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477. Immune Responses Following Salivary Gland Administration of Recombinant Adenoassociated Virus Serotype 2 Vectors
APPLICATIONS IN DISEASE fibers at the regenerating edge of the PC. AAV2/5(80.3%±11.6) followed by AAV2/8 (59.5%±30.3) produced highly efficient gene transfer to the epidermis and both were significantly (p<.05) greater than AAV2 (11.7%±2.9) and AAV2/7 (5.5%±5.4). AAV2/5 expression was in both basilar and suprabasilar keratinocytes, while AAV 2/8 transduction was mainly in suprabasilar keratinocytes. AAV2/5 (3686±424cells/mm²) was significantly more efficient in transducing cells in the remodeling wound matrix than, AAV2 (553±94cells/mm², p<.0001), AAV2/7 (520±128cells/mm², p<.0001), and AAV2/8 (1440±276cells/mm², p<.0001). The change in capsid of the different pseudotyped AAV vectors produces distinct tropism and efficiency profiles in the wound healing model. AAV2/5 and AAV 2/8 produced highly efficient gene transfer compared to AAV2. AAV2/5 was the only vector to efficiently transduce proliferating cells of the epidermis, regenerating myocytes and the spectrum of cells of the wound matrix, suggesting that 2/5 may be the best pseudotyped AAV vector for gene therapy applications in wound repair and regeneration.
administration group. Conversely, we found significant elevations in Hct and serum Epo levels in mice previously treated with saline. We conclude that after a single administration of rAAV2 to murine SGs there is no significant acute immune response. However, rAAV2 administration to murine SGs results in both cellular and humoral immune responses. The latter may interfere with the efficacy of rAAV2 vectors after readministration.
477. Immune Responses Following Salivary Gland Administration of Recombinant Adenoassociated Virus Serotype 2 Vectors
AAV offers multiple advantages over alternative viral vectors, including minimal induction of secondary immune responses, broad tissue tropism and prolonged transgene expression. However, inherent disadvantages have limited the application of AAV. Choice of transgene is limited by a packaging constraint of ~5kb of DNA. It remains costly and technically demanding to produce large quantities of AAV at high titers. Finally, transgene expression is delayed compared to other commonly used viral vectors, a factor that can complicate experiments in vivo. Recently, reports of selfcomplementary (SC) AAV utilizing modified AAV plasmid constructs that permit formation and packaging of double-stranded recombinant AAV genomes which limit the need for second-strand synthesis, have demonstrated more rapid transgene expression and enhanced transduction efficiency. Here we describe the production and function of a SC-AAV encoding the glucose regulated insulin transgene. The SC-AAV backbone was created by removing the Rep Binding Site sequence from one of the viral ITRs and further incorporating a previously described (GlRE)3BP1.INS glucose sensitive insulin transgene or a CMV.EGFP. The population of viruses produced from such constructs are heterogeneous containing ~30% doublelength, double-stranded genomes of the SC-AAV. The remaining viruses contain one or two copies of a single length genome. Standard (SD)-AAV containing the identical but only single stranded sequences were used as controls. To compare requirements for stimulators of second strand synthesis HepG2 cells were transduced with or without helper adenovirus (Ad dl312, MOI - 10), and equal amounts either SC-AAV (GlRE)3BP1.INS or SD-AAV (GlRE)3BP1.INS. Secreted insulin in conditioned medium was detectable following SD-AAV (GlRE)3BP-1INS infection after 4 days. As expected, coinfection with Ad advanced insulin production to 2 days. Most importantly, however, SC-AAV (GlRE)3BP1.INS infection in absence of Ad. produced insulin levels that were greater than SDAAV (GlRE)3BP-1INS infection at the both 2 and 4 days. On day 4 insulin secretion from cultures infected with the self-complementary virus, SC-AAV (GlRE)3BP.1INS, was 7-fold greater than in those infected with the standard AAV. Similar results were obtained with self-complementary viruses expressing EGFP: photomicrographs show earlier and greater GFP expression with SC-AAV CMV.EGFP compared to SD-AAV CMV.EGFP infected cells. Preliminary data indicate the same phenomenon in primary cultures of hepatocytes. In sum, these data indicate that transduction with SC-AAV causes more rapid and enhanced transgene expression compared to that of SD-AAV, and underscore potential benefits of SC-AAV in gene transfer in vivo.
