifier based on support vector machine (SVM) is designed for infrared target recognition. In allusion to the problem how to choose kernel parameter and error penalty factor, quantum genetic algorithm (QGA) is used to optimize the parameters of SVM model, it overcomes the shortcoming of determining its parameters after trial and error in the past. Classification experiments of infrared target features extracted by this method show that the convergence speed is fast and the rate of accurate recognition is high.

  10. Successful malaria elimination strategies require interventions that target changing vector behaviours

    Russell Tanya L


    cannot be restored by rotating the insecticide to one with a different mode of action. The scientific community needs to urgently develop systematic methods for monitoring behavioural resistance and then to work in collaboration with vector control programmes to implement monitoring in sentinel sites. In situations where behavioural resistance is detected, there will be a need to target other bionomic vulnerabilities that may exist in the larval stages, during mating, sugar feeding or another aspect of the life cycle of the vector to continue the drive towards elimination.

  11. Construction of Txt5 Adenovirus Vector and Analysis of Its Expression in Cadiomyocytes%Txt5腺病毒载体构建及其在心肌细胞中的表达

    彭浩; 陈宇; 李佑锋; 朱旋; 周作琼; 吴秀山; 范雄伟; 李永青


    设计小鼠 Txt5基因的特异性引物,将小鼠 cDNA 作为模板,用 PCR 扩增出 mTxt5编码区,并加入 HA标签序列.将这一片段插入 pMD -18T 载体,再亚克隆至 pAdtrack - cmv 穿梭载体上.线性化后,转化 BJ5183感受态细胞,在 BJ5183中发生同源重组获得 pAd - Txt5质粒.pAd - Txt5线性化后转染293A 细胞,包装得到含 Txt5基因的病毒.将病毒溶液感染大鼠原代心肌细胞,一段时间后观察绿色荧光,然后通过 RT - PCR 和免疫印迹法检测 HA 标签蛋白的表达.结果表明成功构建小鼠 Txt5腺病毒表达载体并实现在大鼠原代心肌细胞中的表达.%Specific primers for the mouse Txt5 gene were designed and employed to amplify the coding sequence using mouse cDNAs as the template .HA - tagged sequence was induced into the PCR primers .The PCR product was inserted into the pMD -18T vector and subcloned into pAd - track - cmv shuttle vector .The recombinant plasmid was linearized and transformed into bacteria BJ5183 .After homologous recombination in the bacteria ,plasmid pAd - Txt5 was obtained .Linearized pAd - Txt5 was transfected into 293 A cells .Then , the recombinant virus containing Txt5 was obtained after packaging . Cardiomyocytes were infected with the collected virus fluid .To detect the expression of Txt5 ,green fluorescence protein (GFP) was observed by green fluorescence and RT - PCR , immunoblotting was performed . The results showed that mouse Txt5 adenovirus vector was constructed successfully ,and it could induce the sequence coding Txt5 - HA into cardiomyocytes .

  12. Fusion of the BCL9 HD2 domain to E1A increases the cytopathic effect of an oncolytic adenovirus that targets colon cancer cells

    Pittet Anne-Laure


    Full Text Available Abstract Background The Wnt signaling pathway is activated by mutations in the APC and β-catenin genes in many types of human cancer. β-catenin is stabilized by these mutations and activates transcription in part by acting as a bridge between Tcf/LEF proteins and the HD2 domain of the BCL9 coactivator. We have previously described oncolytic adenoviruses with binding sites for Tcf/LEF transcription factors inserted into the early viral promoters. These viruses replicate selectively in cells with activation of the Wnt pathway. To increase the activity of these viruses we have fused the viral transactivator E1A to the BCL9 HD2 domain. Methods Luciferase assays, co-immunoprecipitation and Western blotting, immunofluorescent cell staining and cytopathic effect assays were used to characterize the E1A-HD2 fusion protein and virus in vitro. Growth curves of subcutaneous SW620 colon cancer xenografts were used to characterize the virus in vivo. Results The E1A-HD2 fusion protein binds to β-catenin in vivo and activates a Tcf-regulated luciferase reporter better than wild-type E1A in cells with activated Wnt signaling. Expression of the E1A-HD2 protein promotes nuclear import of β-catenin, mediated by the strong nuclear localization signal in E1A. Tcf-regulated viruses expressing the fusion protein show increased expression of viral proteins and a five-fold increase in cytopathic effect (CPE in colorectal cancer cell lines. There was no change in viral protein expression or CPE in HeLa cells, indicating that E1A-HD2 viruses retain selectivity for cells with activation of the Wnt signaling pathway. Despite increasing the cytopathic effect of the virus in vitro, fusion of the HD2 domain to E1A did not increase the burst size of the virus in vitro or the anti-tumor effect of the virus in an SW620 xenograft model in vivo. Conclusion Despite an increase in the nuclear pool of β-catenin, the effects on viral activity in colon cancer cells were small

  13. A targeted change-detection procedure by combining change vector analysis and post-classification approach

    Ye, Su; Chen, Dongmei; Yu, Jie


    In remote sensing, conventional supervised change-detection methods usually require effective training data for multiple change types. This paper introduces a more flexible and efficient procedure that seeks to identify only the changes that users are interested in, here after referred to as "targeted change detection". Based on a one-class classifier "Support Vector Domain Description (SVDD)", a novel algorithm named "Three-layer SVDD Fusion (TLSF)" is developed specially for targeted change detection. The proposed algorithm combines one-class classification generated from change vector maps, as well as before- and after-change images in order to get a more reliable detecting result. In addition, this paper introduces a detailed workflow for implementing this algorithm. This workflow has been applied to two case studies with different practical monitoring objectives: urban expansion and forest fire assessment. The experiment results of these two case studies show that the overall accuracy of our proposed algorithm is superior (Kappa statistics are 86.3% and 87.8% for Case 1 and 2, respectively), compared to applying SVDD to change vector analysis and post-classification comparison.

  14. Effects of nasal or pulmonary delivered treatments with an adenovirus vectored interferon (mDEF201 on respiratory and systemic infections in mice caused by cowpox and vaccinia viruses.

    Donald F Smee

    Full Text Available An adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mDEF201 was evaluated for efficacy against lethal cowpox (Brighton strain and vaccinia (WR strain virus respiratory and systemic infections in mice. Two routes of mDEF201 administration were used, nasal sinus (5-µl and pulmonary (50-µl, to compare differences in efficacy, since the preferred treatment of humans would be in a relatively small volume delivered intranasally. Lower respiratory infections (LRI, upper respiratory infections (URI, and systemic infections were induced by 50-µl intranasal, 10-µl intranasal, and 100-µl intraperitoneal virus challenges, respectively. mDEF201 treatments were given prophylactically either 24 h (short term or 56d (long-term prior to virus challenge. Single nasal sinus treatments of 10(6 and 10(7 PFU/mouse of mDEF201 protected all mice from vaccinia-induced LRI mortality (comparable to published studies with pulmonary delivered mDEF201. Systemic vaccinia infections responded significantly better to nasal sinus delivered mDEF201 than to pulmonary treatments. Cowpox LRI infections responded to 10(7 mDEF201 treatments, but a 10(6 dose was only weakly protective. Cowpox URI infections were equally treatable by nasal sinus and pulmonary delivered mDEF201 at 10(7 PFU/mouse. Dose-responsive prophylaxis with mDEF201, given one time only 56 d prior to initiating a vaccinia virus LRI infection, was 100% protective from 10(5 to 10(7 PFU/mouse. Improvements in lung hemorrhage score and lung weight were evident, as were decreases in liver, lung, and spleen virus titers. Thus, mDEF201 was able to treat different vaccinia and cowpox virus infections using both nasal sinus and pulmonary treatment regimens, supporting its development for humans.

  15. Structure, Function and Dynamics in Adenovirus Maturation

    Mangel, Walter F.; Carmen San Martín


    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkabl...

  16. A technique for measurement of vector and tensor polarization in solid spin one polarized targets

    Kielhorn, W.F.


    Vector and tensor polarizations are explicitly defined and used to characterize the polarization states of spin one polarized targets, and a technique for extracting these polarizations from nuclear magnetic resonance (NMR) data is developed. This technique is independent of assumptions about spin temperature, but assumes the target's crystal structure induces a quadrupole interaction with the spin one particles. Analysis of the NMR signals involves a computer curve fitting algorithm implemented with a fast Fourier transform method which speeds and simplifies curve fitting algorithms used previously. For accurate curve fitting, the NMR electronic circuit must be modeled by the fitting algorithm. Details of a circuit, its model, and data collected from this circuit are given for a solid deuterated ammonia target. 37 refs., 19 figs., 3 tabs.

  17. Development of a Canine Adenovirus Type 1 Vaccine Strain E3-deleted Based Expression Vector%犬腺病毒1型疫苗株E3缺失表达载体的构建

    黎皓; 唐七义; 张云; 王树蕙; 郭彩云


    Objective To evaluate canine adenovirus type 1 vaccine strain (Cannaught Laboratory Limited,CLL) as recombinant vaccine and gene transfer vector. Methods Recombinant virus CLLEGFP which contains enhanced green fluorescent protein(EGFP) reporter gene was constructed. CLLEGFP was used to infect various human derived cell lines (293, Hela, CO, SW, Hep-2 and CAM) by inoculating intraperitoneally(IP), intravenously(IV)and intramuscularly (IM)to Kunming mice other than oral administration. Various tissue samples of the mice were collected at multitime point for observing EGFP green fluorescence. Anti-EGFP antibodies were detected by Western blot analysis in the sera after 4 weeks. Results CLLEGFP can infect various human derived cell lines and express EGFP. EGFP green fluorescence were observed in liver tissue cells after IP transducing 3 days. All immune inoculation ways above could induce Kunming mice producing anti-EGFP antibodies which were identified by Western blot analysis. Conclusions These resluts indicate that CLL possess powerful potential as recombinant vaccine and gene transfer vector.%探索以犬腺病毒1型疫苗株(Cannaught Laboratory Limited.CLL)作为病毒重组疫苗和基因转移载体的可行性。方法构建带增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的E3缺失重组病毒CLLEGFP。将CLLEGFP感染各种人源细胞,并以灌胃、腹腔注射、尾静脉注射和肌肉注射等不同途径接种昆明小鼠。多时间点取小鼠组织标本,冷冻干燥切片,观察EGFP的表达。4周后采集小鼠血清,以Western blot分析抗EGFP 抗体的产生。结果 CLLEGFP能够感染各种人源细胞并表达EGFP。在腹腔接种CLLEGFP 3 d的小鼠肝组织细胞中可见转导的EGFP。Western blot分析显示,以各种途径免疫接种重组病毒4周后的小鼠血清中均存在抗EGFP特异抗体。结论 CLL具有开发成为病毒重组疫苗和基因转移载体的潜力。

  18. A viral vectored prime-boost immunization regime targeting the malaria Pfs25 antigen induces transmission-blocking activity.

    Anna L Goodman

    Full Text Available The ookinete surface protein Pfs25 is a macrogamete-to-ookinete/ookinete stage antigen of Plasmodium falciparum, capable of exerting high-level anti-malarial transmission-blocking activity following immunization with recombinant protein-in-adjuvant formulations. Here, this antigen was expressed in recombinant chimpanzee adenovirus 63 (ChAd63, human adenovirus serotype 5 (AdHu5 and modified vaccinia virus Ankara (MVA viral vectored vaccines. Two immunizations were administered to mice in a heterologous prime-boost regime. Immunization of mice with AdHu5 Pfs25 at week 0 and MVA Pfs25 at week 10 (Ad-MVA Pfs25 resulted in high anti-Pfs25 IgG titers, consisting of predominantly isotypes IgG1 and IgG2a. A single priming immunization with ChAd63 Pfs25 was as effective as AdHu5 Pfs25 with respect to ELISA titers at 8 weeks post-immunization. Sera from Ad-MVA Pfs25 immunized mice inhibited the transmission of P. falciparum to the mosquito both ex vivo and in vivo. In a standard membrane-feeding assay using NF54 strain P. falciparum, oocyst intensity in Anopheles stephensi mosquitoes was significantly reduced in an IgG concentration-dependent manner when compared to control feeds (96% reduction of intensity, 78% reduction in prevalence at a 1 in 5 dilution of sera. In addition, an in vivo transmission-blocking effect was also demonstrated by direct feeding of immunized mice infected with Pfs25DR3, a chimeric P. berghei line expressing Pfs25 in place of endogenous Pbs25. In this assay the density of Pfs25DR3 oocysts was significantly reduced when mosquitoes were fed on vaccinated as compared to control mice (67% reduction of intensity, 28% reduction in prevalence and specific IgG titer correlated with efficacy. These data confirm the utility of the adenovirus-MVA vaccine platform for the induction of antibodies with transmission-blocking activity, and support the continued development of this alternative approach to transmission-blocking malaria subunit

  19. In silico models for predicting vector control chemicals targeting Aedes aegypti.

    Devillers, J; Lagneau, C; Lattes, A; Garrigues, J C; Clémenté, M M; Yébakima, A


    Human arboviral diseases have emerged or re-emerged in numerous countries worldwide due to a number of factors including the lack of progress in vaccine development, lack of drugs, insecticide resistance in mosquitoes, climate changes, societal behaviours, and economical constraints. Thus, Aedes aegypti is the main vector of the yellow fever and dengue fever flaviviruses and is also responsible for several recent outbreaks of the chikungunya alphavirus. As for the other mosquito species, the A. aegypti control relies heavily on the use of insecticides. However, because of increasing resistance to the different families of insecticides, reduction of Aedes populations is becoming increasingly difficult. Despite the unquestionable utility of insecticides in fighting mosquito populations, there are very few new insecticides developed and commercialized for vector control. This is because the high cost of the discovery of an insecticide is not counterbalanced by the 'low profitability' of the vector control market. Fortunately, the use of quantitative structure-activity relationship (QSAR) modelling allows the reduction of time and cost in the discovery of new chemical structures potentially active against mosquitoes. In this context, the goal of the present study was to review all the existing QSAR models on A. aegypti. The homology and pharmacophore models were also reviewed. Specific attention was paid to show the variety of targets investigated in Aedes in relation to the physiology and ecology of the mosquito as well as the diversity of the chemical structures which have been proposed, encompassing man-made and natural substances.

  20. Ephrin A2 receptor targeting does not increase adenoviral pancreatic cancer transduction in vivo

    van Geer, M.A.; Bakker, C.T.; Koizumi, N.; Mizuguchi, H.; Wesseling, J.G.; Oude Elferink, R.P.J.; Bosma, P.J.


    AIM: To generate an adenoviral vector specifically targeting the EphA2 receptor (EphA2R) highly expressed on pancreatic cancer cells in vivo. METHODS: YSA, a small peptide ligand that binds the EphA2R with high affinity, was inserted into the HI loop of the adenovirus serotype 5 fiber knob. To furth


    孙茂盛; 昝云红; 马雁冰; 张光明; 杜秋江; 戴长柏


    Objective. Using a recombinant human adenovirus to express modified VP4 gene of rotavirus SA11 strain.``Methods. A whole VP4 gene was obtained with PCR and induced the signal peptide at the gene N terninal.``The chimera gene was cloned into pCMV plasmid that consists of human cytomegalovirus promoter, and then the gene was cloned to the transfer vector of human adenovirus type 5. Homologous recombination was performed by co-transfection to 293 cell lines with recombinant plasmid and viral genome using CaPO4 precipitation.``Results. No mutation was found in the whole VP4 gene sequence of 2362 base pair. The expressed product in recombinant adenovirus was confirmed to be specific and more antigenicity by indirect immunofluorescence assay. Both the Western blot and immunoprecipitation assay showed that the molecular mass of the expressed protein was higher than the wild type VP4 protein, and that the modified product was corresponding to a glycosylation of VP4 protein.``Conclusion. To modify the target gene might be an effective method to enhance the stability, antigenicity and immunogenicity of expressed protein.``

  2. Adenovirus-mediated sphingomyelin synthase 2 increases atherosclerotic lesions in ApoE KO mice

    Zhao Yarui


    Full Text Available Abstract Background Sphingomyelin synthase 2 (SMS2 contributes to de novo sphingomyelin (SM biosynthesis. Its activity is related to SM levels in the plasma and the cell membrane. In this study, we investigated the possibility of a direct relationship between SMS and atherosclerosis. Methods The Adenovirus containing SMS2 gene was given into 10-week ApoE KO C57BL/6J mice by femoral intravenous injection. In the control group, the Adenovirus containing GFP was given. To confirm this model, we took both mRNA level examination (RT-PCR and protein level examination (SMS activity assay. Result We generated recombinant adenovirus vectors containing either human SMS2 cDNA (AdV-SMS2 or GFP cDNA (AdV-GFP. On day six after intravenous infusion of 2 × 1011 particle numbers into ten-week-old apoE KO mice, AdV-SMS2 treatment significantly increased liver SMS2 mRNA levels and SMS activity (by 2.7-fold, 2.3-fold, p Conclusions Our results present direct morphological evidence for the pro-atherogenic capabilities of SMS2. SMS2 could be a potential target for treating atherosclerosis.


    孙茂盛; 昝云红; 马雁冰; 张光明; 杜秋江; 戴长柏


    Objective. Using a recombinant human adenovirus to express modified VP4 gene of rotavirus SA11 strain. Methods. A whole VP4 gene was obtained with PCR and induced the signal peptide at the gene N terminal.The chimera gene Was cloned into pCMV plasmid that consists of human cytomegalovirus promoter, and then the gene was cloned to the transfer vector of human adenovirus type 5. Homologous recombination was performed by co-transfection to 293 cell lines with recombinant plasmid and viral genome using CaPO4 precipitation. Results. No mutation was found in the whole VP4 gene sequence of 2362 base pair. The expressed productin recombinant adenovirus was confirmed to be specific and more antigenicity by indirect immunofluorescence as-say. Both the Western blot and immunoprecipitation assay showed that the molecular mass of the expressed protein was higher than the wild type VP4 protein, and that the modified product was corresponding to a glycosyla-tion of VP4 protein. Conclusion. To modify the target gene might be an effective method to enhance the stability, antigenicityand immunogenicity of expressed protein.

  4. Initiation of adenovirus DNA replication.

    Reiter, T; Fütterer, J; Weingärtner, B; Winnacker, E L


    In an attempt to study the mechanism of initiation of adenovirus DNA replication, an assay was developed to investigate the pattern of DNA synthesis in early replicative intermediates of adenovirus DNA. By using wild-type virus-infected cells, it was possible to place the origin of adenovirus type 2 DNA replication within the terminal 350 to 500 base pairs from either of the two molecular termini. In addition, a variety of parameters characteristic of adenovirus DNA replication were compared ...

  5. Serotype Chimeric Human Adenoviruses for Cancer GeneTherapy

    Akseli Hemminki


    Full Text Available Cancer gene therapy consists of numerous approaches where the common denominator is utilization of vectors for achieving therapeutic effect. A particularly potent embodiment of the approach is virotherapy, in which the replication potential of an oncolytic virus is directed towards tumor cells to cause lysis, while normal cells are spared. Importantly, the therapeutic effect of the initial viral load is amplified through viral replication cycles and production of progeny virions. All cancer gene therapy approaches rely on a sufficient level of delivery of the anticancer agent into target cells. Thus,enhancement of delivery to target cells, and reduction of delivery to non-target cells, in an approach called transductional targeting, is attractive. Both genetic and non-genetic retargeting strategies have been utilized. However, in the context of oncolytic viruses, it is beneficial to have the specific modification included in progeny virions and hence genetic modification may be preferable. Serotype chimerism utilizes serotype specific differences in receptor usage, liver tropism and seroprevalence in order to gain enhanced infection of target tissue. This review will focus on serotype chimeric adenoviruses for cancer gene therapy applications.

  6. siRNA干扰人BMP9重组腺病毒载体的构建及其促进乳腺癌SK-BR-3细胞增殖%Construction of recombinant adenovirus vector interfering the expression of human BMP9 and promoting the proliferation of breast cancer SK-BR-3 cells

    刘月红; 王科; 孙笑笑; 万绍恒; 王维; 陈莹莹; 张彦


    目的 筛选特异性干扰人BMP9基因的siRNA序列并制备重组腺病毒AdsiBMP9,探讨RNAi人BMP9基因后对乳腺癌SK-BR-3细胞增殖能力的影响.方法 设计制备3对干扰人BMP9的双链DNA序列,亚克隆至Pses-Hus质粒中获得Pses-Hus-siBMP9质粒,脂质体转染乳腺上皮细胞HBL-100筛选有效干扰质粒,构建重组腺病毒并感染SK-BR-3细胞,RT-PCR,Western blot检测BMP9表达,MTT检测细胞增殖能力.结果 成功构建并筛选出针对人BMP9基因的有效干扰质粒并包装成腺病毒,病毒滴度为1×1010IU/mL,感染SK-BR-3细胞显示BMP9在转录水平和翻译水平表达量显著低于对照组和空白组(P<0.05),第5天AdsiBMP9组细胞的增殖率显著高于AdsiNC组(P<0.05).结论 成功构建特异性沉默人BMP9基因的siRNA腺病毒载体,可有效抑制SK-BR-3细胞中BMP9基因的表达从而促进该细胞增殖.%Objective To screen specific small interfering RNA(siRNA) target human BMP9 gene and to prepare recombinant adenovirus vector AdsiBMP9 for investigation of its effects on the proliferation of breast cancer SK-BR-3 cells. Methods Three pairs of double-stranded DNA fragments for silencing human BMP9 were designed and synthesized, then subcloned into the shuttle plasmid Pses-Hus. The recombinant plasmids Pses-Hus-siBMP9 were transfected into the breast epithelial cells HBL-100 by lipofectamine transfection reagent, screened the effective interfering plasmid, constructed AdsiBMP9 and infected SK-BR-3 cells. The expression level of BMP9 mRNA and protein were detected by RT-PCR and western blot. The proliferation of SK-BR-3 cells were observed with MTT assay. Results The recombinant plasmid Pses-Hus-siBMP9 and recombinant adenovirus AdsiBMP9 were successfully constructed and its titer was 1 × 1010 IU/mL. Compared to the negative and non-infected controls, the expres- sion of BMP9 gene was significantly inhibited after the SK-BR-3 cells were infected by AdsiBMP9. SK-BR-3 cells infected with

  7. Expression of reconstructive hFⅧ in the hrDNA by using hrDNA targeting vector

    LIU Xionghao; WU Lingqian; DAI Heping; XIA Kun; XIA Jiahui; LIU Mujun; SHE Hua; WEN Lu; XUE Zhigang; LIANG Desheng; CAI Fang; PAN Qian; LONG Zhigao


    Our lab has constructed a new nonviral vector-hrDNA targeting vector(pHrneo). pHrneo is a human derived vector that can target gene into human ribosomal DNA(hrDNA) locus. In this study, we inserted expression cassette of reconstructive hFⅧ (hFVIII-BDDAK39) to pHrneo to construct targeting vector: pHrneo-BDDAK39. Through electroporation of pHrneo-BDDAK39 into HT1080 cells, we identified the homologous recombinants by PCR and Southen blotting, and tested the expression of hFVIII- BDDAK39 in the hrDNA locus. The hFⅧ-BDDAK39 was successfully targeted into hrDNA locus of HT-1080 by pHrneo-BDDAK39, and the efficiency of site-specific integration was 2.0×10-5. hFⅧ-BDDAK39 in hrDNA locus of HT-1080 is found to be able to express efficiently (32±5 ng·106 cells-1·24 h-1). Targeting vector pHrneo-BDDAK39 can find use in gene therapy for hemophilia.

  8. Calcium gluconate in phosphate buffered saline increases gene delivery with adenovirus type 5.

    Marko T Ahonen

    Full Text Available BACKGROUND: Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important. METHODS/RESULTS: We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly. CONCLUSION: In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline.

  9. Calcium Gluconate in Phosphate Buffered Saline Increases Gene Delivery with Adenovirus Type 5

    Ahonen, Marko T.; Diaconu, Iulia; Pesonen, Sari; Kanerva, Anna; Baumann, Marc; Parviainen, Suvi T.; Spiller, Brad


    Background Adenoviruses are attractive vectors for gene therapy because of their stability in vivo and the possibility of production at high titers. Despite exciting preclinical data with various approaches, there are only a few examples of clear efficacy in clinical trials. Effective gene delivery to target cells remains the key variable determining efficacy and thus enhanced transduction methods are important. Methods/Results We found that heated serum could enhance adenovirus 5 mediated gene delivery up to twentyfold. A new protein-level interaction was found between fiber knob and serum transthyretin, but this was not responsible for the observed effect. Instead, we found that heating caused the calcium and phosphate present in the serum mix to precipitate, and this was responsible for enhanced gene delivery. This finding could have relevance for designing preclinical experiments with adenoviruses, since calcium and phosphate are present in many solutions. To translate this into an approach potentially testable in patients, we used calcium gluconate in phosphate buffered saline, both of which are clinically approved, to increase adenoviral gene transfer up to 300-fold in vitro. Gene transfer was increased with or without heating and in a manner independent from the coxsackie-adenovirus receptor. In vivo, in mouse studies, gene delivery was increased 2-, 110-, 12- and 13-fold to tumors, lungs, heart and liver and did not result in increased pro-inflammatory cytokine induction. Antitumor efficacy of a replication competent virus was also increased significantly. Conclusion In summary, adenoviral gene transfer and antitumor efficacy can be enhanced by calcium gluconate in phosphate buffered saline. PMID:20927353

  10. Rapid and sustained CD4(+) T-cell-independent immunity from adenovirus-encoded vaccine antigens

    Holst, Peter J; Bartholdy, Christina; Buus, Anette Stryhn


    Many novel vaccine strategies rely on recombinant viral vectors for antigen delivery, and adenovirus vectors have emerged among the most potent of these. In this report, we have compared the immune response induced through priming with adenovirus vector-encoded full-length viral protein to that e......Many novel vaccine strategies rely on recombinant viral vectors for antigen delivery, and adenovirus vectors have emerged among the most potent of these. In this report, we have compared the immune response induced through priming with adenovirus vector-encoded full-length viral protein...... to that elicited with an adenovirus-encoded minimal epitope covalently linked to beta(2)-microglobulin. We demonstrate that the beta(2)-microglobulin-linked epitope induced an accelerated and augmented CD8(+) T-cell response. Furthermore, the immunity conferred by vaccination with beta(2)-microglobulin...... in the absence of CD4(+) T-cell help were sustained in the long term and able to expand and control a secondary challenge with LCMV. Our results demonstrate that modifications to the antigen used in adenovirus vaccines may be used to improve the induced T-cell response. Such a strategy for CD4(+) T...

  11. Adrenal gland infection by serotype 5 adenovirus requires coagulation factors.

    Lucile Tran

    Full Text Available Recombinant, replication-deficient serotype 5 adenovirus infects the liver upon in vivo, systemic injection in rodents. This infection requires the binding of factor X to the capsid of this adenovirus. Another organ, the adrenal gland is also infected upon systemic administration of Ad, however, whether this infection is dependent on the cocksackie adenovirus receptor (CAR or depends on the binding of factor X to the viral capsid remained to be determined. In the present work, we have used a pharmacological agent (warfarin as well as recombinant adenoviruses lacking the binding site of Factor X to elucidate this mechanism in mice. We demonstrate that, as observed in the liver, adenovirus infection of the adrenal glands in vivo requires Factor X. Considering that the level of transduction of the adrenal glands is well-below that of the liver and that capsid-modified adenoviruses are unlikely to selectively infect the adrenal glands, we have used single-photon emission computed tomography (SPECT imaging of gene expression to determine whether local virus administration (direct injection in the kidney could increase gene transfer to the adrenal glands. We demonstrate that direct injection of the virus in the kidney increases gene transfer in the adrenal gland but liver transduction remains important. These observations strongly suggest that serotype 5 adenovirus uses a similar mechanism to infect liver and adrenal gland and that selective transgene expression in the latter is more likely to be achieved through transcriptional targeting.

  12. Regulation of Human Adenovirus Replication by RNA Interference.

    Nikitenko, N A; Speiseder, T; Lam, E; Rubtsov, P M; Tonaeva, Kh D; Borzenok, S A; Dobner, T; Prassolov, V S


    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy.

  13. New Protein Vector ApE1 for Targeted Delivery of Anticancer Drugs

    N. V. Pozdniakova


    Full Text Available A new chimeric gene ApE1 encoding the receptor-binding domain of the human alpha-fetoprotein fused to a sequence of 22 glutamic acid residues was constructed. A new bacterial producer strain E. coli SHExT7 ApE1 was selected for ApE1 production in a soluble state. A simplified method was developed to purify ApE1 from bacterial biomass. It was shown that the new vector protein selectively interacts with AFP receptors on the tumor cell surface and can be efficiently accumulated in tumor cells. In addition, ApE1 was shown to be stable in storage and during its chemical modification. An increased number of carboxyl groups in the molecule allows the production of cytotoxic compound conjugates with higher drug-loading capacity and enhanced tumor targeting potential.

  14. Derivation of a myeloid cell-binding adenovirus for gene therapy of inflammation.

    Michael O Alberti

    Full Text Available The gene therapy field is currently limited by the lack of vehicles that permit efficient gene delivery to specific cell or tissue subsets. Native viral vector tropisms offer a powerful platform for transgene delivery but remain nonspecific, requiring elevated viral doses to achieve efficacy. In order to improve upon these strategies, our group has focused on genetically engineering targeting domains into viral capsid proteins, particularly those based on adenovirus serotype 5 (Ad5. Our primary strategy is based on deletion of the fiber knob domain, to eliminate broad tissue specificity through the human coxsackie-and-adenovirus receptor (hCAR, with seamless incorporation of ligands to re-direct Ad tropism to cell types that express the cognate receptors. Previously, our group and others have demonstrated successful implementation of this strategy in order to specifically target Ad to a number of surface molecules expressed on immortalized cell lines. Here, we utilized phage biopanning to identify a myeloid cell-binding peptide (MBP, with the sequence WTLDRGY, and demonstrated that MBP can be successfully incorporated into a knob-deleted Ad5. The resulting virus, Ad.MBP, results in specific binding to primary myeloid cell types, as well as significantly higher transduction of these target populations ex vivo, compared to unmodified Ad5. These data are the first step in demonstrating Ad targeting to cell types associated with inflammatory disease.

  15. Target product profile choices for intra-domiciliary malaria vector control pesticide products: repel or kill?

    Moore Sarah J


    Full Text Available Abstract Background The most common pesticide products for controlling malaria-transmitting mosquitoes combine two distinct modes of action: 1 conventional insecticidal activity which kills mosquitoes exposed to the pesticide and 2 deterrence of mosquitoes away from protected humans. While deterrence enhances personal or household protection of long-lasting insecticidal nets and indoor residual sprays, it may also attenuate or even reverse communal protection if it diverts mosquitoes to non-users rather than killing them outright. Methods A process-explicit model of malaria transmission is described which captures the sequential interaction between deterrent and toxic actions of vector control pesticides and accounts for the distinctive impacts of toxic activities which kill mosquitoes before or after they have fed upon the occupant of a covered house or sleeping space. Results Increasing deterrency increases personal protection but consistently reduces communal protection because deterrent sub-lethal exposure inevitably reduces the proportion subsequently exposed to higher lethal doses. If the high coverage targets of the World Health Organization are achieved, purely toxic products with no deterrence are predicted to generally provide superior protection to non-users and even users, especially where vectors feed exclusively on humans and a substantial amount of transmission occurs outdoors. Remarkably, this is even the case if that product confers no personal protection and only kills mosquitoes after they have fed. Conclusions Products with purely mosquito-toxic profiles may, therefore, be preferable for programmes with universal coverage targets, rather than those with equivalent toxicity but which also have higher deterrence. However, if purely mosquito-toxic products confer little personal protection because they do not deter mosquitoes and only kill them after they have fed, then they will require aggressive "catch up" campaigns, with

  16. A new method for rapidly generating gene-targeting vectors by engineering BACs through homologous recombination in bacteria.

    Cotta-de-Almeida, Vinicius; Schonhoff, Susan; Shibata, Tomoyuki; Leiter, Andrew; Snapper, Scott B


    Generating knockout mice is still an expensive and highly time-consuming process. Target construct generation, the first labor-intensive step in this process, requires the manipulation of large fragments of DNA and numerous, and often cumbersome, cloning steps. Here we show the development of a rapid approach for generating targeting constructs that capitalizes on efficient homologous recombination between linear DNA fragments and circular plasmids in Escherichia coli ("recombineering"), the availability of bacterial artificial chromosomes (BACs), and the accessibility of the sequence of the mouse genome. Employing recombineering, we demonstrate with only 1-2 template plasmids, short homologies (40-50bp) between donor and target DNA, and one subcloning step that we can efficiently manipulate BACs in situ to generate a complicated targeting vector. This procedure avoids the need to construct or screen genomic libraries and permits the generation of most standard, conditional, or knock-in targeting vectors, often within two weeks.

  17. Construction of recombinant adenovirus vector for human matrix metalloproteinase-1 gene and detection of collagen type III degradation in vitro%构建人基质金属蛋白酶1基因重组腺病毒载体及体外降解Ⅲ型胶原的检测

    杜超; 蒋明德; 曾维政; 郑淑梅


    BACKGROUND:Matrix metaloproteinase-1 can degrade extracelular matrix, which is mainly colagen type I, and has the potential to reverse fibrosis tissue. OBJECTIVE:To construct the recombinant adenovirus vector containing human matrix metaloproteinase-1 (hMMP-1) gene with GatewayTM Clone Technology, and observe the capacity of degrading colagen type IIIin vitro. METHODS: The gene hMMP-1 was amplified by using PCR from the pcDNA3.1 plasmid and was cut down by the double endonuclease. The linear gene fragment was connected to the entry vector pENTERTM 1A. Then the entry clone and the destination vectors pJTI™ R4 Dest CMV-N-EmGFP pA Vector recombined using the LR reaction to form the expression clone pAd-hMMP-1-eGFP. The linear pAd-hMMP-1-eGFP cut down by endonucleasePac I was transfected into HEK293A cels to packaging the Ad-hMMP-1-eGFP. The transfected situation was observed under a fluorescence microscope, the target protein expression was detected by western-blot assay and RT-PCR. Cels can be divided into three groups: blank control group: HEK293A cels, AD-EGFP group: HEK293A cels were infected by Ad-eGFP, AD-HMMP1-EGF group: HEK293A cels were infected by Ad-hMMP1-eGFP and colagen type III. The content of colagen type III was detected by ELISA kits after 24, 48 and 72 hours. RESULTS AND CONCLUSION: It was confirmed that the entry vector and the destination vector both contained hMMP-1 target gene by restriction analysis and sequencing. The green fluorescent protein was observed in the 293A cels transfected by the Ad-hMMP-1-eGFP at 4 days. The fluorescence intensity was the highest at 10 days. The virus was colected at 12 days, the viral titer was determined as 4.84 × 1010 PFU/mL, the target protein was efficient expressionvia western-blot assay. Blank control group and AD-EGFP group had no obvious change of colagen content with the extension of time. The rate of colagen degradation in AD-HMMP1-EGFP group was 24%, 56% and 81% respectively at 24, 48, 72 hours. AD

  18. Using support vector machine ensembles for target audience classification on Twitter.

    Lo, Siaw Ling; Chiong, Raymond; Cornforth, David


    The vast amount and diversity of the content shared on social media can pose a challenge for any business wanting to use it to identify potential customers. In this paper, our aim is to investigate the use of both unsupervised and supervised learning methods for target audience classification on Twitter with minimal annotation efforts. Topic domains were automatically discovered from contents shared by followers of an account owner using Twitter Latent Dirichlet Allocation (LDA). A Support Vector Machine (SVM) ensemble was then trained using contents from different account owners of the various topic domains identified by Twitter LDA. Experimental results show that the methods presented are able to successfully identify a target audience with high accuracy. In addition, we show that using a statistical inference approach such as bootstrapping in over-sampling, instead of using random sampling, to construct training datasets can achieve a better classifier in an SVM ensemble. We conclude that such an ensemble system can take advantage of data diversity, which enables real-world applications for differentiating prospective customers from the general audience, leading to business advantage in the crowded social media space.

  19. 溶瘤腺病毒肿瘤靶向治疗:从实验室到临床%Oncolytic adenoviruses for targeted cancer therapy: From the laboratory to the clinic



    过去几十年从实验室到临床对溶瘤腺病毒进行了广泛的研究.有多种策略增强溶瘤腺病毒的肿瘤靶向性,包括将腺病毒基因组中参与细胞周期节点调控的功能基因(如E1A或E1B)进行突变或(和)利用肿瘤特异性启动子调控E1A基因的靶向转录调控,利用不同血清型腺病毒或RGD基序改变溶瘤腺病毒进入肿瘤细胞方式的靶向转导调控,以及利用细胞载体将溶瘤腺病毒传送至远处的肿瘤部位.溶瘤腺病毒作为一种载体,输送免疫调节基因或治疗性基因,通过增强抗肿瘤免疫,或引起肿瘤细胞凋亡、自杀等产生协同抗肿瘤效应.溶瘤腺病毒临床试验涉及到多种实体瘤,显示其临床应用是安全的,毒性作用轻至中度,患者能很好耐受,但有明确客观抗肿瘤反应的临床病例较少见,联合诸如化、放疗等治疗方法有助于提高临床效果. 未来的方向应强化溶瘤腺病毒免疫学相关机制的研究、突破妨碍溶瘤腺病毒研究的一些技术性瓶颈、优化细胞载体、提高溶瘤腺病毒远处传递的靶向性,以及寻找更具潜能的肿瘤干细胞作为靶点.此外,需要扩大临床试验的研究范围和加强与其他治疗方式特别是免疫治疗联合应用的研究.%Extensive investigations of oncolytic adenoviruse (OA) from the laboratory to the clinic have been made in the past decades. There are a few strategies for improving the potential targeted therapy of OA in cancer, including tran-scriptional targeting by mutating functional genes (such as El A or E1B) of Ad genome which is involved in the control of cell cycle checkpoints in cells and/or utilizing tumor-specific promoter control of El A expression, transductional targeting by changing tropism of OA with different types or incorporated by RGD motif into tumor cells, and cell carriers for delivery of OA to the distant tumor sites. Enhanced anti-tumor immune response, apoptosis or suicide of tumor

  20. Targeted cancer gene therapy : the flexibility of adenoviral gene therapy vectors

    Rots, MG; Curiel, DT; Gerritsen, WR; Haisma, HJ


    Recombinant adenoviral vectors are promising reagents for therapeutic interventions in humans, including gene therapy for biologically complex diseases like cancer and cardiovascular diseases. In this regard, the major advantage of adenoviral vectors is their superior in vivo gene transfer efficienc



    Objective To investigate in vitro heme oxygenase-1 gene (HO-1) delivery to human pancreatic islets by adenovirus vectors. Methods Recombinant adenovirus containing HO-1 or enhanced green fluorescent protein gene(EGFP) was generated by using the AdEasy System. The purified human pancreatic islets were infected with recombinant adenovirus vectors at various multiplicity of infection (MOI). Transduction was confirmed by fluorescence photographs and Western blot. Glucose-stimulated insulin secretion was detected by using Human insulin radioimmunoassay kits and was used to assess the function of human islets infected by recombinant adenovirus.Results Viral titers of Ad-hHO-1 and Ad-EGFP were 1.96×109 and 1.99×109 pfu/mL, respectively. Human pancreatic islets were efficiently infected by recombinant adenovirus vectors in vitro. Transfection of human islets at an MOI of 20 did not inhibit islet function. Recombinant adenovirus mediated HO-1gene transfer significantly improved the islet function of insulin release when simulated by high level glucose. Conclusion Recombinant adenovirus is efficient to deliver exogenous gene into human pancreatic islets in vitro. HO-1 gene transfection can improve human islet function.

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

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


    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.

