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845. Characterization of a Bipartite rAAV Vector for Site Specific Integration
weeks post surgery animals received peripheral intravenous injections of AAV2-FIX at doses of I x 1012 vglkg or I x 1013 vg/kg. Semen samples were obtained prior to vector injection and weekly thereafter. Vector-derived DNA was assessed by a specific PCR assay with a sensitivity of detecting 8 copies/ug genomic DNA. In the low-dose cohort, AAV-2 sequences were detected only at day 2-time point post-injection in the vasectomized rabbits (n=3), and persistently tested negative thereafter (follow up of 14 weeks). In contrast, non-vasectomized rabbits (n=5) injected with similar dose , vector sequences persisted from 6-8 weeks post-injection. In the high dose cohort, vector sequences were detected up to week lOin the vasectomized rabbits, whereas in the non-vasctomized animals vector sequences persisted up to week 13. We next sought to determine whether by changing theAAV serotype, the kinetics of vector clearance would change. We injected vasectomized rabbits with low (n=3) or high (n=2) AAV-8-FIX and compared to those animals injected with AAV-2 at comparable vector doses. In the high-dose cohort, samples collected from week 6 and week 10 were positive at rates of 22% vs. 61% for rabbits injected with AAV-2 or AAV-8, respectively, whereas after week 12 all samples tested negative. There were no differences in the vector clearance among rabbits from the low-dose eohorts of AAV·2 and AAV-8. We documented time course hFIX expression in all injected rabbits. Circulating hFIX plateau levels 10 week following high doses of AAV-2 or AAV-8 were 1539 ng/ml or2050 ng/ml , respectively. In the low-dose groups, FIX levels vary from 310 nglml to 433 ng/ml followingAAV-2 and AAV-8. Taken together, these data demonstrate that the kinetics of AAV vector clearance seems to be vector serotype-independent and vector shedding to the semen does not require the presence ofgerm cells. Thus the low risk ofgermline transmission ofAAV vectors is likely to be acceptable for many clinical trials.
844. Differential Immune Responses to Transgene Products from rAAV1 and rAAV8 Vectors Yuanqing Lu, Sihong Song. 'Pharmaceutics, University ofFlorida, Gainesville, FL.
Recently developed ncw scrotypcs of recombinant adeno-associated virus (rAAV) vectors have significantly enhanced the usc of rAAV vectors for gene delivery and gene therapy. However, host immune responses to the transgene products from dilTerent serotypes remain elusive. In the present study, we evaluated the dilTerential immune response to the transgene products from rAAV 1 and rAAV8 vectors. In vitro studies using mouse macrophage cells (RAW 264.7) and bone marrow derived mouse dendritic cells showed that rAAV 1CB-hAAT vector infected macrophage and dcndretic cells efficiently, while rAAV8-CB-hAAT mediated no detectable levels of hAAT in the culture medium. In order to evaluate the humoral immune responses, non-obese diabetic (NOD) mice were intraperitoneally injected with rAAVI-CB-hAAT(2xlO" particles/mouse) orrAAV8CB-hAAT (2x 10 11 particles/mouse) vectors at 4 weeks ofage. Serum hAAT levels were 1,000 ug/ml (rAAV8-CB-hAAT group) and 35 ug/ml respectively. Interestingly, 90% (9/10) ofrAAV8-CB-hAAT injected mice had undetectable level ofanti-hAAT antibodies, and only one mouse developed low levels of anti-hAAT antibodies. However, most of(7/10) rAAV I-CB-hAAT injectedmice developed high levels ofanti-hAATantibodies. These results are consistent with our previous observations in C57BL/6 mice and indicate that foreign gene expression may not be essential for host humoral immune response, while dendretic cell transduction by gene therapy vector plays an important role in host humoral immune response.
Molecular Therapy Volume 15 ~ Supplement I. May2007 Coprright © Th e American Society of G ene Th erapy
845. Characterization of a Bipartite rAAV Vector for Site Specific Integration
Chun Zhang, I Nenita Cortez, I Kenneth I. Berns.' 'Department ofMolecular Genetics and Microbiology; University ofFlorida, Gainesville, FL.
