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529. Evaluation of Porcine AAVs as Vaccine Vectors Against Avian Flu and Zaire Ebolavirus Infections
AAV VECTOR DESIGN & APPLICATION injection, 0.06mg/kg of tacrolimus was administered and further continuously given during experiment. Viral solution of 1x1013 v.g./ muscle was intramuscularly injected at bilateral tibialis anterior and biceps brachii muscles. Transduced muscles were biopsied at 8, 16, 24, and 42 weeks after injection, and eventually sacrificed for systemic evaluation. X-gal staining and anti-FLAG immunostaining were performed and systemic profile of inflammation was examined. Results: Without the use of tacrolimus, AAV8LacZ expression was disappeared 8 weeks after transduction and AAV9-M3-FLAG immunostaining lasted up to 24 weeks with mononuclear cell infiltrarion. Using tacrolimus, AAV8-mediated LacZ expression was lasted at least 42 weeks after the injection without remarkable inflammatory cell infiltration. Almost 50% of X-gal positive fibers were seen 42weeks after AAV8 CMVLacZ injection, while 17% of X-gal positive fibers were seen 42weeks after AAV9 CMVLacZ injection. AAV9-M3-FLAG immunostaining lasted up to 42 weeks with minimum infiltrarion of mononuclear cell using tacrolimus. Trough blood level of tacrolimus was monitored and was kept under 20 ng/ml. No expression of LacZ was observed in tissues other than injected skeletal muscles. There was no laboratory abnormality or morbidity in any of the monkeys at any time during the observation period. Also, no pathologic abnormality was detected in major organs including liver and heart. Conclusion: With all the tested monkeys, no significant generalized toxicity was observed with rAAV8 and rAAV9. Tacrolimus administration effectively regulates immune response to LacZ and artificial truncated microdystrophin in normal primate. AAV8 is more likely to be affected by tacrolimus than AAV9.This tacrolimus-assisted transduction strategy would enhance therapeutic benefits of AAV-mediated gene therapy of muscular dystrophy.
529. Evaluation of Porcine AAVs as Vaccine Vectors Against Avian Flu and Zaire Ebolavirus Infections
Alexander J. Bello,1,2 Allan R. Chand,1,2 Gary P. Kobinger.1,2 1 Special Pathogens, Public Health Agency of Canada, Winnipeg, MB, Canada; 2Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada. The ability of recombinant Adeno-associated virus (AAV) to deliver genes to a host in a safe and well tolerated manner has been exploited in a number of different clinical trials with varying degrees of success. The majority of these clinical trials were for the treatment of genetic diseases. The potential of AAV as a vaccine vector has, however, been limited to few disease models mainly in rodents, with the exception of a clinical trial involving the use of AAV as a vaccine vector for Human Immunodeficiency virus (HIV). In this study, we evaluated the protective efficacy of primate and porcine-derived AAVs against two different emerging/re-emerging pathogens: Avian influenza H5N1 and Zaire ebolavirus. Our primary objective was to evaluate AAV’s potential to stimulate humoral and cellular immune responses in parallel to protection following lethal challenge in mice. Porcine-derived AAVpo1, -po4, -po5, and –po6, as well as AAVs -8 and –rh32.33 isolated from non-human primates, all expressed either the influenza hemagglutinin (HA; Hanoi 2005) or the Zaire ebolavirus glycoprotein (ZEBOV-GP). AAV8 or -rh32.33 were used as control benchmarks, since only the latter was shown to generate functional T cells in vaccinated hosts. BALB/C mice were vaccinated intramuscularly (IM) with 2.5E10 of each AAV vector and challenged with 100 or 1000 LD50 28 days later with H5N1 or mouse-adapted ZEBOV respectively. Infected animals were monitored for weight loss and signs of disease for up to 21 days post-challenge. T or B-cell responses were monitored by ELISPOT or neutralizing antibody assays (NAB), total IgGs and hemaglutination inhibition (HI) assays at various time-points respectively. Interestingly, antigen-specific T-cell responses continued to rise up to 40 days post vaccination. In order to identify the best AAV vector for vaccine application among S204
the AAVs tested, a vector dose response experiment was performed. Overall, AAVpo1 performed better at a dose of 1E9 GC/mouse, followed by AAVpo6, while all but one AAV protected 100% of the mice against lethal challenge at a dose of 2.5E10 GC/mouse. These results indicate that porcine AAVs are promising vaccine vector candidates.