  3. Systemic Delivery of an Oncolytic Adenovirus Expressing Decorin for the Treatment of Breast Cancer Bone Metastases.

    Yang, Yuefeng; Xu, Weidong; Neill, Thomas; Hu, Zebin; Wang, Chi-Hsiung; Xiao, Xianghui; Stock, Stuart R; Guise, Theresa; Yun, Chae-Ok; Brendler, Charles B; Iozzo, Renato V; Seth, Prem


    The development of novel therapies for breast cancer bone metastasis is a major unmet medical need. Toward that end, we have constructed an oncolytic adenovirus, Ad.dcn, and a nonreplicating adenovirus, Ad(E1-).dcn, both containing the human decorin gene. Our in vitro studies showed that Ad.dcn produced high levels of viral replication and the decorin protein in the breast tumor cells. Ad(E1-).dcn-mediated decorin expression in MDA-MB-231 cells downregulated the expression of Met, β-catenin, and vascular endothelial growth factor A, all of which are recognized decorin targets and play pivotal roles in the progression of breast tumor growth and metastasis. Adenoviral-mediated decorin expression inhibited cell migration and induced mitochondrial autophagy in MDA-MB-231 cells. Mice bearing MDA-MB-231-luc skeletal metastases were systemically administered with the viral vectors, and skeletal tumor growth was monitored over time. The results of bioluminescence imaging and X-ray radiography indicated that Ad.dcn and Ad(E1-).dcn significantly inhibited the progression of bone metastases. At the terminal time point, histomorphometric analysis, micro-computed tomography, and bone destruction biomarkers showed that Ad.dcn and Ad(E1-).dcn reduced tumor burden and inhibited bone destruction. A nonreplicating adenovirus Ad(E1-).luc expressing the luciferase 2 gene had no significant effect on inhibiting bone metastases, and in several assays, Ad.dcn and Ad(E1-).dcn were better than Ad.luc, a replicating virus expressing the luciferase 2 gene. Our data suggest that adenoviral replication coupled with decorin expression could produce effective antitumor responses in a MDA-MB-231 bone metastasis model of breast cancer. Thus, Ad.dcn could potentially be developed as a candidate gene therapy vector for treating breast cancer bone metastases.

  4. A RGD-Containing Oligopeptide (K)16GRGDSPC: A Novel Vector for Integrin-Mediated Targeted Gene Delivery

    PAN Haitao; ZHENG Qixin; GUO Xiaodong; LIU Yong; LI Changwen; SONG Yulin


    A 23 amino acid, bifunctional integrin-targeted synthetic oligopeptide was evaluated for ex vivo gene delivery to rabbit bone marrow stromal cells (BMSCs). Synthesis of the peptide (K)16GRGDSPC was performed on a solid-phase batch peptide synthesizer. BMSCs were transfected with plasmid DNA coding for luciferase by (K)16GRGDSPC and the transfection efficiency was assayed. The influences of chloroquine and polyethyleneimine on the transfection efficiency were also examined. The target specificity of (K)16GRGDSPC to mediate exogenous gene into BMSCs was analyzed using cell attachment test and gene delivery inhibition test. The results showed that the transfection efficiency of the oligopeptide vector was lower than that of Lipofectamine. But in the presence of endosomal buffer chloroquine or endosomal disrupting agent polyethyleneimine, the transfection efficiency of the vector was greatly enhanced. In addition, RGD-containing peptides inhibited BMSCs' attachment to the 96-well plates pretreated with fibronectin or vitronectin and significantly decreased the transfection efficiency of the oligopeptide vector. These studies demonstrated that oligopeptide (K)16GRGDSPC was an ideal novel targeted non-viral gene delivery vector, which was easy to be synthesized, high efficient and low cytotoxicity. The vector could effectively deliver exogenous gene into rat BMSCs.

  5. Realization to Extend the Orientation Estimation Range of Moving Target on the Ground by a Single Vector Sensor

    Xiaopeng Song


    Full Text Available The DOA (direction of arrival estimation of seismic signals from the moving target on the ground bears great significance for unattended ground systems. The traditional DOA estimation of seismic signals is achieved by a sensor array and its corresponding algorithms. MEMS (Micro-Electro- Mechanical Systems vector vibration sensor, however, gets the vector information over the propagation of seismic signals and therefore can get a DOA estimation within a certain range through a single vector sensor. This paper proposes a new method to extend the orientation range through the rotation of the MEMS vector vibration axis. The experiment shows that this method shares the merits with simple systematic structure, high sensitivity and less than 5 degrees of error on average, which has an extensive wide application prospect.

  6. The inhibition effects of the c-myc targeting CRISPR/Cas9 adenovirus system on hepatoma cells%靶向 c-myc 基因的 CRISPR/Cas9腺病毒系统对肝癌的抑制作用

    连竹生; 梁鑫; 金桂花; 吴领知; 韩苏夏


    目的:设计、构建并包装靶向 c-myc 基因的 CRISPR/Cas9腺病毒;评估靶向 c-myc 基因的 CRISPR/Cas9腺病毒系统对肝癌的抑制作用。方法:采用生物信息学网站设计靶向 c-myc 基因的 gRNA,以 GFP 作为对照设计GFP gRNA;通过 T7E1筛选出编辑效率高的 gRNA 并将筛选的 gRNA 构建 CRISPR/Cas9腺病毒载体并包装腺病毒,通过分析细胞增殖、周期和凋亡及迁移能力的变化来评估靶向 c-myc 基因的 CRISPR/Cas9腺病毒系统对肝癌的抑制作用。结果:成功设计、构建并包装靶向 c-myc 基因的 CRISPR/Cas9腺病毒;靶向 c-myc 基因的 CRISPR/Cas9腺病毒系统能够显著抑制 Hepa1-6细胞的增殖和迁移、阻滞细胞周期进程,但对细胞凋亡无影响。结论:靶向 c-myc 基因的 CRISPR/Cas9腺病毒系统可在细胞水平明显抑制肝癌细胞的生长。%Aim:To design,construct and package the CRISPR/Cas9 adenovirus system for targeting the mouse c-myc gene;To evaluate the inhibition efficiency of the constructed adenovirus system on c-myc gene in hepatoma cells.Methods:We designed gRNAs for targeting the mouse c-myc gcontrolene and used another gene (gRNAs for GFP)as control based on bioinformatics website.The editing efficiencies of these gRNAs were determined by T7E1 cleavage.The gRNAs with higher editing efficiency were se-lected and packed into adenovirus for the following experiments.Firstly,Hepa 1-6 cells were infected with the constructed adenovirus,then cell proliferation,cell cycle,cell apoptosis and migration assays were detected to de the inhibition efficiency of the adenovirus on hepatoma cells.Statistical analyses were performed with SPSS(version 18.0)and t-test,P <0.05 was considered statistically significant.Re-sults:The CRISPR/Cas9 adenovirus system targeting the mouse c-myc gene was successfully designed constructed and packaged.Compared with the control group,the experiment groups (with CRISPR/Cas9

  7. Construction of human 14-3-3 γadenovirus vector and its expression in PC12 cells%人14-3-3γ基因的腺病毒载体的构建及其在PC12细胞中的表达

    陈小武; 陈志斌; 王埮; 袁昆雄; 王淑荣; 孙圣刚


    Objective: To construct the recombinant adenovirus vector carrying human 14-3-3 γ gene, and infect the PC12 cells.Methods: The full 14-3-3 γ DNA sequence was obtained from plasmids Top1O/pHis-14-3-3 γusing PCR.The 14-3-3 γ gene was cloned into pAdTrack-CMV vector which was subsequently homologously recombined with pAdEasy-1 vector in the HEK293 cells to package the recombinant adenovirus vector (pAd-14-3-3 γ) carrying human 14-3-3 γ.After verified by PCR, we amplified pAd/14-3-3 γin HEK293 cells and purified it by CsCI gradient purification,titrated it using 50% tissue culture infective dose (TCID50) assay.Results: PC12 cells were infected with adenoviruses.Protein expressions of EGFP (enhanced green fluorescent protein) and 14-3-3 γwere detected by the intensity of green fluorescence under fluorescence microscope and Western Blot respectively.The 14-3-3 γgene was cloned and verified by sequencing and high tittered virus was produced by a construct carrying 14-3-3 γgene, and 14-3-3 γ was expressed efficiently in the PCI2 cells after infection.Conclusion: The newly constructed adenovirus vector containing human 14-3-3 γ gene provides the basis for genetherapy of Parkinson's disease.%目的:构建携带人14-3-3 γ基因的重组腺病毒表达载体并确定其对PC12细胞的感染效率.方法:采用PCR方法,从Top10/pHis-14-3-3 γ质粒中扩增14-3-3 γ DNA序列,将14-3-3 γ基因定向克隆到穿梭质粒载体pAdTrack-CMV,经与腺病毒骨架质粒pAdEasy-1载体同源重组后得到携带人14-3-3γ基因的重组腺病毒(pAd/14-3-3 γ),采用PCR的方法对重组腺病毒进行鉴定,转染HEK293细胞进行包装和扩增,氯化铯密度梯度离心法纯化,半数组织培养感染剂量(50%tissue culture infective dose,TCID50)方法测定重组腺病毒的滴度.体外感染PC12细胞,荧光显微镜观察绿色荧光蛋白(GFP)和Western Blot检测14-3-3γ蛋白的表达.结果:克隆得到人14-3-3γ基因,经PCR鉴定和测序证实

  8. Structure of Human Adenovirus

    Nemerow, Glen R.; Phoebe L Stewart; Reddy, Vijay S.


    A detailed structural analysis of the entire human adenovirus capsid has been stymied by the complexity and size of this 150 MDa macromolecular complex. Over the past 10 years, the steady improvements in viral genome manipulation concomitant with advances in crystallographic techniques and data processing software has allowed structure determination of this virus by X-ray diffraction at 3.5 Å resolution. The virus structure revealed the location, folds, and interactions of major and minor (ce...

  9. Target Localization in Wireless Sensor Networks Using Online Semi-Supervised Support Vector Regression

    Jaehyun Yoo


    Full Text Available Machine learning has been successfully used for target localization in wireless sensor networks (WSNs due to its accurate and robust estimation against highly nonlinear and noisy sensor measurement. For efficient and adaptive learning, this paper introduces online semi-supervised support vector regression (OSS-SVR. The first advantage of the proposed algorithm is that, based on semi-supervised learning framework, it can reduce the requirement on the amount of the labeled training data, maintaining accurate estimation. Second, with an extension to online learning, the proposed OSS-SVR automatically tracks changes of the system to be learned, such as varied noise characteristics. We compare the proposed algorithm with semi-supervised manifold learning, an online Gaussian process and online semi-supervised colocalization. The algorithms are evaluated for estimating the unknown location of a mobile robot in a WSN. The experimental results show that the proposed algorithm is more accurate under the smaller amount of labeled training data and is robust to varying noise. Moreover, the suggested algorithm performs fast computation, maintaining the best localization performance in comparison with the other methods.

  10. Adenovirus E4orf6 targets pp32/LANP to control the fate of ARE-containing mRNAs by perturbing the CRM1-dependent mechanism.

    Higashino, Fumihiro; Aoyagi, Mariko; Takahashi, Akiko; Ishino, Masaho; Taoka, Masato; Isobe, Toshiaki; Kobayashi, Masanobu; Totsuka, Yasunori; Kohgo, Takao; Shindoh, Masanobu


    E4orf6 plays an important role in the transportation of cellular and viral mRNAs and is known as an oncogene product of adenovirus. Here, we show that E4orf6 interacts with pp32/leucine-rich acidic nuclear protein (LANP). E4orf6 exports pp32/LANP from the nucleus to the cytoplasm with its binding partner, HuR, which binds to an AU-rich element (ARE) present within many protooncogene and cytokine mRNAs. We found that ARE-mRNAs, such as c-fos, c-myc, and cyclooxygenase-2, were also exported to and stabilized in the cytoplasm of E4orf6-expressing cells. The oncodomain of E4orf6 was necessary for both binding to pp32/LANP and effect for ARE-mRNA. C-fos mRNA was exported together with E4orf6, E1B-55kD, pp32/LANP, and HuR proteins. Moreover, inhibition of the CRM1-dependent export pathway failed to block the export of ARE-mRNAs mediated by E4orf6. Thus, E4orf6 interacts with pp32/LANP to modulate the fate of ARE-mRNAs by altering the CRM1-dependent export pathway.

  11. Cre/lox-Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti.

    Häcker, Irina; Harrell Ii, Robert A; Eichner, Gerrit; Pilitt, Kristina L; O'Brochta, David A; Handler, Alfred M; Schetelig, Marc F


    Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting.

  12. Cre/lox-Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti

    Häcker, Irina; Harrell II, Robert A.; Eichner, Gerrit; Pilitt, Kristina L.; O’Brochta, David A.; Handler, Alfred M.; Schetelig, Marc F.


    Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting. PMID:28266580

  13. Retargeted oncolytic adenovirus displaying a single variable domain of camelid heavy-chain-only antibody in a fiber protein.

    van Erp, Elisabeth A; Kaliberova, Lyudmila N; Kaliberov, Sergey A; Curiel, David T


    Conditionally replicative adenoviruses are promising agents for oncolytic virotherapy. Various approaches have been attempted to retarget adenoviruses to tumor-specific antigens to circumvent deficiency of receptor for adenoviral binding and to provide an additional level of tumor specificity. Functional incorporation of highly specific targeting molecules into the viral capsid can potentially retarget adenoviral infection. However, conventional antibodies are not compatible with the cytoplasmic adenovirus capsid synthesis. The goal of this study was to evaluate the utility of single variable domains derived from heavy chain camelid antibodies for retargeting of adenovirus infection. We have combined transcriptional targeting using a tumor-specific promoter with transductional targeting through viral capsid incorporation of antihuman carcinoembryonic antigen single variable domains. Obtained data demonstrated that employment of a single variable domain genetically incorporated into an adenovirus fiber increased specificity of infection and efficacy of replication of single variable domain-targeted oncolytic adenovirus. The double targeting, both transcriptional through the C-X-C chemokine receptor type 4 promoter and transductional using the single variable domain, is a promising means to improve the therapeutic index for these advanced generation conditionally replicative adenoviruses. A successful strategy to transductional retargeting of oncolytic adenovirus infection has not been shown before and therefore we believe this is the first employment of transductional targeting using single variable domains derived from heavy chain camelid antibodies to enhance specificity of conditionally replicative adenoviruses.

  14. Effect of recombinant adenovirus vector expressing human endostatin on endothelial cell proliferation%内皮抑素重组腺病毒表达载体构建及对内皮细胞增殖的影响

    唐辉; 徐永清; 李春晓; 张秀琼; 郑天娥; 刘旭盛; 梁晚益


    certain degree. OBJECTIVE: To construct a recombinant adenovirus vector expressing human endostatin (Ad/hEnd), and to investigate the cooperative effect of Ad/hEnd and keratinocyte on endothelial cell proliferation. DESIGN, TIME AND SETTING: Observational study, which was performed in the State Key Laboratory of Trauma, Burn and Combined Injury, Institute of Burn Research, Southwest Hospital of the Third Military Medical University of Chinese PLA between September 2006 and May 2007. MATERIALS: pAdTrack-CMV and pAdEasy-1 were obtained from Stratagene Company, USA; 293 cell and Ecoli.DH5α were stored in our laboratory. METHODS: The endostatin gene sequence was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and polymerase chain reaction (PCR) based on mRNA of human fetal hepatic tissue and inserted into the adenovims shuttle plasmid pAdTrack-CMV to obtain recombinant plasmid pAdTrack-ES. After identification, positive recon was transformed into pAdeasy 1 recipient virus to screen positive clones. The adenovirus Ad/hEnd was generated from 293 cells and identified by PCR and fluorescence microscope. Then the keratinocytes were infected with Ad/hEnd, and co-cultured with endothelial cells by nest dish culture method. The content of endostatin was detected, and the non-transfection keratinocytes were used as the controls. MAIN OUTCOME MEASURES: Homologous recombination and identification of pAd/hEnd; generation and identification of Ad/hEnd; endostatin expression after 293 cell transfection; purification and titer measurement of Ad/hEnd; content of endostatin in culture solution; apoptotic percentage of endothelial cells; inhibitory ratio of endothelial cells. RESULTS: Ad/hEnd was constructed and the virus titer was generally up to 1.65×1012 PFU/L. Ad/hEnd-infected keratinocytes could effectively express and secrete endostatin of which the content reached 226 μg/L after 3 days of co-culture. The apoptotic percentage and inhibitory ratio of the

  15. 基质金属蛋白酶组织抑制剂1基因重组腺病毒载体的构建%Construction of Recombinant Adenovirus Vector Carrying the Human Tissue Inhibitor of Metalloproteinase 1 cDNA

    周毅; 屠冠军


    Objective To construct recombinant adenovirus vector carrying the human tissue inhibitor of metalloproteinase 1 (T1MP1) cD-NA and explore the feasibility of reversing or delaying intervertebral disc degeneration by gene therapy. Methods The hTIMPl cDNA was amplified from plasmid containing human TIMP1 ORF sequence by PCR. hTIMPl cDNA was subcloned into the adenovirus shuttle plasmid pDC316 of Ad5MaxTM adenovirus vector system,and the products were co-transfected into HEK293 cells with the helper plasmid pBH- Glox_El ,3Cre by lipofectin transfection method. The recombinant adenovirus Ad5-TIMP1 was generated by homologous recombination of the two plasmids in HEK 293 cells. After identification by PCR and enzyme digestion, Ad5-TTMP1 was amplified in HEK 293 adenoviral packaging cells, purified by column chromatography, and virus activity was measured by TCID50 assay. Results Recombinant adenoviral vector carrying hTIMPl(Ad5-hTIMPl) was successfully constructed with virus activity of l×l010 IU/ml,which was confirmed by PCR,restriction enzyme digestion and DNA sequencing. Conclusion The Recombinant adenoviral vector carrying hTIMPl (Ad5-hTlMPl) was successfully constructed, which provided a foundation for further research on reversing or delaying intervertebral disc degeneration by gene therapy.%目的 为探讨基因治疗逆转或延缓椎间盘退变的可行性,构建及制备人基质金属蛋白酶组织抑制剂1 (tissue inhibitor of metalloproteinase 1,TIMP1) cDNA重组腺病毒载体.方法 以含TIMP1 cDNA序列的质粒为模板,通过PCR方法扩增TIMP1cDNA片段,将TIMP1 cDNA全长定向克隆到Ad5MaxTM腺病毒系统的穿梭质粒pDC316上,构建pDC316-TIMP1穿梭质粒;使用Ad5MaxTM腺病毒包装系统,脂质体介导的穿梭质粒及骨架质粒pBHGlox-E1,3Cre共转染HEK293细胞,同源重组构建含TIMP1 cDNA的重组腺病毒Ad5-TIMP1,通过PCR方法鉴定重组腺病毒Ad5-TIM P1的正确性.通过阴离子柱层析

  16. Noninvasive visualization of adenovirus replication with a fluorescent reporter in the E3 region.

    Ono, Hidetaka A; Le, Long P; Davydova, Julia G; Gavrikova, Tatyana; Yamamoto, Masato


    To overcome the inefficacy and undesirable side effects of current cancer treatment strategies, conditionally replicative adenoviruses have been developed to exploit the unique mechanism of oncolysis afforded by tumor-specific viral replication. Despite rapid translation into clinical trials and the established safety of oncolytic adenoviruses, the in vivo function of these agents is not well understood due to lack of a noninvasive detection system for adenovirus replication. To address this issue, we propose the expression of a reporter from the adenovirus E3 region as a means to monitor replication. Adenovirus replication reporter vectors were constructed with the enhanced green fluorescent protein (EGFP) gene placed in the deleted E3 region under the control of the adenoviral major late promoter while retaining expression of the adenovirus death protein to conserve the native oncolytic capability of the virus. Strong EGFP fluorescence was detected from these vectors in a replication-dependent manner, which correlated with viral DNA replication. Fluorescence imaging in vivo confirmed the ability to noninvasively detect fluorescent signal during replication, which generally corresponded with the underlying level of viral DNA replication. EGFP representation of viral replication was further confirmed by Western blot comparison with the viral DNA content in the tumors. Imaging reporter expression controlled by the adenoviral major late promoter provides a viable approach to noninvasively monitor adenovirus replication in preclinical studies and has the potential for human application with clinically relevant imaging reporters.

  17. A sensitive and rapid assay for homologous recombination in mosquito cells: impact of vector topology and implications for gene targeting

    Zhao Yuguang


    Full Text Available Abstract Background Recent progress in insect transgenesis has been dramatic but existing transposon-based approaches are constrained by position effects and potential instability. Gene targeting would bring a number of benefits, however progress requires a better understanding of the mechanisms involved. Much can be learned in vitro since extrachromosomal recombination occurs at high frequency, facilitating the study of multiple events and the impact of structural changes among the recombining molecules. We have investigated homologous recombination in mosquito cells through restoration of luciferase activity from deleted substrates. The implications of this work for the construction of insect gene targeting vectors are discussed. Results We show that linear targeting vectors are significantly more efficient than circular ones and that recombination is stimulated by introducing double-strand breaks into, or near, the region of homology. Single-strand annealing represents a very efficient pathway but may not be feasible for targeting unbroken chromosomes. Using circular plasmids to mimic chromosomal targets, one-sided invasion appears to be the predominant pathway for homologous recombination. Non-homologous end joining reactions also occur and may be utilised in gene targeting if double-strand breaks are first introduced into the target site. Conclusions We describe a rapid, sensitive assay for extrachromosomal homologous recombination in mosquito cells. Variations in substrate topology suggest that single-strand annealing and one-sided invasion represent the predominant pathways, although non-homologous end joining reactions also occur. One-sided invasion of circular chromosomal mimics by linear vectors might therefore be used in vitro to investigate the design and efficiency of gene targeting strategies.

  18. Targeted, homology-driven gene insertion in stem cells by ZFN-loaded 'all-in-one' lentiviral vectors

    Cai, Yujia; Laustsen, Anders; Zhou, Yan;


    Biased integration remains a key challenge for gene therapy based on lentiviral vector technologies. Engineering of next-generation lentiviral vectors targeting safe genomic harbors for insertion is therefore of high relevance. In a previous paper (Cai et et, 2014a), we showed the use of integrase......% of transduced iPSCs. Our findings demonstrate the applicability of nuclease-loaded 'all-in-one' IDLVs for site-directed gene insertion in stem cell based gene therapies.......-defective lentiviral vectors (IDLVs) as carriers of complete gene repair kits consisting of zinc-finger nuclease (ZFN) proteins and repair sequences, allowing gene correction by homologous recombination (HR). Here, we follow this strategy to engineer ZEN-loaded IDLVs that insert transgenes by a homology...

  19. Crystal structure of human adenovirus at 3.5 Å resolution*

    Reddy, Vijay S.; Natchiar, S. Kundhavai; Phoebe L Stewart; Nemerow, Glen R.


    Rational development of adenovirus vectors for therapeutic gene transfer is hampered by the lack of accurate structural information. Here we report the X-ray structure at 3.5 Å resolution of the 150 megadalton adenovirus capsid containing nearly 1 million amino acids. We describe interactions between the major capsid protein (hexon) and several accessory molecules that stabilize the capsid. The virus structure also reveals an altered association between the penton base and the trimeric fiber ...

  20. 人Nanog基因重组腺病毒载体的构建及其在人脐带间充质干细胞中的表达%Construction of recombinant adenovirus vector carrying human Nanog gene and its expression in human umbilical cord mesenchymal stem cells

    孙靖; 徐哲; 钱茜; 严家来; 陈圆; 张徐; 高硕; 钱晖; 许文荣


    Objective To construct recombinant adenovirus vector carrying human Nanog gene and transfect human umbilical cord mesenchymal stem cells (hucMSCs). Methods The amplification products of Nanog in polymernse chain reaction (PCR) by using a pair of primers containing the sites of restriction endonuclease Kpn I and Xho I were subcloned into shuttle plasmid pAdtrack-CMV.After analysis of restriction endonuclease and confirmation by sequencing, the recombinant shuttle plasmid pAdTrack-CMV-Nanog was linearized by Pme I , and then transformed into E. coli. BJ5183 which was transformed by adenoviral backbone plasmid pAdEasy-1.The recombinant plasmid pAd-Nanog obtained from screening was confirmed by PCR and restriction endonuclease analysis. The pAdNanog plasmid was linearized by Pac Ⅰ and transfected into human embryonal kidney cell 293A via liposome. The recombinant adenovirus was packaged and amplified in 293A cells following three amplification. The prepared highly expresed Ad-Nanog was transfected into hucMSCs. Results PCR and restriction endonuclease analysis confirmed that Nanog gene was inserted into the recombinant adenovirus vector successfully. The efficiently expressed green fluorescent protein (GFP) in transfected hucMSCs was visualized by fluorescent microscopy. Conclusion Recombinant adenovirus vector containing Nanog was constructed successfully and efficiently transfected hucMSCs which should be useful in the successive investigation on transgenic mesenchymal stem cells.%目的 构建重组人Nanog基因腺病毒栽体(Ad-Nanog),转染人脐带间充质干细胞(hucMSCs),用于后续研究.方法 设计含有Kpn Ⅰ及Xho Ⅰ酶切位点的引物,PCR扩增Nanog基因,将扩增产物亚克隆到pAdTrack-CMV穿梭质粒上,经双酶切和基因测序鉴定,重组穿梭质粒经Pine Ⅰ线性化后,在含有腺病毒骨架质粒pAdEasy-1的BJ5183中同源重组,筛选获得Ad-Nanog重组腺病毒质粒.经Pac Ⅰ酶切线性化,脂质体转染293A细胞,包

  1. Coacervate microspheres as carriers of recombinant adenoviruses.

    Kalyanasundaram, S; Feinstein, S; Nicholson, J P; Leong, K W; Garver, R I


    The therapeutic utility of recombinant adenoviruses (rAds) is limited in part by difficulties in directing the viruses to specific sites and by the requirement for bolus administration, both of which limit the efficiency of target tissue infection. As a first step toward overcoming these limitations, rAds were encapsulated in coacervate microspheres comprised of gelatin and alginate followed by stabilization with calcium ions. Ultrastructural evaluation showed that the microspheres formed in this manner were 0.8-10 microM in diameter, with viruses evenly distributed. The microspheres achieved a sustained release of adenovirus with a nominal loss of bioactivity. The pattern of release and the total amount of virus released was modified by changes in microsphere formulation. Administration of the adenovirus-containing microspheres to human tumor nodules engrafted in mice showed that the viral transgene was transferred to the tumor cells. It is concluded that coacervate microspheres can be used to encapsulate bioactive rAd and release it in a time-dependent manner.

  2. Sterile immunity to malaria after DNA prime/adenovirus boost immunization is associated with effector memory CD8+T cells targeting AMA1 class I epitopes.

    Martha Sedegah

    Full Text Available BACKGROUND: Fifteen volunteers were immunized with three doses of plasmid DNA encoding P. falciparum circumsporozoite protein (CSP and apical membrane antigen-1 (AMA1 and boosted with human adenovirus-5 (Ad expressing the same antigens (DNA/Ad. Four volunteers (27% demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA did not develop sterile protection. METHODOLOGY/PRINCIPAL FINDINGS: We sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to P. falciparum in humans. CONCLUSIONS/SIGNIFICANCE: We suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of

  3. Construction and Characterization of Lentiviral shRNA Expression Vector Targeting Rat CD40 Gene in Dendritic Cells

    SUN Mei; LI Jin-dong; JIANG Rui; JIN Cheng-yan; GAO Nan; LUO Shu-li; WANG Chun-guang; WANG Bin; WANG Rong-you; ZHANG Xing-yi


    To construct a lentiviral shRNA vector targeting rat CD40 gene and detect its effectiveness of gene silencing in dendritic cells(DCs), specific siRNA targets with short hairpin frame were designed and synthesized according to the mRNA sequence of rat CD40 gene. DNA oligo was cloned into lentiviral expression vector, and then PCR and sequencing analyses were conducted to verify the constructs. The verified plasmids were transfected into 293T cells that over-express recombinant CD40 in order to select the most effective siRNA targets. shRNA lentivi-ruses from the selected constructs were propagated and harvested with a virus packaging system, and the virus titers were determined. Western blot and Real-time PCR were performed to determine CD40 expression level in the virus-infected dendritic cells. PCR and sequencing analyses reveal that shRNA plasmids of four targets were successfully constructed. The optimal interfering target was selected, and the virus with a titer of 5×10~7 TU/mL was successfully packaged. CD40 expression in rat DCs was knockdown at both mRNA and protein levels by virus infection. In comparison to that of control groups, CD40 mRNA expression and protein expression were decreased by 60.9% and 61.2%, respectively. We have successfully constructed recombinant lentiviral shRNA expression vector targeting rat CD40 gene that can effectively down-regulate CD40 gene expression at mRNA and protein levels in rat DC.

  4. Peptide-Mediated Tumor Targeting by a Degradable Nano Gene Delivery Vector Based on Pluronic-Modified Polyethylenimine

    Wu, Zhaoyong; Zhan, Shuyu; Fan, Wei; Ding, Xueying; Wu, Xin; Zhang, Wei; Fu, Yinghua; Huang, Yueyan; Huang, Xuan; Chen, Rubing; Li, Mingjuan; Xu, Ningyin; Zheng, Yongxia; Ding, Baoyue


    Polyethylenimine (PEI) is considered to be a promising non-viral gene delivery vector. To solve the toxicity versus efficacy and tumor-targeting challenges of PEI used as gene delivery vector, we constructed a novel non-viral vector DR5-TAT-modified Pluronic-PEI (Pluronic-PEI-DR5-TAT), which was based on the attachment of low-molecular-weight polyethylenimine (LMW-PEI) to the amphiphilic polymer Pluronic to prepare Pluronic-modified LMW-PEI (Pluronic-PEI). This was then conjugated to a multifunctional peptide containing a cell-penetrating peptide (TAT) and a synthetic peptide that would bind to DR5—a receptor that is overexpressed in cancer cells. The vector showed controlled degradation, favorable DNA condensation and protection performance. The Pluronic-PEI-DR5-TAT/DNA complexes at an N/P ratio of 15:1 were spherical nanoparticles of 122 ± 11.6 nm and a zeta potential of about 22 ± 2.8 mV. In vitro biological characterization results indicated that Pluronic-PEI-DR5-TAT/DNA complexes had a higher specificity for the DR5 receptor and were taken up more efficiently by tumor cells than normal cells, compared to complexes formed with PEI 25 kDa or Pluronic-PEI. Thus, the novel complexes showed much lower cytotoxicity to normal cells and higher gene transfection efficiency in tumor cells than that exhibited by PEI 25 kDa and Pluronic-PEI. In summary, our novel, degradable non-viral tumor-targeting vector is a promising candidate for use in gene therapy.

  5. A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3'-UTR of amplifiable dhfr.

    Kang, Shin-Young; Kim, Yeon-Gu; Lee, Hong Weon; Lee, Eun Gyo


    Gene amplification using dihydrofolate reductase gene (dhfr) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3'-untranslated region (UTR) of endogenous dhfr. Thus, shRNAs were designed to target the 3'-UTR of endogenous dhfr, and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80% in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity (q EPO) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q EPO increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr. This study reveals that an expression vector including shRNA that targets the 3'-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector.

  6. SD大鼠白细胞介素10复制缺陷型重组腺病毒的构建及鉴定术%Construction and identification of recombinant replication- defective adenovirus vector containing interleukin-10 in a rat

    李爽; 宋仁刚; 唐冰; 朱斌; 朱旭红; 朱家源; 毕良宽


    背景:重组腺病毒质粒的构建是重组腺病毒制备过程中的中枢环节,传统的细胞内同源重组法步骤多、成功率低、实验周期长.目的:构建含有大鼠白细胞介素10(rlL-10)基因的重组复制缺陷型腺病毒Ad.rlL-10,为真核表达及其动物模型的研究提供实验基础.设计、时间及地点:开放性实验,于2005-07/2006-04在中山大学附属第一医院完成.材料:SD大鼠1只.AdEasy系统由美国John Hopkins肿瘤研究中心惠赠.ThermoscriptTMRT kit 和Trizol 为Invitrogen产品;HEK-293保存于中山大学动物实验中心细胞库;克隆大鼠白细胞介素10基因引物合成及测序由上海博亚生物技术有限公司完成.方法:采用反转录聚合酶链反应的方法从健康SD大鼠脾脏新鲜组织中提取的总RNA中克隆rIL-10基因,采用AdEasy-1腺病毒载体系统经E.CoilBJ5183细菌内同源重组及HEK.293包装而获得重组腺病毒Ad.rlL-10.主要观察指标:用Western blot和RT-PCR的方法鉴定Ad.rlL-10并进行滴度测定.结果:自健康SD大鼠脾脏组织中克隆rIL-10基因,构建出重组腺病毒Ad.rlL-10.经PCR扩增、酶切鉴定、DNA 测序最终确定插入序列为rlL-1 0,Western blot和RT-PCR均检测出细胞内rlL-10基因及其蛋白表达.再通过扩增、纯化后获得滴度为1.0x1014pfu/mL的重组腺病毒.结论:采用AdEasy-1系统,经细HEK-293细胞包装、扩增,纯化后获得足够数量和质量的病毒,方法简便易行,结果稳定可靠.%BACKGROUND: Construction of recombinant adenovirus plasmid plays a central role in preparation of recombinant adenovirus.However, conventionally intracellular homologous recombination method is limited by complex procedures, low successful ratio,and long experimental cycle.OBJECTIVE: To construct the recombinant replication-defective adenovirus vector containing interleukin-10 (Ad.dL-10) in a SD rat, and to provide experimental evidences for eukaryotic expression and animat model studying

  7. Targeted adenovirus mediated inhibition of NF-kappa B-dependent inflammatory gene expression in endothelial cells in vitro and in vivo

    Kuldo, J. M.; Asgeirsdottir, S. A.; Zwiers, P. J.; Bellu, A. R.; Rots, M. G.; Schalk, J. A. C.; Ogawara, K. I.; Trautwein, C.; Banas, B.; Haisma, H. J.; Molema, G.; Kamps, J. A. A. M.


    In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor kappa B signal transduction could silence the proinflammatory activation status of en

  8. VEGFR-3 siRNA腺病毒表达载体对结肠癌细胞凋亡和侵袭的影响%Effects of the adenovirus expressing the small interfering RNA targeting vascular endothelia growth factor receptor-3 gene on apoptosis and invasion of human colon cancer cells

    吕志诚; 苏芝兰; 马强; 张鑫


    Objective To investigate the effects of the adenovirus expression vector of small interfering RNA ( siR-NA) targeting vascular endothelia growth factor receptor 3 (VEGFR-3) gene on apoptosis and invasion of human colon cancer LoVo cells. Methods VEGFR-3 siRNA adenovirus was transfected into the LoVo cells, and the expressions of VEGFR-3 were measured by Western blotting. The apoptosis was detected by Hoechst 33342 staining and FCM ( flow cytometry) . The invasion ability was assayed by Transwell method. Results Compared with the blank control group and the negative control group, the expression level of VEGFR-3 protein in LoVo cells of experimental group was re-duced after transfection with pAd-VEGFR3-siRNA ( P <0 . 05 ) . The apoptosis rate was increased significantly ( P <0. 05). The proliferation of LoVo cells was inhibited, the invasion ability decreased sinificantly (P<0. 05). Conclu-sion VEGFR-3 siRNA can down-regulate the expression levels of VEGFR-3 protein in LoVo cells, inhibit the invasion abilities of LoVo cells, and induce the apoptosis of LoVo cells effectively. VEGFR-3 can be used as a potential aim of colon cancer targeted therapy.%目的:探讨靶向血管内皮细胞生长因子受体3(vascular endothelial growth factor receptor-3, VEGFR-3)基因的小干扰RNA ( small interfering RNA,siRNA)腺病毒载体对人结肠癌LoVo细胞系凋亡及侵袭的影响。方法将靶向VEGFR-3 siRNA腺病毒转染结肠癌LoVo细胞,以Western blotting检测VEGFR-3蛋白的表达,Hoechst 33342染色法和流式细胞仪检测LoVo细胞的凋亡情况,用Transwell小室测定LoVo细胞的侵袭力。结果实验组与空白对照组和阴性对照组比较,实验组中转染靶向VEGFR-3 siRNA腺病毒后结肠癌LoVo细胞中VEGFR-3蛋白的表达被下调(P<0.05)。 Hoechst 33342染色法和流式细胞仪检测LoVo细胞凋亡率明显升高(P<0.05),Transwell小室测定LoVo细胞侵袭能力下降(P<0.05)。结论靶向VEGFR-3 siRNA腺病毒能

  9. Heterologous Immunity between Adenoviruses and Hepatitis C Virus: A New Paradigm in HCV Immunity and Vaccines.

    Shakti Singh

    Full Text Available Adenoviruses (Ad are commonly used as vectors for gene therapy and/or vaccine delivery. Recombinant Ad vectors are being tested as vaccines for many pathogens. We have made a surprising observation that peptides derived from various hepatitis C virus (HCV antigens contain extensive regions of homology with multiple adenovirus proteins, and conclusively demonstrate that adenovirus vector can induce robust, heterologous cellular and humoral immune responses against multiple HCV antigens. Intriguingly, the induction of this cross-reactive immunity leads to significant reduction of viral loads in a recombinant vaccinia-HCV virus infected mouse model, supporting their role in antiviral immunity against HCV. Healthy human subjects with Ad-specific pre-existing immunity demonstrated cross-reactive cellular and humoral immune responses against multiple HCV antigens. These findings reveal the potential of a previously uncharacterized property of natural human adenovirus infection to dictate, modulate and/or alter the course of HCV infection upon exposure. This intrinsic property of adenovirus vectors to cross-prime HCV immunity can also be exploited to develop a prophylactic and/or therapeutic vaccine against HCV.

  10. Heterologous Immunity between Adenoviruses and Hepatitis C Virus: A New Paradigm in HCV Immunity and Vaccines

    Singh, Shakti; Vedi, Satish; Samrat, Subodh Kumar; Li, Wen; Kumar, Rakesh; Agrawal, Babita


    Adenoviruses (Ad) are commonly used as vectors for gene therapy and/or vaccine delivery. Recombinant Ad vectors are being tested as vaccines for many pathogens. We have made a surprising observation that peptides derived from various hepatitis C virus (HCV) antigens contain extensive regions of homology with multiple adenovirus proteins, and conclusively demonstrate that adenovirus vector can induce robust, heterologous cellular and humoral immune responses against multiple HCV antigens. Intriguingly, the induction of this cross-reactive immunity leads to significant reduction of viral loads in a recombinant vaccinia-HCV virus infected mouse model, supporting their role in antiviral immunity against HCV. Healthy human subjects with Ad-specific pre-existing immunity demonstrated cross-reactive cellular and humoral immune responses against multiple HCV antigens. These findings reveal the potential of a previously uncharacterized property of natural human adenovirus infection to dictate, modulate and/or alter the course of HCV infection upon exposure. This intrinsic property of adenovirus vectors to cross-prime HCV immunity can also be exploited to develop a prophylactic and/or therapeutic vaccine against HCV. PMID:26751211


    Li Jian; Xia Yongjing; Jiang Lei; Li Hongxia; Hu Yajun; Yi Lin; Hu Shixue; Xu Hongji


    Objective: To study the growth suppression of lung adenocarcinoma cell by the introduction of wild-type P53gene and explore a gene therapy approach for lung adenocarcinoma. Methods: A replication-deficient adenovirus vector encoding a wild-type P53 was constructed and transfected into the cultured human lung adenocarcinoma cell line GLC-82. The efficiency of gene transfection and expression was detected by immunochemical staining and polymerase chain reaction. The cell growth rate and cell cycle were analysed by cell-counting and flow cytometry. Results: Wild-type P53 gene could be quickly and effectively transfected into the cells by adenovirus vector. Wild-type P53 expression could inhibit GLC-82 cell proliferation and induce apoptosis.Conclusion: The results indicated that recombinant adenovirus expressing wild-type P53 might be useful vector for gene therapy of human lung adenocarcinoma.

  12. Adenovirus infection in immunocompromised patients

    Sylwia Rynans


    Full Text Available Human adenoviruses belong to the Adenoviridae family and they are divided into seven species, including 56 types. Adenoviruses are common opportunistic pathogens that are rarely associated with clinical symptoms in immunocompetent patients. However, they are emerging pathogens causing morbidity and mortality in recipients of hematopoietic stem cell and solid organ transplants, HIV infected patients and patients with primary immune deficiencies. Clinical presentation ranges from asymptomatic viraemia to respiratory and gastrointestinal disease, haemorrhagic cystitis and severe disseminated illness. There is currently no formally approved therapy for the treatment of adenovirus infections.This article presents current knowledge about adenoviruses, their pathogenicity and information about available methods to diagnose and treat adenoviral infections.

  13. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells.

    Holkers, Maarten; Maggio, Ignazio; Liu, Jin; Janssen, Josephine M; Miselli, Francesca; Mussolino, Claudio; Recchia, Alessandra; Cathomen, Toni; Gonçalves, Manuel A F V


    The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 'safe harbor' locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform.

