31. Gene Therapy of Adult MLD Model Mice by Intrathecal Administration of Type 9 AAV Vector

31. Gene Therapy of Adult MLD Model Mice by Intrathecal Administration of Type 9 AAV Vector

MUSCULO-SKELETAL GENE & CELL THERAPY 31. Gene Therapy of Adult MLD Model Mice by Intrathecal Administration of Type 9 AAV Vector Noriko Miyake,1 Koic...

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MUSCULO-SKELETAL GENE & CELL THERAPY 31. Gene Therapy of Adult MLD Model Mice by Intrathecal Administration of Type 9 AAV Vector

Noriko Miyake,1 Koichi Miyake,1 Atsushi Sakai,2 Motoko Yamamoto,1 Ayumi Endo,1 Hidenori Suzuki,2 Takashi Shimada.1 1 Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan; 2Pharmacology, Nippon Medical School, Tokyo, Japan.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease (LSD) caused by the deficiency of arylsulfatase A (ASA) and characterized by severe neurological symptoms due to widespread demyelination in the both central and peripheral nervous systems. Enzyme replacement therapy (ERT) has been applied to treat certain types of LSDs, but correction of neurological abnormalities is usually hampered by the blood brain barrier (BBB). Recently, we have succeeded to treat ASA knockout MLD model mice by systemic neonatal gene delivery of type 9 adeno-associated viral (AAV) vector expressing ASA (AAV9/ASA). However, intravenous systemic gene delivery by AAV9/ASA is non-effective to treat neurological symptoms in adult MLD model mice because of the established BBB. Moreover, it is unclear whether overt neurological symptoms can be reversed by gene therapy approaches.To address these problems, we examined the possibility of intrathecal (IT) administration of type 9 AAV vectors for the treatment of young adult MLD model mice (6 week-old) which are free of neurological symptoms and old adult MLD mice (1 year-old) which have already neurological abnormalities on the balanced beam test. AAV type 9 vector expressing ASA and GFP (4 x 1011 vg/body in 10ul) was IT injected via a suboccipital puncture into 6 week-old (6wITMLD) and 1 year-old MLD model mice (1yITMLD) (n=5 each). These treated mice were analyzed at age 1.3 year. Broad distribution of GFP expression throughout the brain, a large number of nerve fibers in the dorsal spinal cord, and many neural cell bodies in the dorsal root ganglia were efficiently transduced. Immunohistochemical analysis showed efficient ASA expression was detected in the brain, with the cerebellum and olfactory bulbs having highest activities. Alcian blue staining and quantative analysis of sulfatide contents by biochemical assay showed decrease of the amount of stored sulfatide in 1yITMLD in cerebellum but not in cerebral cortex compared to non-treated MLD mouse (cerebellum: 15.1±2.6 vs. 24.5±2.8, p<0.05; cerebral cortex: 9.2±0.4 vs. 9.5±0.8, mg/mg protein, p>0.05). In the behavior test, AAV9/ASA treated mice did not show a significant improvement in their ability to traverse narrow balance beams, as compared to non-treated MLD mice (Latency: 15.4±3.7 vs. 12.2±4.2 sec, p>0.05; Slips: 12.6±3.3 vs. 9.4±3.8 times, p>0.05). On the other hand, Significant decrease of the amount of stored sulfatide in the whole brain (cerebral cortex: 9.5±2.6 vs. 15.2±2.0, p<0.05; cerebellum: 22.0±4.7 vs. 35.0±4.7 mg/mg protein, p<0.05) and improvement of the behavior test (Latency: 8.7±0.9 vs. 13.9±0.8 sec, P<0.01; Slips: 4±0.4 vs. 8.2±0.6 times, P<0.01) were observed in 6wITMLD compared to non-treated MLD mouse. These data indicate that IT administration of AAV vector is a promising option to treat the central nervous system. However, it is difficult to correct overt neurological symptoms in one-year-old MLD mice probably because some irreversible histological changes have already started at this age. Therefore, it may be essential to start gene therapy before onset of neurological symptoms of MLD.

Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy

32. Normalized Survival and Permanent Restoration of NAGLU Activity in the CNS, PNS and Somatic Tissues in MPS IIIB Mice after a Single Intravenous rAAV9-hNAGLU Gene Delivery

Tierra Ware,1 Darren Murrey,1 Douglas M. McCarty,1,2 Haiyan Fu.1 Center for Gene Therapy, Research Institute at Nationwide Children’s Hospital, Columbus, OH; 2Department of Pediatrics, College of Medicane and Public Health, The Ohio State University, Columbus, OH. 1

The novel ability of AAV9 to cross the blood-brain barrier has provided a powerful tool for the development of a systemic gene delivery approach for treating both the neurological and somatic disease associated with Mucopolysaccharidosis (MPS) IIIB. We previously demonstrated the functional benefits of the approach in an adult MPS IIIB mouse model. Our recent results showed normalized survival (18.8-27.2 months) in MPS IIIB mice receiving a single intravenous injection of rAAV9-CMV-hNAGLU vector at 4-6-weeks of age. The global transduction of the central and peripheral nervous systems and widespread restoration of NAGLU enzyme activity persisted to the endpoints in these mice with the exception of the liver. A significant loss of liver transduction was observed with 3040% rNAGLU-positive hepatocytes at 6mo post-injection reduced to <5% at the endpoints. These data suggest permanent transgene expression in non-dividing cells and gradual loss in dividing cells due to the predominantly episomal status of AAV vector. In addition, the AAV9-mediated NAGLU expression was observed in cells in the stroma of the testes, but not in seminiferous cells, suggesting minimal possibility of vertical transmission of AAV9. This study demonstrates that a single systemic rAAV9-NAGLU gene delivery may offer permanent therapeutic benefits in the treatment of MPS IIIB.

Musculo-skeletal Gene & Cell Therapy 33. Therapeutic Applications of Sleeping Beauty System and iPS technology To Treat Duchenne Muscular Dystrophy

Antonio Filareto,1 Sarah Parker,1 Radbod Darabi,1 Luciene Borges,1 Tory Schaaf,1 Timothy Mayerhofer,1 Jeffrey S. Chamberlain,2 James M. Ervasti,3 R. Scott McIvor,4 Michael Kyba,1 Rita C. R. Perlingeiro.1 1 Lillehei Heart Institute, Department of Medicine, University of Minnesota, Minneapolis, MN; 2Department of Neurology, University of Washington School of Medicine, Seattle, WA; 3 Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN; 4Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN. Duchenne muscular dystrophy (DMD) is a progressive and fatal neuromuscular disease caused by genetic and biochemical defects of the dystrophin-glycoprotein complex (DGC). These alterations lead to cell membrane damage and apoptosis of muscle cells, resulting in chronic tissue degeneration and impaired muscle contractility. To date, there is no cure for DMD. Approaches to date involving cell transplantation and gene therapy have given suboptimal results. An alternative strategy that holds promise is the direct reprogramming of adult fibroblasts to a pluripotent state, generating patient- and disease-specific stem cells, and correcting these in vitro prior to transplantation. Here we show the regenerative potential of myogenic progenitors derived from corrected dystrophic iPS cells generated from fibroblasts of mice lacking both dystrophin and utrophin (dKO). We corrected the phenotype of these dystrophic iPS cells using a Sleeping Beauty transposon carrying the micro-utrophin (μUTRN) gene, differentiated these cells into skeletal muscle progenitors, and assessed whether their transplantation back into S13