Marc R. Kok,1,2 Antonis Voutetakis,1 Seiichi Yamano,1 Jianghua Wang,1 Hisako Katano,1 Ioannis Bossis,1 Sandra Afione,1 Micheal Schmidt,1 John A. Chiorini,1 Paul-Peter Tak,2 Bruce J. Baum.1 1 Gene Therapy and Therapeutics Branch, NIH, Bethesda, MD; 2 Division of Clinical Immunology and Rheumatology, AMC, University of Amsterdam, Amsterdam, Netherlands. Salivary glands (SGs) provide a novel target site for several potentially useful clinical gene transfer applications. The SGs are capable of producing large amounts of proteins, and are a site where gene transfer can be readily accomplished in a minimally invasive manner (intraductal cannulation). Human SGs are well encapsulated, a circumstance likely to minimize the undesirable access of administered vectors and transgenes to other tissues (Baum et al, Int Rev Cytol, 2002). Numerous studies have shown that recombinant adenoassociated virus serotype 2 (rAAV2) vectors are useful for long term gene transfer applications to many tissues, including SGs (e.g., Yamano et al, Hum Gene Ther, 2002). Previous studies have indicated that intravenous, intramuscular and intranasal administration of rAAV2 vectors induces host immune responses. SGs are part of the mucosal immune system, and as yet there are no reported studies on the effects of administration of rAAV2 vectors into SGs on immune responsiveness. To examine this issue, the main excretory ducts of the submandibular glands of Balb/c mice were cannulated and vector administered by retrograde infusion (Baum et al, ibid). On day zero, we delivered a rAAV2 encoding βgalactosidase (rAAV2LacZ; 2.5x109 particles/gland) or saline (control) to adult mice (n=15/group). On day 28, we administered a second rAAV2 vector encoding human erythropoeitin (Epo; rAAV2Epo; 2.5x109 particles/gland) to 5 mice of each group. Immune activities were evaluated in saliva, serum, SGs and spleens collected on days 1, 28, and 56. There were no differences in salivary flow rates at all time points among the control, vector, and vector readministered groups. Histological examination of SGs on day 1 did not indicate any significant mononuclear cell infiltration in any of the treatment groups. Ex-vivo stimulation, with rAAV2, of splenocytes collected from mice sacrificed on days 28 and 56, resulted in elevated interferon γ levels in culture media from cells of mice administered rAAV2 in vivo but not from cells of control mice. In addition, significant titers of neutralizing antibodies to rAAV2 were detected in serum (from 1:200 - 1:6400). Also, we were unable to observe transduction of SG cells by rAAV2Epo in mice previously infected with rAAV2LacZ. No elevations in hematocrit (Hct) and serum Epo levels were seen at day 56 in mice from the virus reMolecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
478. Self-Complementary AAV (SC-AAV) Delivery Accelerates and Enhances Transgenic Insulin Production in Hepatocytes Miroslaw Kozlowski,1,3 Dorota Lyszkowicz,1 Sara A. Paveglio,2 Adam G. Campbell,2 Darrin E. Olson,2 Janet Rubin,1,2 Peter M. Thule.1,2 1 Atlanta VA Medical Center, Atlanta, GA; 2Div. of Endocrinology and Metabolism, EMORY University School of Medicine, Atlanta, GA; 3Dep. of Orthopedics, EMORY University School of Medicine, Atlanta, GA.
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administration group. Conversely, we found significant elevations in Hct and serum Epo levels in mice previously treated with saline. We conclude that after a single administration of rAAV2 to murine SGs there is no significant acute immune response. However, rAAV2 administration to murine SGs results in both cellular and humoral immune responses. The latter may interfere with the efficacy of rAAV2 vectors after readministration.
477. Immune Responses Following Salivary Gland Administration of Recombinant Adenoassociated Virus Serotype 2 Vectors
AAV offers multiple advantages over alternative viral vectors, including minimal induction of secondary immune responses, broad tissue tropism and prolonged transgene expression. However, inherent disadvantages have limited the application of AAV. Choice of transgene is limited by a packaging constraint of ~5kb of DNA. It remains costly and technically demanding to produce large quantities of AAV at high titers. Finally, transgene expression is delayed compared to other commonly used viral vectors, a factor that can complicate experiments in vivo. Recently, reports of selfcomplementary (SC) AAV utilizing modified AAV plasmid constructs that permit formation and packaging of double-stranded recombinant AAV genomes which limit the need for second-strand synthesis, have demonstrated more rapid transgene expression and enhanced transduction efficiency. Here we describe the production and function of a SC-AAV encoding the glucose regulated insulin transgene. The SC-AAV backbone was created by removing the Rep Binding Site sequence from one of the viral ITRs and further incorporating a previously described (GlRE)3BP1.INS glucose sensitive insulin transgene or a CMV.EGFP. The population of viruses produced from such constructs are heterogeneous containing ~30% doublelength, double-stranded genomes of the SC-AAV. The remaining viruses contain one or two copies of a single length genome. Standard (SD)-AAV containing the identical but only single stranded sequences were used as controls. To compare requirements for stimulators of second strand synthesis HepG2 cells were transduced with or without helper adenovirus (Ad dl312, MOI - 10), and equal amounts either SC-AAV (GlRE)3BP1.INS or SD-AAV (GlRE)3BP1.INS. Secreted insulin in conditioned medium was detectable following SD-AAV (GlRE)3BP-1INS infection after 4 days. As expected, coinfection with Ad advanced insulin production to 2 days. Most importantly, however, SC-AAV (GlRE)3BP1.INS infection in absence of Ad. produced insulin levels that were greater than SDAAV (GlRE)3BP-1INS infection at the both 2 and 4 days. On day 4 insulin secretion from cultures infected with the self-complementary virus, SC-AAV (GlRE)3BP.1INS, was 7-fold greater than in those infected with the standard AAV. Similar results were obtained with self-complementary viruses expressing EGFP: photomicrographs show earlier and greater GFP expression with SC-AAV CMV.EGFP compared to SD-AAV CMV.EGFP infected cells. Preliminary data indicate the same phenomenon in primary cultures of hepatocytes. In sum, these data indicate that transduction with SC-AAV causes more rapid and enhanced transgene expression compared to that of SD-AAV, and underscore potential benefits of SC-AAV in gene transfer in vivo.