  14. Molecular Imaging of Biological Gene Delivery Vehicles for Targeted Cancer Therapy: Beyond Viral Vectors

    Min, Jung Joon; Nguyen, Vu H. [Chonnam National University Medical School, Gwangju (Korea, Republic of); Gambhir, Sanjiv S. [Stanford University, California(United States)


    Cancer persists as one of the most devastating diseases in the world. Problems including metastasis and tumor resistance to chemotherapy and radiotherapy have seriously limited the therapeutic effects of present clinical treatments. To overcome these limitations, cancer gene therapy has been developed over the last two decades for a broad spectrum of applications, from gene replacement and knockdown to vaccination, each with different requirements for gene delivery. So far, a number of genes and delivery vectors have been investigated, and significant progress has been made with several gene therapy modalities in clinical trials. Viral vectors and synthetic liposomes have emerged as the vehicles of choice for many applications. However, both have limitations and risks that restrict gene therapy applications, including the complexity of production, limited packaging capacity, and unfavorable immunological features. While continuing to improve these vectors, it is important to investigate other options, particularly nonarrival biological agents such as bacteria, bacteriophages, and bacteria-like particles. Recently, many molecular imaging techniques for safe, repeated, and high-resolution in vivo imaging of gene expression have been employed to assess vector-mediated gene expression in living subjects. In this review, molecular imaging techniques for monitoring biological gene delivery vehicles are described, and the specific use of these methods at different steps is illustrated. Linking molecular imaging to gene therapy will eventually help to develop novel gene delivery vehicles for preclinical study and support the development of future human applications.

  15. Two-dimensional direction finding for low altitude target based on intensity measurement using an acoustic vector-sensor

    CHEN Huawei; ZHAO Junwei


    A method of two-dimensional direction of arrival (DOA) estimation for low altitude target, which is based on intensity measurement using a three-dimensional differential pressure acoustic vector-sensor, is presented. With the perfect characteristics of acoustic vector sensor in the low frequency band, accurate DOA estimation is achieved under small array size. The validity of the proposed method was assessed by experiments on the noise signals radiated by a helicopter. The influence of acoustic sensor size, integral time and signal to noise ratio to the accuracy of DOA estimation were investigated, respectively. The performance comparisons demonstrated that it outperformed the traditional time-delay measurement based method for a small acoustic array.

  16. Efficient and selective gene transfer into primary human brain tumors by using single-chain antibody-targeted adenoviral vectors with native tropism abolished

    van Beusechem, VW; Grill, J; Mastenbroek, DCJ; Wickham, TJ; Roelvink, PW; Haisma, HJ; Lamfers, MLM; Dirven, CMF; Pinedo, HM; Gerritsen, WR


    The application of adenoviral vectors in cancer gene therapy is hampered by low receptor expression on tumor cells and high receptor expression on normal epithelial cells. Targeting adenoviral vectors toward tumor cells may improve cancer gene therapy procedures by providing augmented tumor transduc

  17. 携带TRAIL基因的条件复制型腺病毒载体的构建及其辐射诱导表达%Construction of conditionally replicative adenovirus vector carrying TRAIL gene and its mRNA and protein expressions induced by ionizing radiation

    王宏芳; 吴嘉慧; 刘纯岩; 刘威武; 孙延红; 龚守良; 王志成; 刘扬


    Objective To construct the conditionally replicative adenovirus vector pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K carrying early growth response gene-1 (Egr1)promoter and tumor necrosis factor related apoptosis inducing ligand (TRAIL)gene, and to observe the effects of the vector combined with 2 Gy irradiation on the TRAIL expression in MDA-MB-231 cells.Methods Egr-1 promotor sequence was cloned from pMD18 T-Egr1, TRAIL was constructed the downstream of Egr1 promoter, pShuttle-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K (CRAd.pEgr1-TRAIL)was constructed,after the adenovirus vector was packaged successfully,MDA-MB-231 cells were infected with them and irradiated with X-rays.Real time PCR method and ELISA were used to detect the expression levels of TRAIL mRNA and protein, respectively. Six groups in the experiment were set up:control, 2 Gy,CRAd.p,CRAd.pEgr1-TRAIL,CRAd.p + 2 Gy and CRAd.pEgr1-TRAIL + 2 Gy. Results The recombinant adenovirus vector pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K was constructed and packaged successfully.The expression level of TRAIL mRNA in MDA-MB-231 cells transfected with the vector of 5 MOI for 24 h following 2.0 Gy X-rays irradiation began to increase and arrived to the top 8 h later in various groups,then declined.The expression level of TRAIL protein in MDA-MB-231 cells began to increase 6 h after irradiation and reached to the peak 24 h later,then declined 48 h later.There were significant differences in the expression levels of TRAIL protein between CRAd.pEgr1-TRAIL + 2.0 Gy and other groups at the same time point (P<0.01). Conclusion The recombinant adenovirus vector is obtained successfully, and the TRAIL mRNA and protein expression levels in MDA-MB-231 cells can be increased significantly by the vector combined with 2.0 Gy X-rays irradiation.%目的:构建携带早期生长反应基因-1(Egr-1)启动子和肿瘤坏死因子相关的凋亡诱导配体(TRAIL)基因的条件复制型腺病毒载体 pAd-Egr1-TRAIL-hTERT-E1A-E1Bp-E1B55K

  18. mIL-18腺病毒载体构建及在大鼠骨髓间充质干细胞中的表达%Construction of adenovirus vectors contained mIL-18 and expression in rat mesenchymal stem cells

    许刚; 郭燕舞; 徐如祥; 姜晓丹


    目的 构建表达mIL-18基因的腺病毒载体,并转染大鼠骨髓间充质干细胞(rBMSCs),观察其在rBMSCs中的表达,为脑胶质瘤的基因治疗实验研究奠定基础.方法 mIL-18基因亚克隆于穿梭质粒pAdtrackCMV上,构建的pAdtrackCMV-mIL-18线性化后转化已含有腺病毒骨架质粒pAdEasy-1的大肠杆菌BJ5183感受态细胞,挑选同源重组质粒,线性化后HEK293细胞包装,CsCI纯化.体外分离培养大鼠骨髓间充质干细胞,流式细胞仪检测细胞免疫表型;然后转染rBMSCs,通过荧光显微镜、RT-PCR、ELISA等方法检测mIL-18表达.结果 成功构建了绿色荧光蛋白标记的腺病毒Ad-mIL-18,并在rBMSCs中高效表达.结论 pAdEasy-1是基因转染的高效系统,腺病毒Ad- mIL-18转染的rBMSCs可以作为脑肿瘤基因治疗研究的种子细胞.%Objective To construct adenovirus vectors contained mIL-18 and observation the expression in rBMSCs and explore experiment study for gene therapy of gliorna. Methods mIL-18 gene was subcloned into the shuttle plasmid pAdtrackCMV, then linearized the vector and convert competent cell BJ5183 which contain the adenovirus frame plasmid pAdEasy-1. The homologous recombination plasmid was packaged by HEK293 cells and purified by CsCL The cell im-munophenotype was detected by flow cytometry. The expression of mIL-18 was measured by fluorescence microscope. RT-PCR and ELISA. Results The adenovirus vector Ad- mIL-18 was successfully constructed, which was labeled by green fluorescent protein (GFP) and effectively expressed in rBMSCs, Conclusion pAdEasyl is an effective gene trans-fection system. rBMSCs transfected with Ad- mIL-18 can serve as the seeds cells for the gene therapy of glioma.

  19. Gene therapy for cardiovascular disease: advances in vector development, targeting, and delivery for clinical translation.

    Rincon, Melvin Y; VandenDriessche, Thierry; Chuah, Marinee K


    Gene therapy is a promising modality for the treatment of inherited and acquired cardiovascular diseases. The identification of the molecular pathways involved in the pathophysiology of heart failure and other associated cardiac diseases led to encouraging preclinical gene therapy studies in small and large animal models. However, the initial clinical results yielded only modest or no improvement in clinical endpoints. The presence of neutralizing antibodies and cellular immune responses directed against the viral vector and/or the gene-modified cells, the insufficient gene expression levels, and the limited gene transduction efficiencies accounted for the overall limited clinical improvements. Nevertheless, further improvements of the gene delivery technology and a better understanding of the underlying biology fostered renewed interest in gene therapy for heart failure. In particular, improved vectors based on emerging cardiotropic serotypes of the adeno-associated viral vector (AAV) are particularly well suited to coax expression of therapeutic genes in the heart. This led to new clinical trials based on the delivery of the sarcoplasmic reticulum Ca(2+)-ATPase protein (SERCA2a). Though the first clinical results were encouraging, a recent Phase IIb trial did not confirm the beneficial clinical outcomes that were initially reported. New approaches based on S100A1 and adenylate cyclase 6 are also being considered for clinical applications. Emerging paradigms based on the use of miRNA regulation or CRISPR/Cas9-based genome engineering open new therapeutic perspectives for treating cardiovascular diseases by gene therapy. Nevertheless, the continuous improvement of cardiac gene delivery is needed to allow the use of safer and more effective vector doses, ultimately bringing gene therapy for heart failure one step closer to reality.

  20. Construction of a regulable gene therapy vector targeting for hepatocellular carcinoma

    Shao-Ying Lu; Yan-Fang Sui; Zeng-Shan Li; Cheng-En Pan; Jing Ye; Wen-Yong Wang


    AIM: To construct a gene modified hepatocellular carcinoma (HCC) specific EGFP expression vector regulated by abbreviated cis-acting element of AFP gene.METHODS: The minimal essential DNA segments of AFP gene enhancer and promoter were synthesized through PCR from Genome DNA of HepG2 cells. Gene fragments were then cloned into the multiple cloning site of non-promoter EGFP vector pEGFP-t. Recombinant plasmid was transferred into positive or negative AFP cell lines by means of lipofectamine. The expression of EGFP was tested by fluorescence microscope and flow cytometry. The effect of all-trans retinoic acid (ATRA) on the expression of EGFP was tested in different concentrations.RESULTS: By the methods of restriction digestion and sequence analyses we confirmed that the length, position and orientation of inserted genes of cis-acting element of AFP were all correct. The transcription of EGFP was under the control of AFP cis-acting element. The expressing EGFP can only been detected in AFP producing hepatoma cells.The expression rate of EGFP in G418 screened cell line was 34.9±4.1%. 48 h after adding 1×10-7M retinoic acid, EGFP expression rate was 14.7±3.5%. The activity of AFP gene promoter was significantly suppressed by addition of 1×10-7M retinoic acid (P<0.05, P=0.003, t=6.488).CONCLUSION: This recombinant expression vector can be used as a gene therapy vector for HCC. The expression of tumor killing gene will be confined within the site of tumor and the activity of which can be regulated by retinoic acid.

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

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


    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...

  2. Targeting of breast metastases using a viral gene vector with tumour-selective transcription.

    Rajendran, Simon


    BACKGROUND: Adeno-associated virus (AAV) vectors have significant potential as gene delivery vectors for cancer gene therapy. However, broad AAV2 tissue tropism results in nonspecific gene expression. MATERIALS AND METHODS: We investigated use of the C-X-C chemokine receptor type 4 (CXCR4) promoter to restrict AAV expression to tumour cells, in subcutaneous MCF-7 xenograft mouse models of breast cancer and in patient samples, using bioluminescent imaging and flow cytometric analysis. RESULTS: Higher transgene expression levels were observed in subcutaneous MCF-7 tumours relative to normal tissue (muscle) using the CXCR4 promoter, unlike a ubiquitously expressing Cytomegalovirus promoter construct, with preferential AAVCXCR4 expression in epithelial tumour and CXCR4-positive cells. Transgene expression following intravenously administered AAVCXCR4 in a model of liver metastasis was detected specifically in livers of tumour bearing mice. Ex vivo analysis using patient samples also demonstrated higher AAVCXCR4 expression in tumour compared with normal liver tissue. CONCLUSION: This study demonstrates for the first time, the potential for systemic administration of AAV2 vector for tumour-selective gene therapy.

  3. Anti-Inflammatory Effects of Modified Adenoviral Vectors for Gene Therapy: A View through Animal Models Tested.

    Castañeda-Lopez, M E; Garza-Veloz, I; Lopez-Hernandez, Y; Barbosa-Cisneros, O Y; Martinez-Fierro, M L


    The central dogma of gene therapy relies on the application of novel therapeutic genes to treat or prevent diseases. The main types of vectors used for gene transfer are adenovirus, retrovirus, lentivirus, liposome, and adeno-associated virus vectors. Gene therapy has emerged as a promising alternative for the treatment of inflammatory diseases. The main targets are cytokines, co-stimulatory molecules, and different types of cells from hematological and mesenchymal sources. In this review, we focus on molecules with anti-inflammatory effects used for in vivo gene therapy mediated by adenoviral gene transfer in the treatment of immune-mediated inflammatory diseases, with particular emphasis on autoinflammatory and autoimmune diseases.

  4. Construction and characterization of a hexon-chimeric human adenovirus type 3 vector expressing one major epitope of dengue virus type 1%1型登革病毒抗原表位嵌合人3型腺病毒六邻体重组病毒的构建及免疫学鉴定

    招穗珊; 周志超; 李潇; 樊晔; 廖小红; 周荣; 苏晓波


    Objective To construct a recombinant human adenovirus type 3 ( HAd3 ) vector ex-pressing one major epitope of dengue virus type 1.Methods The gene encoding the envelope protein (304-314 aa) of dengue virus type 1 was inserted into the hypervariable region 1 ( HVR1 ) of HAd3 hexon by using overlap PCR.The recombinant gene was cloned into the shuttle plasmid, then linearized with AsisⅠrestriction enzyme and co-transformed into Escherichia coli BJ5183 strains with the digested backbone plas-mid for homologous recombination.The recombinant plasmid pBRAdΔE3GFP-DENV1 was transfected into AD293 cells to rescue recombinant adenovirus strains (rAdΔE3GFP-DENV1).ELISA and Western blot as-say were performed to evaluate the humoral responses induced in BALB/c mice after the immunization with rAdΔE3GFP-DENV1 strains.Results The recombinant adenovirus strains were successfully rescued. ELISA and Western blot assay showed that the antibodies in serum sample could recognize dengue virus type 1 strains.Conclusion The recombinant adenovirus strains expressing the epitope of dengue virus type 1 were successfully constructed.This study provided evidence for the development of multivalent vaccines against dengue virus.%目的:构建六邻体嵌入1型登革病毒( DENV1)抗原表位的人3型重组腺病毒,鉴定其抗原性。方法以人3型腺病毒骨架质粒pBRAdΔE3GFP为模板,overlap PCR在六邻体高变区HVR1插入DENV1的抗原表位,突变的六邻体片段克隆到穿梭载体,酶切后与线性化的3型腺病毒骨架质粒pBRAdΔE3GFP在大肠杆菌BJ5183同源重组,获得阳性重组腺病毒质粒pBRAdΔE3GFP-DENV1。线性化后转染AD293细胞拯救重组腺病毒rAdΔE3GFP-DENV1并大量培养。纯化后腺病毒免疫BALB/c小鼠,通过ELISA和Western blot检测小鼠的体液免疫应答。结果在人3型腺病毒六邻体成功插入DENV1抗原表位并包装出重组腺病毒,ELISA和Western blot结果显示小鼠免

  5. Preventive and curative effects of Apple latent spherical virus vectors harboring part of the target virus genome against potyvirus and cucumovirus infections.

    Tamura, Akihiro; Kato, Takahiro; Taki, Ayano; Sone, Mikako; Satoh, Nozomi; Yamagishi, Noriko; Takahashi, Tsubasa; Ryo, Bo-Song; Natsuaki, Tomohide; Yoshikawa, Nobuyuki


    Apple latent spherical virus (ALSV)-based vectors experimentally infect a broad range of plant species without causing symptoms and can effectively induce stable virus-induced gene silencing in plants. Here, we show that pre-infection of ALSV vectors harboring part of a target viral genome (we called ALSV vector vaccines here) inhibits the multiplication and spread of the corresponding challenge viruses [Bean yellow mosaic virus, Zucchini yellow mosaic virus (ZYMV), and Cucumber mosaic virus (CMV)] by a homology-dependent resistance. Further, the plants pre-infected with an ALSV vector having genome sequences of both ZYMV and CMV were protected against double inoculation of ZYMV and CMV. More interestingly, a curative effect of an ALSV vector vaccine could also be expected in ZYMV-infected cucumber plants, because the symptoms subsided on subsequent inoculation with an ALSV vector vaccine. This may be due to the invasion of ALSV, but not ZYMV, in the shoot apical meristem of cucumber.

  6. Adenovirus gene transfer to amelogenesis imperfecta ameloblast-like cells.

    Anton V Borovjagin

    Full Text Available To explore gene therapy strategies for amelogenesis imperfecta (AI, a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5 vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including "pK7" and/or "RGD" motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3 fiber "knob" domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both α(vβ3/α(vβ5 integrins and heparan sulfate proteoglycans (HSPGs highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI.

  7. Characterizing clearance of helper adenovirus by a clinical rAAV1 manufacturing process.

    Thorne, Barbara A; Quigley, Paulene; Nichols, Gina; Moore, Christine; Pastor, Eric; Price, David; Ament, Jon W; Takeya, Ryan K; Peluso, Richard W


    Recombinant adeno-associated viral vectors (rAAV) are being developed as gene therapy delivery vehicles and as genetic vaccines, and some of the most scaleable manufacturing methods for rAAV use live adenovirus to induce production. One aspect of establishing safety of rAAV products is therefore demonstrating adequate and reliable clearance of this helper virus by the vector purification process. The ICH Q5A regulatory guidance on viral safety provides recommendations for process design and characterization of viral clearance for recombinant proteins, and these principles were adapted to a rAAV serotype 1 purification process for clinical vectors. Specific objectives were to achieve overall adenovirus clearance factors significantly greater than input levels by using orthogonal separation and inactivation methods, and to segregate adenovirus from downstream operations by positioning a robust clearance step early in the process. Analytical tools for process development and characterization addressed problematic in-process samples, and a viral clearance validation study was performed using adenovirus and two non-specific model viruses. Overall clearance factors determined were >23 LRV for adenovirus, 11 LRV for BVDV, and >23 LRV for AMuLV.

  8. Characterization and vectorization of siRNA targeting RET/PTC1 in human papillary thyroid carcinoma cells

    Massade L.


    Full Text Available RET/PTC1 fusion oncogene is the most common genetic alteration identified to date in thyroid papillary carcinomas (PTC and represents a good target for small interfering RNA (siRNA. Our aim was: i to target the RET/PTC1 oncogene by siRNAs, ii to assess the knockdown effects on cell growth and cell cycle regulation and iii to vectorize it in order to protect it from degradation. Methods. Human cell lines expressing RET/PTC1 were transfected by siRNA RET/PTC1, inhibition of the oncogene expression was assessed by qRT-PCR and by Western blot. Conjugation of siRNA RET/PTC1 to squalene was performed by coupling it to squalene. In vivo studies are performed in nude mice. Conclusion. In this short communication, we report the main published results obtained during last years.

  9. An MRI-visible non-viral vector for targeted Bcl-2 siRNA delivery to neuroblastoma

    Shen M


    Full Text Available Min Shen,1,* Faming Gong,3,* Pengfei Pang,1,* Kangshun Zhu,1 Xiaochun Meng,1 Chun Wu,1 Jin Wang,1 Hong Shan,1,2 Xintao Shuai3,41Molecular Imaging Lab, Department of Radiology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China; 2Institute of Intervention Radiology, Sun Yat-sen University, Guangzhou, China; 3PCFM Lab of Ministry of Education, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, China; 4Center of Biomedical Engineering, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China*These authors contributed equally to this workAbstract: Polyethylene glycol-grafted polyethylenimine (PEG-g-PEI which was functionalized with a neuroblastoma cell-specific ligand, the GD2 single chain antibody (scAbGD2, was synthesized in order to effectively deliver Bcl-2 siRNA into neuroblastoma cells. This polymer was complexed first with superparamagnetic iron oxide nanoparticle (SPION to get a MRI-visible targeted non-viral vector (scAbGD2-PEG-g-PEI-SPION and then with Bcl-2 siRNA to form nanoparticles showing low cytotoxicity. The targeting capacity of scAbGD2-PEG-g-PEI-SPION was successfully verified in vivo and in vitro by magnetic resonance imaging. The single chain antibody encoded targeted polyplex was more effective in transferring Bcl-2 siRNA than the nontargeting one in SK-N-SH cells, a human neuroblastoma cell line, resulting in a 46.34% inhibition in the expression of Bcl-2 mRNA. Consequently, a high level of cell apoptosis up to 50.76% and a significant suppression of tumor growth were achieved, which indicates that scAbGD2-PEG-g-PEI-SPION is a promising magnetic resonance imaging-visible non-viral vector for targeted neuroblastoma siRNA therapy and diagnosis.Keywords: tumor targeting, GD2, non-viral vector, Bcl-2 small interfering RNA, magnetic resonance imaging

  10. Dengue vector management using insecticide treated materials and targeted interventions on productive breeding-sites in Guatemala

    Rizzo Nidia


    Full Text Available Abstract Background In view of the epidemiological expansion of dengue worldwide and the availability of new tools and strategies particularly for controlling the primary dengue vector Aedes aegypti, an intervention study was set up to test the efficacy, cost and feasibility of a combined approach of insecticide treated materials (ITMs alone and in combination with appropriate targeted interventions of the most productive vector breeding-sites. Methods The study was conducted as a cluster randomized community trial using “reduction of the vector population” as the main outcome variable. The trial had two arms: 10 intervention clusters (neighborhoods and 10 control clusters in the town of Poptun Guatemala. Activities included entomological assessments (characteristics of breeding-sites, pupal productivity, Stegomyia indices at baseline, 6 weeks after the first intervention (coverage of window and exterior doorways made of PermaNet 2.0 netting, factory treated with deltamethrin at 55 mg/m2, and of 200 L drums with similar treated material and 6 weeks after the second intervention (combination of treated materials and other suitable interventions targeting productive breeding-sites i.e larviciding with Temephos, elimination etc.. The second intervention took place 17 months after the first intervention. The insecticide residual activity and the insecticidal content were also studied at different intervals. Additionally, information about demographic characteristics, cost of the intervention, coverage of houses protected and satisfaction in the population with the interventions was collected. Results At baseline (during the dry season a variety of productive container types for Aedes pupae were identified: various container types holding >20 L, 200 L drums, washbasins and buckets (producing 83.7% of all pupae. After covering 100% of windows and exterior doorways and a small number of drums (where the commercial cover could be fixed in 970 study

  11. A novel and simple method for construction of recombinant adenoviruses.

    Tan, Rong; Li, Chunhua; Jiang, Sijing; Ma, Lixin


    Recombinant adenoviruses have been widely used for various applications, including protein expression and gene therapy. We herein report a new and simple cloning approach to an efficient and robust construction of recombinant adenoviral genomes based on the mating-assisted genetically integrated cloning (MAGIC) strategy. The production of recombinant adenovirus serotype 5-based vectors was greatly facilitated by the use of the MAGIC procedure and the development of the Adeasy adenoviral vector system. The recombinant adenoviral plasmid can be generated by a direct and seamless substitution, which replaces the stuff fragment in a full-length adenoviral genome with the gene of interest in a small plasmid in Escherichia coli. Recombinant adenoviral plasmids can be rapidly constructed in vivo by using the new method, without manipulations of the large adenoviral genome. In contrast to other traditional systems, it reduces the need for multiple in vitro manipulations, such as endonuclease cleavage, ligation and transformation, thus achieving a higher efficiency with negligible background. This strategy has been proven to be suitable for constructing an adenoviral cDNA expression library. In summary, the new method is highly efficient, technically less demanding and less labor-intensive for constructing recombinant adenoviruses, which will be beneficial for functional genomic and proteomic researches in mammalian cells.

  12. Growth suppression effect of anti-sense miRNA-21/rAV-Tumstatin adenovirus vector on bladder cancer cell%反义miRNA-21/rAV-Tumstatin腺病毒载体对膀胱癌细胞生长的抑制作用

    温都苏; 刘禄成; 魏巍; 李志; 郭航; 张明


    Objective To investigate the suppression effect of anti-sense miRNA-21/rAV-Tutnstatin adenovirus vector two-side target treatment on situ bladder transplanted tumor. Methods The anti-sense miRNA-21/rAV-Tumstatin was used on transplantation cancer by urethra irrigation of bladder, MTT way was applied to evaluate cell proliferation,TUNEL way was used to evaluate cell apoptosis. Results MTT way evaluating showed that nell growth rates of anti-sense miRNA-21, rAV-Tumstatin and combination groups were 48.7% ± 6. 2% 、 35.8% ±9.4% and 27. 8% ±3. 7%, which were obviously lower than those of control group(92. 8% ±7.4% ,P<0. 01). TUNEL way result showed that tumor cell apoptosis rates of 4 groups were in order:drug combination group(79. 3% ±4. 1% ) , anti-sense miRNA-21 group (51.2% ±7.5%), rAV-Tumstatin group(57.8% ±6.9%), control group(8.3% ±2. 6%),and the differences had statistical significance between the four groups (P < 0. 01). The tumor cell growth had been some more inhibited after vector treatment on 3 groups, but in combination group , the tumor cell growth was slowly, the difference had statistical significance ( P < 0.05) compared with the other two groups, furthermore, tumor cell growth was almost stasis state from 28 days. Conclusions Anti-sense miRNA-21 and rAV-Tumstatin alone applying could inhibit tumor cell proliferation and induce apoptosis, accordingly inhibit growth of bladder cancer cell, but two-side target drug which aim at carcinogene and anti-oncogene are significantly more useful than single drug, and more approach ideal treatment%目的 探讨反义miRNA-21/rAV-Tumstatin腺病毒载体双靶向治疗对裸鼠原位膀胱移植瘤生长的抑制作用.方法 应用携带反义miRNA-21/rAV-Tumstatin的载体经尿道膀胱灌注治疗移植癌.采用二苯基溴化四氮唑蓝(MTT)法测定瘤细胞的增殖,原位末端标记(TUNEL)法检测瘤细胞的凋亡.结果 MTI法检测结果显示,反义miRNA-21,rAV-Tumatin和联合应用组

  13. Defining Therapeutic Targets by Using Adenovirus: Blocking NF-kappa B Inhibits Both Inflammatory and Destructive Mechanisms in Rheumatoid Synovium but Spares Anti-Inflammatory Mediators

    Bondeson, Jan; Foxwell, Brian; Brennan, Fionula; Feldmann, Marc


    The role of the transcription factor NF-kappa B in the pathogenesis of rheumatoid arthritis has long been a subject of controversy. We used an adenoviral technique of blocking NF-kappa B through overexpression of the inhibitory subunit Ikappa Bα , which has the advantage that it can be used in the diseased tissue itself, with >90% of the synovial macrophages, fibroblasts, and T cells infected. We found that the spontaneous production of tumor necrosis factor α and other pro-inflammatory cytokines is NF-kappa B-dependent in rheumatoid synovial tissue, in contrast to the main anti-inflammatory mediators, like IL-10 and -11, and the IL-1 receptor antagonist. Of even more interest, Ikappa Bα overexpression inhibited the production of matrix metalloproteinases 1 and 3 while not affecting their tissue inhibitor. Blocking NF-kappa B in the rheumatoid joint thus has a very beneficial profile, reducing both the inflammatory response and the tissue destruction. The adenoviral technique described here has widespread applicability, allowing rapid testing of the effects of blocking a potential therapeutic target in either cultures of normal cells or in the diseased tissue itself.

  14. Split vector systems for ultra-targeted gene delivery: a contrivance to achieve ethical assurance of somatic gene therapy in vivo.

    Tolmachov, Oleg E


    Tightly controlled spatial localisation of therapeutic gene delivery is essential to maximize the benefits of somatic gene therapy in vivo and to reduce its undesired effects on the 'bystander' cell populations, most importantly germline cells. Indeed, complete ethical assurance of somatic gene therapy can only be achieved with ultra-targeted gene delivery, which excludes the risk of inadvertent germline gene transfer. Thus, it is desired to supplement existing strategies of physical focusing and biological (cell-specific) targeting of gene delivery with an additional principle for the rigid control over spread of gene transfer within the body. In this paper I advance the concept of 'combinatorial' targeting of therapeutic gene transfer in vivo. I hypothesize that it is possible to engineer complex gene delivery vector systems consisting of several components, each one of them capable of independent spread within the human body but incapable of independent facilitation of gene transfer. As the gene delivery augmented by such split vector systems would be reliant on the simultaneous availability of all the vector system components at a predetermined body site, it is envisaged that higher order reaction kinetics required for the assembly of the functional gene transfer configuration would sharpen spatial localisation of gene transfer via curtailing the blurring effect of the vector spread within the body. A particular implementation of such split vector system could be obtained through supplementing a viral therapeutic gene vector with a separate auxiliary vector carrying a non-integrative and non-replicative form of a gene (e.g., mRNA) coding for a cellular receptor of the therapeutic vector component. Gene-transfer-enabling components of the vector system, which would be delivered separately from the vector component loaded with the therapeutic gene cargo, could also be cell-membrane-insertion-proficient receptors, elements of artificial transmembrane channels

  15. Adenovirus Type 11 Uses CD46 as a Cellular Receptor

    Segerman, Anna; Atkinson, John P.; Marttila, Marko; Dennerquist, Veronica; Wadell, Göran; Arnberg, Niklas


    The 51 human adenovirus serotypes are divided into six species (A to F). Many adenoviruses use the coxsackie-adenovirus receptor (CAR) for attachment to host cells in vitro. Species B adenoviruses do not compete with CAR-binding serotypes for binding to host cells, and it has been suggested that species B adenoviruses use a receptor other than CAR. Species B adenoviruses mainly cause disease in the respiratory tract, the eyes, and in the urinary tract. Here we demonstrate that adenovirus type...

  16. Protection of non-human primates against rabies with an adenovirus recombinant vaccine

    Xiang, Z.Q. [The Wistar Institute of Anatomy and Biology, Philadelphia, PA (United States); Greenberg, L. [Centers for Disease Control and Prevention, Atlanta, GA (United States); Ertl, H.C., E-mail: [The Wistar Institute of Anatomy and Biology, Philadelphia, PA (United States); Rupprecht, C.E. [The Global Alliance for Rabies Control, Manhattan, KS (United States); Ross University School of Veterinary Medicine, Basseterre (Saint Kitts and Nevis)


    Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials. - Highlights: • Pre-exposure vaccination with vaccine based on a chimpanzee derived adenovirus protects against rabies. • Protection is sustained. • Protection is achieved with single low-dose of vaccine given intramuscularly. • Protection is not affected by pre-existing antibodies to common human serotypes of adenovirus.

  17. [Downregulation of Human Adenovirus DNA Polymerase Gene by Modified siRNAs].

    Nikitenko, N A; Speiseder, T; Chernolovskaya, E L; Zenkova, M A; Dobner, T; Prassolov, V S


    Human adenoviruses, in particular D8, D19, and D37, cause ocular infections. Currently, there is no available causally directed treatment, which efficiently counteracts adenoviral infectious diseases. In our previous work, we showed that gene silencing by means of RNA interference is an effective approach for downregulation of human species D adenoviruses replication. In this study, we compared the biological activity of siRNAs and their modified analogs targeting human species D adenoviruses DNA polymerase. We found that one of selectively 2'-O-methyl modified siRNAs mediates stable and long-lasting suppression of the target gene (12 days post transfection). We suppose that this siRNA can be used as a potential therapeutic agent against human species D adenoviruses.

  18. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae.

    Hammond, Andrew; Galizi, Roberto; Kyrou, Kyros; Simoni, Alekos; Siniscalchi, Carla; Katsanos, Dimitris; Gribble, Matthew; Baker, Dean; Marois, Eric; Russell, Steven; Burt, Austin; Windbichler, Nikolai; Crisanti, Andrea; Nolan, Tony


    Gene drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years. We describe CRISPR-Cas9 endonuclease constructs that function as gene drive systems in Anopheles gambiae, the main vector for malaria. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female-sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene drive constructs designed to target and edit each gene. For each targeted locus we observed a strong gene drive at the molecular level, with transmission rates to progeny of 91.4 to 99.6%. Population modeling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to suppress mosquito populations to levels that do not support malaria transmission.

  19. Targeted gene disruption by use of a group Ⅱ intron (tarqetron)vector in Clostridium acetobutylicum

    Lijun Shao; Shiyuan Hu; Yi Yang; Yang Gu; Jun Chen; Yunliu Yang; Weihong Jiang; Sheng Yang


    @@ Dear Editor: Clostridium acetobutylicum,a gram-positive,anaerobic,spore-forming bacterium,is capable of using a wide variety of carbon sources to produce acetone,butanol and ethanol.To improve solvent productivity of C.acetobutylicum.metabolic engineering is considered as a useful tool in developing strains with industrially desirable characteristics.However,to date,there are few useful methods for genetic manipulation of C.acetobutylicum,especially for gene disruption.To our knowledge,two types of vectors,including non-replicative and replicative integrative plasmids,have been developed for gene-inactivation in C.acetobutylicum.By using non-replicative integrative plasmids,buk and solR genes of C. acetobutylicum were inactivated[1,2].

  20. Chitosan-graft-polyethylenimine/DNA nanoparticles as novel non-viral gene delivery vectors targeting osteoarthritis.

    Lu, Huading; Dai, Yuhu; Lv, Lulu; Zhao, Huiqing


    The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes.

  1. Construction and identification of an RNA interference lentivirus vector targeting the Ras homology C gene of melanoma cells

    Wang Qiying; Wang Ximei; Zhai Xiaomei; Zhang Jianwen; Chen Minjing; Liu Linbo


    Background Melanoma has the highest mortality among all superficial malignant tumors.The poor prognosis is due to its high metastasis rate and the lack of therapeutic targets.As a molecular switch that controls tumor metastasis,Ras homology C (RhoC) has been correlated with tumor progression,especially tumor invasion and metastasis.However,little research has been done about the effects of RNA interference (RNAi) targeting RhoC on the invasion and metastasis of melanoma.In this study,we constructed an RNAi lentivirus vector targeting the RhoC gene of melanoma cells and identified its silencing effects on the RhoC gene.Methods Based on the RhoC gene encoding information,three pGPU6/GFP/Neo-short hairpin (shRNA) plasmids were constructed.After detecting their silencing effects on the RhoC gene of A375 cells,the most effective pGPU6/GFP/ Neo-shRNA plasmid was packed with lentivirus to construct the recombinant pLenti6.3-EGFP-453 targeting RhoC.The lentivirus vector was used to infect A375 cells,and then the expression of RhoC mRNA and protein were determined with real-time PCR and Western blotting.Results The plasmids pGPU6/GFP/Neo-shRNA 336,pGPU6/GFP/Neo-shRNA 453,and pGPU6/GFP/Neo-shRNA 680 were constructed.After they were transfected into A375 cells,the expressions of RhoC mRNA and protein were 1.47±0.26,1.13±0.16,1.39±0.11 and 70.98±9.21,50.67±6.06,65.77±4.06,respectively.pGPU6/GFP/Neo-shRNA 453 was the most effective sequence,and was used to successfully construct the pLenti6.3-EGFP-453 lentiviral vector targeting RhoC.pLenti6.3-EGFP-453 was used to infect A375 cells.The expression of RhoC mRNA and protein were 1.05±0.05 and 62.04±15.86 in the lentivirus group,4.21±0.24 and 220.86±24.07 in the negative lentivirus control group,and 4.63±0.32 and 257.39±12.30 in the normal control group respectively with the difference between the lentivirus group and the control groups being statistically significant (P <0.05).Conclusion The successfully constructed

  2. 重组GGPPS腺病毒载体的构建及其调节肝星状细胞活化的研究%Construction of Recombinant Adenovirus Vector Containing Human ggpps Gene and Its Effect on Activation of HSCs

    来珊珊; 陈卫波; 徐康; 丁尧; 沈宁; 潘飞燕; 李朝军; 薛斌


    目的:构建携带人源ggpps基因的重组腺病毒并用其研究GGPPS对人肝星状细胞的活化作用.方法:利用PCR方法扩增ggpps基因片段,并亚克隆至腺病毒穿梭质粒pAdTrack-CMV中,形成穿梭质粒pAdTrack-CMV-GGPPS.测序正确后,经PmeⅠ单酶切线性化的pAdTrack-CMV-GGPPS与腺病毒骨架质粒pAdEasy-1用电穿孔法共转化大肠杆菌BJ5183,进行同源重组.经PacⅠ酶切鉴定正确后,用磷酸钙法转染QBI-293A细胞,包装成重组体腺病毒Ad-GGPPS颗粒.利用穿梭质粒pAdTrack-CMV中GFP报告基因的表达,观察重组腺病毒的产生.用TCID50法测定病毒颗粒的浓度,并用免疫蛋白印迹和实时荧光定量PCR方法鉴定目的基因的表达.然后利用该病毒感染人肝星状细胞LX-2,观察对肝星状细胞的活化以及细胞外基质合成的影响.结果:成功地构建了携带ggpps基因的重组腺病毒,得到的病毒滴度经TCID50法检测为2.5×109 PFU/mL.该病毒能够在人源的肝脏细胞中成功表达GGPPS蛋白.在LX-2细胞中过量表达GGPPS可以促进肝星状细胞(Hepatic stellate cells,HSCs)转分化成表达平滑肌肌动蛋白α(Alpha-Smooth muscle actin,α-SMA)的肌成纤维细胞(Myofibroblast,MF),同时检测到胞外基质成分纤连蛋白( Fibronectin,FN)的表达量显著增加.结论:成功构建的重组GGPPS腺病毒能促进肝星状细胞的活化.%Objective: To construct the recombinant adenovirus vector containing human ggpps gene and study its effect on activation of HSCs. Methods: ggpps gene was obtained by PCR amplification and subcloned into adenovirus shuttle plas-mid of pAdTrack-CMV to form the shuttle vector of pAdTrack-CMV-GGPPS. After sequencing, it was linearized with Pmel and co-transfected into E. Coli BJ5183 cells with adenovirus genomtc plasmid of pAdEasy-1 to achieve homologous recombination. The DNA of identified recombinant plasmid was digested with Pacl and then transfected into QBI-293A cells to package adenovirus

  3. Targeting the immune system to fight cancer using chemical receptor homing vectors carrying Poly Inosine/Cytosine (PolyIC

    Alexander eLevitzki


    Full Text Available Cancer researchers have been looking for ways to harness the immune system and to reinstate immune surveillance, to kill cancer cells without collateral damage. Here we scan current approaches to targeting the immune system against cancer, and emphasize our own approach. We are using chemical vectors attached to a specific ligand, to introduce synthetic dsRNA, poly Inosine/Cytosine (polyIC, into tumors. The ligand binds to a receptor protein that is overexpressed on the surface of the tumor cells. Upon ligand binding, the receptor complex is internalized, introducing the polyIC into the cell. In this fashion a large amount of synthetic dsRNA can be internalized, leading to the activation of dsRNA binding proteins, such as dsRNA dependent protein kinase (PKR, Toll-3 receptor (TLR3, retinoic acid–inducible gene I (RIG-1 and melanoma differentiation–associated gene 5 (MDA5. The simultaneous activation of these signaling proteins leads to the rapid demise of the targeted cell and to cytokine secretion. The cytokines lead to a strong bystander effect and to the recruitment of immune cells that converge upon the targeted cells. The bystander effects lead to the destruction of neighboring tumor cells not targeted themselves by the vector. Normal cells, being more robust than tumor cells, survive. This strategy has several advantages: (1 Recruitment of the immune system is localized to the tumor. (2 The response is rapid, leading to fast tumor eradication. (3 The bystander effects lead to the eradication of tumor cells not harboring the target. (4 The multiplicity of pro-death signaling pathways elicited by PolyIC minimizes the likelihood of the emergence of resistance. In this chapter we focus on EGFR as the targeted receptor, which is overexpressed in many tumors. In principle, the strategy can be extended to other tumors that overexpress a protein that can be internalized by a ligand, which be a small molecule, a single chain antibody or an

  4. Targeting the Immune System to Fight Cancer Using Chemical Receptor Homing Vectors Carrying Polyinosine/Cytosine (PolyIC).

    Levitzki, Alexander


    Cancer researchers have been looking for ways to harness the immune system and to reinstate immune surveillance, to kill cancer cells without collateral damage. Here we scan current approaches to targeting the immune system against cancer, and emphasize our own approach. We are using chemical vectors attached to a specific ligand, to introduce synthetic dsRNA, polyinosine/cytosine (polyIC), into tumors. The ligand binds to a receptor protein that is overexpressed on the surface of the tumor cells. Upon ligand binding, the receptor complex is internalized, introducing the polyIC into the cell. In this fashion a large amount of synthetic dsRNA can be internalized, leading to the activation of dsRNA-binding proteins, such as dsRNA dependent protein kinase (PKR), Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I (RIG-1), and melanoma differentiation-associated gene 5 (MDA5). The simultaneous activation of these signaling proteins leads to the rapid demise of the targeted cell and to cytokine secretion. The cytokines lead to a strong bystander effect and to the recruitment of immune cells that converge upon the targeted cells. The bystander effects lead to the destruction of neighboring tumor cells not targeted themselves by the vector. Normal cells, being more robust than tumor cells, survive. This strategy has several advantages: (1) recruitment of the immune system is localized to the tumor. (2) The response is rapid, leading to fast tumor eradication. (3) The bystander effects lead to the eradication of tumor cells not harboring the target. (4) The multiplicity of pro-death signaling pathways elicited by PolyIC minimizes the likelihood of the emergence of resistance. In this chapter we focus on EGFR as the targeted receptor, which is overexpressed in many tumors. In principle, the strategy can be extended to other tumors that overexpress a protein that can be internalized by a ligand, which can be a small molecule, a single chain antibody, or an affibody.

