34. Augmenting CNS Glucocerebrosidase Activity as a Therapeutic Strategy for Parkinsonism and Other Gaucher-Related Synucleinopathies

NEUROLOGICAL THERAPY: MONOGENEIC DISORDERS that cause accumulation of autophagosomes by inhibiting lysosomal fusion and acidification. Treatments with these small molecules demonstrated that blocking autophagy decreased transduction by 3-fold, whereas disrupting autophagy flux decreased transduction up to 3.5-fold. These findings imply that the Rapamycin-mediated increase in LV HSC transduction was dependent upon autophagy. Taken together, these findings demonstrate that short-term exposure to Rapamycin, a clinically approved drug, significantly enhances LV transduction of murine and human HSC without affecting the ability of these cells to engraft, suggesting that this approach might be rapidly translated for use in clinical gene therapy applications. Drs. Wang and Sather contributed equally to this work.

Neurological Therapy: Monogeneic Disorders 33. Intravenous AAV8-MTM1 Prolongs Life and Ameliorates Severe Muscle Pathology in Mouse and Dog Models of X-Linked Myotubular Myopathy

Martin K. Childers,1,2 Romain Joubert,3 Melanie N. Holder,4 Robert W. Grange,5 Jon Doering,5 Michael W. Lawlor,6,11 Christelle Moal,3 Thibaud Jamet,3 Nathalie Daniele,3 Samia Martin,3 Christel Riviere,3 Kimberly Poppante,3 Thomas Soker,4 Caroline Hammer,3 Laetitia Van Wittenberghe,3 Melissa Goddard,7 Erin Mitchell,4 Jane Barber,4 Mark E. Furth,8 Alban Vignaud,3 Carole Masurier,3 Philippe Moullier,3,9,10 Alan H. Beggs,11 Alan H. Beggs,11 Anna BujBello.3 1 Rehabilitation Medicine, U Washington, Seattle; 2Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle; 3Genethon, Evry, France; 4Wake Forest Institute for Regenerative Medicine, Winston-Salem; 5Virginia Tech, Blacksburg; 6Medical College of Wisconsin, Milwaukee; 7Wake Forest University, Winston-Salem; 8Comprehensive Cancer Center, Winston-Salem; 9INSERM, Nantes, France; 10University of Florida, Gainesville; 11Harvard Medical School, Boston. Mutations in the myotubularin gene (MTM1) result in X-linked myotubular myopathy (XLMTM), a fatal pediatric disease of skeletal muscle. Patients with XLMTM typically present with severe hypotonia, muscle weakness and respiratory failure. Here we report long-term survival data in mice and dogs following intravenous delivery of an adeno-associated virus serotype 8 (AAV8) vector expressing myotubularin under the muscle-specific desmin promoter. XLMTM dogs (n=3) were treated at 8 weeks of age with full-length canine MTM1 cDNA using an AAV8 vector. Affected XLMTM dogs were infused with AAV8-MTM1 (2.5 x 10 13 vg/kg), IV under high pressure into the saphenous vein while a tourniquet was applied around the upper thigh. While AAV8-MTM1 was administered by hindlimb infusion, effects were observed systemically in dogs. Mice were given intravenous AAV8-MTM1 by tail vein injection. In both murine and canine models, myotubularin transgene was expressed in all limb muscles and this translated into marked improvement of contractile force in mice and dogs.

All mutant mice, including those injected at a late stage of the disease, survived until the end of the 6-month study. The lifespan of MTM1 mutant dogs that normally survive to 4 months was prolonged beyond 1 year in the first treated animal. Our findings in these two species provide proof-of-principle that intravenous AAV-mediated myotubularin replacement can rescue the severe phenotype of both small and large animal models of myotubular myopathy.

34. Augmenting CNS Glucocerebrosidase Activity as a Therapeutic Strategy for Parkinsonism and Other Gaucher-Related Synucleinopathies

Pablo S. Sardi,1 Lamya S. Shihabuddin,1 Richard L. Sidman,2 Seng H. Cheng.1 1 Rare Diseases Science, Genzyme, a Sanofi Company, Framingham, MA; 2Beth Israel Deaconess Medical Center, Boston, MA. Mutations of GBA1, the gene encoding glucocerebrosidase, represent a common genetic risk factor for developing the synucleinopathies Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). PD patients with or without GBA1 mutations also exhibit lower enzymatic levels of glucocerebrosidase in the central nervous system (CNS), suggesting a possible link between the enzyme and the development of the disease. Previously, we have shown that early treatment with glucocerebrosidase can modulate alpha-synuclein aggregation in a pre-symptomatic mouse model of Gaucher-related synucleinopathy (Gba1D409V/D409V) and ameliorate

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Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

NEUROLOGICAL THERAPY: MONOGENEIC DISORDERS the associated cognitive deficit. To probe this link further, we have now evaluated the efficacy of augmenting glucocerebrosidase activity in the CNS of symptomatic Gba1D409V/D409V mice and in a transgenic mouse model overexpressing A53T alpha-synuclein. Adeno-associated virus-mediated expression of glucocerebrosidase in the CNS of symptomatic Gba1D409V/D409V mice completely corrected the aberrant accumulation of the toxic lipid glucosylsphingosine and reduced the levels of ubiquitin, tau and proteinase-K-resistant alpha-synuclein aggregates. Importantly, hippocampal expression of glucocerebrosidase in Gba1D409V/D409V mice (starting at 4 or 12 months of age) also reversed their cognitive impairment when examined using a novel object recognition test. Correspondingly, overexpression of glucocerebrosidase in the CNS of A53T alpha-synuclein mice reduced the levels of soluble alpha-synuclein, suggesting that increasing the glycosidase activity can modulate alpha-synuclein processing and may modulate the progression of alpha-synucleinopathies. Hence, increasing glucocerebrosidase activity in the CNS represents a potential therapeutic strategy for GBA1-related and non-GBA1associated synucleinopathies, including PD.

35. Efficacious and Sustained Canavan Gene Therapy by Single rAAV Injections Via IV at Postnatal Day 21 or ICV at a 100 Fold Lower Dose Seemin S. Ahmed,1 Reuben Matalon,2 Guangping Gao.1 UMass Medical School, Worcester; 2UTMB, Galveston.

1

Genetic loss of functional aspartoacylase (AspA) causes Canavan’s disease (CD); a lethal pediatric leukodystrophy characterized by dysmyelination, hydrocephalus, progressive central nervous system (CNS) vacuolation and psychomotor retardation. To date, there is no efficacious therapy for CD. Gene therapy using recombinant AAVs (rAAVs) in an AspA knockout mouse model which authentically recapitulates the severest clinical CD phenotypes is an ideal platform to test the efficacy of CD therapeutics. A recent follow-up study on CD gene therapy using first generation rAAV2 emphasized the long-term safety of rAAV and showed encouraging though marginal clinical improvements in patients. This study suggests possible improvement in therapeutic efficacy for global CNS transduction using second generation rAAVs with stronger transduction efficiency and ability to cross the blood-brain-barrier, which we demonstrated in our previous study using intravenously (IV) injected novel rAAVs in early postnatal CD mice. In our current study, we characterized progressive postnatal neurodegeneration and retinopathy in CD mice. In an attempt to define the therapeutic window to rescue lethality in CD mice by IV delivered rAAVs, we evaluated treatments at postnatal day (PD) 0, 6, 13 and 20. We found a complete rescue of lethality, extending the lifespan from <4 weeks to >7 months and restoration of growth even when treated as late as PD 20, 5 days before their death. Improvement of motor functions and reduction of hydrocephaly in the treated animals positively correlated with the age of treatment, highlighting the benefits of earlier therapeutic intervention. Importantly, improvement in disease phenotypes was well correlated with reduced CNS histopathology in treated groups. To minimize potential systemic vector toxicity and reduce vector manufacturing demands, we tested if single intracerebroventricular (ICV) injections of CD mice at P0 with a 100-fold lower dose can efficiently rescue CD phenotypes. All ICV injections of rAAVs resulted in extended survival (>12 months), normalized growth, improved motor function and clinical symptoms; which also correlated well with amelioration of neuro-histopathology and reduction of hydrocephaly in injected groups. However ICV injections were less effective than IV in improving motor functions which suggests the importance of peripheral AspA gene transfer. In addition, at later ages, ICV injected animals developed paralysis earlier than the IV injected animals. Biochemical studies are underway to compare effectiveness of two methods of rAAV injections. Collectively, we achieved complete Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

