365. Enzyme Replacement Therapy Enhanced a Therapeutic Efficacy of Bone Marrow Transplantation in MPS II Mouse

GENE & CELL THERAPY OF DIABETES, METABOLIC AND GENETIC DISEASES I received 1x1012 vector genomes (vg) each (n=5) while AAV8-hFIX fetuses received 2x1012-1x1013vg (n=3). Fetuses treated with AAVhFX received 1x1013vg each (AAV5, n=2; AAV8, n=3). AAV5-FIX subjects expressed median levels of 2.1% at 0.6G and 0.02-2.6% over 16 months during which time weight increased by 5-fold. AAV8-FIX expression was 1.6% at 0.6G and ranged from 6.0-32.4% over the first year with a 5-fold weight gain. AAV8-mediated hFIX expression was higher overall (medians 15.3 vs. 2.1%, p<0.0001). Median AAV5FX expression ranged from 0-4.8% while AAV8-FX expression ranged from 0.6-24.2% in the first year, with a weight gain of up to 4-fold. Overall hFX expression was higher with AAV8 (median 14.0 vs. 0.6%, p<0.01) at the same dose. Offspring demonstrated mild non-neutralising humoral responses against AAV and transgene that peaked in the first three months before steadily declining. Maternal humoral response followed similar patterns but was sustained for at least six months. AAV5 was the more immunogenic vector. Treated infants had normal transaminases and no hepatic inflammation. Median vector copies ranged from 0.02-2.0 copies/cell in all organs apart from the diaphragm (35 copies/cell). A log-fold lower VCN was found in maternal tissues but no AAV DNA was found in maternal oocytes. Therapeutic transgene levels were thus achieved with a single dose of AAV at 0.4G, sustained for a year despite continued growth and an AAV- and transgene-directed humoral response. AAV8 was more efficient and less immunogenic at the same dose. Despite the preimmune gestation, fetal antibodies were detected from one month post-FGT. This transient response that mirrored maternal immune development may be reflective of acquired maternal antibodies rather than an endogenous reaction. Considering the substantial post-FGT growth and accompanying dilution of AAV, sustained transgene levels suggest long-term episomal persistence or integration. AAV-mediated FGT at 0.4G carries a reassuring safety profile, although the immune response warrants further study, and may be a beneficial strategy for early-onset pathology.

364. AAV Vector-Mediated Iduronidase Gene Delivery in a Murine Model of Mucopolysaccharidosis Type I: Comparing Different Routes of Delivery to the CNS

Lalitha Belur,1 Zhenhong Nan,2 Kelly M. Podetz-Pedersen,1 Carolyn A. Fairbanks,3 Leah R. Hanson,4 William H. Frey,4 Karen Kozarsky,5 Walter C. Low,2 R. Scott McIvor.1 1 Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN; 2Neurosurgery, University of Minnesota, Minneapolis, MN; 3Pharmaceutics, University of Minnesota, Minneapolis, MN; 4Alzheimer’s Research Center, Regions Hospital, St. Paul, MN; 5ReGenX Biosciences, Washington, DC. Mucopolysaccharidosis type I (MPS I) is an inherited metabolic disorder caused by deficiency of the lysosomal enzyme alphaL-iduronidase (IDUA). Systemic and abnormal accumulation of glycosaminoglycans is associated with growth delay, organomegaly, skeletal dysplasia, and cardiopulmonary disease. Individuals with the most severe form of the disease (Hurler syndrome) suffer from neurodegeneration, mental retardation, and early death. The two current treatments for MPS I (hematopoietic stem cell transplantation and enzyme replacement therapy) cannot effectively treat all central nervous system (CNS) manifestations of the disease. We have previously reported that intravenous administration as well as direct administration of AAV vectors to the CNS in newborn IDUA-deficient mice reduced or prevented the emergence of neurological dysfunction (Molecular Therapy 9:866, 2004; Neurobiology of Disease 43:123, 2011). We have subsequently extended the applicability of AAVmediated IDUA gene transfer to adult mice by direct infusion of AAV-IDUA to the central nervous system using different routes of administration. AAV-IDUA serotype 9 was administered by direct S140