  5. Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

    Motoharu Ono

    Full Text Available We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2.

  6. Adenovirus-based vaccine against Listeria monocytogenes

    Jensen, Søren; Steffensen, Maria Abildgaard; Jensen, Benjamin Anderschou Holbech


    The use of replication-deficient adenoviruses as vehicles for transfer of foreign genes offers many advantages in a vaccine setting, eliciting strong cellular immune responses involving both CD8(+) and CD4(+) T cells. Further improving the immunogenicity, tethering of the inserted target Ag to MHC...... class II-associated invariant chain (Ii) greatly enhances both the presentation of most target Ags, as well as overall protection against viral infection, such as lymphocytic choriomeningitis virus (LCMV). The present study extends this vaccination concept to include protection against intracellular...... bacteria, using Listeria monocytogenes as a model organism. Protection in C57BL/6 mice against recombinant L. monocytogenes expressing an immunodominant epitope of the LCMV glycoprotein (GP33) was greatly accelerated, augmented, and prolonged following vaccination with an adenoviral vaccine encoding GP...

  7. Epidermal growth factor receptor targeting enhances adenoviral vector based suicide gene therapy of osteosarcoma

    Witlox, M.A.; van Beusechem, V.W.; Grill, J.; Haisma, H.J.; Schaap, G.; Bras, J.; Van Diest, P.; De Gast, A.; Curiel, D.T.; Pinedo, H.M.; Gerritsen, W.R.; Wuisman, P.I.


    Background Despite improvements in the treatment of osteosarcoma (OS) there are still too many patients who cannot benefit from current treatment modalities. Therefore, new therapeutic approaches are warranted. Here we explore the efficacy of targeted adenoviral based suicide gene therapy. Methods a

  8. Intratracheal administration of recombinant adenovirus containing IL-18 gene in treatment of experimental metastatic lung carcinoma

    CHEN Ji-quan; GAO Xue-tao; XIU Qing-yu; YU Yi-zhi; LUO Wen-tong


    To study the treatment of experimental metastatic lung carcinoma by intratracheal injection of IL-18 gene recombinant adenovirus. Methods: (1)The mouse IL-18 mRNA was detected by RT-PCR, and the concentration of IL-18 and associated cytokines in lung lavages and blood were determined by ELISA at different time points after intratracheal injection of IL-18 recombinant adenovirus. (2)The lung metastasis nodes, mouse survival periods and survival rates were evaluated. NK activity and CTL activity were determined by 51Cr 4 h release method. Results: (1)IL-18 mRNA was detectable in lung tissue 6 h after intratracheal use of IL-18 recombinant adenovirus, and the concentration of IL-18 in lung lavage was higher than that in pelipheral blood. Neither IL-18 mRNA nor IL-18 was detectable in control group. (2) Intratracheal use of IL-18 recombinant adenovirus resulted in increased CTL and NK activity, longer survival time and higher survival rates compared with the control group, showing significant therapeutic effect on experimental lung metastasis. Conclusion: Intratracheal use of adenovirus vector containing IL-18 gene has therapeutic effect on the lung metastasis, denoting that gene therapy of lung diseases could be applied through airway directly with recombinant adenovirus.

  9. 去甲基化酶腺病毒载体构建及其在人颌下腺细胞中的表达%Cloning and expression of methyl-CpG binding domain 2 gene in human submandibular gland cell line by adenovirus vector transfection

    李莹; 刘淑丹; 杨彦春; 董世武; 熊剑; 李方; 周继祥


    目的 构建去甲基化酶(methyl-CpG binding domain 2,MBD2)基因腺病毒载体,并观测其在人颌下腺(human submandibular gland,HSG)细胞中的表达.方法 以HSG细胞总RNA为模板,RT-PCR法扩增MBD2基因全长编码序列,克隆入载体pMD18-T后,亚克隆入pAdTrack-CMV腺病毒穿梭载体,构建pAdTrack-MBD2重组体.该重组体与腺病毒骨架质粒pAdEmy-1于BJ5183菌中同源重组,产生重组腺病毒质粒Ad-MBD2,该质粒经293细胞包装,获得具有感染力的Ad/MBD2重组腺病毒颗粒,将该病毒颗粒感染HSG细胞,倒置显微镜检测转染后HSG细胞生长变化及MBD2在其中的表达等情况.图像分析法计算感染效率.RT-PCR法检测MBD2基因在该细胞中的表达变化.结果 成功克隆到909 bp大小的MBD2基因,构建了其腺病毒载体Ad/MBD2,该腺病毒载体经293细胞成功包装,并获具感染力的Ad/MBD2重组腺病毒颗粒,其感染效率约70%.感染的HSG高表达MBD2.结论 成功克隆到MBD2基因,构建了其重组腺病毒载体Ad/MBD2,并证实了其感染HSG细胞的有效表达.%Objective To construct the adenovirus vector carrying methyl-CpG binding domain 2 (MBD2) gene and express this gene in human submandibular gland (HSG)cell line. Methods cDNA fragment encoding human MBD2 gene was amplified by reverse transcriptase-polynierase chain reaction (RT-PCR) with the total mRNA isolated from HSG cells as template. The PCR amplified product was first cloned into pMD18-T vector, and then subcloned into the shuttle vector pAdTrack-CMV in order to construct the recombi-nant plasmids pAdTrack-MBD2, which homologously recombinated with the adenoviral backbone vectors Adeasy-1 in BJ5183 bacterial cells, so as to generate recombinant adenoviral plasmids Ad-MBD2. The recombi-nant adenoviruses Ad/MBD2, which were employed to infect HSG cells, were generated by transfecting the recombinant adenoviral DNA into 293 cells. The growth of transfected HSG cells and the expression of enhanced

  10. Dengue vector dynamics (Aedes aegypti influenced by climate and social factors in Ecuador: implications for targeted control.

    Anna M Stewart Ibarra

    Full Text Available BACKGROUND: Dengue fever, a mosquito-borne viral disease, is now the fastest spreading tropical disease globally. Previous studies indicate that climate and human behavior interact to influence dengue virus and vector (Aedes aegypti population dynamics; however, the relative effects of these variables depends on local ecology and social context. We investigated the roles of climate and socio-ecological factors on Ae. aegypti population dynamics in Machala, a city in southern coastal Ecuador where dengue is hyper-endemic. METHODS/PRINCIPAL FINDINGS: We studied two proximate urban localities where we monitored weekly Ae. aegypti oviposition activity (Nov. 2010-June 2011, conducted seasonal pupal surveys, and surveyed household to identify dengue risk factors. The results of this study provide evidence that Ae. aegypti population dynamics are influenced by social risk factors that vary by season and lagged climate variables that vary by locality. Best-fit models to predict the presence of Ae. aegypti pupae included parameters for household water storage practices, access to piped water, the number of households per property, condition of the house and patio, and knowledge and perceptions of dengue. Rainfall and minimum temperature were significant predictors of oviposition activity, although the effect of rainfall varied by locality due to differences in types of water storage containers. CONCLUSIONS: These results indicate the potential to reduce the burden of dengue in this region by conducting focused vector control interventions that target high-risk households and containers in each season and by developing predictive models using climate and non-climate information. These findings provide the region's public health sector with key information for conducting time and location-specific vector control campaigns, and highlight the importance of local socio-ecological studies to understand dengue dynamics. See Text S1 for an executive summary in

  11. Dengue Vector Dynamics (Aedes aegypti) Influenced by Climate and Social Factors in Ecuador: Implications for Targeted Control

    Stewart Ibarra, Anna M.; Ryan, Sadie J.; Beltrán, Efrain; Mejía, Raúl; Silva, Mercy; Muñoz, Ángel


    Background Dengue fever, a mosquito-borne viral disease, is now the fastest spreading tropical disease globally. Previous studies indicate that climate and human behavior interact to influence dengue virus and vector (Aedes aegypti) population dynamics; however, the relative effects of these variables depends on local ecology and social context. We investigated the roles of climate and socio-ecological factors on Ae. aegypti population dynamics in Machala, a city in southern coastal Ecuador where dengue is hyper-endemic. Methods/Principal findings We studied two proximate urban localities where we monitored weekly Ae. aegypti oviposition activity (Nov. 2010-June 2011), conducted seasonal pupal surveys, and surveyed household to identify dengue risk factors. The results of this study provide evidence that Ae. aegypti population dynamics are influenced by social risk factors that vary by season and lagged climate variables that vary by locality. Best-fit models to predict the presence of Ae. aegypti pupae included parameters for household water storage practices, access to piped water, the number of households per property, condition of the house and patio, and knowledge and perceptions of dengue. Rainfall and minimum temperature were significant predictors of oviposition activity, although the effect of rainfall varied by locality due to differences in types of water storage containers. Conclusions These results indicate the potential to reduce the burden of dengue in this region by conducting focused vector control interventions that target high-risk households and containers in each season and by developing predictive models using climate and non-climate information. These findings provide the region's public health sector with key information for conducting time and location-specific vector control campaigns, and highlight the importance of local socio-ecological studies to understand dengue dynamics. See Text S1 for an executive summary in Spanish. PMID:24324542

  12. Classification of a target analyte in solid mixtures using principal component analysis, support vector machines, and Raman spectroscopy

    O'Connell, Marie-Louise; Howley, Tom; Ryder, Alan G.; Leger, Marc N.; Madden, Michael G.


    The quantitative analysis of illicit materials using Raman spectroscopy is of widespread interest for law enforcement and healthcare applications. One of the difficulties faced when analysing illicit mixtures is the fact that the narcotic can be mixed with many different cutting agents. This obviously complicates the development of quantitative analytical methods. In this work we demonstrate some preliminary efforts to try and account for the wide variety of potential cutting agents, by discrimination between the target substance and a wide range of excipients. Near-infrared Raman spectroscopy (785 nm excitation) was employed to analyse 217 samples, a number of them consisting of a target analyte (acetaminophen) mixed with excipients of different concentrations by weight. The excipients used were sugars (maltose, glucose, lactose, sorbitol), inorganic materials (talcum powder, sodium bicarbonate, magnesium sulphate), and food products (caffeine, flour). The spectral data collected was subjected to a number of pre-treatment statistical methods including first derivative and normalisation transformations, to make the data more suitable for analysis. Various methods were then used to discriminate the target analytes, these included Principal Component Analysis (PCA), Principal Component Regression (PCR) and Support Vector Machines.

  13. Efficiency of adenoviral vector mediated CTLA4Ig gene delivery into mesenchymal stem cells

    邓宇斌; 郭小荑; 原清涛; 李树浓


    Objective To prevent Graft-versus-host disease (GVHD) in rat model, we evaluated the feasibility of mesenchymal stem cells (MSCs) as a gene transfer target and studied the efficiency of recombinant adenovirus mediated gene therapy. Methods We constructed the recombinant adenovirus containing CTLA4Ig gene. Rat MSCs of passages 3-5 were infected by the adenovirus, and the transfection efficiency was monitored by GFP markers. We performed flow cytometric analysis, immunohistochemical and Western blotting analysis to identify the CTLA4Ig expression. The gene transferred MSCs were tested for their ability to inhibit the allogeneic lymphocyte response in vitro and to prevent GVHD in a rat model. Results Recombinant adenovirus pAd-CTLA4Ig was correctly constructed and confirmed. After MSCs were infected by the adenovirus, the CTLA4Ig protein was detected not only in transgenic MSCs, but also in the culture medium. In a mixed lymphocytes response (MLR) test, the transgenic MSCs could significantly inhibit the allogeneic lymphocyte response compared with the control groups (P<0.05). A model of GVHD was developed by transplanting bone marrow cells and spleen lymphocytes of F344 rats to lethally irradiated SD rats. The onset of GVHD could be ameliorated or prevented by co-administration of transgenic MSCs. All the rats in the control groups suffered severe acute GVHD. CTLA4Ig expression was observed in the liver, intestine, kidney and spleen 30 days post- transplantation. Conclusions Our results indicate that adenoviral vectors could efficiently transfer CTLA4Ig gene into MSCs and sustain long-term stable expression in vitro and in vivo.

  14. Use of microRNA Let-7 to control the replication specificity of oncolytic adenovirus in hepatocellular carcinoma cells.

    Huajun Jin

    Full Text Available Highly selective therapy for hepatocellular carcinoma (HCC remains an unmet medical need. In present study, we found that the tumor suppressor microRNA, let-7 was significantly downregulated in a proportion of primary HCC tissues (12 of 33, 36.4% and HCC cell lines. In line with this finding, we have engineered a chimeric Ad5/11 fiber oncolytic adenovirus, SG7011(let7T, by introducing eight copies of let-7 target sites (let7T into the 3' untranslated region of E1A, a key gene associated with adenoviral replication. The results showed that the E1A expression (both RNA and protein levels of the SG7011(let7T was tightly regulated according to the endogenous expression level of the let-7. As contrasted with the wild-type adenovirus and the control virus, the replication of SG7011(let7T was distinctly inhibited in normal liver cells lines (i.e. L-02 and WRL-68 expressing high level of let-7 (>300 folds, whereas was almost not impaired in HCC cells (i.e. Hep3B and PLC/PRF/5 with low level of let-7. Consequently, the cytotoxicity of SG7011(let7T to normal liver cells was successfully decreased while was almost not attenuated in HCC cells in vitro. The antitumor ability of SG7011(let7Tin vivo was maintained in mice with Hep3B xenograft tumor, whereas was greatly decreased against the SMMC-7721 xenograft tumor expressing a high level of let-7 similar with L-02 when compared to the wild-type adenovirus. These results suggested that SG7011(let7T may be a promising anticancer agent or vector to mediate the expression of therapeutic gene, broadly applicable in the treatment for HCC and other cancers where the let-7 gene is downregulated.

  15. Intraosseous delivery of lentiviral vectors targeting factor VIII expression in platelets corrects murine hemophilia A.

    Wang, Xuefeng; Shin, Simon C; Chiang, Andy F J; Khan, Iram; Pan, Dao; Rawlings, David J; Miao, Carol H


    Intraosseous (IO) infusion of lentiviral vectors (LVs) for in situ gene transfer into bone marrow may avoid specific challenges posed by ex vivo gene delivery, including, in particular, the requirement of preconditioning. We utilized IO delivery of LVs encoding a GFP or factor VIII (FVIII) transgene directed by ubiquitous promoters (a MND or EF-1α-short element; M-GFP-LV, E-F8-LV) or a platelet-specific, glycoprotein-1bα promoter (G-GFP-LV, G-F8-LV). A single IO infusion of M-GFP-LV or G-GFP-LV achieved long-term and efficient GFP expression in Lineage(-)Sca1(+)c-Kit(+) hematopoietic stem cells and platelets, respectively. While E-F8-LV produced initially high-level FVIII expression, robust anti-FVIII immune responses eliminated functional FVIII in circulation. In contrast, IO delivery of G-F8-LV achieved long-term platelet-specific expression of FVIII, resulting in partial correction of hemophilia A. Furthermore, similar clinical benefit with G-F8-LV was achieved in animals with pre-existing anti-FVIII inhibitors. These findings further support platelets as an ideal FVIII delivery vehicle, as FVIII, stored in α-granules, is protected from neutralizing antibodies and, during bleeding, activated platelets locally excrete FVIII to promote clot formation. Overall, a single IO infusion of G-F8-LV was sufficient to correct hemophilia phenotype for long term, indicating that this approach may provide an effective means to permanently treat FVIII deficiency.

  16. 牛新孢子虫NcSRS2基因腺病毒穿梭载体的构建及在293细胞中的表达%Construction of bovine Neospora caninum NcSRS2 adenovirus shuttle vector and expression of NcSRS2 gene in 293 cells

    贾立军; 张守发; 刘明明


    应用PCR技术扩增牛新孢子虫NcSRS2基因,纯化PCR产物后与克隆载体pMD18-T Simple Vector连接,将PCR、酶切鉴定及测序分析正确的pMD-18T-NcSRS2重组质粒进行EcoRⅠ和XbaⅠ双酶切,克隆至相同酶切回收后的腺病毒穿梭载体pCR259中,再将PCR、酶切鉴定正确的pCR259-NcSRS2重组质粒转染293细胞,应用IF-AT和Western-blotting技术检测重组质粒在293细胞中的表达情况。结果显示,扩增的牛新孢子虫NcSRS2基因长度为1 227bp,与GenBank中发表的NcSRS2(AF061249)核苷酸序列同源性为99%,构建的pCR259-NcSRS2重组质粒在293细胞中得到瞬时表达,表达蛋白的相对分子质量为43 000,具有较好的反应原性。本试验为新孢子虫病腺病毒载体疫苗的构建奠定了基础。%In this research,bovine Neospora caninum NcSRS2 gene was amplified by PCR.The purified PCR products were ligated with pMD18-T simple vector.After enzyme digestion and sequence analysis,the correct pMD-18T-NcSRS2 gene was digested by EcoRⅠand XbaⅠ,and cloned into the adenovirus shuttle vector pCR259 which was digested by the same enzymes.The correct pCR259-NcSRS2 recombinant plasmid was transfected into 293 cells.The expression of recombinant plasmid in 293 cells was detected by IFAT and Western-blotting.The result shows that the length of bovine Neospora caninum NcSRS2 gene is 1 227 bp.which has 99% homology with sequence and sequence in GenBank.The pCR259-NcSRS2 recombinant plasmid could transiently express in 293 cells.The molecular weight of the expressed protein which has good reactionogenicity is 43 000.This research laid the foundation for the construction of Neospora caninum vector-adenovirus vaccine

  17. Adenoviral Vector Driven by a Minimal Rad51 Promoter Is Selective for p53-Deficient Tumor Cells

    Fong, Vincent; Osterbur, Marika; Capella, Cristina; Kim, Yo-El; Hine, Christopher; Gorbunova, Vera; Seluanov, Andrei; Dewhurst, Stephen


    Background The full length Rad51 promoter is highly active in cancer cells but not in normal cells. We therefore set out to assess whether we could confer this tumor-selectivity to an adenovirus vector. Methodology/Principal Findings Expression of an adenovirally-vectored luciferase reporter gene from the Rad51 promoter was up to 50 fold higher in cancer cells than in normal cells. Further evaluations of a panel of truncated promoter mutants identified a 447 bp minimal core promoter element that retained the full tumor selectivity and transcriptional activity of the original promoter, in the context of an adenovirus vector. This core Rad51 promoter was highly active in cancer cells that lack functional p53, but less active in normal cells and in cancer cell lines with intact p53 function. Exogenous expression of p53 in a p53 null cell line strongly suppressed activity of the Rad51 core promoter, underscoring the selectivity of this promoter for p53-deficient cells. Follow-up experiments showed that the p53-dependent suppression of the Rad51 core promoter was mediated via an indirect, p300 coactivator dependent mechanism. Finally, transduction of target cells with an adenovirus vector encoding the thymidine kinase gene under transcriptional control of the Rad51 core promoter resulted in efficient killing of p53 defective cancer cells, but not of normal cells, upon addition of ganciclovir. Conclusions/Significance Overall, these experiments demonstrated that a small core domain of the Rad51 promoter can be used to target selective transgene expression from adenoviral vectors to tumor cells lacking functional p53. PMID:22174876

  18. Adenoviral vector driven by a minimal Rad51 promoter is selective for p53-deficient tumor cells.

    Vincent Fong

    Full Text Available BACKGROUND: The full length Rad51 promoter is highly active in cancer cells but not in normal cells. We therefore set out to assess whether we could confer this tumor-selectivity to an adenovirus vector. METHODOLOGY/PRINCIPAL FINDINGS: Expression of an adenovirally-vectored luciferase reporter gene from the Rad51 promoter was up to 50 fold higher in cancer cells than in normal cells. Further evaluations of a panel of truncated promoter mutants identified a 447 bp minimal core promoter element that retained the full tumor selectivity and transcriptional activity of the original promoter, in the context of an adenovirus vector. This core Rad51 promoter was highly active in cancer cells that lack functional p53, but less active in normal cells and in cancer cell lines with intact p53 function. Exogenous expression of p53 in a p53 null cell line strongly suppressed activity of the Rad51 core promoter, underscoring the selectivity of this promoter for p53-deficient cells. Follow-up experiments showed that the p53-dependent suppression of the Rad51 core promoter was mediated via an indirect, p300 coactivator dependent mechanism. Finally, transduction of target cells with an adenovirus vector encoding the thymidine kinase gene under transcriptional control of the Rad51 core promoter resulted in efficient killing of p53 defective cancer cells, but not of normal cells, upon addition of ganciclovir. CONCLUSIONS/SIGNIFICANCE: Overall, these experiments demonstrated that a small core domain of the Rad51 promoter can be used to target selective transgene expression from adenoviral vectors to tumor cells lacking functional p53.

  19. Safety profile, efficacy, and biodistribution of a bicistronic high-capacity adenovirus vector encoding a combined immunostimulation and cytotoxic gene therapy as a prelude to a phase I clinical trial for glioblastoma

    Puntel, Mariana [Department of Neurosurgery, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689 (United States); Department of Cell and Developmental Biology, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689 (United States); Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Ghulam, Muhammad A.K.M. [Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Farrokhi, Catherine [Department of Psychiatry and Behavioral Neurosciences, Cedars Sinai Medical Center, Los Angeles, CA 90048 (United States); VanderVeen, Nathan; Paran, Christopher; Appelhans, Ashley [Department of Neurosurgery, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689 (United States); Department of Cell and Developmental Biology, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689 (United States); Kroeger, Kurt M.; Salem, Alireza [Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Lacayo, Liliana [Department of Psychiatry and Behavioral Neurosciences, Cedars Sinai Medical Center, Los Angeles, CA 90048 (United States); Pechnick, Robert N. [Department of Psychiatry and Behavioral Neurosciences, Cedars Sinai Medical Center, Los Angeles, CA 90048 (United States); Department of Psychiatry and Behavioral Neurosciences, David Geffen School of Medicine, University of California, Los Angeles, CA (United States); Kelson, Kyle R.; Kaur, Sukhpreet; Kennedy, Sean [Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Palmer, Donna; Ng, Philip [Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030 (United States); and others


    Adenoviral vectors (Ads) are promising gene delivery vehicles due to their high transduction efficiency; however, their clinical usefulness has been hampered by their immunogenicity and the presence of anti-Ad immunity in humans. We reported the efficacy of a gene therapy approach for glioma consisting of intratumoral injection of Ads encoding conditionally cytotoxic herpes simplex type 1 thymidine kinase (Ad-TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Ad-Flt3L). Herein, we report the biodistribution, efficacy, and neurological and systemic effects of a bicistronic high-capacity Ad, i.e., HC-Ad-TK/TetOn-Flt3L. HC-Ads elicit sustained transgene expression, even in the presence of anti-Ad immunity, and can encode large therapeutic cassettes, including regulatory elements to enable turning gene expression “on” or “off” according to clinical need. The inclusion of two therapeutic transgenes within a single vector enables a reduction of the total vector load without adversely impacting efficacy. Because clinically the vectors will be delivered into the surgical cavity, normal regions of the brain parenchyma are likely to be transduced. Thus, we assessed any potential toxicities elicited by escalating doses of HC-Ad-TK/TetOn-Flt3L (1 × 10{sup 8}, 1 × 10{sup 9}, or 1 × 10{sup 10} viral particles [vp]) delivered into the rat brain parenchyma. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points. The results indicate that doses up to 1 × 10{sup 9} vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered into the normal rat brain and underpin further developments for its implementation in a phase I clinical trial for glioma. - Highlights: ► High capacity Ad vectors elicit sustained therapeutic gene expression in the brain. ► HC-Ad-TK/TetOn-Flt3L encodes two therapeutic genes and a transcriptional switch. ► We performed a dose escalation study at

  20. Dielectrophoresis and dielectrophoretic impedance detection of adenovirus and rotavirus

    Nakano, Michihiko; Ding, Zhenhao; Suehiro, Junya


    The aim of this study is the electrical detection of pathogenic viruses, namely, adenovirus and rotavirus, using dielectrophoretic impedance measurement (DEPIM). DEPIM consists of two simultaneous processes: dielectrophoretic trapping of the target and measurement of the impedance change and increase in conductance with the number of trapped targets. This is the first study of applying DEPIM, which was originally developed to detect bacteria suspended in aqueous solutions, to virus detection. The dielectric properties of the viruses were also investigated in terms of their dielectrophoretic behavior. Although their estimated dielectric properties were different from those of bacteria, the trapped viruses increased the conductance of the microelectrode in a manner similar to that in bacteria detection. We demonstrated the electrical detection of viruses within 60 s at concentrations as low as 70 ng/ml for adenovirus and 50 ng/ml for rotavirus.

  1. 腺病毒介导的秦川牛固醇调节元件结合蛋白2基因(SREBP2)过表达载体的构建与鉴定%Construction and Identification of Recombinant Adenovirus Vector Specific to Qinchuan Cattle(Bos taurus) Derived Sterol Regulatory Element Binding Protein 2 Gene (SREBP2)

    付常振; 刘扬; 昝林森; 王虹; 成功; 王嘉力


    Sterol regulatory element binding protein 2(SERBP2) is a basic-helix-loop-helix-luecine zipper factor which regulates the metabolization process of cholesterol.We cloned the SREBP2 gene from Qinchuan cattle (Bos taurus) and constructed the overexpression adenoviral vector,and packed and amplified the virus for a high titer,as a antecedent work for the further study of cellular level function of SERBP2 gene.Total RNA was extracted from the adipose tissue of Qinchuan cattle and then reversely transcripted to cDNA.A pair of exclusive primers were designed according to the GenBank sequence information of SREBP2 gene(Accession No.NM_001205600) to amplify the complete coding sequence(CDS) area of SREBP2 gene by polymerase chain reaction (PCR).The fragments containing CDS area of SREBP2 gene were inserted into the shuttle vector to construct the pAdTrack-CMV-SREBP2 plasmid.The recombinant plasmid and the blank control pAdTrack-CMV were linearized by digesting with restriction endonuclease Pine Ⅰ and subsequently transformed into Escherichia coli B J5183 containing pAdEasy-1 to homologous recombine and obtain the recombinant adenovirus plasmid pAd-SREBP2 and pAd-CMV.And then,the confirmed recombinant adenovirus plasmid pAd-SREBP2 was digested with Pac Ⅰ and transfected into 293A cell line to package and amplify the recombinant adenovirus Ad-SREBP2 and Ad-CMV,and to collect virus of high titer.The viral titer of AdSREBP2 and Ad-CMV was 7 × 108 and 1.3 × 109 GFU/mL respectively,measured by green fluorescent protein (GFP) labelled method.Qinchuan cattle derived preadipocyte was infected by Ad-SREBP2 and Ad-CMV to verify the availability of the virues.The expression of SREBP2 increased by 102.3 times after infected with the recombinant adenovirus for 48 h,determined by quantitative Real-time PCR.The cloning of SERBP2 gene of Qinchuan cattle obtaining of recombinant adenoviru and virus of high titer are set as foundation for the studies of the gene function on cellular

  2. Human immunodeficiency virus type 1-mediated syncytium formation is compatible with adenovirus replication and facilitates efficient dispersion of viral gene products and de novo-synthesized virus particles.

    Li, H; Haviv, Y S; Derdeyn, C A; Lam, J; Coolidge, C; Hunter, E; Curiel, D T; Blackwell, J L


    Conditionally replicative adenovirus (CRAd) vectors are designed for specific oncolytic replication in tumor tissues with concomitant sparing of normal cells. As such, CRAds offer an unprecedented level of anticancer potential for malignancies that have been refractory to previous cancer gene therapy interventions. CRAd efficacy may, however, be compromised by inefficient dispersion of the replicating vector within the tumor tissue. To address this issue, we evaluated the utility of a fusogenic membrane glycoprotein (FMG), which induces the fusion of neighboring cellular membranes to form multinucleated syncytia. We hypothesized that the FMG-mediated syncytia would facilitate dispersion of the adenovirus (Ad) gene products and viral progeny. To test this, human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, which induce syncytia in the presence of CD4+ target cells, were expressed by an Ad (Ad5HIVenv) in permissive (CD4-positive) and nonpermissive (CD4-negative) cell lines. After validating this Ad-FMG model, the efficiency of Ad replication in the presence or absence of syncytia was evaluated. The results demonstrated that syncytium formation was compatible with Ad replication and dramatically increased the dispersion of virus gene products within the cytoplasm of the syncytia as well as viral particles in the nuclei of the syncytial mass. Moreover, progeny virions were released more efficiently from syncytia compared with nonsyncytial cells. These data demonstrate the utility of FMGs as a dispersion agent and suggest that FMGs can improve the efficacy of CRAd gene therapy.

  3. Three-Dimensional Structure of Canine Adenovirus Serotype 2 Capsid▿

    Schoehn, Guy; El Bakkouri, Majida; Fabry, Céline M. S.; Billet, Oliver; Leandro F. Estrozi; Le, Van Long; Curiel, David T.; Kajava, Andrey V; Ruigrok, Rob W. H.; Eric J Kremer


    There are more than 100 known adenovirus (AdV) serotypes, including 50 human serotypes. Because AdV-induced disease is relatively species specific, vectors derived from nonhuman serotypes may have wider clinical potential based, in part, on the lack of ubiquitous memory immunity. Whereas a few of the human serotype capsids have been studied at the structural level, none of the nonhuman serotypes has been analyzed. The basis laid by the analysis of human AdV (hAdV) has allowed us to determine ...

  4. Construction and identification of recombinant adenovirus-mediated gene transfer system for rat vascular endothelial growth factor

    Hongyu Yang; Hong Qi; Junjie Zou; Xiwei Zhang


    Objective: To construct the recombinant adenovirus vector carrying rat vascular endothelial growth factor(VEGF), as preparation for genetic transfection that follows. Methods: Rat VEGF was obtained by using RT-PCR amplification and then cloned into the shutter plasmid pDC316. Subsequently, this newly constructed plasmid pDC316-VEGF, after identification by nuclease digestion analysis and sequencing analysis, was transfected into human embryonic kidney cells HEK293 by Lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGE3. Based on the homologous recombination of the two plasmids within HEK293 cells, the recombinant adenovirus vector carrying VEGF and VDC316-VEGF was created. VDC316-VEGF was subsequently identified using PCR, purified using repeated plaque passages, proliferated using freezing and melting within HEK293 cells, and titrated using 50% Tissue Culture Infective Dose(TCID50) assay. Results:The newly constructed recombinant adenovirus was confirmed to carry rat VEGF based on PCR results, and its titration value determined based on TCID50 assay was 3×109 pfu/ml. Conclusion:The recombinant adenovirus carrying rat VEGF was successfully constructed. The newly constructed adenovirus can produce a sufficiently high titration value within HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.

  5. Targeted delivery of human VEGF gene via complexes of magnetic nanoparticle-adenoviral vectors enhanced cardiac regeneration.

    Yue Zhang

    Full Text Available This study assessed the concept of whether delivery of magnetic nanobeads (MNBs/adenoviral vectors (Ad-encoded hVEGF gene (Ad(hVEGF could regenerate ischaemically damaged hearts in a rat acute myocardial infarction model under the control of an external magnetic field. Adenoviral vectors were conjugated to MNBs with the Sulfo-NHS-LC-Biotin linker. In vitro transduction efficacy of MNBs/Ad-encoded luciferase gene (Ad(luc was compared with Ad(luc alone in human umbilical vein endothelial cells (HUVECs under magnetic field stimulation. In vivo, in a rat acute myocardial infarction (AMI model, MNBs/Ad(hVEGF complexes were injected intravenously and an epicardial magnet was employed to attract the circulating MNBs/Ad(hVEGF complexes. In vitro, compared with Ad(luc alone, MNBs/Ad(luc complexes had a 50-fold higher transduction efficiency under the magnetic field. In vivo, epicardial magnet effectively attracted MNBs/Ad(hVEGF complexes and resulted in strong therapeutic gene expression in the ischemic zone of the infarcted heart. When compared to other MI-treated groups, the MI-M(+/Ad(hVEGF group significantly improved left ventricular function (p<0.05 assessed by pressure-volume loops after 4 weeks. Also the MI-M(+/Ad(hVEGF group exhibited higher capillary and arteriole density and lower collagen deposition than other MI-treated groups (p<0.05. Magnetic targeting enhances transduction efficiency and improves heart function. This novel method to improve gene therapy outcomes in AMI treatment offers the potential into clinical applications.

  6. Multiple insecticide resistance mechanisms involving metabolic changes and insensitive target sites selected in anopheline vectors of malaria in Sri Lanka

    Karunaratne SHP Parakrama


    Full Text Available Abstract Background The current status of insecticide resistance and the underlying resistance mechanisms were studied in the major vector of malaria, Anopheles culicifacies, and the secondary vector, Anopheles subpictus in five districts (Anuradhapura, Kurunegala, Moneragala, Puttalam and Trincomalee of Sri Lanka. Eight other anophelines, Anopheles annularis, Anopheles barbirostris, Anopheles jamesii, Anopheles nigerrimus, Anopheles peditaeniatus, Anopheles tessellatus, Anopheles vagus and Anopheles varuna from Anuradhapura district were also tested. Methods Adult females were exposed to the WHO discriminating dosages of DDT, malathion, fenitrothion, propoxur, λ-cyhalothrin, cyfluthrin, cypermethrin, deltamethrin, permethrin and etofenprox. The presence of metabolic resistance by esterase, glutathione S-transferase (GST and monooxygenase-based mechanisms, and the sensitivity of the acetylcholinesterase target site were assessed using synergists, and biochemical, and metabolic techniques. Results All the anopheline species had high DDT resistance. All An. culicifacies and An. subpictus populations were resistant to malathion, except An. culicifacies from Kurunegala, where there was no malathion carboxylesterase activity. Kurunegala and Puttalam populations of An. culicifacies were susceptible to fenitrothion. All the An. culicifacies populations were susceptible to carbamates. Both species were susceptible to the discriminating dosages of cypermethrin and cyfluthrin, but had different levels of resistance to other pyrethroids. Of the 8 other anophelines, only An. nigerrimus and An. peditaeniatus were resistant to all the insecticides tested, probably due to their high exposure to the insecticides used in agriculture. An. vagus showed some resistance to permethrin. Esterases, GSTs and monooxygenases were elevated in both An. culicifacies and An. subpictus. AChE was most sensitive to insecticides in Kurunegala and Trincomalee An. culicifacies

  7. A one-step cloning method for the construction of somatic cell gene targeting vectors: application to production of human knockout cell lines

    Liu Yi


    Full Text Available Abstract Background Gene targeting is a powerful method that can be used for examining the functions of genes. Traditionally, the construction of knockout (KO vectors requires an amplification step to obtain two homologous, large fragments of genomic DNA. Restriction enzymes that cut at unique recognitions sites and numerous cloning steps are then carried out; this is often a time-consuming and frustrating process. Results We have developed a one-step cloning method for the insertion of two arms into a KO vector using exonuclease III. We modified an adeno-associated virus KO shuttle vector (pTK-LoxP-NEO-AAV to yield pAAV-LIC, which contained two cassettes at the two multiple-cloning sites. The vector was digested with EcoRV to give two fragments. The two homologous arms, which had an overlap of 16 bases with the ends of the vector fragments, were amplified by polymerase chain reaction. After purification, the four fragments were mixed and treated with exonuclease III, then transformed into Escherichia coli to obtain the desired clones. Using this method, we constructed SirT1 and HDAC2 KO vectors, which were used to establish SirT1 KO cells from the colorectal cancer cell line (HCT116 and HDAC2 KO cells from the colorectal cancer cell line (DLD1. Conclusions Our method is a fast, simple, and efficient technique for cloning, and has great potential for high-throughput construction of KO vectors.

  8. Analysis of the expression of coxsackievirus and adenovirus receptor in five colon cancer cell lines


    AIM: To investigate the expression of coxsackievirus and adenovirus receptor (CAR) and adenovirus-mediated reporter gene transfer in five human colon cancer cell lines.METHODS: Expression of CAR-specific mRNA and protein was analyzed by reverse transcriptase polymerase chain reaction and Western blotting, respectively. Adenovirusbased gene delivery was evaluated by infection of cells with adenoviral vector carrying the green fluorescent protein (GFP) gene.RESULTS: All the colon cancer cell lines examined (HT29,LS180, SW480, SW948 and SW1116) expressed CAR full-length mRNA and an alternatively-spliced variant that lacks the transmembrane coding exon. All cell lines were detected as CAR-positive by Western blot analysis.Further, all cells we examined were efficiently infected with adenoviral vector-GFP.CONCLUSION: The data indicated that the five colon cancer cell lines tested expressed adenovirus primary receptor and could be efficiently infected by adenoviral vectors. Therefore, these cell lines will be useful for adenovirus-based gene transfer and research.

  9. Adenovirus sequences required for replication in vivo.

    Wang, K.; Pearson, G D


    We have studied the in vivo replication properties of plasmids carrying deletion mutations within cloned adenovirus terminal sequences. Deletion mapping located the adenovirus DNA replication origin entirely within the first 67 bp of the adenovirus inverted terminal repeat. This region could be further subdivided into two functional domains: a minimal replication origin and an adjacent auxillary region which boosted the efficiency of replication by more than 100-fold. The minimal origin occup...

  10. Reassessing culture media and critical metabolites that affect adenovirus production.

    Shen, Chun Fang; Voyer, Robert; Tom, Roseanne; Kamen, Amine


    Adenovirus production is currently operated at low cell density because infection at high cell densities still results in reduced cell-specific productivity. To better understand nutrient limitation and inhibitory metabolites causing the reduction of specific yields at high cell densities, adenovirus production in HEK 293 cultures using NSFM 13 and CD 293 media were evaluated. For cultures using NSFM 13 medium, the cell-specific productivity decreased from 3,400 to 150 vp/cell (or 96% reduction) when the cell density at infection was increased from 1 to 3 x 10(6) cells/mL. In comparison, only 50% of reduction in the cell-specific productivity was observed under the same conditions for cultures using CD 293 medium. The effect of medium osmolality was found critical on viral production. Media were adjusted to an optimal osmolality of 290 mOsm/kg to facilitate comparison. Amino acids were not critical limiting factors. Potential limiting nutrients including vitamins, energy metabolites, bases and nucleotides, or inhibitory metabolites (lactate and ammonia) were supplemented to infected cultures to further investigate their effect on the adenovirus production. Accumulation of lactate and ammonia in a culture infected at 3 x 10(6) cells/mL contributed to about 20% reduction of the adenovirus production yield, whereas nutrient limitation appeared primarily responsible for the decline in the viral production when NSFM 13 medium was used. Overall, the results indicate that multiple factors contribute to limiting the specific production yield at cell densities beyond 1 x 10(6) cells/mL and underline the need to further investigate and develop media for better adenoviral vector productions.

  11. A Plasmodium vivax plasmid DNA- and adenovirus-vectored malaria vaccine encoding blood stage antigens AMA1 and MSP142 in a prime/boost heterologous immunization regimen partially protects Aotus monkeys against blood stage challenge.

    Obaldia, Nicanor; Stockelman, Michael G; Otero, William; Cockrill, Jennifer A; Ganeshan, Harini; Abot, Esteban N; Zhang, Jianfeng; Limbach, Keith; Charoenvit, Yupin; Doolan, Denise L; Tang, De-Chu C; Richie, Thomas L


    Malaria is caused by parasites of the genus Plasmodium that are transmitted to humans by the bites of Anopheles mosquitoes. After the elimination of P. falciparum it is predicted that Plasmodium vivax will remain an important cause of morbidity and mortality outside of Africa, stressing the importance of developing a vaccine against malaria. In this study we assess the immunogenicity and protective efficacy of two P. vivax antigens, AMA1 and MSP142 in a recombinant DNA plasmid prime/adenoviral vector (Ad) boost regimen in Aotus monkeys. Groups of 4 to 5 monkeys were immunized with DNA alone, Ad alone, prime/boost regimens of each antigen, prime/boost with both antigens, and empty vector controls, and then subjected to blood stage challenge. The heterologous immunization regimen with the antigen pair was more protective than either antigen alone or both antigens delivered with a single vaccine platform, based on their ability to induced the longest pre-patent period and time to peak parasitemia; the lowest peak and mean parasitemia; the smallest area under the parasitemia curve and the highest self-cured rate. Overall, pre-challenge MSP1 antibody titers strongly correlated with decreased parasite burden. Nevertheless, a significant proportion of immunized animals developed anemia. In conclusion, P. vivax plasmid DNA/Ad5 vaccine encoding blood stage parasite antigens AMA1 and MSP142 in a heterologous prime/boost immunization regimen, provided significant protection against blood-stage challenge in Aotus monkeys, indicating the suitability of these antigens and regimen for further development.