AAV2 is the only virus known to integrate into a specific locus in the human genome. The locus, AAVSI , is on the q arm ofchromosome 19 at position 13.4. AAV is currently a popular vector for human gene therapy. However, current vectors do not contain two important elements needed for site specific integration; i.e., the rep gene or the P5 promoter, although they do integrate with low frequency at random locations in the human genome. We have designed a bipartite vector which does insert the transgene into AAVS I. One component contains the rep gene, but has the SV40 early promoter substituted for the P5 promoter, Thus the insertion enhancer clement (lEE) within P5 which greatly enhances site specific integration has been deleted. The other component contains the lEE plus the transgene with associated regulatory elements. We have created clones of transduced HeLa cells, most of which appear to have the transgene inserted in AAVS I. We have not detected any clones which have rep inserted anywhere. With the optimal MOl and ratio of rAAVSVAV2 and rAAVP5UF 1I, the transgene integrated at AAVS I specifically with high efficiency (63 .63%). Most importantly, the cloned cell lines with the AAVS I site specific integrated GFP were healthy and continuously and stably expressed GFP for 35 passages. An AAV vector which would integrate at a specific site with a high frequency could offer significant advantage in the transduction of progenitor cells and stem cells ex vivo and engineered cells could be used for human gene therapy. AAV site specific integration gene therapy could provide a novel approach for the diseases that need long-term gene expression.
846. Strategies for Improving AdenoAssociated Virus Vector-Mediated Gene Transfer in Primary Murine Hematopoietic Stem Cells In Vivo Njeri Maina.P Zongchao Han,' Xiaomiao LV Weihong Zhao,':' Li Zhong,' Zhongbo Hu,' Wenqin Ma,' William Slayton,' Arun Srivastava: 'Pediatrics, University ofFlorida College ofMedicine, Gainesville, FL; lMolecl/lar Genetics and Microbiology; University of Florida College ofMedicine , Gainesville, FL; 'Mkrobtotogy and Immunology; Indiana University School ofMedicine, Indianapolis, IN; 'Nephrology, The First Affiliated Hospital ofNanjing Medical University. Nanjing. Jiangsu , China. Several factors limit high-efficiency transduction of hematopoietic stem cells by recombinant adeno-associated virus serotype 2 (AAV2) vectors. These include: (i) inadequate expression of cellular receptor/co-receptors for AAV2; (ii) impaired intracellular trafficking and failure to undergo efficient uncoating in the nucleus; (iii) the single-stranded nature of the viral genome, which is transcriptionally inactive, and failure to undergo second-strand DNA synthesis; and (iv) the use of sub-optimal promoters, such as CMY. We have undertaken systematic studies to develop alternative strategies to achieve AAV-mediated high-efficiency transduction of primary murine hematopoietic stem cells and lineage-restricted transgenc expression in a bone marrow transplant model in vivo. These include the use of additional AAV serotype vectors (AAVI, AAV7, AAV8, and AAV 10); the use ofdouble-stranded, self-complementary AAV (scAAV) serotype vectors; and the use oferythroid cell-specific promoters such as the human l3-globin gene and the human parvovirus B 19 promoters. Double-stranded recombinant AAV genomes containing the EGFP gene under the control ofeither the human l3-globin promoter or the human parvovirus B 19 promoter were packaged in S323
844. Differential Immune Responses to Transgene Products from rAAV1 and rAAV8 Vectors Yuanqing Lu, Sihong Song. 'Pharmaceutics, University ofFlorida, Gainesville, FL.
Recently developed ncw scrotypcs of recombinant adeno-associated virus (rAAV) vectors have significantly enhanced the usc of rAAV vectors for gene delivery and gene therapy. However, host immune responses to the transgene products from dilTerent serotypes remain elusive. In the present study, we evaluated the dilTerential immune response to the transgene products from rAAV 1 and rAAV8 vectors. In vitro studies using mouse macrophage cells (RAW 264.7) and bone marrow derived mouse dendritic cells showed that rAAV 1CB-hAAT vector infected macrophage and dcndretic cells efficiently, while rAAV8-CB-hAAT mediated no detectable levels of hAAT in the culture medium. In order to evaluate the humoral immune responses, non-obese diabetic (NOD) mice were intraperitoneally injected with rAAVI-CB-hAAT(2xlO" particles/mouse) orrAAV8CB-hAAT (2x 10 11 particles/mouse) vectors at 4 weeks ofage. Serum hAAT levels were 1,000 ug/ml (rAAV8-CB-hAAT group) and 35 ug/ml respectively. Interestingly, 90% (9/10) ofrAAV8-CB-hAAT injected mice had undetectable level ofanti-hAAT antibodies, and only one mouse developed low levels of anti-hAAT antibodies. However, most of(7/10) rAAV I-CB-hAAT injectedmice developed high levels ofanti-hAATantibodies. These results are consistent with our previous observations in C57BL/6 mice and indicate that foreign gene expression may not be essential for host humoral immune response, while dendretic cell transduction by gene therapy vector plays an important role in host humoral immune response.