530. An AAV Vector-Based Toolbox for Somatic Reprogramming and for iPS Cell Tracking and Purging
Elena Senís,1 Eike Kienle,1 Carlos A. Fajardo,1 Inci Aydin,1 Dirk Grimm.1 1 Inf. Diseases/Virology, Cluster of Excellence CellNetworks, Heidelberg University, Heidelberg, Germany.
Induced pluripotent stem cells (iPSC), derived by deliberate expression of a set of reprogramming factors (transcription factors and/or miRNAs) in adult somatic cells, have tremendous potential for regenerative medicine. Still, several challenges remain to be overcome prior to clinical translation of iPS cells and technologies. Particular concerns pertain to the vectors used for delivering the reprogramming cocktail and hence to the safety and robustness of the entire process. For instance, integrating retroviral vectors bear a high risk of transforming iPSC and their progeny, while adenoviral vectors may adversely perturb cellular miRNA and gene expression. Non-viral alternatives, such as plasmids, pure mRNAs or proteins, are safer but exhibit slower reprogramming kinetics and lower efficiencies. Another concern in the context of human cell therapy is that iPSC which escape the differentiation step into new somatic cells remain pluripotent and may form tumors following cell transplantation. To overcome these two issues - lack of a safe yet potent reprogramming vector and of means to purge non-differentiated iPSC - we started to assemble a comprehensive toolbox based on non-pathogenic and non-integrating Adeno-associated viral (AAV) vectors. In a first step, we engineered single- or double-stranded AAV vectors to express the four genuine reprogramming factors (Oct4, Klf4, Sox2, c-myc) as well as the miR302/367 miRNA cluster. Moreover, the wealth of natural AAV serotypes allowed us to moleculary evolve synthetic variants tailored to classic donor cells for reprogramming or to iPSC, respectively. We thus screened our collection of AAV capsid libraries and indeed isolated several chimeric candidates that outperformed natural AAV isolates on fibroblasts as well as on murine and human iPSC. Here, we present our findings from our evaluation of various ensuing combinations of AAV expression cassettes, vector genomes and optimized capsids regarding their efficiency for iPSC generation in direct comparison to a widely used lentiviral vector-based reprogramming system. Moreover, we created AAV vectors that can distinguish fully reprogrammed iPSC from their non-differentiated ancestors based on the down-regulation of let-7 miRNA in stem cells. Accordingly, our vectors encode a gfp reporter gene that we tagged with perfect let-7 binding sites, resulting in exclusive vector expression in pluripotent cells and hence allowing iPSC identification and tracking in somatic cell cultures undergoing reprogramming. Notably, as AAV vectors remain episomal, our constructs are eventually lost during cell reprogramming which adds to the safety of our approach. Finally, we also present AAV vectors that we designed to express suicide genes in a miRNA-sensitive manner whereby differentiated iPSC progeny are protected from cell killing due to the presence of specific miRNAs. In contrast, partly or nondifferentiated iPSC are eradicated from the culture as they lack these selected miRNA. As a whole, our new toolbox of AAV vectors for iPSC generation, tracking and purging should complement or even replace existing technologies and markedly advance the blooming field of somatic reprogramming.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy
529. Evaluation of Porcine AAVs as Vaccine Vectors Against Avian Flu and Zaire Ebolavirus Infections
Alexander J. Bello,1,2 Allan R. Chand,1,2 Gary P. Kobinger.1,2 1 Special Pathogens, Public Health Agency of Canada, Winnipeg, MB, Canada; 2Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada. The ability of recombinant Adeno-associated virus (AAV) to deliver genes to a host in a safe and well tolerated manner has been exploited in a number of different clinical trials with varying degrees of success. The majority of these clinical trials were for the treatment of genetic diseases. The potential of AAV as a vaccine vector has, however, been limited to few disease models mainly in rodents, with the exception of a clinical trial involving the use of AAV as a vaccine vector for Human Immunodeficiency virus (HIV). In this study, we evaluated the protective efficacy of primate and porcine-derived AAVs against two different emerging/re-emerging pathogens: Avian influenza H5N1 and Zaire ebolavirus. Our primary objective was to evaluate AAV’s potential to stimulate humoral and cellular immune responses in parallel to protection following lethal challenge in mice. Porcine-derived AAVpo1, -po4, -po5, and –po6, as well as AAVs -8 and –rh32.33 isolated from non-human primates, all expressed either the influenza hemagglutinin (HA; Hanoi 2005) or the Zaire ebolavirus glycoprotein (ZEBOV-GP). AAV8 or -rh32.33 were used as control benchmarks, since only the latter was shown to generate functional T cells in vaccinated hosts. BALB/C mice were vaccinated intramuscularly (IM) with 2.5E10 of each AAV vector and challenged with 100 or 1000 LD50 28 days later with H5N1 or mouse-adapted ZEBOV respectively. Infected animals were monitored for weight loss and signs of disease for up to 21 days post-challenge. T or B-cell responses were monitored by ELISPOT or neutralizing antibody assays (NAB), total IgGs and hemaglutination inhibition (HI) assays at various time-points respectively. Interestingly, antigen-specific T-cell responses continued to rise up to 40 days post vaccination. In order to identify the best AAV vector for vaccine application among S204
the AAVs tested, a vector dose response experiment was performed. Overall, AAVpo1 performed better at a dose of 1E9 GC/mouse, followed by AAVpo6, while all but one AAV protected 100% of the mice against lethal challenge at a dose of 2.5E10 GC/mouse. These results indicate that porcine AAVs are promising vaccine vector candidates.
530. An AAV Vector-Based Toolbox for Somatic Reprogramming and for iPS Cell Tracking and Purging
Elena Senís,1 Eike Kienle,1 Carlos A. Fajardo,1 Inci Aydin,1 Dirk Grimm.1 1 Inf. Diseases/Virology, Cluster of Excellence CellNetworks, Heidelberg University, Heidelberg, Germany.
Induced pluripotent stem cells (iPSC), derived by deliberate expression of a set of reprogramming factors (transcription factors and/or miRNAs) in adult somatic cells, have tremendous potential for regenerative medicine. Still, several challenges remain to be overcome prior to clinical translation of iPS cells and technologies. Particular concerns pertain to the vectors used for delivering the reprogramming cocktail and hence to the safety and robustness of the entire process. For instance, integrating retroviral vectors bear a high risk of transforming iPSC and their progeny, while adenoviral vectors may adversely perturb cellular miRNA and gene expression. Non-viral alternatives, such as plasmids, pure mRNAs or proteins, are safer but exhibit slower reprogramming kinetics and lower efficiencies. Another concern in the context of human cell therapy is that iPSC which escape the differentiation step into new somatic cells remain pluripotent and may form tumors following cell transplantation. To overcome these two issues - lack of a safe yet potent reprogramming vector and of means to purge non-differentiated iPSC - we started to assemble a comprehensive toolbox based on non-pathogenic and non-integrating Adeno-associated viral (AAV) vectors. In a first step, we engineered single- or double-stranded AAV vectors to express the four genuine reprogramming factors (Oct4, Klf4, Sox2, c-myc) as well as the miR302/367 miRNA cluster. Moreover, the wealth of natural AAV serotypes allowed us to moleculary evolve synthetic variants tailored to classic donor cells for reprogramming or to iPSC, respectively. We thus screened our collection of AAV capsid libraries and indeed isolated several chimeric candidates that outperformed natural AAV isolates on fibroblasts as well as on murine and human iPSC. Here, we present our findings from our evaluation of various ensuing combinations of AAV expression cassettes, vector genomes and optimized capsids regarding their efficiency for iPSC generation in direct comparison to a widely used lentiviral vector-based reprogramming system. Moreover, we created AAV vectors that can distinguish fully reprogrammed iPSC from their non-differentiated ancestors based on the down-regulation of let-7 miRNA in stem cells. Accordingly, our vectors encode a gfp reporter gene that we tagged with perfect let-7 binding sites, resulting in exclusive vector expression in pluripotent cells and hence allowing iPSC identification and tracking in somatic cell cultures undergoing reprogramming. Notably, as AAV vectors remain episomal, our constructs are eventually lost during cell reprogramming which adds to the safety of our approach. Finally, we also present AAV vectors that we designed to express suicide genes in a miRNA-sensitive manner whereby differentiated iPSC progeny are protected from cell killing due to the presence of specific miRNAs. In contrast, partly or nondifferentiated iPSC are eradicated from the culture as they lack these selected miRNA. As a whole, our new toolbox of AAV vectors for iPSC generation, tracking and purging should complement or even replace existing technologies and markedly advance the blooming field of somatic reprogramming.
Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy