605. A Mucopolysaccharidosis-Like Phenotype in Feline GM2 Gangliosidosis and Partial Amelioration After Intracranial AAV-Gene Therapy

NEUROLOGICAL DISEASES II the peripheral features of the disease, it does not address CNS complications. Using domestic cats carrying an IDUA mutation–a naturally occurring disease model that recapitulates the clinical, histological and biochemical features of MPSI–we tested the capacity of intrathecal AAV9-mediated delivery of the IDUA gene to induce enzyme expression and correct storage pathology in the CNS. Cats treated with a single intrathecal injection of 1 x 1012 GC/kg of an AAV9 vector demonstrated robust IDUA activity in the CSF and serum, with nearly complete correction of lysosomal ganglioside storage throughout the CNS, as well as normalization of the aberrantly overexpressed unrelated lysosomal enzymes characteristic of this disease. Antibody responses against IDUA were detected in 2 out of 3 treated cats, with a concomitant decrease in circulating enzyme activity in the animal with the highest titers. Antibody induction was not associated with clinical sequelae or abnormalities in CSF protein or cell counts, and only modestly affected histological improvement. These results strongly support the development of intrathecal AAV9mediated gene delivery as a therapy for the CNS features of MPSI.

605. A Mucopolysaccharidosis-Like Phenotype in Feline GM2 Gangliosidosis and Partial Amelioration After Intracranial AAV-Gene Therapy

Heather L. Gray-Edwards,1 Patricia M. Beadlescomb,1,3 Ashley N. Randle,1 Brandon L. Brunson,3 Merrilee Holland,1 Allison M. Bradbury,1,3 Victoria J. McCurdy,1,3 Aime K. Johnson,1 Nancy R. Cox,1 Diane U. Wilson,1 Judith A. Hudson,1 Miguel Sena-Esteves,2 Douglas R. Martin.1,3 1 College of Veterinary Medicine, Auburn University, Auburn, AL; 2 Department Lof Neurology and Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA; 3Anatomy, Physiology & Pharmacology, Auburn University, Auburn, AL. Introduction: Sandhoff disease (SD) results from a deficiency of the beta subunit (HEXB) of N-acetylhexosaminidase (EC 3.2.1.52). In addition to cleavage of GM2 ganglioside, HexB is also responsible for hydrolysis of glycosaminoglycans (GAGs), namely dermatan sulfate (DS) and keratan sulfate (KS). Human Sandhoff patients experience skeletal malformations, cardiac abnormalities, and impaired catabolism of DS and KS in isolated fibroblasts, but do not exhibit urinary mucopolysaccaridosis. Intracranial (IC) adenoassociated viral (AAV) gene replacement of hexosaminidase alpha and beta subunits results in a >four-fold increase in lifespan of Sandhoff cats, with marked attenuation of neurologic signs, but the dramatic increase in life span has permitted otherwise subclinical MPS-like pathology to emerge. Methods: Monocistronic AAVrh8 vectors expressing feline Hex alpha and beta subunits (1:1 ratio; 4.4×1012 g.c. total) were injected bilaterally into the thalamus (Thal) and either the deep cerebellar nuclei (DCN) or intracerebroventricularly (ICV) into the lateral ventricle of SD cats at one month of age. Results: 3D CT shows mild skeletal malformations in untreated animals at humane endpoint and prominent malformations in AAV-treated animals such as: hemivertebrae with vertebral bridging, and spinal cord compression. Urinary glycosylaminoglycans (GAG) concentrations were 4.21 sd 1.02 fold of normal for untreated SD cats, 5.36 sd 3.02 fold of normal for Thal+DCN AAV- treated SD cats and 4.31 sd 2.36 fold of normal in Thal +ICV SD cats. The SD cat exhibited cortical GAG concentrations of 1.90 sd 0.32 mcg GAG/mg of tissue (dry weight). Intracranial AAV gene therapy resulted in correction (p<0.001 all groups) of GAG storage in the occipital cortex in both Thal +DCN (0.15sd 0.05 mcg GAG/mg) and Thal+ICV (0.31 sd 0.13 mcg/mg) treatment groups compared to normal (0.10 sd 0.10 mcg/mg). Significant correction was observed in the liver in the Thal+DCN (0.33 sd 0.23 mcg/mg, p<0.05) and Thal+ICV groups (0.63 sd 0.65 mcg/mg, p<0.05) compared to untreated (2.07 sd 0.56 mcg/mg, p<0.01) and normal (undetectable). Partial correction, which trended toward significance, was noted in the heart, kidney, lung, S234