rescue of lethality and effective alleviation of hydrocephalus and motor dysfunction by novel rAAV gene therapy irrespective of administration route or time of intervention. Our study is the first to demonstrate efficacious and sustained CNS gene therapy not only by a single IV dose of rAAVs as late as PD20 but also by ICV at a 100 fold lower dose. Our study emphasizes the potential to further refine approaches for CD gene therapy using the second generation of rAAVs and opens up newer vistas for the effective treatment of other currently untreatable monogenic CNS disorders.

36. Dramatic Phenotypic Improvement after Adeno-Associated Virus Gene Therapy in a Feline Model of Sandhoff Disease

Victoria J. McCurdy,1 Heather L. Gray-Edwards,1 Ashley N. Randle,1 Allison M. Bradbury,1 Aime K. Johnson,1 Patricia M. Beadlescomb,1 Nancy Morrison,1 Misako Hwang,1 Miguel SenaEsteves,2 Douglas R. Martin.1 1 College of Veterinary Medicine, Auburn University, Auburn, AL; 2 Gene Therapy Center, University of Massachussetts Medical School, Worcester, MA.

The GM2 gangliosidoses, Tay-Sachs and Sandhoff disease, result from lysosomal hexosaminidase A (HexA) alpha or beta subunit deficiency, respectively. Neuronal storage of GM2 ganglioside causes progressive neurological deterioration and early death, often by age 5. Adeno-associated virus (AAV) gene therapy has shown success in the GM2 mouse model (Proc Natl Acad Sci USA, 103:10373, 2006). Prior to initiating human clinical trials, AAV gene therapy is being evaluated in the GM2 feline model ( subunit deficient), which has a brain size and complexity more closely resembling humans. One GM2 cat treated before disease onset with bilateral thalamus and deep cerebellar nuclei injections of AAV-fHex is currently 31.9 months old, and eight other AAV-treated GM2 cats reached humane endpoint between 9.8-29.9 months old (untreated humane endpoint, 4.4 ±0.6 months, n=9). Because cerebellar injection is risky in human patients, it is important to explore alternative routes to deliver sufficient HexA activity to the cerebellum. In the current study, monocistronic AAV2/ rh8 vectors expressing feline Hex  and  subunit cDNAs (2e12 g.c. per vector) were injected bilaterally into the thalamus and lateral ventricle of 1-month old GM2 cats (disease onset 1.4 ±0.4 months, n=8). In four cats euthanized 16 weeks post-treatment, HexA activity was present at several fold above normal levels throughout the brain (5.0-72.3 fold normal) and spinal cord (5.3-14.7 fold normal), and PAS staining demonstrated substantial clearance of storage material. Activity of lysosomal -galactosidase and -mannosidase, elevated in untreated GM2 cats, was significantly normalized throughout the entire CNS of AAV-treated cats, demonstrating restoration of lysosomal function. In long term therapeutic experiments, four AAVtreated GM2 cats are currently between 7.1-21.1 months old. All remain ambulatory but with diminished mobility caused by hind limb weakness (similar to cats treated by thalamic + cerebellar injection), and all are generally healthy and able to maintain body weight. Another 2 AAV-treated GM2 cats reached humane endpoint at 25.9 and 11.2 months old, though not according to stereotypical disease progression. In contrast to debilitating whole-body tremors and balance problems in untreated cats, AAV-treated cats lost the ability to stand due to hind limb weakness, with or without joint abnormalities. In the AAV-treated brains collected at humane endpoint, HexA activity was distributed throughout the entire cerebrum (2.0-20.9 fold normal), cerebellum (2.6-5.5 fold normal) and spinal cord (5.2-17.7 fold normal). AAV vector was detected by qPCR at varying levels throughout the CNS. These translational studies show that lateral ventricular injection may effectively treat the cerebellum, and they provide strong support for the initiation of AAV-based clinical trials for human GM2 gangliosidosis. S15