Molecular Therapy Volume 15 ~ Supplement I. May2007 Coprright © Th e American Society of G ene Th erapy
845. Characterization of a Bipartite rAAV Vector for Site Specific Integration
Chun Zhang, I Nenita Cortez, I Kenneth I. Berns.' 'Department ofMolecular Genetics and Microbiology; University ofFlorida, Gainesville, FL.
AAV2 is the only virus known to integrate into a specific locus in the human genome. The locus, AAVSI , is on the q arm ofchromosome 19 at position 13.4. AAV is currently a popular vector for human gene therapy. However, current vectors do not contain two important elements needed for site specific integration; i.e., the rep gene or the P5 promoter, although they do integrate with low frequency at random locations in the human genome. We have designed a bipartite vector which does insert the transgene into AAVS I. One component contains the rep gene, but has the SV40 early promoter substituted for the P5 promoter, Thus the insertion enhancer clement (lEE) within P5 which greatly enhances site specific integration has been deleted. The other component contains the lEE plus the transgene with associated regulatory elements. We have created clones of transduced HeLa cells, most of which appear to have the transgene inserted in AAVS I. We have not detected any clones which have rep inserted anywhere. With the optimal MOl and ratio of rAAVSVAV2 and rAAVP5UF 1I, the transgene integrated at AAVS I specifically with high efficiency (63 .63%). Most importantly, the cloned cell lines with the AAVS I site specific integrated GFP were healthy and continuously and stably expressed GFP for 35 passages. An AAV vector which would integrate at a specific site with a high frequency could offer significant advantage in the transduction of progenitor cells and stem cells ex vivo and engineered cells could be used for human gene therapy. AAV site specific integration gene therapy could provide a novel approach for the diseases that need long-term gene expression.
846. Strategies for Improving AdenoAssociated Virus Vector-Mediated Gene Transfer in Primary Murine Hematopoietic Stem Cells In Vivo Njeri Maina.P Zongchao Han,' Xiaomiao LV Weihong Zhao,':' Li Zhong,' Zhongbo Hu,' Wenqin Ma,' William Slayton,' Arun Srivastava: 'Pediatrics, University ofFlorida College ofMedicine, Gainesville, FL; lMolecl/lar Genetics and Microbiology; University of Florida College ofMedicine , Gainesville, FL; 'Mkrobtotogy and Immunology; Indiana University School ofMedicine, Indianapolis, IN; 'Nephrology, The First Affiliated Hospital ofNanjing Medical University. Nanjing. Jiangsu , China. Several factors limit high-efficiency transduction of hematopoietic stem cells by recombinant adeno-associated virus serotype 2 (AAV2) vectors. These include: (i) inadequate expression of cellular receptor/co-receptors for AAV2; (ii) impaired intracellular trafficking and failure to undergo efficient uncoating in the nucleus; (iii) the single-stranded nature of the viral genome, which is transcriptionally inactive, and failure to undergo second-strand DNA synthesis; and (iv) the use of sub-optimal promoters, such as CMY. We have undertaken systematic studies to develop alternative strategies to achieve AAV-mediated high-efficiency transduction of primary murine hematopoietic stem cells and lineage-restricted transgenc expression in a bone marrow transplant model in vivo. These include the use of additional AAV serotype vectors (AAVI, AAV7, AAV8, and AAV 10); the use ofdouble-stranded, self-complementary AAV (scAAV) serotype vectors; and the use oferythroid cell-specific promoters such as the human l3-globin gene and the human parvovirus B 19 promoters. Double-stranded recombinant AAV genomes containing the EGFP gene under the control ofeither the human l3-globin promoter or the human parvovirus B 19 promoter were packaged in S323