  12. Construction and identification of recombinant adenovirus vector co-expressing VEGF121 and BMP2 genes and its expression in HEK293 cells%VEGF121和BMP2双基因共表达重组腺病毒载体的构建及其在HEK293中的表达

    栗刚; 吴秀成; 钟声; 王巍; 李媛; 刘丹平


    目的 构建人血管内皮生长因子121(VEGF121)与人骨形态发生蛋白2(BMP2)双基因共表达腺病毒载体Adv-BMP2-IRES-VEGF121,并观察其在人胚肾细胞株(HEK293)中的表达情况.方法 对腺病毒质粒pShuttle-CMV-BMP2的目的基因BMP2进行PCR扩增.腺病毒质粒pShuttle-CMV-VEGF121-IRES-hrGFP-1经Kpn I/Xba I酶切后,将BMP2片段定向导入pShuttle-CMV-VEGF121-IRES,构建pShuttle-CMV-V EGF121-IRES-BMP2,并注入大肠杆菌DH5a中扩增,提取质粒.通过酶切分析、PCR检测和序列分析进行鉴定.将构建所得的质粒转染HEK293,采用RT-PCR法检测HEK293中的BMP2、VEGF121 mRNA,Western blot法检测其蛋白.结果 成功构建了Adv-BMP2-IRES-VEGF121.酶切分析及DNA序列测定证实重组质粒构建正确.质粒转染后的HEK293 BMP2和VEGF121表达阳性.结论 成功构建了Adv-BMP2-IRES-VEGF121,其转染HEK293后,VEGF121、BMP2在HEK293中共表达阳性.%Objective To construct and identify the adenovirus shuttle plasmid pShuttle-CMV-VEGF121-IRES-BMP2 and its express in HEK293 cells.Methods The DNA fragments of human BMP2 gene were changed restriction sites and subcloned by PCR.The human BMP2 genes and pShuttle-CMV-VEGF121-IRES were ligated into the plasmid by directional cloning method.The inserted target genes in the plasmid were verified by restriction enzyme digestion and nucleotide sequencing.The correct recombinant express plasmid was transfected to HEK293 cells.The expression of VEGF121, BMP2 mRNA were detected by RT-PCR, the VEGF121, BMP2 protein were detected by Western blotting.Results The adenovirus shuttle plasmid was constructed correctly.The VEGF121, BMP2 mRNA and protein were expressed in HEK293 cells.Conclusion The adenovirus shuttle plasmid is constructed, VEGF121, BMP2 mRNA and protein are successfully expressed in HEK293 cells.

  13. Chapter three--Syrian hamster as an animal model to study oncolytic adenoviruses and to evaluate the efficacy of antiviral compounds.

    Wold, William S M; Toth, Karoly


    The Syrian (golden) hamster (Mesocricetus auratus) has served as a useful model for different aspects of biology for at least 50 years, and its use has been expanding recently. In earlier years, among other things, it was a model for cancer development. More recently, it has become a model for many different infectious diseases. It has also become an alternative model for the study of oncolytic adenovirus vectors for cancer gene therapy. Among several other human pathogens, the hamster is permissive for the replication of human species C adenoviruses, which are the parental virus for the majority of adenovirus vectors in use today. These vectors replicate in some of the established hamster tumor cell lines that can be used to generate tumors in vivo, that is, one can study oncolytic (replication competent) adenoviruses in a permissive, immunocompetent model. This has afforded the opportunity to study the effect of the host immune system on the vector-infected tumor and has allowed the use of a more relevant animal model to determine the safety and biodistribution of replication-competent adenoviruses. The hamster has also been used to evaluate antiviral compounds and vaccines against many viruses, including adenoviruses, flaviviruses, alphaviruses, arenaviruses, bunyaviruses, and paramyxoviruses.

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

    Marino Michael P


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

  15. Evaluation of a combinatorial RNAi lentivirus vector targeting foot-and-mouth disease virus in vitro and in vivo.

    Zhang, Xiaoxi; Zheng, Haixue; Xu, Minjun; Zhou, Yu; Li, Xiangping; Yang, Fan; Liu, Qingyou; Shi, Deshun


    Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven‑hoofed animals, which leads to serious economical losses. FMDV is not adequately controlled by vaccination or biosecurity measures. To generate genetically modified FMDV‑resistant animals, a combinatorial expression cassette producing three short hairpin (sh)RNAs was constructed using the lentivirus (LV) vector, LV‑3shRNA. The three shRNAs were expressed under the regulation of DNA polymerase III promoters from a buffalo and a bovine source, with one targeted to the non‑structural protein 3B, and the other two targeted to the viral polymerase protein 3D of FMDV, respectively. The role of LV‑3shRNA in the inhibition of the replication of FMDV was determined in BHK‑21 cells and in suckling mice. The results revealed that LV‑3shRNA reduced viral growth 3‑fold (24 h post‑infection) when the cells were challenged with 107‑times the tissue culture infective dose (TCID50)/ml of O serotype FMDV. The suckling mice pretreated with LV‑3shRNA were completely protected on administration of 5‑times the dose of FMDV otherwise sufficient to kill 50% of the experimental animals (LD50). These results demonstrated that the LV‑mediated dual expression of three FMDV‑specific shRNAs provided a novel strategy towards combating FMDV, which facilitates the permanent introduction of novel disease-resistance traits into the buffalo and bovine genomes in the future.

  16. The Comparative and Functional Study between Two Construction Methods of shRNA Expression Vector Targeted LMP1 Gene Encoded by EBV

    Yi-qin WANG; Yu-cheng YANG; Wen-lu ZHANG; Su-ling HONG


    To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1) encoded by Epstein-Barr virus(pshLMP1), and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells, we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis, RT-PCR and western blot. pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method. Furthermore, the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors. According to our research, we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells, and also provides a novel application of RNA interference technology against-EBV.

  17. Chemical composition, toxicity and non-target effects of Pinus kesiya essential oil: An eco-friendly and novel larvicide against malaria, dengue and lymphatic filariasis mosquito vectors.

    Govindarajan, Marimuthu; Rajeswary, Mohan; Benelli, Giovanni


    Mosquitoes (Diptera: Culicidae) are vectors of important parasites and pathogens causing death, poverty and social disability worldwide, with special reference to tropical and subtropical countries. The overuse of synthetic insecticides to control mosquito vectors lead to resistance, adverse environmental effects and high operational costs. Therefore, the development of eco-friendly control tools is an important public health challenge. In this study, the mosquito larvicidal activity of Pinus kesiya leaf essential oil (EO) was evaluated against the malaria vector Anopheles stephensi, the dengue vector Aedes aegypti and the lymphatic filariasis vector Culex quinquefasciatus. The chemical composition of the EO was analyzed by gas chromatography-mass spectroscopy. GC-MS revealed that the P. kesiya EO contained 18 compounds. Major constituents were α-pinene, β-pinene, myrcene and germacrene D. In acute toxicity assays, the EO showed significant toxicity against early third-stage larvae of An. stephensi, Ae. aegypti and Cx. quinquefasciatus, with LC50 values of 52, 57, and 62µg/ml, respectively. Notably, the EO was safer towards several aquatic non-target organisms Anisops bouvieri, Diplonychus indicus and Gambusia affinis, with LC50 values ranging from 4135 to 8390µg/ml. Overall, this research adds basic knowledge to develop newer and safer natural larvicides from Pinaceae plants against malaria, dengue and filariasis mosquito vectors.

  18. Selective effects of a fiber chimeric conditionally replicative adenovirus armed with hep27 gene on renal cancer cell.

    Fang, Lin; Cheng, Qian; Liu, Wenshun; Zhang, Jie; Ge, Yan; Zhang, Qi; Li, Liantao; Liu, Junjie; Zheng, Junnian


    ASBTARCT Adenoviruses mediated cancer gene therapies are widely investigated and show a promising effect on cancer treatment. However, efficient gene transfer varies among different cancer cell lines based on the expression of coxsakie adenovirus receptor (CAR). Hep27, a member of dehydrogenase/reductase (SDR) family, can bind to Mdm2, resulting in the attenuation of Mdm2-mediated p53 degradation. Here we constructed a fiber chimeric adenovirus carrying hep27 gene (F5/35-ZD55-Hep27), in which the fiber protein of 5-serotype adenovirus (Ad5) was substituted by that of 35-serotype adenovirus (Ad35), aiming to facilitate the infection for renal cancer cells and develop the role of hep27 in cancer therapy. We evaluated the CAR and CD46 (a membrane cofactor protein for Ad35) expression in four kinds of renal cancer cells and assessed the relationship between receptors and infection efficiency. 5/35 fiber-modified adenovirus had a much promising infectivity compared with Ad5-based vector in renal cancer cells. F5/35-ZD55-Hep27 had enhanced antitumor activity against human renal cancer cells compared to the other groups. Further, hep27 mediated p53 and cleaved-PARP upregulation and mdm2 downregulation was involved and caused increased apoptosis. Moreover, F5/35-ZD55-Hep27 significantly suppressed tumor growth in subcutaneous renal cancer cell xenograft models. Our data demonstrated that 5/35 fiber-modified adenovirus F5/35-ZD55-Hep27 transferred into renal cancers efficiently and increased p53 to induce cancer cell apoptosis. Thus 5/35 fiber-modified adenoviral vector F5/35-ZD55-Hep27 might a promising vector and antitumor reagent for renal cancer gene therapy.

  19. Oncolytic adenovirus-mediated therapy for prostate cancer

    Sweeney, Katrina; Halldén, Gunnel


    Prostate cancer is a leading cause of cancer-related death and morbidity in men in the Western world. Tumor progression is dependent on functioning androgen receptor signaling, and initial administration of antiandrogens and hormone therapy (androgen-deprivation therapy) prevent growth and spread. Tumors frequently develop escape mechanisms to androgen-deprivation therapy and progress to castration-resistant late-stage metastatic disease that, in turn, inevitably leads to resistance to all current therapeutics, including chemotherapy. In spite of the recent development of more effective inhibitors of androgen–androgen receptor signaling such as enzalutamide and abiraterone, patient survival benefits are still limited. Oncolytic adenoviruses have proven efficacy in prostate cancer cells and cause regression of tumors in preclinical models of numerous drug-resistant cancers. Data from clinical trials demonstrate that adenoviral mutants have limited toxicity to normal tissues and are safe when administered to patients with various solid cancers, including prostate cancer. While efficacy in response to adenovirus administration alone is marginal, findings from early-phase trials targeting local-ized and metastatic prostate cancer suggest improved efficacy in combination with cytotoxic drugs and radiation therapy. Here, we review recent progress in the development of multimodal oncolytic adenoviruses as biological therapeutics to improve on tumor elimination in prostate cancer patients. These optimized mutants target cancer cells by several mechanisms including viral lysis and by expression of cytotoxic transgenes and immune-stimulatory factors that activate the host immune system to destroy both infected and noninfected prostate cancer cells. Additional modifications of the viral capsid proteins may support future systemic delivery of oncolytic adenoviruses. PMID:27579296

  20. Impact of endogenous Inflammation caused by adenovirus as a vector in gene therapy:experiment with mice models of acute lung injury%以腺病毒为载体的基因治疗对急性肺损伤治疗效果的影响及其意义

    王燕; 陆权; 滕国良; Feistein S.Sheldon; Micheal F.Beers; Aron B.Fisher


    Objective To evaluate the impact of endogenous inflammation caused by adenovims as a vector of gene therapy on monse model of acute lung injury(ALI).Methods black C57/B6 mice randomly divided into 4 research groups:(1)adenovirus encoded-lacZ genetreatment group(AdLacZ):n=AdLscZ reagent through intranasal administration to infect mice lungs at 48h before 100%O2 inhalation to induce ALI mouse model,which companied by mice survival rates recorded.B-gal protein activity in lung wag detected to show the level of LacZ DNA transgenic protein activity;meanwhile,the indices of lung wet/dry ratio and bronchial alveolar lavage liquid(BALF)analysis with protein concentration and cell elagsification were detected.Results The method of adenovirus-mediated gene therapy with intranagal administration restthed in IJacZ DNA transgenic protein activity to keep effective highly expression in lung.and the expression level maintained 2.fold increusing even after 72 h of O.inhalation compared to that before O2 inhalation(3.688 U/mg vs 1.589 U/mg);AdLacZ mice had more subjective to O2 inhalation compared to other groups.the 50%mice survival time of this group wag shorter compared to that of the PBS group [(86±3)h vs(94±7)h];also in AdLacZ group,the level of nucleated cell counting in BALF was statistically higher compared to other groups at 48 h of O2 inhalation,which following with 50%-level decreased within anther 24 h O2 inhalation;on the contrary,the level of lung wet/dry ratio and protein concentration in BALF didn,t show remarkably decreasing.Conclusion The endogenous inflammation caused by adenovirus as a vector in ALl gene therapy is temporary and rapidly weaken after getting a peak,however,enough attention still should be paid attention when evaluating the effect of gene therapy.%目的 评价腺病毒自身的致炎作用在以其为载体的基因治疗中,对小鼠急性肺损伤(ALI)治疗效果的影响及意义.方法 C57/B6雄性小鼠随机分为4组:(1)以腺病毒为

  1. Low-Affinity Neurotrophin Receptor p75 Promotes the Transduction of Targeted Lentiviral Vectors to Cholinergic Neurons of Rat Basal Forebrain.

    Antyborzec, Inga; O'Leary, Valerie B; Dolly, James O; Ovsepian, Saak V


    Basal forebrain cholinergic neurons (BFCNs) are one of the most affected neuronal types in Alzheimer's disease (AD), with their extensive loss documented at late stages of the pathology. While discriminatory provision of neuroprotective agents and trophic factors to these cells is thought to be of substantial therapeutic potential, the intricate topography and structure of the forebrain cholinergic system imposes a major challenge. To overcome this, we took advantage of the physiological enrichment of BFCNs with a low-affinity p75 neurotrophin receptor (p75(NTR)) for their targeting by lentiviral vectors within the intact brain of adult rat. Herein, a method is described that affords selective and effective transduction of BFCNs with a green fluorescence protein (GFP) reporter, which combines streptavidin-biotin technology with anti-p75(NTR) antibody-coated lentiviral vectors. Specific GFP expression in cholinergic neurons was attained in the medial septum and nuclei of the diagonal band Broca after a single intraventricular administration of such targeted vectors. Bioelectrical activity of GFP-labeled neurons was proven to be unchanged. Thus, proof of principle is obtained for the utility of the low-affinity p75(NTR) for targeted transduction of vectors to BFCNs in vivo.

  2. A Targeted Multifunctional Platform for Imaging and Treatment of Breast Cancer and Its Metastases Based on Adenoviral Vectors and Magnetic Nanoparticles


    MNPs ) added to HeLa and MDA-MB-435 cancer cells Sample # Vector (MOI 5000 vp/cell) MNP (volume added per well) 1 - - 2 - 0.2 uL 3 - 2 uL 4 - 20...modified set-up. We analyzed cells incubated with Ad vectors and/or MNPs resuspended in low-melting agarose rather than adhered to the bottom Lab-Tek...converting enzyme (9B9), which is a membrane-bound ectopeptidase expres- sed on pulmonary vascular endothelium. For transcrip- tional targeting, the

  3. Ephrin A2 receptor targeting does not increase adenoviral pancreatic cancer transduction in vivo

    Michael A van Geer; Conny T Bakker; Naoya Koizumi; Hiroyuki Mizuguchi; John G Wesseling; Ronald PJ Oude Elferink; Piter J Bosma


    AIM:To generate an adenoviral vector specifically targeting the EphA2 receptor (EphA2R) highly expressed on pancreatic cancer cells in vivo.METHODS:YSA,a small peptide ligand that binds the EphA2R with high affinity,was inserted into the HI loop of the adenovirus serotype 5 fiber knob.To further increase the specificity of this vector,binding sites for native adenoviral receptors,the coxsackie and adenovirus receptor (CAR) and integrin,were ablated from the viral capsid.The ablated retargeted adenoviral vector was produced on 293T cells.Specific targeting of this novel adenoviral vector to pancreatic cancer was investigated on established human pancreatic cancer cell lines.Upon demonstrating specific in vitro targeting,in vivo targeting to subcutaneous growing human pancreatic cancer was tested by intravenous and intraperitoneal administration of the ablated adenoviral vector.RESULTS:Ablation of native cellular binding sites reduced adenoviral transduction at least 100-fold.Insertion of the YSA peptide in the HI loop restored adenoviral transduction of EphA2R-expressing cells but not of cells lacking this receptor.YSA-mediated transduction was inhibited by addition of synthetic YSA peptide.The transduction specificity of the ablated retargeted vector towards human pancreatic cancer cells was enhanced almost 10-fold in vitro.In a subsequent in vivo study in a nude (nu/nu) mouse model however,no increased adenoviral targeting to subcutaneously growing human pancreas cancer nodules was seen upon injection into the tail vein,nor upon injection into the peritoneum.CONCLUSION:Targeting the EphA2 receptor increases specificity of adenoviral transduction of human pancreatic cancer cells in vitro but fails to enhance pancreatic cancer transduction in vivo.

  4. High expression level of soluble SARS spike protein mediated by adenovirus in HEK293 cells

    Fei Zhong; Zhen-Yu Zhong; Shuang Liang; Xiu-Jin Li


    AIM: To develop a highly efficacious method for preparation of soluble SARS S-protein using adenovirus vector to meet the requirement for S-protein investigation.METHODS: The human adenovirus vector was used to express the soluble S-protein (corresponding to 1~1190 amino acids) fused with Myc/His tag using codon-optimized gene construct in HEK239 cells. The recombinant adenovirus bearing S-protein gene was generated by ligation method. The expressed S-protein with Myc/His tag was purified from culture medium with Ni-NTA agarose beads followed by dialysis. The S-protein was detected by Western blot and its biologic activity was analyzed by binding to Vero cells.RESULTS: Under the conditions of infection dose (MOI of 50) and expression time (48 h), the high-level expression of S-protein was obtained. The expression level was determined to be approximately 75 μg/106cells after purification. Purified soluble S-protein was readily detected by Western blot with anti-Myc antibody and showed the ability to bind to surface of Vero cells,demonstrating that the soluble S-protein could remain the biologic activity in the native molecule.CONCLUSION: The high-level expression of S-protein in HEK293 cells mediated by adenovirus can be achieved under the optimized expression conditions. The proteins possess the biologic activity, which lays a foundation for further investigation of S-protein biological function.

  5. Mechanisms of pyrethroid resistance in the dengue mosquito vector, Aedes aegypti: target site insensitivity, penetration, and metabolism.

    Shinji Kasai


    Full Text Available Aedes aegypti is the major vector of yellow and dengue fevers. After 10 generations of adult selection, an A. aegypti strain (SP developed 1650-fold resistance to permethrin, which is one of the most widely used pyrethroid insecticides for mosquito control. SP larvae also developed 8790-fold resistance following selection of the adults. Prior to the selections, the frequencies of V1016G and F1534C mutations in domains II and III, respectively, of voltage-sensitive sodium channel (Vssc, the target site of pyrethroid insecticide were 0.44 and 0.56, respectively. In contrast, only G1016 alleles were present after two permethrin selections, indicating that G1016 can more contribute to the insensitivity of Vssc than C1534. In vivo metabolism studies showed that the SP strain excreted permethrin metabolites more rapidly than a susceptible SMK strain. Pretreatment with piperonyl butoxide caused strong inhibition of excretion of permethrin metabolites, suggesting that cytochrome P450 monooxygenases (P450s play an important role in resistance development. In vitro metabolism studies also indicated an association of P450s with resistance. Microarray analysis showed that multiple P450 genes were over expressed during the larval and adult stages in the SP strain. Following quantitative real time PCR, we focused on two P450 isoforms, CYP9M6 and CYP6BB2. Transcription levels of these P450s were well correlated with the rate of permethrin excretion and they were certainly capable of detoxifying permethrin to 4'-HO-permethrin. Over expression of CYP9M6 was partially due to gene amplification. There was no significant difference in the rate of permethrin reduction from cuticle between SP and SMK strains.

  6. Amplified and Persistent Immune Responses Generated by Single-Cycle Replicating Adenovirus Vaccines

    Crosby, Catherine M.; Nehete, Pramod; Sastry, K. Jagannadha


    ABSTRACT Replication-competent adenoviral (RC-Ad) vectors generate exceptionally strong gene-based vaccine responses by amplifying the antigen transgenes they carry. While they are potent, they also risk causing adenovirus infections. More common replication-defective Ad (RD-Ad) vectors with deletions of E1 avoid this risk but do not replicate their transgene and generate markedly weaker vaccine responses. To amplify vaccine transgenes while avoiding production of infectious progeny viruses, we engineered “single-cycle” adenovirus (SC-Ad) vectors by deleting the gene for IIIa capsid cement protein of lower-seroprevalence adenovirus serotype 6. In mouse, human, hamster, and macaque cells, SC-Ad6 still replicated its genome but prevented genome packaging and virion maturation. When used for mucosal intranasal immunization of Syrian hamsters, both SC-Ad and RC-Ad expressed transgenes at levels hundreds of times higher than that of RD-Ad. Surprisingly, SC-Ad, but not RC-Ad, generated higher levels of transgene-specific antibody than RD-Ad, which notably climbed in serum and vaginal wash samples over 12 weeks after single mucosal immunization. When RD-Ad and SC-Ad were tested by single sublingual immunization in rhesus macaques, SC-Ad generated higher gamma interferon (IFN-γ) responses and higher transgene-specific serum antibody levels. These data suggest that SC-Ad vectors may have utility as mucosal vaccines. IMPORTANCE This work illustrates the utility of our recently developed single-cycle adenovirus (SC-Ad6) vector as a new vaccine platform. Replication-defective (RD-Ad6) vectors produce low levels of transgene protein, which leads to minimal antibody responses in vivo. This study shows that replicating SC-Ad6 produces higher levels of luciferase and induces higher levels of green fluorescent protein (GFP)-specific antibodies than RD in a permissive Syrian hamster model. Surprisingly, although a replication-competent (RC-Ad6) vector produces more luciferase

  7. Adenovirus-mediated nitric oxide synthase gene transfer.

    Raman, Kathleen G; Shapiro, Richard A; Tzeng, Edith; Kibbe, Melina R


    The varied biological effects of nitric oxide (NO) have led to intense research into its diverse physiologic and pathophysiologic roles in multiple disease processes. It has been implicated in the development of altered vasomotor tone, intimal hyperplasia, atherosclerosis, impotence, host defense, and wound healing. Using the modern technologies of recombinant DNA and gene transfer using adenoviral vectors, the effects of NO derived from various NO synthase (NOS) enzymes can be studied in a variety of tissues and the therapeutic applications of NOS is possible. Such uses of NOS gene transfer have been investigated extensively in the vasculature where NO is critical to regulating vascular homeostasis. NOS gene therapy has the theoretical advantage of allowing NO delivery to be localized, thereby limiting potential adverse effects of NO. The benefits of adenoviral vectors in gene transfer include relatively high transduction efficiencies, both replicating and nonreplicating cells may be infected, and the high titers of adenovirus that can be produced. The methods described in this chapter include the cloning of the iNOS cDNA into a recombinant adenoviral vector, large-scale production of that vector AdiNOS preparation, and the use of the vector to transduce tissue in vitro and in vivo.

  8. One-pot fabrication of silver nanocrystals using Nicandra physalodes: A novel route for mosquito vector control with moderate toxicity on non-target water bugs.

    Govindarajan, Marimuthu; Khater, Hanem F; Panneerselvam, Chellasamy; Benelli, Giovanni


    Mosquitoes (Diptera: Culicidae) as vectors for important diseases and parasites causing millions of deaths every year. The use of synthetic pesticides against Culicidae leads to resistance and environmental concerns. Therefore, eco-friendly control tools are a priority. In this research, Nicandra physalodes-mediated synthesis of silver nanoparticles (Ag NPs) was conducted, in order to control larval populations of three important mosquito vectors, Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. Biofabricated Ag NPs were characterized using UV-vis spectrophotometry, XRD, FTIR spectroscopy, SEM, and TEM analyses. Ag NPs were highly toxic against the three mosquito vectors. Maximum efficacy was detected against A. stephensi (LC50=12.39μg/mL), followed by Ae. aegypti (LC50=13.61μg/mL) and Cx. quinquefasciatus (LC50=14.79μg/mL). Interestingly, Ag NPs were safer for the non-target aquatic organism Diplonychus indicus sharing the same aquatic habitats of mosquito larvae. LC50 and LC90 values were 1032.81 and 19,076.59μg/mL, respectively. Overall, our results highlight that N. physalodes-fabricated Ag NPs are a promising for development of eco-friendly larvicides against mosquito vectors, with negligible toxicity against non-target aquatic water bugs.

  9. 鼠胰岛素样生长因子-1重组腺病毒的构建及其在胰岛β细胞的表达%Construction of Recombinant Adenovirus Vector Containing Rat Insulin-like Growth Factor 1 Gene and Its Expression in Islet β-cells

    陈志红; 李堂; 胡素娟; 王桂琴


    This study was aimed to construct the recombinant adenovirus containing rat insulin-like growth factor 1(rIGF-1),and then to investigate its expression in islet β-cells. RNA was extracted using Trizol from rat livers. rIGF-1 cDNA was obtained using RT-PCR. The purified RT-PCR products and pAdTrack-CMV were digested using Bgl II and EcoR V and religated by T4 DNA ligase,then transformed into electro-competent JM109 bacteria and selected on Kanamycin LB plates. This plasmid pAd-CMV-rIGF-1 was linearized by PmeI and co-transformed into electro-competent BJ5183 bacteria with pAdEasy-1 and selected on Kanamycin LB plates. After having been screened,the extracted plasmid of positive bacteria was transfected into HEK 293 cells with liposome and was identified by the green fluorescence protein (GFP) expression. The recombinant adenovirus encoding rIGF-1 was named Ad-rIGF-1,and the viral particles were further amplified,purified,and its titer was about 4.0×10~8pfu/ml. Ad-rIGF-1 was transfected into rat pancreatic β cell lines-RINm5F cells,RT-PCR was carried out to detect the transfer genes. rIGF-1 protein in cells culture supernatants was detected by ELISA method,and its concentration was 91.6±26.8ng/ml. rIGF-1 was present in Ad-rIGF-1-infected RINm5F cells as measured by Western blotting. The recombinant adenovirus vector containing rIGF-1 was constructed successfully,and the rIGF-1 protein was expressed by RINm5F cells. This method provided the mechanism of rIGF-1 to prevent β cell from impairmentand to treat the case of type 1 diabetes.%构建含有鼠胰岛素样生长因子1(rIGF-1)重组腺病毒,研究其在胰岛β细胞的表达.Trizol 一步法提取大鼠肝脏组织总RNA,RT-PCR法合成鼠胰岛素样生长因1(rIGF-1) cDNA;PCR纯化产物和pAdTrack-CMV分别经BglⅡ和EcoRⅤ双酶切后纯化,T4 DNA 连接酶连接,转化JM109感受态细菌,卡那霉素平板筛选阳性克隆扩增培养,抽提质粒;经BglⅡ和EcoRⅤ双酶切及测序鉴定后,

  10. Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

    Bilbao, R; Reay, D P; Hughes, T; Biermann, V; Volpers, C; Goldberg, L; Bergelson, J; Kochanek, S; Clemens, P R


    High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle.

  11. Research and development of cancer-targeting vectors: An exploration on oncotropism and oncosuppression of autonomous parvoviruses


    Autonomous parvoviruses are single-stranded DNA viruses with a genome size of about 5 kb. They are characterized by their oncotropism and oncosuppression to the hosts. After infection the viral genes are not integrated into the chromosome of host cells. Autonomous parvoviruses could be used as potential vectors carrying heterologous therapeutic genes for the construction of recombinant autonomous parvoviruses. The specific advantages of these viral vectors to cancer gene therapy are discussed.

  12. One-oligonucleotide method for constructing vectors for RNA interference



    Aim: To develop an easy, fast, automated, and inexpensive method for constructing short-hairpin-RNA cassettes for RNAi studies. Methods: Using single oligonucleotides, a variety of DNA cassettes for RNAi vectors were constructed in only few minutes in an automated manner. The cassettes, targeting the eGFP,were cloned into plasmids driven by RNA polymerase Ⅲ promoter H 1. Then, the plasmids were transfected into HeLa cells that were later infected with a recombinant adenovirus encoding the eGFP gene. The level of eGFP fluorescence was evaluated by confocal imaging and flow cytometry. Results: The plasmids constructed with the DNA cassettes made by the one-oligonucleotide method inhibited eGFP with different potencies, ranging from 55% to 75%. Conclusion: By using the method reported here, it is possible to simultaneously construct hundreds of different DNA cassettes for RNAi experiments in an inexpensive, automated way. This method will facilitate functional genomics studies on mammalian cells.

  13. Inhibitory Effect of Pulmonary Carcinoma by Adenovirus-Mediated CD/UPRT Gene

    HUANG Qi; CHEN Dayu; FU Xiangning; ZU Yukun


    The cell killing effects and bystander effects of double suicide gene on pulmonary carcinoma cells were explored. Lung adenocarcinoma cells (A549) were transfected with different titers of adenovirus vector and followed with different concentrations of 5-FC after a recombinant adenovirus vector carrying CD/UPRT gene (Ad-CD/UPRT) was constructed. The cell viability was measured by MTT assay 4 days later. The cell viability was dropped to 30.57 %-8.62 % after 10 MOI of Ad-CD/UPRT transfected and 5-FC (10-1000 μg/mL) administration. Furthermore, Ad-CD/UPRT-infected A549 cells showed a profound neighbor cell killing effect in the same methods. These results suggested that Ad-CD/UPRT/5-FC system can effectively suppress growth of lung adenocarcinoma cells, which may provide a novel and powerful candidate for lung cancer gene therapy strategies.

  14. Anti-Viral Drugs for Human Adenoviruses

    Chor Wing Sing


    Full Text Available There are many stages in the development of a new drug for viral infection and such processes are even further complicated for adenovirus by the fact that there are at least 51 serotypes, forming six distinct groups (A–F, with different degree of infectivity. This review attempts to address the importance of developing pharmaceuticals for adenovirus and also review recent development in drug discovery for adenovirus, including newer strategies such as microRNA approaches. Different drug screening strategies will also be discussed.

  15. Mechanism of ad5 vaccine immunity and toxicity: fiber shaft targeting of dendritic cells.

    Cheng Cheng


    Full Text Available Recombinant adenoviral (rAd vectors elicit potent cellular and humoral immune responses and show promise as vaccines for HIV-1, Ebola virus, tuberculosis, malaria, and other infections. These vectors are now widely used and have been generally well tolerated in vaccine and gene therapy clinical trials, with many thousands of people exposed. At the same time, dose-limiting adverse responses have been observed, including transient low-grade fevers and a prior human gene therapy fatality, after systemic high-dose recombinant adenovirus serotype 5 (rAd5 vector administration in a human gene therapy trial. The mechanism responsible for these effects is poorly understood. Here, we define the mechanism by which Ad5 targets immune cells that stimulate adaptive immunity. rAd5 tropism for dendritic cells (DCs was independent of the coxsackievirus and adenovirus receptor (CAR, its primary receptor or the secondary integrin RGD receptor, and was mediated instead by a heparin-sensitive receptor recognized by a distinct segment of the Ad5 fiber, the shaft. rAd vectors with CAR and RGD mutations did not infect a variety of epithelial and fibroblast cell types but retained their ability to transfect several DC types and stimulated adaptive immune responses in mice. Notably, the pyrogenic response to the administration of rAd5 also localized to the shaft region, suggesting that this interaction elicits both protective immunity and vector-induced fevers. The ability of replication-defective rAd5 viruses to elicit potent immune responses is mediated by a heparin-sensitive receptor that interacts with the Ad5 fiber shaft. Mutant CAR and RGD rAd vectors target several DC and mononuclear subsets and induce both adaptive immunity and toxicity. Understanding of these interactions facilitates the development of vectors that target DCs through alternative receptors that can improve safety while retaining the immunogenicity of rAd vaccines.

  16. High efficient generation of replication-defective adenoviruses containing thymidine kinase by homogeneous recombination in bacteria

    CONG Tie-chuan; LU Zhe-ming; LI Yong; ZHENG Li; QIN Yong


    Background Suicide gene therapy is a widely used molecular treatment for head and neck cancer. In this study, we try to use the method of homogenous recombination in bacteria to clone thymidine kinase gene (tk)-a kind of suicide gene to adenovirus backbone vectors for the construction of replication-defective adenoviruses.Methods pAdTrack-CMV/tk was constructed through subclone of a restriction endonuclease fragment including thymidine kinase gene from plasmid pCMV-tk to another plasmid pAdTrack-CMV, and then co-transfected with supercoiled pAdEasy-1, which was an adenoviral backbone vector except for deletions of E1 and E3, to competent E.coli BJ5183 for homogenous recombination using electroporation procedure. With the same method, pAdTrack-CMV was also co-transformed with pAdEasy-1 for homogenous recombination in BJ5183. Identified with restriction endonuclease Pacl and polymerase chain reaction (PCR), plasmids pAd-GFP/tk and pAd-GFP were successfully constructed. Each of them was digested with Pacl and sequently transfected into human embryo kidney 293 cells (HEK293) using Lipofectamine 2000.Results Comet-like adenovirus-producing foci of Ad-GFP/tk and Ad-GFP were observed after 5 to 7 days of cell culture.After twelve days of packaging, the replication-defective adenoviruses were collected. Identified with PCR, thymidine kinase gene was successfully constructed into Ad-GFP/tk.Conclusion The replication-defective adenoviruses containing thymidine kinase can be constructed more easily by homogenous recombination in bacteria than conventional techniques.

  17. 绵羊FecB基因打靶载体构建%Construction of a new gene targeting vector in the sheep

    吴伟伟; 王力俭; 田可川; 徐新明; 张廷虎; 张艳花; 哈尼克孜


    In order to construct a sheep FecB gene targeting vector, plasmids of peGFP-Cl, pRET. IL. PGK-TK and pMD19-T Simple and pMD19-T Simple cloning vector as the carrier frame with a positive selection marker eGFP gene and a negative selection marker gene HSV-tk gene were used. The correct structure of the targeting vector was confirmed by restriction enzyme digestion and sequencing analysis. Our experiment indicated that a FecB gene targeting vector of sheep was successfully constructed.%利用peGFP-C1,pRET.IL.PGK-TK和pMD19-T Simple等基本质粒,构建绵羊FecB基因打靶载体.以克隆载体pMD19-T Simple为载体骨架,插入一个正选择标记eGFP基因、一个负选择标记基因HSV-tk基因,并在eGFP基因两侧各添加一个同源长臂和同源短臂,从而构建绵羊FecB基因打靶载体.结果:经多个限制性内切核酸酶酶切鉴定和测序证实,所构建的基因打靶载体符合设计要求,表明成功构建了绵羊FecB基因打靶载体.

  18. Adenoviral vectors stimulate glucagon transcription in human mesenchymal stem cells expressing pancreatic transcription factors.

    Arnaud Zaldumbide

    Full Text Available Viral gene carriers are being widely used as gene transfer systems in (transdifferentiation and reprogramming strategies. Forced expression of key regulators of pancreatic differentiation in stem cells, liver cells, pancreatic duct cells, or cells from the exocrine pancreas, can lead to the initiation of endocrine pancreatic differentiation. While several viral vector systems have been employed in such studies, the results reported with adenovirus vectors have been the most promising in vitro and in vivo. In this study, we examined whether the viral vector system itself could impact the differentiation capacity of human bone-marrow derived mesenchymal stem cells (hMSCs toward the endocrine lineage. Lentivirus-mediated expression of Pdx-1, Ngn-3, and Maf-A alone or in combination does not lead to robust expression of any of the endocrine hormones (i.e. insulin, glucagon and somatostatin in hMSCs. Remarkably, subsequent transduction of these genetically modified cells with an irrelevant early region 1 (E1-deleted adenoviral vector potentiates the differentiation stimulus and promotes glucagon gene expression in hMSCs by affecting the chromatin structure. This adenovirus stimulation was observed upon infection with an E1-deleted adenovirus vector, but not after exposure to helper-dependent adenovirus vectors, pointing at the involvement of genes retained in the E1-deleted adenovirus vector in this phenomenon. Lentivirus mediated expression of the adenovirus E4-ORF3 mimics the adenovirus effect. From these data we conclude that E1-deleted adenoviral vectors are not inert gene-transfer vectors and contribute to the modulation of the cellular differentiation pathways.

  19. Modulation of breast cancer resistance protein mediated atypical multidrug resistance using RNA interference delivered by adenovirus

    LI Wen-tong; ZHOU Geng-yin; WANG Chun-ling; GUO Cheng-hao; SONG Xian-rang; CHI Wei-ling


    @@ Clinical multidrug resistance (MDR) of malignancies to many antineoplastic agents is the major obstacle in the successful treatment of cancer. The emergence of breast cancer resistance protein (BCRP), a member of the adenosine triphosphate (ATP) binding cassette (ABC) transporter family, has necessitated the development of antagonists. To overcome the BCRP-mediated atypical MDR, RNA interference (RNAi) delivered by adenovirus targeting BCRP mRNA was used to inhibit the atypical MDR expression by infecting MCF-7/MX100 cell lines with constructed RNAi adenovirus.

  20. New Conditionally Replicating Adenovirus Vectors for Breast Cancer Therapy


    Baculovirus Construct Description of Mutation I664V Ad5 Pol – pAcgp67A Protein Production in SF9 insect cells I664M Ad5 Pol – pAcgp67A “” I664S...out in SF9 insect cells (Figure 3). Figure 3: Expression of Ad pol proteins in insect cells LEGEND: 3µg of recombinant baculovirus DNA was...transfected with Cellfectin (Invitrogen) in SF9 insect cells. Three days later the recombinant baculoviruses were collected and used for viral

  1. 表达人IL-17F重组腺病毒载体的构建及其表达产物的功能研究%Construction of recombinant adenovirus vector expressing hIL-17F and functional study of expressed IL-17 F

    盛伟华; 任苏勤; 谢宇锋; 缪竞诚; 刘铁连; 杨吉成


    目的构建表达人白细胞介素IL-17F (hIL-17F)的重组腺病毒载体(Ad-hIL-17F),为进行hIL-17F基因表达抑制血管形成和抑瘤效应的研究奠定基础。方法以pUCm-T/hIL-17F重组质粒为模板PCR扩增hIL-17F,酶切连接到带有GFP标记的pAdTrack-CMV转移质粒上,PmeⅠ线性化重组质粒pAdTrack-CMV-hIL-17F,与腺病毒骨架质粒pAdEasy-1共转化BJ5183细菌,经同源重组,获得同源重组腺病毒质粒pAdEasy-1-pTrack-CMV-hIL-17F经PacⅠ线性化后转染QBI-293A细胞,收获Ad-hIL-17F,用RT-PCR和间接免疫荧光法鉴定人IL-17F基因表达。 MTT法检测hIL-17F基因表达对ECV304细胞的生长抑制作用, ELISA法检测人血管内皮生长因子( VEGF)和血管生成素( Ang-1)基因在293A细胞、ECV304细胞中的表达水平;实时定量RT-PCR检测Ad-hIL-17F对293A细胞中人VEGF转录的影响。结果测序显示hIL-17F序列正确,RT-PCR和间接免疫荧光法检测到了IL-17 F基因的表达。 Ad-hIL-17 F能显著抑制ECV304细胞的生长,抑制人VEGF和Ang-1基因在293 A细胞、ECV304细胞中的表达。结论成功构建和获得了hIL-17F的重组腺病毒载体(Ad-hIL-17F), Ad-hIL-17 F可通过下调VEGF和Ang-1的分泌而抑制血管形成。%Objective To construct a recombinant adenovirus vector ( Ad-hIL-17F) expressing human interleukin 17F (hIL-17F) and to investigate the effects of expressed hIL-17F on angiogenesis. Methods The hIL-17F fragments was amplified by PCR using pUCm-T/hIL-17F plasmids as templates and then cloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-hIL-17F.The pAdTrack-CMV-hIL-17F transfer vector was linearized with PmeI digestion and then transformed into competent BJ 5183 with pAdEasy-1 backbone vector for homologous recombination .Then it was linearized with PacI digestion and transfected into human embryonic kidney 293 (QBI-293A) cells to construct Ad-hIL-17F.RT-PCR analysis and indirect

  2. Development of Nonviral Vectors Targeting the Brain as a Therapeutic Approach For Parkinson's Disease and Other Brain Disorders.

    Javed, Hayate; Menon, Sindhu A; Al-Mansoori, Karima M; Al-Wandi, Abdelmojib; Majbour, Nour K; Ardah, Mustafa T; Varghese, Shiji; Vaikath, Nishant N; Haque, M Emdadul; Azzouz, Mimoun; El-Agnaf, Omar Ma


    Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by tremor, rigidity, bradykinesia, and postural instability, for which there is no effective treatment available till date. Here, we report the development of nonviral vectors specific for neuronal cells that can deliver short interfering RNA (siRNA) against the α-synuclein gene (SNCA), and prevent PD-like symptoms both in vitro and in vivo. These vectors not only help siRNA duplexes cross the blood-brain barrier in mice, but also stabilize these siRNAs leading to a sustainable 60-90% knockdown of α-synuclein protein. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine rapidly develop PD-like symptoms which were significantly alleviated when SNCA was knocked down using our vectors. Together, our data not only confirm the central role of α-synuclein in the onset of PD, but also provide a proof of principle that these nonviral vectors can be used as novel tools to design effective strategies to combat central nervous system diseases.

  3. Critical Role of Autophagy in the Processing of Adenovirus Capsid-Incorporated Cancer-Specific Antigens.

    Sarah R Klein

    Full Text Available Adenoviruses are highly immunogenic and are being examined as potential vectors for immunotherapy. Infection by oncolytic adenovirus is followed by massive autophagy in cancer cells. Here, we hypothesize that autophagy regulates the processing of adenoviral proteins for antigen presentation. To test this hypothesis, we first examined the presentation of viral antigens by infected cells using an antibody cocktail of viral capsid proteins. We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation. Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner. We then hypothesize that this mechanism can be utilized to generate an efficient cancer vaccine. To this end, we constructed an oncolytic virus encompassing an EGFRvIII cancer-specific epitope in the adenoviral fiber. Infection of cancer cells with this fiber-modified adenovirus resulted in recognition of infected cancer cells by a specific anti-EGFRvIII antibody. However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells. Our data suggest that combination of adenoviruses with autophagy inducers may enhance the processing and presentation of cancer-specific antigens incorporated into capsid proteins.

  4. Tumor growth inhibition effect of hIL-6 on colon cancer cells transfected with the target gene by retroviral vector

    Bing Xiao; Bo Jing; Ya Li Zhang; Dian Yuan Zhou; Wan Dai Zhang


    AIM To observe the tumor inhibitory effects by transfecting IL-6 cDNA into colon cancer cell line HT-29 with retroviral vector pZIP cDNA.METHODS Human IL-6 gene was reconstructed in retrovirus vector and transfected into incasing cells PA317 by lipofectamine mediated method,the clones of the cells transferred with hIL-6 were selected by G418, and targeted HT-29 cells were infected with the virus granules secreted from PA317 and also selected by G418. Test gene transcription and expression level by hybridization, ELISA and MTT assay, etc.Analyze tumor inhibitory effects according to the cell growth curve, plating forming rate and tumorigenicity in nude mice.RESULT Successfully constructed and transfected recombinant expressing vectors pZIPIL-6 cDNA and got positive transfected cell lines. The colon cancer cell line (HT-29 IL-6)transfected with the hIL-6 gene by retroviral vector was established. The log proliferation period and the doubling time of this cell line was between 4 to 7 days and 2.5 days according to the direct cell count, the cell proliferation was obviously inhibited with MTT assay, the plating inhibitory rate was 50% by plating efficiency test. When HT-29 IL-6 cells were inoculated into the nude mice subcutaneously, carcinogenic activity of the solid tumor was found superior to the control group and the size of tumor was not significantly enlarged. Injection of combination virus fluid containing IL-6 gene into transplantation tumors could inhibit the growth and development of the tumor.CONCLUSION IL-6 could inhibit the growth and proliferation of colon cancer cells by retroviral vector-mediated transduction.

  5. Adenovirus-mediated shRNA interference against HSV-1 replication in vitro.

    Song, Bo; Liu, Xinjing; Wang, Qingzhi; Zhang, Rui; Yang, Ting; Han, Zhiqiang; Xu, Yuming


    The UL29 and UL28 proteins encoded by herpes simplex virus type 1 (HSV-1) are critical for its replication and packaging, respectively. Research has demonstrated that synthesized siRNA molecules targeting the UL29 gene are able to suppress HSV-2 replication and the UL28-null HSV-1 gene cannot form infectious viruses in vitro. Silencing the UL28 and UL29 genes by RNAi might lead to the development of novel antiviral agents for the treatment of HSV-1 infections. Two kinds of short hairpin RNAs (shRNAs) targeting the UL29 and UL28 genes were chemically synthesized and then delivered into cells by a replication-defective human adenovirus type 5 (Adv5) vector. (-) shRNAs targeting none of the genome of HSV-1 were used as the control. Vero cells were inoculated with Ad-UL28shRNA or Ad-UL29shRNA at a multiplicity of infection (MOI) of 100 and challenged 24 h later with HSV-1 at an MOI of 0.01 to inhibit HSV-1 replication, as measured by the level of the corresponding RNA and proteins. In addition, the amount of progeny virus was assessed at daily intervals. The antiviral effects of Ad-shRNAs at ongoing HSV-1 infection were explored at 12 h after inoculation of the HSV-1. The results showed that the shRNAs delivered by Adv5 significantly suppressed HSV-1 replication in vitro, as determined by the levels of viral RNA transcription, viral protein synthesis, and viral production. The Ad-UL28shRNA and Ad-UL29shRNA suppressed the replication of HSV-1, respectively, compared with the control group (P HSV-1 infection (P HSV-1 infection.

  6. Enfermedad neurologica por adenovirus Neurologic disease due to adenovirus infection

    Cristina L. Lema


    Full Text Available El objetivo de este trabajo fue determinar la prevalencia de adenovirus (ADV en las infecciones del sistema nervioso central (SNC. Se analizaron 108 muestras de líquido cefalorraquídeo (LCR provenientes de 79 casos de encefalitis, 7 meningitis y 22 de otras patologías neurológicas, recibidas en el período 2000-2002. Cuarenta y nueve (47.35% se obtuvieron de pacientes inmunocomprometidos. La presencia de ADV se investigó mediante reacción en cadena de la polimerasa en formato anidado (Nested-PCR. La identificación del genogrupo se realizó mediante análisis filogenético de la secuencia nucleotídica parcial de la región que codifica para la proteína del hexón. Se detectó la presencia de ADV en 6 de 108 (5.5% muestras de LCR analizadas. Todos los casos positivos pertenecieron a pacientes con encefalitis que fueron 79, (6/79, 7.6%. No se observó diferencia estadísticamente significativa entre los casos de infección por ADV en pacientes inmunocomprometidos e inmunocompetentes (p>0.05. Las cepas de ADV detectadas se agruparon en los genogrupos B1 y C. En conclusión, nuestros resultados describen el rol de los ADV en las infecciones neurológicas en Argentina. La información presentada contribuye al conocimiento de su epidemiología, en particular en casos de encefalitis.The aim of this study was to assess the prevalence of adenovirusm (ADV infections in neurological disorders. A total of 108 cerebrospinal fluid (CSF samples from 79 encephalitis cases, 7 meningitis and 22 other neurological diseases analysed in our laboratory between 2000 and 2002 were studied. Forty nine (47.4% belonged to immunocompromised patients. Viral genome was detected using nested polymerase chain reaction (Nested-PCR and ADV genotypes were identified using partial gene sequence analysis of hexon gene. Adenovirus were detected in 6 of 108 (5.5% CSF samples tested. All of these were from encephalitis cases, 6/79, representing 7.6% of them. No statistically

  7. Improving Adenovirus Based Gene Transfer: Strategies to Accomplish Immune Evasion

    Andrea Amalfitano


    Full Text Available Adenovirus (Ad based gene transfer vectors continue to be the platform of choice for an increasing number of clinical trials worldwide. In fact, within the last five years, the number of clinical trials that utilize Ad based vectors has doubled, indicating growing enthusiasm for the numerous positive characteristics of this gene transfer platform. For example, Ad vectors can be easily and relatively inexpensively produced to high titers in a cGMP compliant manner, can be stably stored and transported, and have a broad applicability for a wide range of clinical conditions, including both gene therapy and vaccine applications. Ad vector based gene transfer will become more useful as strategies to counteract innate and/or pre-existing adaptive immune responses to Ads are developed and confirmed to be efficacious. The approaches attempting to overcome these limitations can be divided into two broad categories: pre-emptive immune modulation of the host, and selective modification of the Ad vector itself. The first category of methods includes the use of immunosuppressive drugs or specific compounds to block important immune pathways, which are known to be induced by Ads. The second category comprises several innovative strategies inclusive of: (1 Ad-capsid-display of specific inhibitors or ligands; (2 covalent modifications of the entire Ad vector capsid moiety; (3 the use of tissue specific promoters and local administration routes; (4 the use of genome modified Ads; and (5 the development of chimeric or alternative serotype Ads. This review article will focus on both the promise and the limitations of each of these immune evasion strategies, and in the process delineate future directions in developing safer and more efficacious Ad-based gene transfer strategies.

  8. Coupling of a bifunctional peptide R13 to OTMCS-PEI copolymer as a gene vector increases transfection efficiency and tumor targeting

    Lv H


    Full Text Available Hui Lv,1,* Qing Zhu,1,* Kewu Liu,2 Manman Zhu,1 Wenfang Zhao,1 Yuan Mao,1 Kehai Liu1 1Department of Biopharmaceutics, College of Food Science and Technology, Shanghai Ocean University, Shanghai, People's Republic of China; 2Heilongjiang Forest By-Product and Speciality Institute, Mudanjiang, People's Republic of China *These authors contributed equally to this work Background: A degradable polyethylenimine (PEI derivative coupled to a bifunctional peptide R13 was developed to solve the transfection efficiency versus cytotoxicity and tumor-targeting problems of PEI when used as a gene vector. Methods: We crossed-linked low molecular weight PEI with N-octyl-N-quaternary chitosan (OTMCS to synthesize a degradable PEI derivative (OTMCS-PEI, and then used a bifunctional peptide, RGDC-Tat (49–57 called R13 to modify OTMCS-PEI so as to prepare a new gene vector, OTMCS-PEI-R13. This new gene vector was characterized by various physicochemical methods. Its cytotoxicity and gene transfection efficiency were also determined both in vitro and in vivo. Results: The vector showed controlled degradation and excellent buffering capacity. The particle size of the OTMCS-PEI-R13/DNA complexes was around 150–250 nm and the zeta potential ranged from 10 mV to 30 mV. The polymer could protect plasmid DNA from being digested by DNase I at a concentration of 23.5 U DNase I/µg DNA. Further, the polymer was resistant to dissociation induced by 50% fetal bovine serum and 400 µg/mL sodium heparin. Compared with PEI 25 kDa, the OTMCS-PEI-R13/DNA complexes showed higher transfection efficiency both in vitro and in vivo. Further, compared with OTMCS-PEI, distribution of OTMCS-PEI-R13 at tumor sites was markedly enhanced, indicating the tumor-targeting specificity of R13. Conclusion: OTMCS-PEI-R13 could be a potential candidate as a safe and efficient gene delivery carrier for gene therapy. Keywords: nonviral gene vector, polyethylenimine, R13, transfection efficiency

  9. Interaction between mouse adenovirus type 1 and cell surface heparan sulfate proteoglycans.

    Liesbeth Lenaerts

    Full Text Available Application of human adenovirus type 5 (Ad5 derived vectors for cancer gene therapy has been limited by the poor cell surface expression, on some tumor cell types, of the primary Ad5 receptor, the coxsackie-adenovirus-receptor (CAR, as well as the accumulation of Ad5 in the liver following interaction with blood coagulation factor X (FX and subsequent tethering of the FX-Ad5 complex to heparan sulfate proteoglycan (HSPG on liver cells. As an alternative vector, mouse adenovirus type 1 (MAV-1 is particularly attractive, since this non-human adenovirus displays pronounced endothelial cell tropism and does not use CAR as a cellular attachment receptor. We here demonstrate that MAV-1 uses cell surface heparan sulfate proteoglycans (HSPGs as primary cellular attachment receptor. Direct binding of MAV-1 to heparan sulfate-coated plates proved to be markedly more efficient compared to that of Ad5. Experiments with modified heparins revealed that the interaction of MAV-1 to HSPGs depends on their N-sulfation and, to a lesser extent, 6-O-sulfation rate. Whereas the interaction between Ad5 and HSPGs was enhanced by FX, this was not the case for MAV-1. A slot blot assay demonstrated the ability of MAV-1 to directly interact with FX, although the amount of FX complexed to MAV-1 was much lower than observed for Ad5. Analysis of the binding of MAV-1 and Ad5 to the NCI-60 panel of different human tumor cell lines revealed the preference of MAV-1 for ovarian carcinoma cells. Together, the data presented here enlarge our insight into the HSPG receptor usage of MAV-1 and support the development of an MAV-1-derived gene vector for human cancer therapy.

  10. In vitro effect of p21WAF-1/CIP1 gene on growth of human glioma cells mediated by EGFR targeted non-viral vector GE7 system

    陈永新; 许秀兰; 张光霁; 王韦; 金海英; 卢亦成; 朱诚; 顾健人


    Objective: To construct the EGFR targeted non-viral vector GE7 system and explore the in vitro effect of p21WAF-1/CIP1 gene on growth of human glioma cells mediated by the GE7 system. Methods: The EGFR targeted non-viral vector GE7 gene delivery system was constructed. The malignant human glioma cell line U251MG was transfected in vitro with β-galactosidase gene(reporter gene) and p21WAF-1/CIP1 gene (therapeutic gene) using the GE7 system. By means of X-gal staining, MTS and FACS, the transfection efficiency of exogenous gene and apoptosis rate of tumor cells were examined. The expression of p21WAF-1/CIP1 gene in transfected U251MG cell was examined by immunohistochemistry staining. Results: The highest transfer rate of exogenous gene was 70%. After transfection with p21WAF-1/CIP1 gene, the expression of WAF-1 increased remarkably and steadily; the growth of U251MG cells were inhibited evidently. FACS examination showed G1 arrest. The average apoptosis rate was 25.2%. Conclusion: GE7 system has the ability to transfer exogenous gene to targeted cells efficiently, and expression of p21WAF-1/CIP1 gene can induce apoptosis of glioma cell and inhibit its growth.

  11. Priorities and needs for research on urban interventions targeting vector-borne diseases: rapid review of scoping and systematic reviews

    Bermudez-Tamayo, Clara; Mukamana, Olive; Carabali, Mabel; Osorio, Lyda; Fournet, Florence; Dabiré, Kounbobr Roch; Turchi Marteli, Celina; Contreras, Adolfo; Ridde, Valéry


    This paper highlights the critical importance of evidence on vector-borne diseases (VBD) prevention and control interventions in urban settings when assessing current and future needs, with a view to setting policy priorities that promote inclusive and equitable urban health services. Research should produce knowledge about policies and interventions that are intended to control and prevent VBDs at the population level and to reduce inequities. Such interventions include policy, program, and ...

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

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


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

  13. 表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用%The antitumor effects of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy in nude mouse model

    薛松; 刘斌; 高超; 顾玉明; 郑骏年; 李望; 李海龙; 朱海涛; 辛勇; 刘俊杰; 徐为; 宋文哲


    目的 观察表达针对端粒酶逆转录酶(hTERT)基因的小干扰RNA(hTERT siRNA)的溶瘤腺病毒(ZD55-hTERT)抑制肾癌移植瘤生长作用.方法 荷肾癌裸鼠随机分4组,每组8只.瘤体内分别注射ZD55-hTERT、增殖缺陷型腺病毒(Ad-hTERT)、溶瘤腺病毒ZD55-EGFP及磷酸盐缓冲液(PBS),每次注射病毒7×108pfu/只,连续注射3 d.注射后第7天,每组处死3只取肿瘤组织,免疫组织化学检测肿瘤hTERT、E1A表达及凋亡.第50天时处死动物测量肿瘤体积.结果 ZD55-hTERT、Ad-hTERT、ZD55-EGFP及PBS处理组肿瘤体积(mm3)分别为:124.1±27.5、609.0±102.5、499.8±77.1、1552.1±206.4,ZD55-hTERT处理组与各组之间差异有统计学意义(P<0.01).Ad-hTERT处理组肿瘤无E1A表达,ZD55-hTERT处理组E1A大量表达,表明病毒复制.ZD55-hTERT处理组肿瘤hTERT表达显著低于Ad-hTERT处理组,凋亡细胞阳性率均显著高于Ad-hTERT处理组.结论 表达hTERT siRNA的溶瘤腺病毒ZD55-hTERT具有更强的抑制肾癌生长作用.%Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups

  14. The Use of Viral Vectors in Gene Transfer Therapy

    A. Dziaková


    Full Text Available Gene therapy is strategy based on using genes as pharmaceuticals. Gene therapy is a treatment that involves altering the genes inside body's cells to stop disease. Genes contain DNA- the code controlling body form and function. Genes that do not work properly can cause disease. Gene therapy replaces a faulty gene or adds a new gene in an attempt to cure disease or improve the ability of the body to fight disease. Gene therapy holds promise for treating a wide range of diseases, including cancer, cystic fibrosis, heart disease, diabetes, hemophilia and AIDS. Various types of genetic material are used in gene therapy; double-stranded DNA (dsDNA, single-stranded DNA (ssDNA, plasmid DNA and antisense oligodeoxynucleotides (ASON. The success of gene therapy depends on assuring the entrance of the therapeutic gene to targeted cells without any form of biodegradation. Commonly used vectors in gene therapy are: adenoviruses (400 clinical studies; 23.8%, retroviruses (344 clinical studies; 20.5%, unenveloped/plasmid DNA (304 clinical studies, 17.7%, adeno-associated viruses (75 clinical studies; 4.5% and others. In this paper, we have reviewed the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors.

  15. Tumor-Specific Targeting With Modified Sindbis Viral Vectors: Evaluation with Optical Imaging and Positron Emission Tomography In Vivo

    Stelter, Lars; Tseng, Jen-Chieh; Torosjan, Armen; Levin, Brandi; Longo, Valerie A.; Pillarsetty, Nagavarakishore; Zanzonico, Pat; Meruelo, Daniel; Daniel, Steven M.


    Purpose Sindbis virus (SINV) infect tumor cells specifically and systemically throughout the body. Sindbis vectors are capable of expressing high levels of transduced suicide genes and thus efficiently produce enzymes for prodrug conversion in infected tumor cells. The ability to monitor suicide gene expression levels and viral load in patients, after administration of the vectors, would significantly enhance this tumor-specific therapeutic option. Procedures The tumor specificity of SINV is mediated by the 67-kDa laminin receptor (LR). We probed different cancer cell lines for their LR expression and, to determine the specific role of LR-expression in the infection cycle, used different molecular imaging strategies, such as bioluminescence, fluorescence molecular tomography, and positron emission tomography, to evaluate SINV-mediated infection in vitro and in vivo. Results All cancer cell lines showed a marked expression of LR. The infection rates of the SINV particles, however, differed significantly among the cell lines. Conclusion We used novel molecular imaging techniques to visualize vector delivery to different neoplatic cells. SINV infection rates proofed to be not solely dependent on cellular LR expression. Further studies need to evaluate the herein discussed ways of cellular infection and viral replication. PMID:22847302

  16. Efficient downregulation of multiple mRNA targets with a single shRNA-expressing lentiviral vector.

    Chumakov, Stepan P; Kravchenko, Julia E; Prassolov, Vladimir S; Frolova, Elena I; Chumakov, Peter M


    Gene silencing based on RNA interference is widely used in fundamental research and in practical applications. However, a commonly incomplete functional suppression represents a serious drawback of this technology. We describe a series of lentiviral vectors each containing a single or multiple shRNA-expression cassette(s) driven by a RNA-polymerase III specific promoter and localized within the 3'-LTR of the lentiviral DNA backbone. The vectors also contain an antibiotic-resistance gene that allows positive selection of recipient cells. The combined expression of three different shRNAs specific to a single mRNA was shown to improve dramatically the level of mRNA inhibition, while the use of three different RNA-polymerase III specific promoters avoids the loss of shRNA-expression cassettes through the homologous recombination. The vector system was used for successful simultaneous suppression of three related SESN1, SESN2 and SESN3 genes, which suggests its particular value for testing phenotypes of functionally redundant genes.

  17. Molecular epidemiological study of adenovirus infecting western lowland gorillas and humans in and around Moukalaba-Doudou National Park (Gabon).

    Nkogue, Chimène Nze; Horie, Masayuki; Fujita, Shiho; Ogino, Michiko; Kobayashi, Yuki; Mizukami, Keijiro; Masatani, Tatsunori; Ezzikouri, Sayeh; Matsuu, Aya; Mizutani, Tetsuya; Ozawa, Makoto; Yamato, Osamu; Ngomanda, Alfred; Yamagiwa, Juichi; Tsukiyama-Kohara, Kyoko


    Adenoviruses are widespread in human population as well as in great apes, although the data about the naturally occurring adenovirus infections remain rare. We conducted the surveillance of adenovirus infection in wild western lowland gorillas in Moukalaba-Doudou National Park (Gabon), in order to investigate naturally occurring adenovirus in target gorillas and tested specifically a possible zoonotic transmission with local people inhabiting the vicinity of the park. Fecal samples were collected from western lowland gorillas and humans, and analyzed by PCR. We detected adenoviral genes in samples from both gorillas and the local people living around the national park, respectively: the overall prevalence rates of adenovirus were 24.1 and 35.0 % in gorillas and humans, respectively. Sequencing revealed that the adenoviruses detected in the gorillas were members of Human mastadenovirus B (HAdV-B), HAdV-C, or HAdV-E, and those in the humans belonged to HAdV-C or HAdV-D. Although HAdV-C members were detected in both gorillas and humans, phylogenetic analysis revealed that the virus detected in gorillas are genetically distinct from those detected in humans. The HAdV-C constitutes a single host lineage which is compatible with the host-pathogen divergence. However, HAdV-B and HAdV-E are constituted by multiple host lineages. Moreover, there is no evidence of zoonotic transmission thus far. Since the gorilla-to-human transmission of adenovirus has been shown before, the current monitoring should be continued in a broader scale for getting more insights in the natural history of naturally occurring adenoviruses and for the safe management of gorillas' populations.

  18. Regulation of human adenovirus replication by RNA interference

    Nikitenko, N. A.; SPEISEDER T.; Lam, E; Rubtsov, P. M.; TONAEVA KH. D.; S. A. Borzenok; Dobner, T; Prassolov, V.S.


    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to...

  19. Plant microRNA-target interaction identification model based on the integration of prediction tools and support vector machine.

    Jun Meng

    Full Text Available Confident identification of microRNA-target interactions is significant for studying the function of microRNA (miRNA. Although some computational miRNA target prediction methods have been proposed for plants, results of various methods tend to be inconsistent and usually lead to more false positive. To address these issues, we developed an integrated model for identifying plant miRNA-target interactions.Three online miRNA target prediction toolkits and machine learning algorithms were integrated to identify and analyze Arabidopsis thaliana miRNA-target interactions. Principle component analysis (PCA feature extraction and self-training technology were introduced to improve the performance. Results showed that the proposed model outperformed the previously existing methods. The results were validated by using degradome sequencing supported Arabidopsis thaliana miRNA-target interactions. The proposed model constructed on Arabidopsis thaliana was run over Oryza sativa and Vitis vinifera to demonstrate that our model is effective for other plant species.The integrated model of online predictors and local PCA-SVM classifier gained credible and high quality miRNA-target interactions. The supervised learning algorithm of PCA-SVM classifier was employed in plant miRNA target identification for the first time. Its performance can be substantially improved if more experimentally proved training samples are provided.

  20. Antitumor bioactivity of adenovirus-mediated p27mt in colorectal cancer cell line SW480

    Ze-Qun sun; Chang-Sheng Deng; Shao-Yong Xu; Yong Du


    AIM: To explore the antitumor bioactivity of adenovirus-mediated mutant type p27kip1 gene in a colorectal cancer cell line SW480.METHODS: We constructed recombinant adenovirus vector expressing a mutant type p27kip1 gene (ad-p27mt), with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC), and transduced into SW480 cells. Then we detected expression of p27, Bcl-2 and Bax protein in the transductants by Western blotting, cell cycle of transductants by a digital flow cytometric system, migrating potential with Boyden Chamber end SW480 tumor cell growth inhibition in vitro and in vivo.RESULTS: We found that a recombinant adenovirus vector of expressing ad-p27mt, with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC) has potent inhibition of SW480 tumor cell growth in vitro and in vivo. Furthermore, ed-p27mt induced cell apoptosis via regulating bax and bcl-2 expressions, and G1/S arrest in SW480 cells and inhibited celt migration.CONCLUSION: ad-p27mt has a strong anti-tumor bioactivity and has the potential to develop into new therapeutic agents for colorectal cancer.

  1. Components of Adenovirus Genome Packaging

    Ahi, Yadvinder S.; Mittal, Suresh K.


    Adenoviruses (AdVs) are icosahedral viruses with double-stranded DNA (dsDNA) genomes. Genome packaging in AdV is thought to be similar to that seen in dsDNA containing icosahedral bacteriophages and herpesviruses. Specific recognition of the AdV genome is mediated by a packaging domain located close to the left end of the viral genome and is mediated by the viral packaging machinery. Our understanding of the role of various components of the viral packaging machinery in AdV genome packaging has greatly advanced in recent years. Characterization of empty capsids assembled in the absence of one or more components involved in packaging, identification of the unique vertex, and demonstration of the role of IVa2, the putative packaging ATPase, in genome packaging have provided compelling evidence that AdVs follow a sequential assembly pathway. This review provides a detailed discussion on the functions of the various viral and cellular factors involved in AdV genome packaging. We conclude by briefly discussing the roles of the empty capsids, assembly intermediates, scaffolding proteins, portal vertex and DNA encapsidating enzymes in AdV assembly and packaging. PMID:27721809

  2. Effective inhibition of foot-and-mouth disease virus (FMDV replication in vitro by vector-delivered microRNAs targeting the 3D gene

    Cai Xuepeng


    Full Text Available Abstract Background Foot-and-mouth disease virus (FMDV causes an economically important and highly contagious disease of cloven-hoofed animals. RNAi triggered by small RNA molecules, including siRNAs and miRNAs, offers a new approach for controlling viral infections. There is no report available for FMDV inhibition by vector-delivered miRNA, although miRNA is believed to have more potential than siRNA. In this study, the inhibitory effects of vector-delivered miRNAs targeting the 3D gene on FMDV replication were examined. Results Four pairs of oligonucleotides encoding 3D-specific miRNA of FMDV were designed and selected for construction of miRNA expression plasmids. In the reporter assays, two of four miRNA expression plasmids were able to significantly silence the expression of 3D-GFP fusion proteins from the reporter plasmid, p3D-GFP, which was cotransfected with each miRNA expression plasmid. After detecting the silencing effects of the reporter genes, the inhibitory effects of FMDV replication were determined in the miRNA expression plasmid-transfected and FMDV-infected cells. Virus titration and real-time RT-PCR assays showed that the p3D715-miR and p3D983-miR plasmids were able to potently inhibit the replication of FMDV when BHK-21 cells were infected with FMDV. Conclusion Our results indicated that vector-delivered miRNAs targeting the 3D gene efficiently inhibits FMDV replication in vitro. This finding provides evidence that miRNAs could be used as a potential tool against FMDV infection.

  3. Introducing Vectors.

    Roche, John


    Suggests an approach to teaching vectors that promotes active learning through challenging questions addressed to the class, as opposed to subtle explanations. Promotes introducing vector graphics with concrete examples, beginning with an explanation of the displacement vector. Also discusses artificial vectors, vector algebra, and unit vectors.…

  4. Adenovirus-mediated transfer of RA538 gene and its antitumor effect

    程金科; 林晨; 隗玥; 张雪艳; 邢嵘; 牟巨伟; 王秀琴; 吴旻


    The RA538 cDNA was transferred into human ovarian cancer cell line SK-OV-3 and human melanoma cell line WM-983A by its recombinant adenoviral vector constructed through homologous recombination. It was demonstrated that the recombinant adenovirus could transfer RA538 gene with high efficiency, and could obviously inhibit tumor growth, with the inhibiting rates of 85% and 73% respectively, at the same time greatly repress the colony forming ability of the cells. The therapeutic experiments on transplanted subcutaneous tumor model in nude mice demonstrated that RA538 could significantly inhibit tumor growth. Flow cytometry and DNA fragmentation analysis indicated that RA538 could induce the cell cycle G1 arrest/apoptosis of the tumor cells. The expression of cmyc gene was found pronouncedly reduced by Western blot analysis. These results suggest that the RA538 recombinant adenovirus could be a promising drug in cancer gene therapy.

  5. The Influence of Delivery Vectors on HIV Vaccine Efficacy

    Beatrice Omusiro Ondondo


    Full Text Available Development of an effective HIV/AIDS vaccine remains a big challenge, largely due to the enormous HIV diversity which propels immune escape. Thus novel vaccine strategies are targeting multiple variants of conserved antibody and T cell epitopic regions which would incur a huge fitness cost to the virus in the event of mutational escape. Besides immunogen design, the delivery modality is critical for vaccine potency and efficacy, and should be carefully selected in order to not only maximise transgene expression, but to also enhance the immuno-stimulatory potential to activate innate and adaptive immune systems. To date, five HIV vaccine candidates have been evaluated for efficacy and protection from acquisition was only achieved in a small proportion of vaccinees in the RV144 study which used a canarypox vector for delivery. Conversely, in the STEP study (HVTN 502 where human adenovirus serotype 5 (Ad5 was used, strong immune responses were induced but vaccination was more associated with increased risk of HIV acquisition than protection in vaccinees with pre-existing Ad5 immunity. The possibility that pre-existing immunity to a highly promising delivery vector may alter the natural course of HIV to increase acquisition risk is quite worrisome and a huge setback for HIV vaccine development. Thus, HIV vaccine development efforts are now geared towards delivery platforms which attain superior immunogenicity while concurrently limiting potential catastrophic effects likely to arise from pre-existing immunity or vector-related immuno-modulation. However, it still remains unclear whether it is poor immunogenicity of HIV antigens or substandard immunological potency of the safer delivery vectors that has limited the success of HIV vaccines. This article discusses some of the promising delivery vectors to be harnessed for improved HIV vaccine efficacy.

  6. One-pot biogenic fabrication of silver nanocrystals using Quisqualis indica: Effectiveness on malaria and Zika virus mosquito vectors, and impact on non-target aquatic organisms.

    Govindarajan, Marimuthu; Vijayan, Periasamy; Kadaikunnan, Shine; Alharbi, Naiyf S; Benelli, Giovanni


    Currently, mosquito vector control is facing a number of key challenges, including the rapid development of resistance to synthetic pesticides and the recent spread of aggressive arbovirus outbreaks. The biosynthesis of silver nanoparticles (AgNPs) is currently considered an environmental friendly alternative to the employ of pyrethroids, carbamates and microbial agents (e.g. Bacillus thuringiensis var. israelensis), since AgNPs are easy to produce, effective and stable in the aquatic environment. However, their biophysical features showed wide variations according to the botanical agent using for the green synthesis, outlining the importance of screening local floral resources used as reducing and stabilizing agents. In this study, we focused on the biophysical properties and the mosquitocidal action of Quisqualis indica-fabricated AgNPs. AgNPs were characterized using spectroscopic (UV, FTIR, XRD) and microscopic (AFM, SEM, TEM and EDX) techniques. AFM, SEM and TEM confirmed the synthesis of poly-dispersed AgNPs with spherical shape and size ranging from 1 to 30nm. XRD shed light on the crystalline structure of these AgNPs. The acute toxicity of Quisqualis indica extract and AgNPs was evaluated against malaria, arbovirus, and filariasis vectors, Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus, as well as on three important non-target aquatic organisms. The Q. indica leaf extract showed moderate larvicidal effectiveness on Cx. quinquefasciatus (LC50=220.42), Ae. aegypti (LC50=203.63) and An. stephensi (LC50=185.98). Q. indica-fabricated AgNPs showed high toxicity against Cx. quinquefasciatus (LC50=14.63), Ae. aegypti (LC50=13.55) and An. stephensi (LC50=12.52), respectively. Notably, Q. indica-synthesized AgNPs were moderately toxic to non-target aquatic mosquito predators Anisops bouvieri (LC50=653.05μg/mL), Diplonychus indicus (LC50=860.94μg/mL) and Gambusia affinis (LC50=2183.16μg/mL), if compared to the targeted mosquitoes. Overall, the

  7. Rapid Construction of EGFP Labled Recombinant Adenovirus Containing hVEGF165 and Its Expression in Haematopoietic Cells

    仲照东; 邹萍; 黄士昂; 胡中波; 刘凌波; 卢运萍


    By using AdEasy system, which is based on the homologous recombination in bacteria, an EGFP labeled recombinant adenovirus vector containing hVEGF165 was constructed quickly and efficiently expressed in mouse haematopoietic cells. First, hVEGF165 coding sequence was subcloned into shuttle plasmid pAdTrack-CMV, then cotransformed with adenoviral backbone vector pAdEasy-1 into E. coli strain BJ5183. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly replication-defective adenovirus Ad-EGFP/hVEGF165. The expression of EGFP could be easily detected. The rate of EGFP positive mouse bone marrow mononuclear cells by flow cytometric analysis was 27.3 % (MOI= 100), and the expression of hVEGF165 protein in the supernatant was (1385+332) pg/106 cells. These results suggest that the construction of adenovirus vector by homologous recombination in bacteria features high efficiency and simplicity. The prepared high titer AdEGFP/hVEGF165 can be used an efficient helpful vector to infect hematopoietic cells.

  8. Targeting Gene-Virotherapy for Cancer

    Xin-Yuan LIU; Jing-Fa GU; Wen-Fang SHI


    Gene therapy and viral therapy for cancer have therapeutic effects, but there has been no significant breakthrough in these two forms of therapy. Therefore, a new strategy called "targeting genevirotherapy", which combines the advantages of gene therapy and viral therapy, has been formulated. This new therapy has stronger antitumor effects than either gene therapy or viral therapy. A tumor-specific replicative adenovirus vector ZD55 (E1B55KD deleted Adv.) was constructed and various single therapeutic genes were inserted into ZD55 to form ZD55-gene. These are the targeting gene-virotherapy genes. But experiments showed that a single gene was not effective in eliminating the tumor mass, and therefore two genes were separately inserted into ZD55. This strategy is called "targeting dual gene-virotherapy" (with PCT patent). Better results were obtained with this strategy, and all the xenograft tumor masses were completely eliminated in all mice when two suitable genes producing a synergetic or compensative effect were chosen. Twenty-six papers on these strategies have been published by researchers in our laboratory.Furthermore, an adenoviral vector with two targeting promoters harboring two antitumor genes has been constructed for cancer therapy. Promising results have been obtained with this adenoviral vectorand another patent has been applied for. This antitumor strategy can be used to kill tumor cells completely with minimum damage to normal cells.

  9. Rational and random approaches to adenoviral vector engineering

    Uil, Taco Gilles


    The overall aim of this thesis is to contribute to the engineering of more selective and effective oncolytic Adenovirus (Ad) vectors. Two general approaches are taken for this purpose: (i) genetic capsid modification to achieve Ad retargeting (Chapters 2 to 4), and (ii) directed evolution to improve

  10. Adenoviral vector-mediated gene transfer of IL-13Ralpha2 chain followed by IL-13 cytotoxin treatment offers potent targeted therapy for cytotoxin-resistant cancers.

    Saito, Makoto; Murata, Takashi; Watanabe, Ken; Kawakami, Koji; Suzuki, Motoyoshi; Koji, Takehiko; Puri, Raj K; Kitazato, Kaio; Kobayashi, Nobuyuki


    Previous studies demonstrated that IL-13Ralpha2 chain-overexpressing cancer cells were highly sensitive to IL-13 cytotoxin (IL13-PE38QQR) and could be targeted by cytotoxin treatment. However, the majority of human tumors do not express high levels of IL-13Ralpha2 chain. To expand the IL-13 cytotoxin-mediated cancer targeting therapy, we combined cytotoxin treatment with gene transfer of IL-13Ralpha2 chain. We constructed a recombinant adenoviral vector carrying the human IL-13Ralpha2 gene (Ad-IL-13Ralpha2), which expresses high levels of IL-13Ralpha2 chain on infected cells. Human cancer cell lines A549 and HOS, which originally show no IL-13Ralpha2 expression and little sensitivity to IL-13 cytotoxin, were effectively converted to become sensitive to this cytotoxin after Ad-IL-13Ralpha2 infection. The CC(50) of IL-13 cytotoxin for Ad-IL-13Ralpha2-infected A549 cells was 500 ng/ml. We also examined the antitumor activity of IL-13 cytotoxin in an established xenograft model of cytotoxin-resistant human lung tumor. Only a single i.t. injection of Ad-IL-13Ralpha2 markedly enhanced the sensitivity of established tumors to IL-13 cytotoxin treatment; furthermore, this antitumor effect was significantly sustained for more than 1 month after the last treatment with IL-13 cytotoxin. Taken together, these results suggest the combination of adenoviral vector-mediated IL-13Ralpha2 gene transfer and IL-13 cytotoxin administration can be an effective targeting approach for several types of IL-13 cytotoxin-resistant cancers which show no or little expression of IL-13Ralpha2 chain.

  11. Adenovirus with DNA Packaging Gene Mutations Increased Virus Release

    Wechman, Stephen L.; Rao, Xiao-Mei; McMasters, Kelly M.; Zhou, Heshan Sam


    Adenoviruses (Ads) have been extensively manipulated for the development of cancer selective replication, leading to cancer cell death or oncolysis. Clinical studies using E1-modified oncolytic Ads have shown that this therapeutic platform was safe, but with limited efficacy, indicating the necessity of targeting other viral genes for manipulation. To improve the therapeutic efficacy of oncolytic Ads, we treated the entire Ad genome repeatedly with UV-light and have isolated AdUV which efficiently lyses cancer cells as reported previously (Wechman, S. L. et al. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection. Viruses 2016, 8, 6). In this report, we show that no mutations were observed in the early genes (E1 or E4) of AdUV while several mutations were observed within the Ad late genes which have structural or viral DNA packaging functions. This study also reported the increased release of AdUV from cancer cells. In this study, we found that AdUV inhibits tumor growth following intratumoral injection. These results indicate the potentially significant role of the viral late genes, in particular the DNA packaging genes, to enhance Ad oncolysis. PMID:27999391

  12. Enhanced protection against Ebola virus mediated by an improved adenovirus-based vaccine.

    Jason S Richardson

    Full Text Available BACKGROUND: The Ebola virus is transmitted by direct contact with bodily fluids of infected individuals, eliciting death rates as high as 90% among infected humans. Currently, replication defective adenovirus-based Ebola vaccine is being studied in a phase I clinical trial. Another Ebola vaccine, based on an attenuated vesicular stomatitis virus has shown efficacy in post-exposure treatment of nonhuman primates to Ebola infection. In this report, we modified the common recombinant adenovirus serotype 5-based Ebola vaccine expressing the wild-type ZEBOV glycoprotein sequence from a CMV promoter (Ad-CMVZGP. The immune response elicited by this improved expression cassette vector (Ad-CAGoptZGP and its ability to afford protection against lethal ZEBOV challenge in mice was compared to the standard Ad-CMVZGP vector. METHODOLOGY/PRINCIPAL FINDINGS: Ad-CMVZGP was previously shown to protect mice, guinea pigs and nonhuman primates from an otherwise lethal challenge of Zaire ebolavirus. The antigenic expression cassette of this vector was improved through codon optimization, inclusion of a consensus Kozak sequence and reconfiguration of a CAG promoter (Ad-CAGoptZGP. Expression of GP from Ad-CAGoptZGP was substantially higher than from Ad-CMVZGP. Ad-CAGoptZGP significantly improved T and B cell responses at doses 10 to 100-fold lower than that needed with Ad-CMVZGP. Additionally, Ad-CAGoptZGP afforded full protections in mice against lethal challenge at a dose 100 times lower than the dose required for Ad-CMVZGP. Finally, Ad-CAGoptZGP induced full protection to mice when given 30 minutes post-challenge. CONCLUSIONS/SIGNIFICANCE: We describe an improved adenovirus-based Ebola vaccine capable of affording post-exposure protection against lethal challenge in mice. The molecular modifications of the new improved vaccine also translated in the induction of significantly enhanced immune responses and complete protection at a dose 100 times lower than with the

  13. 串联表达Fas shRNA腺病毒载体的构建%Construction of recombinant adenovirus vector with two fas-targeted shRNA

    刘明社; 赵中夫; 王兰; 张国英; 张芸; 杨慧; 封江南


    目的 利用串联表达质粒pEGFP6-1-siFas1+siFas2和BD Adeno-X系统构建腺病毒表达Fas-shRNA载体.方法 双酶切pEGFP6-1-siFas1+siFas2串联表达质粒和pShuttle2质粒,T4 DNA连接酶连接胶回收片段,转化感受态Escherichia coli(E.coli)DH5α菌株,挑取单克隆菌落LB(Luria-Bertani)培养基中扩增培养;小量质粒试剂盒提取pShuttle2-siFas1+siFas2质粒,酶切鉴定;双酶切pShuttle2-siFas1+siFas2质粒,T4 DNA连接酶连接酶切产物和腺病毒BD Adeno-X Viral DNA.回收连接产物,SwaI酶切去除未重组的腺病毒质粒,再回收酶切产物,转化感受态E.coli DH5α;聚合酶链反应(PCR)筛选鉴定阳性克隆;提取pAdeno-siFas1+siFas2质粒,进一步酶切鉴定;重组腺病毒pAdeno-siFas1+siFas2质粒线性化,经脂质体转染HEK 293A细胞;收细胞进行裂解收病毒.结果 构建表达2个Fas-shRNA的串联重组腺病毒载体pAdeno-siFas1+siFas2,经酶切鉴定和PCR分析,重组腺病毒质粒构建正确;经HEK293A细胞包装成功.结论 成功构建表达2个Fas-shRNA的串联重组腺病毒载体pAdeno-siFas1+siFas2,为进一步转染细胞和感染小鼠抑制Fas基因表达奠定了基础.

  14. RNAi沉默Fas受体表达的腺病毒载体构建%Construction of Recombinant Adenovirus Vector expressing Two Fas-Targeted shRNA

    刘明社; 赵中夫; 王兰; 张国英; 张芸; 封江南


    目的:利用pEGFP6-1和pGenesil-1质粒构建针对小鼠Fas的2个短发卡状RNA (short hairpin RNA,shRNA) 串联表达的载体pEGFP6-1-siFas1+siFas2,进一步通过BD Adeno-X系统构建腺病毒表达Fas-shRNA载体.方法:设计表达2个短发卡状RNA结构的互补DNA序列分别克隆至质粒载体pEGFP6-1和pGenesil-1中,酶切pEGFP6-1-siFas1大片段和酶切pGenesil-1-siFas2小片段,经T4 DNA Ligase连接得到pEGFP6-1-siFas1+siFas2重组质粒.双酶切重组质粒和pShuttle2穿梭质粒,经T4 DNA连接酶连接得到pShuttle2-siFas1+siFas2质粒.双酶切重组穿梭质粒,连接酶切产物和腺病毒转化感受态E.coil DH5а,PCR筛选鉴定阳性克隆,进一步线性化重组腺病毒pAdeno-siFas1+siFas2质粒,经脂质体转染HEK 293A细胞;收细胞进行裂解收病毒. 结果:构建的重组质粒载体经酶切鉴定和测序分析,shRNA编码序列与设计的片段完全一致;重组腺病毒载体经酶切鉴定和PCR分析正确;经HEK293A细胞包装成功,能够表达绿色荧光蛋白.结论:成功构建Fas-shRNA腺病毒串联表达载体.

  15. Biodistribution and inflammatory profiles of novel penton and hexon double-mutant serotype 5 adenoviruses

    Bradshaw, Angela C.; Coughlan, Lynda; Miller, Ashley M.; Alba, Raul; van Rooijen, Nico; Nicklin, Stuart A.; Baker, Andrew H.


    The use of adenovirus serotype 5 (Ad5) vectors in the clinical setting is severely hampered by the profound liver tropism observed after intravascular delivery coupled with the pronounced inflammatory and innate immune response elicited by these vectors. Liver transduction by circulating Ad5 virions is mediated by a high-affinity interaction between the capsid hexon protein and blood coagulation factor X (FX), whilst penton–αvintegrin interactions are thought to contribute to the induction of anti-Ad5 inflammatory and innate immune responses. To overcome these limitations, we sought to develop and characterise for the first time novel Ad5 vectors possessing mutations ablating both hexon:FX and penton:integrin interactions. As expected, intravascular administration of the FX binding-ablated Ad5HVR5*HVR7*E451Q vector (AdT*) resulted in significantly reduced liver transduction in vivo compared to Ad5. In macrophage-depleted mice, increased spleen uptake of AdT* was accompanied by an elevation in the levels of several inflammatory mediators. However ablation of the penton RGD motif in the AdT* vector background (AdT*RGE) resulted in a significant 5-fold reduction in spleen uptake and attenuated the antiviral inflammatory response. A reduction in spleen uptake and inflammatory activation was also observed in animals after intravascular administration of Ad5RGE compared to the parental Ad5 vector, with reduced co-localisation of the viral beta-galactosidase transgene with MAdCAM-1 + sinus-lining endothelial cells. Our detailed assessment of these novel adenoviruses indicates that penton base RGE mutation in combination with FX binding-ablation may be a viable strategy to attenuate the undesired liver uptake and pro-inflammatory responses to Ad5 vectors after intravascular delivery. PMID:22626939


    李贺书; 李殿俊; 刘旭; 李大林


    Objective: To observe the mutagenicity of retrovirus and adenovirus as transgenic vectors to evaluate the safety of transgenic tumor cells as tumor vaccines. Methods: Cells were cultured together with the virus. Then DNA and supernatant were tested for mutagenicity by means of genetic toxicological laboratory technique. Results: The results indicated that DNA and supernatant of transgenic cells had no mutagenicity through both in vivo and in vitro tests. Conclusion: The modified virus had no mutagenicity as a transgenic vector.

  17. Structure and Uncoating of Immature Adenovirus

    Perez-Berna, A.J.; Mangel, W.; Marabini, R.; Scheres, S. H. W., Menendez-Conejero, R.; Dmitriev, I. P.; Curiel, D. T.; Flint, S. J.; San Martin, C.


    Maturation via proteolytic processing is a common trait in the viral world and is often accompanied by large conformational changes and rearrangements in the capsid. The adenovirus protease has been shown to play a dual role in the viral infectious cycle: (a) in maturation, as viral assembly starts with precursors to several of the structural proteins but ends with proteolytically processed versions in the mature virion, and (b) in entry, because protease-impaired viruses have difficulties in endosome escape and uncoating. Indeed, viruses that have not undergone proteolytic processing are not infectious. We studied the three-dimensional structure of immature adenovirus particles as represented by the adenovirus type 2 thermosensitive mutant ts1 grown under non-permissive conditions and compared it with the mature capsid. Our three-dimensional electron microscopy maps at subnanometer resolution indicate that adenovirus maturation does not involve large-scale conformational changes in the capsid. Difference maps reveal the locations of unprocessed peptides pIIIa and pVI and help define their role in capsid assembly and maturation. An intriguing difference appears in the core, indicating a more compact organization and increased stability of the immature cores. We have further investigated these properties by in vitro disassembly assays. Fluorescence and electron microscopy experiments reveal differences in the stability and uncoating of immature viruses, both at the capsid and core levels, as well as disassembly intermediates not previously imaged.

  18. Deaths from Adenovirus in the US Military


    Dr. Joel Gaydos, science advisor for the Armed Forces Health Surveillance Center, and Dr. Robert Potter, a research associate for the Armed Forces Medical Examiner System, discuss deaths from adenovirus in the US military.  Created: 3/26/2012 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 3/29/2012.

  19. MHC class II-associated invariant chain linkage of antigen dramatically improves cell-mediated immunity induced by adenovirus vaccines

    Holst, Peter Johannes; Mandrup Jensen, Camilla Maria; Orskov, Cathrine


    The ideal vaccine induces a potent protective immune response, which should be rapidly induced, long-standing, and of broad specificity. Recombinant adenoviral vectors induce potent Ab and CD8+ T cell responses against transgenic Ags within weeks of administration, and they are among the most...... potent and versatile Ag delivery vehicles available. However, the impact of chronic infections like HIV and hepatitis C virus underscore the need for further improvements. In this study, we show that the protective immune response to an adenovirus-encoded vaccine Ag can be accelerated, enhanced......, broadened, and prolonged by tethering of the rAg to the MHC class II-associated invariant chain (Ii). Thus, adenovirus-vectored vaccines expressing lymphocytic choriomeningitis virus (LCMV)-derived glycoprotein linked to Ii increased the CD4+ and CD8+ T cell stimulatory capacity in vitro and in vivo...

  20. The enzyme 3-hydroxykynurenine transaminase as potential target for 1,2,4-oxadiazoles with larvicide activity against the dengue vector Aedes aegypti.

    Oliveira, Vanessa S; Pimenteira, Cecília; da Silva-Alves, Diana C B; Leal, Laylla L L; Neves-Filho, Ricardo A W; Navarro, Daniela M A F; Santos, Geanne K N; Dutra, Kamilla A; dos Anjos, Janaína V; Soares, Thereza A


    The mosquito Aedes aegypti is the vector agent responsible for the transmission of yellow fever and dengue fever viruses to over 80 million people in tropical and subtropical regions of the world. Exhaustive efforts have lead to a vaccine candidate with only 30% effectiveness against the dengue virus and failure to protect patients against the serotype 2. Hence, vector control remains the most viable route to dengue fever control programs. We have synthesized a class of 1,2,4-oxadiazole derivatives whose most biologically active compounds exhibit potent activity against Aedes aegypti larvae (ca. of 15 ppm) and low toxicity in mammals. Exposure to these larvicides results in larvae pigmentation in a manner correlated with the LC50 measurements. Structural comparisons of the 1,2,4-oxadiazole nucleus against known inhibitors of insect enzymes allowed the identification of 3-hydroxykynurenine transaminase as a potential target for these synthetic larvicides. Molecular docking calculations indicate that 1,2,4-oxadiazole compounds can bind to 3-hydroxykynurenine transaminase with similar conformation and binding energies as its crystallographic inhibitor 4-(2-aminophenyl)-4-oxobutanoic acid.

  1. Advancing vector biology research

    Kohl, Alain; Pondeville, Emilie; Schnettler, Esther; Crisanti, Andrea; Supparo, Clelia; Christophides, George K.; Kersey, Paul J.; Maslen, Gareth L.; Takken, Willem; Koenraadt, Constantianus J.M.; Oliva, Clelia F.; Busquets, Núria; Abad, F.X.; Failloux, Anna Bella; Levashina, Elena A.; Wilson, Anthony J.; Veronesi, Eva; Pichard, Maëlle; Arnaud Marsh, Sarah; Simard, Frédéric; Vernick, Kenneth D.


    Vector-borne pathogens impact public health, animal production, and animal welfare. Research on arthropod vectors such as mosquitoes, ticks, sandflies, and midges which transmit pathogens to humans and economically important animals is crucial for development of new control measures that target t

  2. Adenoviral targeting using genetically incorporated camelid single variable domains.

    Kaliberov, Sergey A; Kaliberova, Lyudmila N; Buggio, Maurizio; Tremblay, Jacqueline M; Shoemaker, Charles B; Curiel, David T


    The unique ability of human adenovirus serotype 5 (Ad5) to accomplish efficient transduction has allowed the use of Ad5-based vectors for a range of gene therapy applications. Several strategies have been developed to alter tropism of Ad vectors to achieve a cell-specific gene delivery by using fiber modifications via genetic incorporation of targeting motifs. In this study, we have explored the utility of novel anti-human carcinoembryonic antigen (hCEA) single variable domains derived from heavy chain (VHH) camelid family of antibodies to achieve targeted gene transfer. To obtain anti-CEA VHHs, we produced a VHH-display library from peripheral blood lymphocytes RNA of alpacas at the peak of immune response to the hCEA antigen (Ag). We genetically incorporated an anti-hCEA VHH into a de-knobbed Ad5 fiber-fibritin chimera and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of target cells. We report that the anti-hCEA VHH used in this study retains Ag recognition functionality and provides specificity for gene transfer of capsid-modified Ad5 vectors. These studies clearly demonstrated the feasibility of retargeting of Ad5-based gene transfer using VHHs.

  3. Suppression of Adenovirus Replication by Cardiotonic Steroids.

    Grosso, Filomena; Stoilov, Peter; Lingwood, Clifford; Brown, Martha; Cochrane, Alan


    The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yields of four human adenoviruses (HAdV-A31, -B35, and -C5 and a species D conjunctivitis isolate) by at least 2 to 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein hexon is compromised. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that both drugs altered E1A RNA splicing (favoring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on the continued presence of the drug and was rapidly reversible. RIDK34, a derivative of convallotoxin, although having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin and digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies.

  4. Methylation of PLCD1 and adenovirus-mediated PLCD1 overexpression elicits a gene therapy effect on human breast cancer

    Mu, Haixi [Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Department of Endocrine and breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Wang, Na; Zhao, Lijuan; Li, Shuman; Li, Qianqian; Chen, Ling; Luo, Xinrong; Qiu, Zhu [Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Li, Lili [Cancer Epigenetics Laboratory, Department of Clinical Oncology, Sir YK Pao Center for Cancer and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong and CUHK Shenzhen Research Institute (Hong Kong); Ren, Guosheng [Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Department of Endocrine and breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Xu, Yongzhu [Chongqing Health Service Center, Chongqing 400020 (China); Zhou, Xiangyang [The Wistar Institute, Philadelphia, PA (United States); Xiang, Tingxiu, E-mail: [Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China)


    Our previous study showed that PLCD1 significantly decreases cell proliferation and affects cell cycle progression in breast cancer cells. In the present study, we aimed to investigate its functional and molecular mechanisms, and whether or not can become a new target for gene therapies. We found reduced PLCD1 protein expression in breast tumor tissues compared with paired surgical margin tissues. PLCD1 promoter CpG methylation was detected in 55 of 96 (57%) primary breast tumors, but not in surgical-margin tissues and normal breast tissues. Ectopic expression of PLCD1 inhibited breast tumor cell proliferation in vivo by inducing apoptosis and suppressed tumor cell migration by regulating cytoskeletal reorganization proteins including RhoA and phospho-cofilin. Furthermore, we found that PLCD1 induced p53 accumulation, increased p27 and p21 protein levels, and cleaved PARP. Finally, we constructed an adenoviral vector expressing PLCD1 (AdH5-PLCD1), which exhibited strong cytotoxicity in breast cancer cells. Our findings provide insights into the development of PLCD1 gene therapies for breast cancer and perhaps, other human cancers. - Highlights: • PLCD1 is downregulated via hypermethylation in breast cancer. • PLCD1 suppressed cell migration by regulating cytoskeletal reorganization proteins. • Adenovirus AdHu5-PLCD1 may be a novel therapeutic option for breast cancer.

  5. Adenovirus Activates Complement by Distinctly Different Mechanisms In Vitro and In Vivo: Indirect Complement Activation by Virions In Vivo▿

    Tian, Jie; Xu, Zhili; Jeffrey S Smith; Hofherr, Sean E.; Barry, Michael A.; Byrnes, Andrew P.


    Understanding innate immunity is key to improving the safety of adenovirus (Ad) vectors for systemic gene therapy. Ad has been shown to activate complement in vitro, but activation of complement after Ad injection in vivo has not been directly measured. Using complement protein C3a as a marker of complement activation, we show that types 2 and 5 human Ads cause rapid complement activation after intravenous injection in mice. Unexpectedly, the mechanisms in vivo were different than those in vi...

  6. Selective modification of adenovirus replication can be achieved through rational mutagenesis of the adenovirus type 5 DNA polymerase.

    Capella, Cristina; Beltejar, Michael-John; Brown, Caitlin; Fong, Vincent; Daddacha, Waaqo; Kim, Baek; Dewhurst, Stephen


    Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates.

  7. A novel strategy for cancer treatment:Targeting cancer stem cells

    LIU Jia; MA LeiNa; WANG YiGang; LIU XinYuan; QIAN QiJun


    Cancer stem cell/tumor-initiating cell (CSC/TIC) is a subclass of cancer cells possessing parts of properties of normal stem cell. It has a high capacity of proliferation and plays a pivotal role in tumor recurrence and tumor resistance to radiotherapy and chemotherapy. At present, small molecule in-hibitors and fusion proteins are widely used in the CSC-targeting strategy. Gene-virotherapy, which uses oncolytic adenovirus as a vector to mediate the expression of therapeutic gene, shows a signifi-cant superiority to other regimens of cancer treatment and has a good efficacy in the treatment of solid tumors. Thus, it is a promising choice to apply gene-virotherapy into the CSC-targeting treatment. Based on the molecular mechanism underlying CSC self-renewal, a series of effective strategies for targeting CSC have been established. This review will summarize the recent research progresses on CSC-targeting treatment.

  8. Preparation of folate-conjugated starch nanoparticles and its application to tumor-targeted drug delivery vector

    XIAO Suyao; TONG Chunyi; LIU Xuanming; YU Danmi; LIU Qiaoling; XUE Changgang; TANG Dongyin; ZHAO Lijian


    Anion starch nanoparticles (StNP) were prepared in water-in-oil microemulsion. Folate modified with PEG was conjugated to the surface of StNP to obtain the folate-conjugated starch nanoparticles (FA-PEG/StNP). The average diameter of FA-PEG/StNP determined by AFM and Zeta-Sizer apparatus was about 130 nm. Doxorubicin (DOX)-loaded FA-PEG/StNP was obtained via infiltrating combination.The result of UV spectrophotometer showed that the saturation concentration of DOX-loaded FA-PEG/StNP was 28 μg/mg, which was effective for the controlled release of anticancer drug DOX. The cell experiments showed that the Lc50 of DOX-loaded FA-PEG/StNP and DOX-loaded StNP was higher than that of DOX, which indicates that FA-PEG/StNP and StNP can decrease the toxicity of DOX; while the lethal rate of DOX-loaded FA-PEG/StNP was 3 times that of DOX-loaded StNP with the same quantity of DOX, which indicates that FA in FA-PEG/StNP is effective for improving the targeting function of nanoparticles, thus enhancing the inhibition effect of anticancer drug to cancer cell. This work demonstrates that the FA-PEG/StNP system is a potentially useful system for the targeted delivery of anticancer drug DOX.

  9. Ligand efficiency-based support vector regression models for predicting bioactivities of ligands to drug target proteins.

    Sugaya, Nobuyoshi


    The concept of ligand efficiency (LE) indices is widely accepted throughout the drug design community and is frequently used in a retrospective manner in the process of drug development. For example, LE indices are used to investigate LE optimization processes of already-approved drugs and to re-evaluate hit compounds obtained from structure-based virtual screening methods and/or high-throughput experimental assays. However, LE indices could also be applied in a prospective manner to explore drug candidates. Here, we describe the construction of machine learning-based regression models in which LE indices are adopted as an end point and show that LE-based regression models can outperform regression models based on pIC50 values. In addition to pIC50 values traditionally used in machine learning studies based on chemogenomics data, three representative LE indices (ligand lipophilicity efficiency (LLE), binding efficiency index (BEI), and surface efficiency index (SEI)) were adopted, then used to create four types of training data. We constructed regression models by applying a support vector regression (SVR) method to the training data. In cross-validation tests of the SVR models, the LE-based SVR models showed higher correlations between the observed and predicted values than the pIC50-based models. Application tests to new data displayed that, generally, the predictive performance of SVR models follows the order SEI > BEI > LLE > pIC50. Close examination of the distributions of the activity values (pIC50, LLE, BEI, and SEI) in the training and validation data implied that the performance order of the SVR models may be ascribed to the much higher diversity of the LE-based training and validation data. In the application tests, the LE-based SVR models can offer better predictive performance of compound-protein pairs with a wider range of ligand potencies than the pIC50-based models. This finding strongly suggests that LE-based SVR models are better than pIC50-based

  10. Stimulation of innate immunity by in vivo cyclic di-GMP synthesis using adenovirus.

    Koestler, Benjamin J; Seregin, Sergey S; Rastall, David P W; Aldhamen, Yasser A; Godbehere, Sarah; Amalfitano, Andrea; Waters, Christopher M


    The bacterial second messenger cyclic di-GMP (c-di-GMP) stimulates inflammation by initiating innate immune cell recruitment and triggering the release of proinflammatory cytokines and chemokines. These properties make c-di-GMP a promising candidate for use as a vaccine adjuvant, and numerous studies have demonstrated that administration of purified c-di-GMP with different antigens increases protection against infection in animal models. Here, we have developed a novel approach to produce c-di-GMP inside host cells as an adjuvant to exploit a host-pathogen interaction and initiate an innate immune response. We have demonstrated that c-di-GMP can be synthesized in vivo by transducing a diguanylate cyclase (DGC) gene into mammalian cells using an adenovirus serotype 5 (Ad5) vector. Expression of DGC led to the production of c-di-GMP in vitro and in vivo, and this was able to alter proinflammatory gene expression in murine tissues and increase the secretion of numerous cytokines and chemokines when administered to animals. Furthermore, coexpression of DGC modestly increased T-cell responses to a Clostridium difficile antigen expressed from an adenovirus vaccine, although no significant differences in antibody titers were observed. This adenovirus c-di-GMP delivery system offers a novel method to administer c-di-GMP as an adjuvant to stimulate innate immunity during vaccination.

  11. Hyaluronic Acid Conjugates as Vectors for the Active Targeting of Drugs, Genes and Nanocomposites in Cancer Treatment

    Silvia Arpicco


    Full Text Available Hyaluronic acid (HA is a naturally-occurring glycosaminoglycan and a major component of the extracellular matrix. Low levels of the hyaluronic acid receptor CD44 are found on the surface of epithelial, hematopoietic, and neuronal cells; it is overexpressed in many cancer cells, and in particular in tumor-initiating cells. HA has recently attracted considerable interest in the field of developing drug delivery systems, having been used, as such or encapsulated in different types of nanoassembly, as ligand to prepare nano-platforms for actively targeting drugs, genes, and diagnostic agents. This review describes recent progress made with the several chemical strategies adopted to synthesize conjugates and prepare novel delivery systems with improved behaviors.

  12. Alpha interferon-induced antiviral response noncytolytically reduces replication defective adenovirus DNA in MDBK cells.

    Guo, Ju-Tao; Zhou, Tianlun; Guo, Haitao; Block, Timothy M


    Although alpha interferon (IFN-alpha) is of benefit in the treatment of viral hepatitis B, HBV replication has been refractory to the cytokine in commonly used hepatocyte-derived cell lines. In search for a cell culture system to study the mechanism by which IFN-alpha inhibits HBV replication, we infected a variety of cell lines with an adenoviral vector containing a replication competent 1.3-fold genome length HBV DNA (AdHBV) and followed by incubation with IFN-alpha. We found that IFN-alpha efficiently decreased the level of HBV DNA replicative intermediates in AdHBV infected Madin-Darby bovine kidney (MDBK) cells. Further analysis revealed, surprisingly, that IFN-alpha did not directly inhibit HBV replication, rather the amount of adenovirus DNA in the nuclei of MDBK cells was reduced. As a consequence, HBV RNA transcription and DNA replication were inhibited. Experiments with adenoviral vector expressing a green fluorescent protein (GFP) further supported the notion that IFN-alpha treatment noncytolytically eliminated adenovirus DNA, but did not kill the vector infected MDBK cells. Our data suggest that IFN-alpha-induced antiviral program is able to discriminate host cellular DNA from episomal viral DNA and might represent a novel pathway of interferon mediate innate defense against DNA virus infections.

  13. A concept of eliminating nonhomologous recombination for scalable and safe AAV vector generation for human gene therapy.

    Dong, Biao; Moore, Andrea R; Dai, Jihong; Roberts, Sean; Chu, Kirk; Kapranov, Philipp; Moss, Bernard; Xiao, Weidong


    Scalable and efficient production of high-quality recombinant adeno-associated virus (rAAV) for gene therapy remains a challenge despite recent clinical successes. We developed a new strategy for scalable and efficient rAAV production by sequestering the AAV helper genes and the rAAV vector DNA in two different subcellular compartments, made possible by using cytoplasmic vaccinia virus as a carrier for the AAV helper genes. For the first time, the contamination of replication-competent AAV particles (rcAAV) can be completely eliminated in theory by avoiding ubiquitous nonhomologous recombination. Vector DNA can be integrated into the host genomes or delivered by a nuclear targeting vector such as adenovirus. In suspension HeLa cells, the achieved vector yield per cell is similar to that from traditional triple-plasmid transfection method. The rcAAV contamination was undetectable at the limit of our assay. Furthermore, this new concept can be used not only for production of rAAV, but also for other DNA vectors.

  14. Adenovirus-mediated Gene Transfer of MMP-2 into Cultured Porcine Trabecular Meshwork Cells


    This study aimed to use adenoviral gene transfer to express matrix metalloproteinase (MMP)-2 in cultured porcine trabecular meshwork cells and to evaluate the duration of adenovirus-mediated MMP-2 expression and its enzymatic activity. MMP-2 cDNA was synthesized by ligating three segments of MMP-2 cDNA obtained by reverse transcription-polymerase chain reaction (RT-PCR) with mRNA extracted from mouse lungs. MMP-2 cDNA was inserted into replication-deficient adenoviral vectors. Western blottin...

  15. Effect of Preexisting Immunity to Adenovirus Human Serotype 5 Antigens on the Immune Responses of Nonhuman Primates to Vaccine Regimens Based on Human- or Chimpanzee-Derived Adenovirus Vectors▿

    McCoy, Kimberly; Tatsis, Nia; Korioth-Schmitz, Birgit; Lasaro, Marcio O; Hensley, Scott E.; Lin, Shih-Wen; Li, Yan; Giles-Davis, Wynetta; Cun, Ann; Zhou, Dongming; Xiang, Zhiquan; Letvin, Norman L.; Ertl, Hildegund C J


    In this study we compared a prime-boost regimen with two serologically distinct replication-defective adenovirus (Ad) vectors derived from chimpanzee serotypes C68 and C1 expressing Gag, Pol, gp140, and Nef of human immunodeficiency virus type 1 with a regimen in which replication-defective Ad vectors of the human serotype 5 (AdHu5) were given twice. Experiments were conducted in rhesus macaques that had or had not been preexposed to antigens of AdHu5. There was no significant difference in T...

  16. Phylogenetic analysis of the genomes of two strains of human adenovirus type 3



    Human adenovirus type 3 (HAdV-3) is widely prevalent all over the world, especially in Asia. The objective of this study is to carry out complete genomic DNA sequencing and the phylogenetic analysis for two strains (Guangzhou01 and Guangzhou02) of HAdV-3 wild virus isolated from South China. Nasopharyngeal secretion aspirate specimens of sick children were inoculated into HEp-2 and HeLa culture tubes, and the cultures were identified by neutralization assay with type-specific reference rabbit antisermn. Type-specific primers were also utilized to confirm the serotype. The restriction fragments of HAdV genome DNA were cloned into pBlueScript SK ( + ) vectors and sequenced, and the 5' and 3'ends of the linear HAdV-3 genome were directly sequenced with double purified genomic DNA as templates. General features of the HAdV-3 genome sequences were explored by using several bio-software.Phylogenetic analysis was done with MEGA 3.0 software. The genomic sequences of Guangzhou01 and Guangzhou02 possess the same 4 early regions and 5 late regions and have 39 ceding sequences and two RNA coding sequences. Other non-ceding regions are conservative. Inverted repeats and palindromes were identified in the genome sequences. The genomes of group B human adenovirus as well as HAdV-3have close phylogenetic relationship with that of chimpanzee adenovirus type 21. The genomie lengths of these two isolated strains are 35 273 bp and 35 269 bp, respectively. The phylogenetie analysis showed that HAdV-B species has some relationship with eertain types of chimpanzee adenovirus.

  17. Gene therapy of cancer and development of therapeutic target gene

    Kim, Chang Min; Kwon, Hee Chung


    We applied HSV-tk/GCV strategy to orthotopic rat hepatoma model and showed anticancer effects of hepatoma. The increased expression of Lac Z gene after adenovirus-mediated gene delivery throughout hepatic artery was thought that is increased the possibility of gene therapy for curing hepatoma. With the construction of kGLP-laboratory, it is possible to produce a good quantity and quality of adenovirus in lage-scale production and purification of adenovirus vector. Also, the analysis of hepatoma related genes by PCR-LOH could be used for the diagnosis of patients and the development of therapeutic gene.

  18. Comparison of polystyrene nanoparticles and UV-inactivated antigen-displaying adenovirus for vaccine delivery in mice


    Background Inert nanoparticles are attracting attention as carriers for protein-based vaccines. Here we evaluate the immunogenicity of the model antigen ovalbumin delivered on polystyrene particles and directly compare particulate delivery with adenovirus-based immunization. Findings Mice were vaccinated with soluble ovalbumin, ovalbumin-coated polystyrene particles of different sizes, or an adenovirus-based expression-display vector that encodes and displays a pIX-ovalbumin fusion protein. Antibody responses were clearly higher when ovalbumin was administered on polystyrene particles compared to soluble protein administration, regardless of the particle size. Compared to adenovirus-based immunization, antibody levels were lower if an equivalent amount of protein was delivered, and no cellular immune response was detectable. Conclusions We demonstrate in a side-by-side comparison that inert nanoparticles allow for the reduction of the administered antigen amount compared to immunization with soluble protein and induce strongly enhanced antibody responses, but responses are lower compared to adenovirus-based immunization. PMID:23560981

  19. In vitro and in vivo characterization of microRNA-targeted alphavirus replicon and helper RNAs.

    Kamrud, Kurt I; Coffield, V McNeil; Owens, Gary; Goodman, Christin; Alterson, Kim; Custer, Max; Murphy, Michael A; Lewis, Whitney; Timberlake, Sarah; Wansley, Elizabeth K; Berglund, Peter; Smith, Jonathan


    Alphavirus-based replicon vector systems (family Togaviridae) have been developed as expression vectors with demonstrated potential in vaccine development against both infectious diseases and cancer. The single-cycle nature of virus-like replicon particles (VRP), generated by supplying the structural proteins from separate replicable helper RNAs, is an attractive safety component of these systems. MicroRNAs (miRNAs) have emerged as important cellular RNA regulation elements. Recently, miRNAs have been employed as a mechanism to attenuate or restrict cellular tropism of replication-competent viruses, such as oncolytic adenoviruses, vesicular stomatitis virus, and picornaviruses as well as nonreplicating lentiviral and adenoviral vectors. Here, we describe the incorporation of miRNA-specific target sequences into replicable alphavirus helper RNAs that are used in trans to provide the structural proteins required for VRP production. VRP were found to be efficiently produced using miRNA-targeted helper RNAs if miRNA-specific inhibitors were introduced into cells during VRP production. In the absence of such inhibitors, cellular miRNAs were capable of downregulating helper RNA replication in vitro. When miRNA targets were incorporated into a replicon RNA, cellular miRNAs were capable of downregulating replicon RNA replication upon delivery of VRP into animals, demonstrating activity in vivo. These data provide the first example of miRNA-specific repression of alphavirus replicon and helper RNA replication and demonstrate the feasibility of miRNA targeting of expression vector helper functions that are provided in trans.

  20. Construction and identification of replication-competent adenovirus expressing siRNA targeting CD133 gene regulated by survivin promoter and its inhibition of liver cancer cell growth%survivin 启动子调控肿瘤干细胞标记 CD133基因 siRNA增殖型溶瘤腺病毒的构建及对肝癌细胞生长的抑制作用

    牛坚; 王月; 刘斌; 王人颢; 朱志军; 申海莲


    目的:构建 survivin 启动子调控的靶向 CD133基因的 siRNA 增殖型溶瘤腺病毒,研究其对肝癌细胞生长的影响。方法RT-PCR 法扩增 survivin 启动子,测序鉴定,双酶切连接,获得 pH-XC2-survivin。酶切 pH-XC2-survivin、pZD55-CD133-siRNA 获得 survivin 启动子表达框的亚克隆和CD133-siRNA 基因表达框的亚克隆,连接获得 survivin 启动子调控的 siRNA 增殖型溶瘤腺病毒表达载体质粒 pT-ZD55-CD133-siRNA。增殖型溶瘤腺病毒 survivin-T-ZD55-CD133-siRNA 经 PCR 和测序鉴定。 qRT-PCR 法检测 CD133表达, Western blot 法检测 E1A,CCK-8法检测细胞生长,流式细胞术检测细胞凋亡。结果成功构建增殖型溶瘤腺病毒 sur-vivin-T-ZD55-CD133-siRNA。 qRT-PCR 法检测 CD133 mRNA明显下降, Western blot 证实 survivin-T-ZD55-CD133-siRNA在肿瘤细胞中表达 E1A 能抑制肝癌细胞 CD133表达及生长。结论构建的增殖型溶瘤腺病毒可有效降低肝癌细胞CD133的表达,用于肝癌基因治疗的进一步研究。%Objective To construct a replication-competent adenovirus expressing siRNA targeting CD133 gene regulated by survivin promoter and investigate its inhibitory effect on Hep 3B cells.Methods The fragment of the survivin promoter was amplified by PCR and inserted into pH -XC2 to reconstruct a recombinant plasmid pH -XC2-survivin.Complete digestion pH-XC2-survivin and pZD55-CD133-siRNA, combinational joining the subclones, then getting replication-competent adenovirus expressing short interference RNA targeting CD 133 gene regulated by survivin promoter, replication-competent adenovirus was constructed .The recombined adenoviruses ( T-ZD55-CD133-siRNA) were verified by PCR and sequencing .The effect of T-ZD55-CD133-siRNA on CD133 expression in Hep3B cells was detected by qRT-PCR.The expression of E1A was detected by Western blot.The antitumor po-tential of replication

  1. Carbon and silver nanoparticles in the fight against the filariasis vector Culex quinquefasciatus: genotoxicity and impact on behavioral traits of non-target aquatic organisms.

    Murugan, Kadarkarai; Nataraj, Devaraj; Madhiyazhagan, Pari; Sujitha, Vasu; Chandramohan, Balamurugan; Panneerselvam, Chellasamy; Dinesh, Devakumar; Chandirasekar, Ramachandran; Kovendan, Kalimuthu; Suresh, Udaiyan; Subramaniam, Jayapal; Paulpandi, Manickam; Vadivalagan, Chithravel; Rajaganesh, Rajapandian; Wei, Hui; Syuhei, Ban; Aziz, Al Thabiani; Alsalhi, Mohamad Saleh; Devanesan, Sandhanasamy; Nicoletti, Marcello; Canale, Angelo; Benelli, Giovanni


    Mosquito-borne diseases represent a deadly threat for millions of people worldwide. The Culex genus, with special reference to Culex quinquefasciatus, comprises the most common vectors of filariasis across urban and semi-urban areas of Asia. In recent years, important efforts have been conducted to propose green-synthesized nanoparticles as a valuable alternative to synthetic insecticides. However, the mosquitocidal potential of carbon nanoparticles has been scarcely investigated. In this study, the larvicidal and pupicidal activity of carbon nanoparticle (CNP) and silver nanoparticle (AgNP) was tested against Cx. quinquefasciatus. UV-Vis spectrophotometry, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray (EDX) spectroscopy, and Raman analysis confirmed the rapid and cheap synthesis of carbon and silver nanoparticles. In laboratory assays, LC50 (lethal concentration that kills 50 % of the exposed organisms) values ranged from 8.752 ppm (first-instar larvae) to 18.676 ppm (pupae) for silver nanoparticles and from 6.373 ppm (first-instar larvae) to 14.849 ppm (pupae) for carbon nanoparticles. The predation efficiency of the water bug Lethocerus indicus after a single treatment with low doses of silver and carbon nanoparticles was not reduced. Moderate evidence of genotoxic effects induced by exposure to carbon nanoparticles was found on non-target goldfish, Carassius auratus. Lastly, the plant extract used for silver nanosynthesis was tested for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activity. Overall, our results pointed out that AgNP and CNP can be a candidate for effective tools to reduce larval and pupal populations of filariasis vectors, with reduced genotoxicity and impact on behavioral traits of other aquatic organisms sharing the same ecological

  2. Locally-Delivered T-Cell-Derived Cellular Vehicles Efficiently Track and Deliver Adenovirus Delta24-RGD to Infiltrating Glioma

    Rutger K. Balvers


    Full Text Available Oncolytic adenoviral vectors are a promising alternative for the treatment of glioblastoma. Recent publications have demonstrated the advantages of shielding viral particles within cellular vehicles (CVs, which can be targeted towards the tumor microenvironment. Here, we studied T-cells, often having a natural capacity to target tumors, for their feasibility as a CV to deliver the oncolytic adenovirus, Delta24-RGD, to glioblastoma. The Jurkat T-cell line was assessed in co-culture with the glioblastoma stem cell (GSC line, MGG8, for the optimal transfer conditions of Delta24-RGD in vitro. The effect of intraparenchymal and tail vein injections on intratumoral virus distribution and overall survival was addressed in an orthotopic glioma stem cell (GSC-based xenograft model. Jurkat T-cells were demonstrated to facilitate the amplification and transfer of Delta24-RGD onto GSCs. Delta24-RGD dosing and incubation time were found to influence the migratory ability of T-cells towards GSCs. Injection of Delta24-RGD-loaded T-cells into the brains of GSC-bearing mice led to migration towards the tumor and dispersion of the virus within the tumor core and infiltrative zones. This occurred after injection into the ipsilateral hemisphere, as well as into the non-tumor-bearing hemisphere. We found that T-cell-mediated delivery of Delta24-RGD led to the inhibition of tumor growth compared to non-treated controls, resulting in prolonged survival (p = 0.007. Systemic administration of virus-loaded T-cells resulted in intratumoral viral delivery, albeit at low levels. Based on these findings, we conclude that T-cell-based CVs are a feasible approach to local Delta24-RGD delivery in glioblastoma, although efficient systemic targeting requires further improvement.

  3. Latest Insights on Adenovirus Structure and Assembly

    Carmen San Martín


    Adenovirus (AdV) capsid organization is considerably complex, not only because of its large size (~950 Å) and triangulation number (pseudo T = 25), but also because it contains four types of minor proteins in specialized locations modulating the quasi-equivalent icosahedral interactions. Up until 2009, only its major components (hexon, penton, and fiber) had separately been described in atomic detail. Their relationships within the virion, and the location of minor coat p...

  4. Chromatin structure of adenovirus DNA throughout infection

    Giberson, Andrea N.; Davidson, Adam R.; Parks, Robin J.


    For more than half a century, researchers have studied the basic biology of Adenovirus (Ad), unraveling the subtle, yet profound, interactions between the virus and the host. These studies have uncovered previously unknown proteins and pathways crucial for normal cell function that the virus manipulates to achieve optimal virus replication and gene expression. In the infecting virion, the viral DNA is tightly condensed in a virally encoded protamine-like protein which must be remodeled within...




    The mechanism of adenovirus attachment to the host cell plasma membrane has been revealed in detail by research over the past 10 years. It has long been known that receptor binding activity is associated with the viral fibers, trimeric spike proteins that protrude radially from the vertices of the icosahedral capsid (Philipson et al. 1968). In some adenovirus serotypes, fiber and other virus structural proteins are synthesized in excess and accumulate in the cell nucleus during late stages of infection. Fiber protein can be readily purified from lysates of cells infected with subgroup C viruses, for example Ad2 and Ad5 (Boulanger and Puvion 1973). Addition of purified fiber protein to virus suspensions during adsorption strongly inhibits infection, indicating that fiber and intact virus particles compete for binding sites on host cells (Philipson et al. 1968; Hautala et al. 1998). Cell binding studies using purified radiolabeled fiber demonstrated that fiber binds specifically and with high affinity to the cell plasma membrane, and that cell lines typically used for laboratory propagation of adenovirus have approximately 10{sup 4} high-affinity receptor sites per cell (Persson et al. 1985; Freimuth 1996). Similar numbers of high-affinity binding sites for radiolabeled intact virus particles also were observed (Seth et al. 1994).