injection into the lateral ventricles of IDUA-deficient mice that were either immunocompetent, immunodeficient (NOD-SCID/ IDUA-/-), immunosuppressed with cyclophosphamide (CP), or immunotolerized by weekly injection of human iduronidase protein (Aldurazyme) starting at birth. CP immunosuppressed animals were also administered AAV9-IDUA by intranasal infusion, by intrathecal injection, and by endovascular infusion with and without mannitol to disrupt the blood-brain barrier. Animals were sacrificed at 8 weeks after vector administration, and brains were harvested and microdissected for evaluation of IDUA enzymatic activity, tissue glycosaminoglycans, and IDUA vector sequences in comparison with normal and affected control mice. Results from these studies will provide an assessment of the most effective route of AAV vector administered directly to the CNS as a model for achieving higher levels of IDUA in the treatment of human MPS I.

365. Enzyme Replacement Therapy Enhanced a Therapeutic Efficacy of Bone Marrow Transplantation in MPS II Mouse

Kazumasa Akiyama,1,7 Yohta Shimada,1 Takashi Higuchi,3 Sayoko Iizuka,1 Makoto Otsu,4 Hiromitsu Nakauchi,4 Jillian R. Brown,5 Brett E. Crawford,5 Takahiro Fukuda,6 Hiroshi Kobayashi,1,2,3 Yoshikatsu Eto,3 Hiroyuki Ida,1,2,3 Toya Ohashi.1,2,3 1 Department of Gene Therapy, Institute of DNA Medicine, The Jikei University School of Medicine, Tokyo, Japan; 2Department of Pediatrics, The Jikei University School of Medicine, Tokyo, Japan; 3 Department of Genetic Diseases and Genomic Science, The Jikei University School of Medicine, Tokyo, Japan; 4Division of Stem Cell Therapy, Center for Stem Cell and Regenerative Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan; 5 Zacharon Pharmaceuticals Inc., San Diego, CA; 6Department of Neuropathology, The Jikei University School of Medicine, Tokyo, Japan; 7Department of Pediatrics, The Kitasato University, Sagamihara, Japan. Mucopolysaccharidosis type II (MPS II), is characterized by deficient activity of iduronate-2-sulfatase (IDS), resulting in accumulation of glycosaminoglycans (GAGs) in tissues. This deficiency causes various clinical symptoms including CNS symptoms. Enzyme Replacement Therapy (ERT) and bone marrow transplantation (BMT) were available. In this study, we compared the efficacy of BMT, ERT and combination therapy (BMT+ERT) to murine model of MPS II. In brain, liver and heart, we assayed total GAG (tGAG) and pathological GAG (pGAG) accumulating due to the deficient IDS at 27 weeks after treatment. Brain function was evaluated by Y-maze test. All treatment suppressed the overgrowth of MPS II. Urinary tGAG was decreased to normal level in all treatment groups. In brain, there was significant difference in amount of pGAG accumulation between wild type mice and MPS II mice, but not in tGAG. All of the treatment failed to reduce the pGAG in brain. Y-maze test failed to demonstrate functional recovery of brain by all treatments. This observation was consistent with the result of tissue pGAG. In liver, all of the treatment reduced tGAG and pGAG and ERT had additive effect to BMT in clearance of tGAG and pGAG. In heart, all of the treatment reduced tGAG and pGAG and ERT had additive effect to BMT in clearance of pGAG, not of tGAG. In lung we only assay the tGAG and ERT had also additive effect of clearance of tGAG. If we compared effectiveness of ERT and BMT, ERT reduced GAG more profoundly in heart, liver and liver than BMT. In conclusion, additive effect of ERT to BMT in clearance of GAG was observed in liver and heart. ERT reduced GAG more profoundly than BMT in some tissues; however we did not detect any antibody formation against enzyme. In human, antibody was usually developed in ERT, thus we overestimated efficacy of ERT in murine system. In addition pGAG is better biomaker to evaluate efficacy of treatment than tGAG. Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy