Expression of acid ?_glucosidase in nonhuman primate diaphragm and peritoneum after direct in utero delivery of recombinant adeno_associated virus type 1

GENE THERAPY FOR MUSCULOSKELETAL DISEASES Compared to vector derived from a longer, AAV2(GlRE)3BP-1 2furGFP, or similarly short sequence, AAV2(GlRE)3BP-1 2fur, infection with SC-AAV2(GlRE)3BP-1 2fur accelerated, and more than tripled secretion of human insulin from primary hepatocytes. Co-transfection of 293 cells with pSC-AAV2(GlRE)3BP-1 2fur, and a REP2/CAP8 expressing plasmid produced pseudotyped SCAAV2/8 vector. Twenty-three male, STZ-diabetic CD-1 (BG>200mg/dl) mice received a portal vein injection of either SCAAV2(GlRE) 3BP-1 2fur, or increasing doses of SC-AAV2/ 8(GlRE)3BP-1 2fur. Despite enhanced transgene expression in vitro, SC-AAV2(GlRE)3BP-1 2fur administration (3.4-4.8x1010vg, n=4) failed to reduce hyperglycemic by 18 days. In contrast, SC-AAV2/ 8(GlRE)3BP-1 2fur administration lowered blood glucose in 18 of 19 diabetic mice by 7 days, beginning with doses as low as 1.3x1010vg. Six of seven mice receiving the three highest doses (5.2x1010vg n=3, 7.8x1010vg n=1, 1.3x1011vg n=3) succumbed to hypoglycemia within 4 days. Two animals receiving 2.6x10 10 vg (n=6) died of hypoglycemia at day 34. However, 9 of the remaining 10 mice receiving 1.3-2.6x1010vg continue to grow normally and maintain near euglycemia (105±6 mg/dl). In conclusion, we have combined SC-AAV, AAV2/8 pseudotyping techniques, and metabolically responsive hepatic insulin gene therapy to treat STZ-diabetic mice in vivo. Effective viral doses may be 10-fold less than standard, non-pseudotyped vector delivering the same transgene. On-going investigations will determine the long-stability of glycemic control in this model.

377. Expression of Acid α-Glucosidase in Nonhuman Primate Diaphragm and Peritoneum after Direct In Utero Delivery of Recombinant Adeno-Associated Virus Type 1 Mary B. Rucker,1 Alice F. Tarantal,2 Thomas Conlon,1 Terence R. Flotte,1 Barry J. Byrne.1 1 Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL; 2California National Primate Research Center, University of California at Davis, Davis, CA. Genetic based neuromuscular disorders such as glycogen storage disease type II (GSDII), results in early and sometimes irreversible cellular damage. Deficiency in acid α-glucosidase (GAA) leads to lysosomal accumulation of glycogen in all cell types resulting in abnormal myofibrillogenesis and reduced contractile function in striated muscle. Previous studies in our laboratory showed that in utero delivery of recombinant adeno-associated virus (rAAV) encoding GAA to the peritoneal cavity of GAA-/- mice resulted in high-level transduction of diaphragm tissue. In addition, GAA-/diaphragm transduced with rAAV-GAA in utero demonstrated near normal diaphragm muscle function when assayed at 6 months (submitted). In this study, 3 groups of 2 rhesus monkey fetuses per group (total N=6) were injected IP in utero under ultrasound guidance with 4.5x1012 particles of rAAV type 1 expressing either human GAA (rAAV1-CMV-hGAA), human minidystrophin (rAAV1CMV-3858Dys or human α-1-antitrypsin (rAAV1-CBA-hAATmyc) at 50 days gestation (term 165±10 days). Fetuses were monitored sonographically during gestation then delivered at term for postnatal studies. Animals remained healthy during the study period (growth, hematology, clinical chemistry), with no evidence of adverse effects. Blood samples were collected weekly to determine immunologic responses to human GAA, human AAT, and rAAV capsid proteins using ELISAs that detect primate IgG, IgM, and IgA. Low levels of antibody against GAA were detected in one of the two animals treated with rAAV-GAA at 7 weeks, but was no longer present at 11 weeks. No antibodies were detected against AAT in either rAAV-AAT treated animal. A high level of antibodies against rAAV capsid proteins were detected at birth in three of the S144