  6. SPOC1-Mediated Antiviral Host Cell Response Is Antagonized Early in Human Adenovirus Type 5 Infection

    Schreiner, Sabrina; Kinkley, Sarah; Bürck, Carolin; Mund, Andreas; Wimmer, Peter; Schubert, Tobias; Groitl, Peter; Will, Hans; Dobner, Thomas


    Little is known about immediate phases after viral infection and how an incoming viral genome complex counteracts host cell defenses, before the start of viral gene expression. Adenovirus (Ad) serves as an ideal model, since entry and onset of gene expression are rapid and highly efficient, and mechanisms used 24–48 hours post infection to counteract host antiviral and DNA repair factors (e.g. p53, Mre11, Daxx) are well studied. Here, we identify an even earlier host cell target for Ad, the chromatin-associated factor and epigenetic reader, SPOC1, recently found recruited to double strand breaks, and playing a role in DNA damage response. SPOC1 co-localized with viral replication centers in the host cell nucleus, interacted with Ad DNA, and repressed viral gene expression at the transcriptional level. We discovered that this SPOC1-mediated restriction imposed upon Ad growth is relieved by its functional association with the Ad major core protein pVII that enters with the viral genome, followed by E1B-55K/E4orf6-dependent proteasomal degradation of SPOC1. Mimicking removal of SPOC1 in the cell, knock down of this cellular restriction factor using RNAi techniques resulted in significantly increased Ad replication, including enhanced viral gene expression. However, depletion of SPOC1 also reduced the efficiency of E1B-55K transcriptional repression of cellular promoters, with possible implications for viral transformation. Intriguingly, not exclusive to Ad infection, other human pathogenic viruses (HSV-1, HSV-2, HIV-1, and HCV) also depleted SPOC1 in infected cells. Our findings provide a general model for how pathogenic human viruses antagonize intrinsic SPOC1-mediated antiviral responses in their host cells. A better understanding of viral entry and early restrictive functions in host cells should provide new perspectives for developing antiviral agents and therapies. Conversely, for Ad vectors used in gene therapy, counteracting mechanisms eradicating incoming

  7. A broadly applicable method to characterize large DNA viruses and adenoviruses based on the DNA polymerase gene

    Montgomery Roy D


    Full Text Available Abstract Background Many viral pathogens are poorly characterized, are difficult to culture or reagents are lacking for confirmatory diagnoses. We have developed and tested a robust assay for detecting and characterizing large DNA viruses and adenoviruses. The assay is based on the use of degenerate PCR to target a gene common to these viruses, the DNA polymerase, and sequencing the products. Results We evaluated our method by applying it to fowl adenovirus isolates, catfish herpesvirus isolates, and largemouth bass ranavirus (iridovirus from cell culture and lymphocystis disease virus (iridovirus and avian poxvirus from tissue. All viruses with the exception of avian poxvirus produced the expected product. After optimization of extraction procedures, and after designing and applying an additional primer we were able to produce polymerase gene product from the avian poxvirus genome. The sequence data that we obtained demonstrated the simplicity and potential of the method for routine use in characterizing large DNA viruses. The adenovirus samples were demonstrated to represent 2 types of fowl adenovirus, fowl adenovirus 1 and an uncharacterized avian adenovirus most similar to fowl adenovirus 9. The herpesvirus isolate from blue catfish was shown to be similar to channel catfish virus (Ictalurid herpesvirus 1. The case isolate of largemouth bass ranavirus was shown to exactly match the type specimen and both were similar to tiger frog virus and frog virus 3. The lymphocystis disease virus isolate from largemouth bass was shown to be related but distinct from the two previously characterized lymphocystis disease virus isolates suggesting that it may represent a distinct lymphocystis disease virus species. Conclusion The method developed is rapid and broadly applicable to cell culture isolates and infected tissues. Targeting a specific gene for in the large DNA viruses and adenoviruses provide a common reference for grouping the newly identified

  8. About vectors

    Hoffmann, Banesh


    From his unusual beginning in ""Defining a vector"" to his final comments on ""What then is a vector?"" author Banesh Hoffmann has written a book that is provocative and unconventional. In his emphasis on the unresolved issue of defining a vector, Hoffmann mixes pure and applied mathematics without using calculus. The result is a treatment that can serve as a supplement and corrective to textbooks, as well as collateral reading in all courses that deal with vectors. Major topics include vectors and the parallelogram law; algebraic notation and basic ideas; vector algebra; scalars and scalar p

  9. Vector analysis

    Newell, Homer E


    When employed with skill and understanding, vector analysis can be a practical and powerful tool. This text develops the algebra and calculus of vectors in a manner useful to physicists and engineers. Numerous exercises (with answers) not only provide practice in manipulation but also help establish students' physical and geometric intuition in regard to vectors and vector concepts.Part I, the basic portion of the text, consists of a thorough treatment of vector algebra and the vector calculus. Part II presents the illustrative matter, demonstrating applications to kinematics, mechanics, and e

  10. Recombinant interferon-γ lentivirus co-infection inhibits adenovirus replication ex vivo.

    Ling Zhang

    Full Text Available Recombinant interferon-γ (IFNγ production in cultured lentivirus (LV was explored for inhibition of target virus in cells co-infected with adenovirus type 5 (Ad5. The ability of three different promoters of CMV, EF1α and Ubiquitin initiating the enhanced green fluorescence protein (GFP activities within lentiviruses was systematically assessed in various cell lines, which showed that certain cell lines selected the most favorable promoter driving a high level of transgenic expression. Recombinant IFNγ lentivirus carrying CMV promoter (LV-CMV-IFNγ was generated to co-infect 293A cells with a viral surrogate of recombinant GFP Ad5 in parallel with LV-CMV-GFP control. The best morphologic conditions were observed from the two lentiviruses co-infected cells, while single adenovirus infected cells underwent clear pathologic changes. Viral load of adenoviruses from LV-CMV-IFNγ or LV-CMV-GFP co-infected cell cultures was significantly lower than that from adenovirus alone infected cells (P=0.005-0.041, and the reduction of adenoviral load in the co-infected cells was 86% and 61%, respectively. Ad5 viral load from LV-CMV-IFNγ co-infected cells was significantly lower than that from LV-CMV-GFP co-infection (P=0.032, which suggested that IFNγ rather than GFP could further enhance the inhibition of Ad5 replication in the recombinant lentivirus co-infected cells. The results suggest that LV-CMV-IFNγ co-infection could significantly inhibit the target virus replication and might be a potential approach for alternative therapy of severe viral diseases.

  11. Recombinant interferon-γ lentivirus co-infection inhibits adenovirus replication ex vivo.

    Zhang, Ling; Yin, Sen; Tan, Wanlong; Xiao, Dong; Weng, Yunceng; Wang, Wenjing; Li, Tingting; Shi, Junwen; Shuai, Lifang; Li, Hongwei; Zhou, Jianhua; Allain, Jean-Pierre; Li, Chengyao


    Recombinant interferon-γ (IFNγ) production in cultured lentivirus (LV) was explored for inhibition of target virus in cells co-infected with adenovirus type 5 (Ad5). The ability of three different promoters of CMV, EF1α and Ubiquitin initiating the enhanced green fluorescence protein (GFP) activities within lentiviruses was systematically assessed in various cell lines, which showed that certain cell lines selected the most favorable promoter driving a high level of transgenic expression. Recombinant IFNγ lentivirus carrying CMV promoter (LV-CMV-IFNγ) was generated to co-infect 293A cells with a viral surrogate of recombinant GFP Ad5 in parallel with LV-CMV-GFP control. The best morphologic conditions were observed from the two lentiviruses co-infected cells, while single adenovirus infected cells underwent clear pathologic changes. Viral load of adenoviruses from LV-CMV-IFNγ or LV-CMV-GFP co-infected cell cultures was significantly lower than that from adenovirus alone infected cells (P=0.005-0.041), and the reduction of adenoviral load in the co-infected cells was 86% and 61%, respectively. Ad5 viral load from LV-CMV-IFNγ co-infected cells was significantly lower than that from LV-CMV-GFP co-infection (P=0.032), which suggested that IFNγ rather than GFP could further enhance the inhibition of Ad5 replication in the recombinant lentivirus co-infected cells. The results suggest that LV-CMV-IFNγ co-infection could significantly inhibit the target virus replication and might be a potential approach for alternative therapy of severe viral diseases.

  12. Lentivirus vectors construction of SiRNA targeting interferenceGPC3 gene and its biological effects on liver cancer cell lines Huh-7

    Chang-Jiang Lei; Chun Yao; Qing-Yun Pan; Hao-Cheng Long; Lei Li; Shu-Ping Zheng; Cheng Zeng; Jian-Bin Huang


    Objective:To buildGPC3 gene short hairpin interferenceRNA(shRNA) slow virus vector, observe expression ofHuh-7GPC3 gene in human liver cell line proliferation apoptosis and the effect ofGPC3 gene influencing on liver cancer cell growth, and provide theoretical basis for gene therapy of liver cancer.Methods:Hepatocellular carcinoma cell lineHuh-7 was transfected by aRNA interference technique.GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitativePCR.TargetedGPC3 gene sequences of small interfering RNA(siRNA)PGC-shRNA-GPC3 were restructured.Stable expression cell lines of siRNA were screened and established with the help of liposomes(lipofectamineTM2000) as carrier transfection of human liver cell lines.In order to validate siRNA interference efficiency,GPC3 siRNA mRNA expression was detected after transfection by usingRT-PCR andWestern blot.The absorbance value of the cells of blank group, untransfection group and transfection group, the cell cycle and cell apoptosis were calculated, and effects ofGPC3 gene onHuh-7 cell proliferation and apoptosis were observed.Results:In the liver cancer cell linesHuh-7,GPC3 gene showed high expression.PGC-shRNA-GPC3 recombinant plasmid was constructed successfully via sequencing validation.Stable recombinant plasmid transfected into liver cancer cell linesHuh-7 can obviously inhibitGPC3 mRNA expression level.Conclusions:The targetedGPC3 siRNA can effectively inhibit the expression ofGPC3.

  13. Multi Target Classification and Recognition Based on Support Vector Machine%基于支持向量机的多目标分类和识别

    侯小丽; 王建国; 王佳丽


    Support Vector Machine(SVM),which was developed for binary classification initially, now is widely used in many research fields like pattern recognition. However,its application to multi-classification is inefficient. This paper did research on the extension algorithms of SVM from binary classification to multi-classification,and found that Directed Acyclic Graph(DAG-SVM)is one of the most popularly used. Therefore,this paper focuses on its application of multi-classification in military area,and achieves recognition of many military objects,such as soldiers,armored cars,low altitude targets,and so on.%支持向量机(SVM)算法广泛应用于模式识别等领域,但是SVM最初是针对二类别分类提出,在多分类识别中稍显逊色。对将SVM由二分类扩展到多分类的算法进行了研究,发现有向无环图(DAG-SVM)是其中用的最多的算法之一。因此,针对军事领域图像的多目标分类,选择有向无环图算法来实现军事图像中单兵、装甲、低空等多目标的分类识别。

  14. A novel adenovirus in Chinstrap penguins (Pygoscelis antarctica) in Antarctica.

    Lee, Sook-Young; Kim, Jeong-Hoon; Park, Yon Mi; Shin, Ok Sarah; Kim, Hankyeom; Choi, Han-Gu; Song, Jin-Won


    Adenoviruses (family Adenoviridae) infect various organ systems and cause diseases in a wide range of host species. In this study, we examined multiple tissues from Chinstrap penguins (Pygoscelis antarctica), collected in Antarctica during 2009 and 2010, for the presence of novel adenoviruses by PCR. Analysis of a 855-bp region of the hexon gene of a newly identified adenovirus, designated Chinstrap penguin adenovirus 1 (CSPAdV-1), showed nucleotide (amino acid) sequence identity of 71.8% (65.5%) with South Polar skua 1 (SPSAdV-1), 71% (70%) with raptor adenovirus 1 (RAdV-1), 71.4% (67.6%) with turkey adenovirus 3 (TAdV-3) and 61% (61.6%) with frog adenovirus 1 (FrAdV-1). Based on the genetic and phylogenetic analyses, CSPAdV-1 was classified as a member of the genus, Siadenovirus. Virus isolation attempts from kidney homogenates in the MDTC-RP19 (ATCC® CRL-8135™) cell line were unsuccessful. In conclusion, this study provides the first evidence of new adenovirus species in Antarctic penguins.

  15. A Novel Adenovirus in Chinstrap Penguins (Pygoscelis antarctica in Antarctica

    Sook-Young Lee


    Full Text Available Adenoviruses (family Adenoviridae infect various organ systems and cause diseases in a wide range of host species. In this study, we examined multiple tissues from Chinstrap penguins (Pygoscelis antarctica, collected in Antarctica during 2009 and 2010, for the presence of novel adenoviruses by PCR. Analysis of a 855-bp region of the hexon gene of a newly identified adenovirus, designated Chinstrap penguin adenovirus 1 (CSPAdV-1, showed nucleotide (amino acid sequence identity of 71.8% (65.5% with South Polar skua 1 (SPSAdV-1, 71% (70% with raptor adenovirus 1 (RAdV-1, 71.4% (67.6% with turkey adenovirus 3 (TAdV-3 and 61% (61.6% with frog adenovirus 1 (FrAdV-1. Based on the genetic and phylogenetic analyses, CSPAdV-1 was classified as a member of the genus, Siadenovirus. Virus isolation attempts from kidney homogenates in the MDTC-RP19 (ATCC® CRL-8135™ cell line were unsuccessful. In conclusion, this study provides the first evidence of new adenovirus species in Antarctic penguins.

  16. Molecular architecture and function of adenovirus DNA polymerase

    Brenkman, A.B. (Arjan Bernard)


    Central to this thesis is the role of adenovirus DNA polymerase (Ad pol) in adenovirus DNA replication. Ad pol is a member of the family B DNA polymerases but belongs to a distinct subclass of polymerases that use a protein as primer. As Ad pol catalyses both the initiation and elongation phases and

  17. Luciferase imaging for evaluation of oncolytic adenovirus replication in vivo.

    Guse, K; Dias, J D; Bauerschmitz, G J; Hakkarainen, T; Aavik, E; Ranki, T; Pisto, T; Särkioja, M; Desmond, R A; Kanerva, A; Hemminki, A


    Oncolytic viruses kill cancer cells by tumor-selective replication. Clinical data have established the safety of the approach but also the need of improvements in potency. Efficacy of oncolysis is linked to effective infection of target cells and subsequent productive replication. Other variables include intratumoral barriers, access to target cells, uptake by non-target organs and immune response. Each of these aspects relates to the location and degree of virus replication. Unfortunately, detection of in vivo replication has been difficult, labor intensive and costly and therefore not much studied. We hypothesized that by coinfection of a luciferase expressing E1-deleted virus with an oncolytic virus, both viruses would replicate when present in the same cell. Photon emission due to conversion of D-Luciferin is sensitive and penetrates tissues well. Importantly, killing of animals is not required and each animal can be imaged repeatedly. Two different murine xenograft models were used and intratumoral coinjections of luciferase encoding virus were performed with eight different oncolytic adenoviruses. In both models, we found significant correlation between photon emission and infectious virus production. This suggests that the system can be used for non-invasive quantitation of the amplitude, persistence and dynamics of oncolytic virus replication in vivo, which could be helpful for the development of more effective and safe agents.

  18. Generation of an adenovirus-parvovirus chimera with enhanced oncolytic potential.

    El-Andaloussi, Nazim; Bonifati, Serena; Kaufmann, Johanna K; Mailly, Laurent; Daeffler, Laurent; Deryckère, François; Nettelbeck, Dirk M; Rommelaere, Jean; Marchini, Antonio


    In this study, our goal was to generate a chimeric adenovirus-parvovirus (Ad-PV) vector that combines the high-titer and efficient gene transfer of adenovirus with the anticancer potential of rodent parvovirus. To this end, the entire oncolytic PV genome was inserted into a replication-defective E1- and E3-deleted Ad5 vector genome. As we found that parvoviral NS expression inhibited Ad-PV chimera production, we engineered the parvoviral P4 early promoter, which governs NS expression, by inserting into its sequence tetracycline operator elements. As a result of these modifications, P4-driven expression was blocked in the packaging T-REx-293 cells, which constitutively express the tetracycline repressor, allowing high-yield chimera production. The chimera effectively delivered the PV genome into cancer cells, from which fully infectious replication-competent parvovirus particles were generated. Remarkably, the Ad-PV chimera exerted stronger cytotoxic activities against various cancer cell lines, compared with the PV and Ad parental viruses, while being still innocuous to a panel of tested healthy primary human cells. This Ad-PV chimera represents a novel versatile anticancer agent which can be subjected to further genetic manipulations in order to reinforce its enhanced oncolytic capacity through arming with transgenes or retargeting into tumor cells.

  19. Construction of recombinant human nerve growth factor beta adenovirus and evaluation of its function An in vitro and in vivo study

    En-Feng Gao; Jong-Ho Lee; Si-Ho Choi; Mi-Ae Sung; Bo-Han Li; Samir Jabaiti; Sang Bae Yoo; Sung-June Kim; Soung-Min Kim; Jeong Won Jahng


    Exogenous delivery of nerve growth factor(NGF)promotes neural regeneration.However,the short half-life limits delivery efficacy.Therefore,a long-term,efficient,local delivery tool or scheme is needed.The purpose of this study was to construct a functioning,recombinant,adenoviral vector carrying human NGF-β(hNGF-β)DNA,and to measure expression of the constructed vector in vitro and in vivo.rhNGF-β adenoviral vector containing full-length hNGF-β cDNA was generated by homologous recombination in Escherichia Coli.The rhNGF-β adenovirus was packaged and amplified in human embryonic kidney HEK293 cells.Transformation efficiency,expression and function of rhNGF-β adenovirus for primary Schwann cells,Schwann cell lines,human embryonic kidney HEK 293 cells,CRH myoblasts,and NIH3T3 fibroblasts were evaluated.Subsequently,expression of rhNGF-β adenovirus at the peripheral nerve of rat was also assessed.Recombinant adenoviral vector carrying hNGF-β was successfully constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis.Green fluorescent protein expression was observed in 90% of rhNGF-β adenovirus-infected cells(primary Schwann cells,Schwann cell line,human embryonic kidney HEK 293 cells,CRH myoblasts,and NIH3T3 fibroblasts)compared with non-infected cells.Total mRNA isolated from rhNGF-β adenovirus-infected cells exhibited strong expression.Maximum NGF release was induced by primary cultured Schwann cells at 4 days after infection,which steadily continued for 14 days.PC-12 cells exposed to media conditioned with rhNGF-β adenovirus-infected Schwann cells exhibited increased neurite extension.In vivo experiment revealed that the injected rhNGF-β adenovirus was transfected into the cells at the injected site and promoted expression of NGF,p75NTR and brain derived neurotrophic factor at the sciatic nerve and dorsal root ganglia.

  20. Molecular confirmation of an adenovirus in brushtail possums (Trichosurus vulpecula).

    Thomson, Darelle; Meers, Joanne; Harrach, Balázs


    Partial genome characterisation of a non-cultivable marsupial adenovirus is described. Adenovirus-like particles were found by electron microscopy (EM) in the intestinal contents of brushtail possums (Trichosurus vulpecula) in New Zealand. Using degenerate PCR primers complementary to the most conserved genome regions of adenoviruses, the complete nucleotide sequence of the penton base gene, and partial nucleotide sequences of the DNA polymerase, hexon, and pVII genes were obtained. Phylogenetic analysis of the penton base gene strongly suggested that the brushtail possum adenovirus (candidate PoAdV-1) belongs to the recently proposed genus Atadenovirus. Sequence analysis of the PCR products amplified from the intestinal contents of brushtail possums originating from different geographical regions of New Zealand identified a single genotype. This is the first report of molecular confirmation of an adenovirus in a marsupial.

  1. Enhanced nigrostriatal neuron-specific, long-term expression by using neural-specific promoters in combination with targeted gene transfer by modified helper virus-free HSV-1 vector particles

    Kong Lingxin


    Full Text Available Abstract Background Direct gene transfer into neurons has potential for developing gene therapy treatments for specific neurological conditions, and for elucidating neuronal physiology. Due to the complex cellular composition of specific brain areas, neuronal type-specific recombinant gene expression is required for many potential applications of neuronal gene transfer. One approach is to target gene transfer to a specific type of neuron. We developed modified Herpes Simplex Virus (HSV-1 particles that contain chimeric glycoprotein C (gC – glial cell line-derived neurotrophic factor (GDNF or brain-derived neurotrophic factor (BDNF proteins. HSV-1 vector particles containing either gC – GDNF or gC – BDNF target gene transfer to nigrostriatal neurons, which contain specific receptors for GDNF or BDNF. A second approach to achieve neuronal type-specific expression is to use a cell type-specific promoter, and we have used the tyrosine hydroxylase (TH promoter to restrict expression to catecholaminergic neurons or a modified neurofilament heavy gene promoter to restrict expression to neurons, and both of these promoters support long-term expression from HSV-1 vectors. To both improve nigrostriatal-neuron specific expression, and to establish that targeted gene transfer can be followed by long-term expression, we performed targeted gene transfer with vectors that support long-term, neuronal-specific expression. Results Helper virus-free HSV-1 vector packaging was performed using either gC – GDNF or gC – BDNF and vectors that contain either the TH promoter or the modified neurofilament heavy gene promoter. Vector stocks were injected into the midbrain proximal to the substantia nigra, and the rats were sacrificed at either 4 days or 1 month after gene transfer. Immunofluorescent costaining was performed to detect both recombinant gene products and nigrostriatal neurons. The combination of targeted gene transfer with neuronal

  2. Combination Adenovirus-Mediated HSV-tk/GCV and Antisense IGF-1 Gene Therapy for Rat Glioma


    Objective To investigate the effects of combination adenovirus-mediated HSV-tk/GCV system and antisense IGF-1 gene therapy for rat glioma and analyze the mechanism.Methods Using the recombinant adenovirus vector,GCV killing effeciency after combined gene transfer of HSV-tk and antisense IGF-1 was observed in vitro.Rat glioma was treated with HSV-tk/GCV and antisense IGF-1 and the survival rate of rats was observed.Results C6 cells transfected with tk and antisense IGF-1 gene were more sensitive to GCV than that transfected with tk gene alone.The survival of the combination gene therapy group was prolonged significantly and large amounts of CD+4,CD+8 lymphocytes were detected in the tumor tissues.Conclusion Antisense IGF-1 gene may enhance the tumor-killing effects of HSV-tk/GCV.

  3. Protection of guinea pigs and swine by a recombinant adenovirus expressing O serotype of foot-and-mouth disease virus whole capsid and 3C protease.

    Lu, Zengjun; Bao, Huifang; Cao, Yimei; Sun, Pu; Guo, Jianhun; Li, Pinghua; Bai, Xingwen; Chen, Yingli; Xie, Baoxia; Li, Dong; Liu, Zaixin; Xie, Qingge


    Two recombinant adenoviruses were constructed expressing foot-and-mouth disease virus (FMDV) capsid and 3C/3CD proteins in replicative deficient human adenovirus type 5 vector. Guinea pigs vaccinated with 1-3 x 10(8)TCID(50) Ad-P12x3C recombinant adenovirus were completely protected against 10,000GID(50) homologous virulent FMDV challenge 25 days post vaccination (dpv). Ad-P12x3CD vaccinated guinea pigs were only partially protected. Swine were vaccinated once with 1x10(9)TCID(50) Ad-P12x3C hybrid virus and challenged 28 days later. Three of four vaccinated swine were completely protected against 200 pig 50% infectious doses (ID(50)) of homologous FMDV challenge, and vaccinated pigs developed specific cellular and humoral immune responses. The immune effect of Ad-P12x3C in swine further indicated that the recombinant adenovirus was highly efficient in transferring the foreign gene. This approach may thus be a very hopeful tool for developing FMD live virus vector vaccine.

  4. Elementary vectors

    Wolstenholme, E Œ


    Elementary Vectors, Third Edition serves as an introductory course in vector analysis and is intended to present the theoretical and application aspects of vectors. The book covers topics that rigorously explain and provide definitions, principles, equations, and methods in vector analysis. Applications of vector methods to simple kinematical and dynamical problems; central forces and orbits; and solutions to geometrical problems are discussed as well. This edition of the text also provides an appendix, intended for students, which the author hopes to bridge the gap between theory and appl

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

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


    . 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...

  6. Adenovirus replication is coupled with the dynamic properties of the PML nuclear structure.

    Doucas, V; Ishov, A M; Romo, A; Juguilon, H; Weitzman, M D; Evans, R M; Maul, G G


    Wild-type PML and at least four other novel proteins are localized within discrete nuclear structures known as PODs. We demonstrate here that during adenovirus infection, immediate early viral proteins from the E1 and E4 transcription units associate with the POD, which in turn undergoes a dramatic morphological change. During this process, the auto-antigen Sp-100 and NDP55 but not PML, relocate from the POD to the viral inclusion bodies, the sites of adenovirus DNA replication and late RNA transcription. The E4-ORF3 11-kD protein alone will induce this reorganization and reciprocally, viruses carrying mutations in the E4-domain fail to do so. These same viral mutants are defective in viral replication as well as the accumulation of late viral mRNAs and host cell transcription shutoff. We show that interferon (INF) treatment enhances the expression of PML, reduces or blocks PODs reorganization, and inhibits BrdU incorporation into viral inclusion bodies. In addition, cell lines engineered to overexpress PML prevent PODs from viral-induced reorganization and block or severely delay adenovirus replication. These results suggest that viral replication relies on components of the POD and that the structure is a target of early viral proteins.

  7. Inactivation of adenovirus using low-dose UV/H2O2 advanced oxidation.

    Bounty, Sarah; Rodriguez, Roberto A; Linden, Karl G


    Adenovirus has consistently been observed to be the most resistant known pathogen to disinfection by ultraviolet light. This has had an impact on regulations set by the United States Environmental Protection Agency regarding the use of UV disinfection for virus inactivation in groundwater and surface water. In this study, enhancement of UV inactivation of adenovirus was evaluated when hydrogen peroxide was added to create an advanced oxidation process (AOP). While 4 log reduction of adenovirus was determined to require a UV dose (UV fluence) of about 200 mJ/cm(2) from a low pressure (LP) UV source (emitting at 253.7 nm), addition of 10 mg/L H(2)O(2) achieved 4 log inactivation at a dose of 120 mJ/cm(2). DNA damage was assessed using a novel nested PCR approach, and similar levels of DNA damage between the two different treatments were noted, suggesting the AOP enhancement in inactivation was not due to additional DNA damage. Hydroxyl radicals produced in the advanced oxidation process are likely able to damage parts of the virus not targeted by LPUV, such as attachment proteins, enhancing the UV-induced inactivation. The AOP-enhanced inactivation potential was modeled in three natural waters. This research sheds light on the inactivation mechanisms of viruses with ultraviolet light and in the presence of hydroxyl radicals and provides a practical means to enhance inactivation of this UV-resistant virus.

  8. Participatory mapping of target areas to enable operational larval source management to suppress malaria vector mosquitoes in Dar es Salaam, Tanzania

    Dongus Stefan


    Full Text Available Abstract Background Half of the population of Africa will soon live in towns and cities where it can be protected from malaria by controlling aquatic stages of mosquitoes. Rigorous but affordable and scaleable methods for mapping and managing mosquito habitats are required to enable effective larval control in urban Africa. Methods A simple community-based mapping procedure that requires no electronic devices in the field was developed to facilitate routine larval surveillance in Dar es Salaam, Tanzania. The mapping procedure included (1 community-based development of sketch maps and (2 verification of sketch maps through technical teams using laminated aerial photographs in the field which were later digitized and analysed using Geographical Information Systems (GIS. Results Three urban wards of Dar es Salaam were comprehensively mapped, covering an area of 16.8 km2. Over thirty percent of this area were not included in preliminary community-based sketch mapping, mostly because they were areas that do not appear on local government residential lists. The use of aerial photographs and basic GIS allowed rapid identification and inclusion of these key areas, as well as more equal distribution of the workload of malaria control field staff. Conclusion The procedure developed enables complete coverage of targeted areas with larval control through comprehensive spatial coverage with community-derived sketch maps. The procedure is practical, affordable, and requires minimal technical skills. This approach can be readily integrated into malaria vector control programmes, scaled up to towns and cities all over Tanzania and adapted to urban settings elsewhere in Africa.

  9. Exclusive and common targets of neostriatofugal projections of rat striosome neurons: a single neuron-tracing study using a viral vector.

    Fujiyama, Fumino; Sohn, Jaerin; Nakano, Takashi; Furuta, Takahiro; Nakamura, Kouichi C; Matsuda, Wakoto; Kaneko, Takeshi


    The rat neostriatum has a mosaic organization composed of striosome/patch compartments embedded in a more extensive matrix compartment, which are distinguished from each other by the input-output organization as well as by the expression of many molecular markers. The matrix compartment gives rise to the dual γ-aminobutyric acid (GABA)ergic striatofugal systems, i.e. direct and indirect pathway neurons, whereas the striosome compartment is considered to involve direct pathway neurons alone. Although the whole axonal arborization of matrix striatofugal neurons has been examined in vivo by intracellular staining, that of striosome neurons has never been studied at the single neuron level. In the present study, the axonal arborizations of single striosome projection neurons in rat neostriatum were visualized in their entirety using a viral vector expressing membrane-targeted green fluorescent protein, and compared with that of matrix projection neurons. We found that not only matrix but also striosome compartments contained direct and indirect pathway neurons. Furthermore, only striatonigral neurons in the striosome compartment projected directly to the substantia nigra pars compacta (SNc), although they sent a substantial number of axon collaterals to the globus pallidus, entopeduncular nucleus and/or substantia nigra pars reticulata. These results suggest that striosome neurons play a more important role in the formation of reward-related signals of SNc dopaminergic neurons than do matrix neurons. Together with data from previous studies in the reinforcement learning theory, our results suggest that these direct and indirect striosome-SNc pathways together with nigrostriatal dopaminergic neurons may help striosome neurons to acquire the state-value function.

  10. Silencing of hepatitis C virus replication by a non-viral vector based on solid lipid nanoparticles containing a shRNA targeted to the internal ribosome entry site (IRES).

    Torrecilla, Josune; Del Pozo-Rodríguez, Ana; Solinís, María Ángeles; Apaolaza, Paola S; Berzal-Herranz, Beatriz; Romero-López, Cristina; Berzal-Herranz, Alfredo; Rodríguez-Gascón, Alicia


    Gene silencing mediated by RNAi has gained increasing interest as an alternative for the treatment of infectious diseases such as refractory hepatitis C virus (HCV) infection. In this work we have designed and evaluated a non-viral vector based on solid lipid nanoparticles (SLN) bearing hyaluronic acid, protamine and a short hairpin RNA (shRNA74) targeted to the Internal Ribosome Entry Site (IRES) of the HCV. The vector was able to inhibit the expression of the HCV IRES in Huh-7 cells, with the inhibition level dependent on the shRNA74 to SLN ratio and on the shRNA74 dose added to the culture cells. The nanocarrier was also able to inhibit the replication in human hepatoma cells supporting a subgenomic HCV replicon (Huh-7 NS3-3'). The vector was quickly and efficiently internalized by the cells, and endocytosis was the most productive uptake mechanism for silencing. Clathrin-mediated endocytosis and to a lesser extent caveolae/lipid raft-mediated endocytosis were identified as endocytic mechanisms involved in the cell uptake. Internalization via the CD44 receptor was also involved, although this entry route seems to be less productive for silencing than endocytosis. The vector did not induce either hemolysis or agglutination of red cells in vitro, which was indicative of good biocompatibility. In summary, we have shown for the first time the ability of a non-viral SLN-based vector to silence a HCV replicon.

  11. Adenovirus entry from the apical surface of polarized epithelia is facilitated by the host innate immune response.

    Kotha, Poornima L N; Sharma, Priyanka; Kolawole, Abimbola O; Yan, Ran; Alghamri, Mahmoud S; Brockman, Trisha L; Gomez-Cambronero, Julian; Excoffon, Katherine J D A


    Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection. The primary receptor for most adenoviruses is the coxsackievirus and adenovirus receptor (CAR), a cell-cell adhesion protein normally localized at the basolateral surface of polarized epithelia and involved in neutrophil transepithelial migration. Recently, an alternate isoform of CAR, CAREx8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that the endogenous role of CAREx8 may be to facilitate host innate immunity. We show that IL-8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAREx8 via activation of AKT/S6K and inhibition of GSK3β. Apical CAREx8 tethers infiltrating neutrophils at the apical surface of a polarized epithelium. Moreover, neutrophils present on the apical-epithelial surface enhance adenovirus entry into the epithelium. These findings suggest that adenovirus evolved to co-opt an innate immune response pathway that stimulates the expression of its primary receptor, apical CAREx8, to allow the initial infection the intact epithelium. In addition, CAREx8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection.

  12. Adenovirus entry from the apical surface of polarized epithelia is facilitated by the host innate immune response.

    Poornima L N Kotha


    Full Text Available Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection. The primary receptor for most adenoviruses is the coxsackievirus and adenovirus receptor (CAR, a cell-cell adhesion protein normally localized at the basolateral surface of polarized epithelia and involved in neutrophil transepithelial migration. Recently, an alternate isoform of CAR, CAREx8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that the endogenous role of CAREx8 may be to facilitate host innate immunity. We show that IL-8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAREx8 via activation of AKT/S6K and inhibition of GSK3β. Apical CAREx8 tethers infiltrating neutrophils at the apical surface of a polarized epithelium. Moreover, neutrophils present on the apical-epithelial surface enhance adenovirus entry into the epithelium. These findings suggest that adenovirus evolved to co-opt an innate immune response pathway that stimulates the expression of its primary receptor, apical CAREx8, to allow the initial infection the intact epithelium. In addition, CAREx8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection.

  13. DNA prime/Adenovirus boost malaria vaccine encoding P. falciparum CSP and AMA1 induces sterile protection associated with cell-mediated immunity.

    Ilin Chuang

    Full Text Available BACKGROUND: Gene-based vaccination using prime/boost regimens protects animals and humans against malaria, inducing cell-mediated responses that in animal models target liver stage malaria parasites. We tested a DNA prime/adenovirus boost malaria vaccine in a Phase 1 clinical trial with controlled human malaria infection. METHODOLOGY/PRINCIPAL FINDINGS: The vaccine regimen was three monthly doses of two DNA plasmids (DNA followed four months later by a single boost with two non-replicating human serotype 5 adenovirus vectors (Ad. The constructs encoded genes expressing P. falciparum circumsporozoite protein (CSP and apical membrane antigen-1 (AMA1. The regimen was safe and well-tolerated, with mostly mild adverse events that occurred at the site of injection. Only one AE (diarrhea, possibly related to immunization, was severe (Grade 3, preventing daily activities. Four weeks after the Ad boost, 15 study subjects were challenged with P. falciparum sporozoites by mosquito bite, and four (27% were sterilely protected. Antibody responses by ELISA rose after Ad boost but were low (CSP geometric mean titer 210, range 44-817; AMA1 geometric mean micrograms/milliliter 11.9, range 1.5-102 and were not associated with protection. Ex vivo IFN-γ ELISpot responses after Ad boost were modest (CSP geometric mean spot forming cells/million peripheral blood mononuclear cells 86, range 13-408; AMA1 348, range 88-1270 and were highest in three protected subjects. ELISpot responses to AMA1 were significantly associated with protection (p = 0.019. Flow cytometry identified predominant IFN-γ mono-secreting CD8+ T cell responses in three protected subjects. No subjects with high pre-existing anti-Ad5 neutralizing antibodies were protected but the association was not statistically significant. SIGNIFICANCE: The DNA/Ad regimen provided the highest sterile immunity achieved against malaria following immunization with a gene-based subunit vaccine (27%. Protection

  14. 腺病毒介导的人表皮生长因子基因转染对人表皮细胞生物学特性的影响%Effects of transfection of human epidermal growth factor gene with adenovirus vector on biological characteristics of human epidermal cells

    尹凯; 马丽; 申传安; 尚玉茹; 李大伟; 李龙珠; 赵东旭; 程文凤


    Objective To investigate the suitable transfection condition of human epidermal cells (hECs) with human epidermal growth factor (EGF) gene by adenovirus vector (Ad-hEGF) and its effects on the biological characteristics of hECs.Methods hECs were isolated from deprecated human fresh prepuce tissue of circumcision by enzyme digestion method and then sub-cultured.hECs of the third passage were used in the following experiments.(1) Cells were divided into non-transfection group and 5,20,50,100,150,and 200 fold transfection groups according to the random number table (the same grouping method below),with 3 wells in each group.Cells in non-transfection group were not transfected with Ad-hEGF gene,while cells in the latter six groups were transfected with Ad-hEGF gene in multiplicities of infection (MOI) of 5,20,50,100,150,and 200 respectively.The morphology of the cells was observed with inverted phase contrast microscope,and expression of green fluorescent protein of the cells was observed with inverted fluorescence microscope at transfection hour (TH) 24,48,and 72.(2) Another three batches of cells were collected,grouped,and treated as above,respectively.Then the transfection rate of Ad-hEGF gene was detected by flow cytometer (n =3),the mass concentration of EGF in culture supernatant of cells was detected by enzyme-linked immunosorbent assay (n =6),and the proliferation activity of cells was detected by cell counting kit 8 (CCK8) and microplate reader (n =6) at TH 24,48,and 72,respectively.(3) Cells were collected and divided into non-transfection group and transfection group,with 6 wells in each group.Cells in non-transfection group were cultured with culture supernatant of cells without transfection,while cells in transfection group were cultured with culture supernatant of cells which were transfected with Ad-hEGF gene in the optimum MOI (50).CCK8 and microplate reader were used to measure the biological activity of EGF secreted by cells on culture day 1,3,and 5

  15. Effects of recombinant adenoviral vector containing IRE1α gene on proliferation and apoptosis of ATDC5 stem cells

    Xiang-zhu LI


    Full Text Available Objective To construct the recombinant adenoviral vector containing human IRE1α (type I transmembrane protein kinase/endoribonucleasegene, and investigate its effects on proliferation and apoptosis of ATDC5 stem cells. Methods  By using pAdEasyTM adenovirus vector system, the recombinant shuttle vectors of IRE1α full-length gene(pAdTrack-IRE1αand RNase+Kinasedomain(pAdTrack-R+Kwere constructed, and then transferred with pAdEasy-1 to generate recombinant adenovirus plasmid pAd-IRE1α and pAd-R+K by electroporation method. Subsequently, the plasmids were transfected into HEK-293 cells to pack and amplify the recombinant adenovirus Ad-IRE1α and Ad-R+K. The expression of recombinant adenovirus was detected by PCR. The ATDC5 cells wereinfected in vitro by recombinant adenovirus Ad-IRE1α and Ad-R+K, the infection efficiency of green fluorescent protein(GFPwas observed, and the influence of Ad-IRE1α and Ad-R+K on the proliferation and apoptosis of ATDC5 cells under endoplasmic reticulum stress(ERS or non-ERS was detected by flow cytometry(FCM. Results Restriction endonuclease digestion analysis and PCR indicated that the recombinant adenovirus vector Ad-IRE1α andAd-R+K was successfully constructed. FCM detection showed that under ERS conditions, the G1 phasedcreased and S phase increased in ATDC5 cells after transfected by Ad-IRE1α and Ad-R+K, meanwhile the apoptosis rate increased significantly(P<0.05. Conclusion Infection of recombinant adenovirus containing IRE1α gene may promote the proliferation and apoptosis of ATDC5cells.

  16. Vaxvec: The first web-based recombinant vaccine vector database and its data analysis.

    Deng, Shunzhou; Martin, Carly; Patil, Rasika; Zhu, Felix; Zhao, Bin; Xiang, Zuoshuang; He, Yongqun


    A recombinant vector vaccine uses an attenuated virus, bacterium, or parasite as the carrier to express a heterologous antigen(s). Many recombinant vaccine vectors and related vaccines have been developed and extensively investigated. To compare and better understand recombinant vectors and vaccines, we have generated Vaxvec (, the first web-based database that stores various recombinant vaccine vectors and those experimentally verified vaccines that use these vectors. Vaxvec has now included 59 vaccine vectors that have been used in 196 recombinant vector vaccines against 66 pathogens and cancers. These vectors are classified to 41 viral vectors, 15 bacterial vectors, 1 parasitic vector, and 1 fungal vector. The most commonly used viral vaccine vectors are double-stranded DNA viruses, including herpesviruses, adenoviruses, and poxviruses. For example, Vaxvec includes 63 poxvirus-based recombinant vaccines for over 20 pathogens and cancers. Vaxvec collects 30 recombinant vector influenza vaccines that use 17 recombinant vectors and were experimentally tested in 7 animal models. In addition, over 60 protective antigens used in recombinant vector vaccines are annotated and analyzed. User-friendly web-interfaces are available for querying various data in Vaxvec. To support data exchange, the information of vaccine vectors, vaccines, and related information is stored in the Vaccine Ontology (VO). Vaxvec is a timely and vital source of vacc