Marc R. Kok,1,2 Antonis Voutetakis,1 Seiichi Yamano,1 Jianghua Wang,1 Hisako Katano,1 Ioannis Bossis,1 Sandra Afione,1 Micheal Schmidt,1 John A. Chiorini,1 Paul-Peter Tak,2 Bruce J. Baum.1 1 Gene Therapy and Therapeutics Branch, NIH, Bethesda, MD; 2 Division of Clinical Immunology and Rheumatology, AMC, University of Amsterdam, Amsterdam, Netherlands. Salivary glands (SGs) provide a novel target site for several potentially useful clinical gene transfer applications. The SGs are capable of producing large amounts of proteins, and are a site where gene transfer can be readily accomplished in a minimally invasive manner (intraductal cannulation). Human SGs are well encapsulated, a circumstance likely to minimize the undesirable access of administered vectors and transgenes to other tissues (Baum et al, Int Rev Cytol, 2002). Numerous studies have shown that recombinant adenoassociated virus serotype 2 (rAAV2) vectors are useful for long term gene transfer applications to many tissues, including SGs (e.g., Yamano et al, Hum Gene Ther, 2002). Previous studies have indicated that intravenous, intramuscular and intranasal administration of rAAV2 vectors induces host immune responses. SGs are part of the mucosal immune system, and as yet there are no reported studies on the effects of administration of rAAV2 vectors into SGs on immune responsiveness. To examine this issue, the main excretory ducts of the submandibular glands of Balb/c mice were cannulated and vector administered by retrograde infusion (Baum et al, ibid). On day zero, we delivered a rAAV2 encoding βgalactosidase (rAAV2LacZ; 2.5x109 particles/gland) or saline (control) to adult mice (n=15/group). On day 28, we administered a second rAAV2 vector encoding human erythropoeitin (Epo; rAAV2Epo; 2.5x109 particles/gland) to 5 mice of each group. Immune activities were evaluated in saliva, serum, SGs and spleens collected on days 1, 28, and 56. There were no differences in salivary flow rates at all time points among the control, vector, and vector readministered groups. Histological examination of SGs on day 1 did not indicate any significant mononuclear cell infiltration in any of the treatment groups. Ex-vivo stimulation, with rAAV2, of splenocytes collected from mice sacrificed on days 28 and 56, resulted in elevated interferon γ levels in culture media from cells of mice administered rAAV2 in vivo but not from cells of control mice. In addition, significant titers of neutralizing antibodies to rAAV2 were detected in serum (from 1:200 - 1:6400). Also, we were unable to observe transduction of SG cells by rAAV2Epo in mice previously infected with rAAV2LacZ. No elevations in hematocrit (Hct) and serum Epo levels were seen at day 56 in mice from the virus reMolecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
478. Self-Complementary AAV (SC-AAV) Delivery Accelerates and Enhances Transgenic Insulin Production in Hepatocytes Miroslaw Kozlowski,1,3 Dorota Lyszkowicz,1 Sara A. Paveglio,2 Adam G. Campbell,2 Darrin E. Olson,2 Janet Rubin,1,2 Peter M. Thule.1,2 1 Atlanta VA Medical Center, Atlanta, GA; 2Div. of Endocrinology and Metabolism, EMORY University School of Medicine, Atlanta, GA; 3Dep. of Orthopedics, EMORY University School of Medicine, Atlanta, GA.
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