vector-treated animals. The level of antibodies decreased over time and was indistinguishable from untreated controls by 3 months of age. Further studies are underway to determine the source of the immune response. Tissues were collected at three months postnatal age (7 months post-gene transfer) and multiple specimens from each animal as well as untreated controls were analyzed by immunohistochimistry for human GAA and AAT. Both diaphragm and peritoneum from vector-treated animals were strongly positive for expression of human GAA or AAT. Expression was evenly distributed over multiple diaphragm and peritoneum sections collected from each animal that was analyzed. Heart, muscle, and brain were negative for transgene expression. However, hepatocytes near the surface of the liver were positive for human GAA and AAT. Studies in progress include real-time PCR to determine tissue distribution of vector genomes. In conclusion, in utero delivery of recombinant AAV vectors provide stable transduction of diaphragm while providing little evidence of antibody response to transgenes. Since prenatal diagnosis is feasible for GSDII, the findings in this study support the potential application of human in utero transduction of the diaphragm to treat these diseases, provided that long-term safety can be shown.

378. An AAV Vector-Mediated Micro-Dystrophin Expression in Relatively Small Percentage of Dystrophin-Deficient mdx Myofibers Still Improved the mdx Phenotype through Compensatory Hypertrophy Madoka Ikemoto, Madoka Yoshimura, Miki Sakamoto, Yasushi Mochizuki, Katsutoshi Yuasa, Toshifumi Yokota, Yuko MiyagoeSuzuki, Shin’ichi Takeda. 1 Department of Molecular Therapy, National Institute of Neuroscience,NCNP, Kodaira, Tokyo, Japan. [Background] Duchenne muscular dystrophy (DMD) is an Xlinked, lethal muscle disorder caused by mutations in the dystrophin gene. An adeno-associated virus (AAV) vector-mediated gene transfer is one of attractive approaches to the treatment of DMD, though it has a limitation in insertion size up to 4.9 kb. Therefore, a fulllength dystrophin cDNA (14 kb) cannot be incorporated into an AAV vector. We previously generated micro-dystrophin transgenic mdx mice. Micro-dystrophin CS1 transgenic mdx mice showed almost complete amelioration of dystrophic phenotypes (BBRC. 293: 1265, 2002). [Objective] We constructed an AAV vector expressing microdystrophin ∆CS1, and introduced it into skeletal muscles of mdx mice and examined whether the dystrophic process had been ameliorated or not. [Method] To incorporate micro-dystrophin CS1 cDNA (4.9 kb) into an AAV vector, we deleted 5’- and 3’-UTRs and exons 71-78 (alternative splicing regions), resulting 3.8 kb ∆CS1 cDNA. We produced type 2 AAV vector expressing ∆CS1 under the control of muscle specific MCK promoter to avoid immune response against transgene product (Gene Ther. 9: 1576, 2002), designated AAV2MCK∆CS1. The vector was injected into anterior tibial (TA) muscles of 10-day-old and 5-week-old mdx mice. Mdx muscles show no obvious changes of degeneration at 10-day, whereas 5-week-old mdx muscles exhibit active cycles of muscle degeneration/ regeneration. [Result] When the AAV2-MCK∆CS1 was injected at 5 weeks of age, dystrophin-positive fibers were 51.5 ± 17.3% at 24 weeks after the injection. The ratio of centrally nucleated fibers in ∆CS1-positive fibers was significantly reduced compared with that of ∆CS1-negative fibers, indicating protective function of ∆CS1 against muscle degeneration. Furthermore, AAV-injected muscles revealed complete recovery of the specific tetanic force. When injected at 10-day-old, ∆CS1-positive fibers was 16.5 ± Molecular Therapy Volume 9, Supplement 1, Ma y 2004

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