pancreas and small intestine. Echocardiography of SD cats revealed truncation and distortion of valves with valvular insufficiency and dilation of the proximal aorta, which progresses with age after AAV gene therapy. Conclusions: Feline gangliosidosis exhibits many of the mucopolysccharidosis like phenotypes exhibited by human Sandhoff patients, and this phenotype results in progressive morbidity after the extension of lifespan afforded by IC AAV gene therapy. As we approach clinical trials, these findings may be valuable in deciding therapeutic routes for Sandhoff patients.

606. Overexpression of a Non-Aggregatable Form of Alpha Synuclein Protects Against Loss of Endogenous Alpha Synucelin in Nigrostriatal Neurons

Matthew J. Benskey,1 Nathan C. Kuhn,1 Sudhish Mishra,1 Nicholas M. Kanaan,1 Caryl E. Sortwell,1 C. Jiang,2 Fredric P. Manfredsson.1 1 Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI; 2Center for Proteomics and Systems Biology, University of Texas, Health Science Center, Houston, TX. Parkinson Disease (PD) is characterized by the progressive loss of nigrostriatal dopaminergic neurons and the presence of proteinacious inclusions known as Lewy bodies. Lewy bodies are highly enriched in the protein alpha-synuclein (α-syn). Mutations or multiplications of the gene encoding α-syn result in familial forms of PD. Thus, α-syn has been proposed to directly contribute to dopaminergic cell loss in PD. The predominant theory posits that α-syn-mediated PD pathology arises due to a toxic gain-of-function. Accordingly, current therapeutic strategies are centered on eliminating the protein from DA neurons. In contrast, the overarching hypothesis in our laboratory is that α-syn is not the primary toxic species in PD; rather, α-syn mutations or aggregation produce pathology by effectively decreasing the pool of functional α-syn available to the cell. To first determine if loss of functional α-syn alone leads to dopaminergic degeneration, adult male rats received unilateral stereotaxic injections of recombinant adeno associated virus (rAAV) expressing a scrambled control short hairpin RNA (shRNA) or shRNA directed toward α-syn to the substantia nigra pars compacta (SNpc). Administration of α-syn shRNA resulted in loss of tyrosine hydroxylase immunoreactive (THir) cells in the SNpc whereas the scrambled shRNA had no effect. The loss of THir cells in the SNpc following α-syn shRNA was found to be both dose and time dependent, occurring as early as 14 days post injection. To further confirm that loss of α-syn produces dopaminergic neurodegeneration, the ability of wild-type human alpha synuclein (WT α-syn) or a non-aggregatable mutant form of α-syn (α-synC6) to rescue α-syn shRNA induced neurodegeneration was explored. The non-aggregatable form of α-syn was produced by inserting 6 cysteine residues into the primary amino acid sequence of α-syn, increasing intramolecular interactions and stabilizing the protein in a partially folded conformation that renders the amyloidogenic portion of the protein inaccessible. The α-synC6 mutant decreases the ability of α-syn to aggregate and thus ensures that the protein will remain in the accessible soluble pool of protein. Adult male rats received unilateral stereotaxic injections of rAAV expressing WT α-syn, α-synC6 or a GFP control one month prior to administration of the α-syn shRNA. Stereological cell counts one month following the α-syn shRNA administration in animals receiving rAAV-GFP or rAAV-WT α-syn showed a significant loss of THir neurons in the injected SNpc. In contrast, animals that were injected with α-synC6 showed a partial rescue of THir neurons in the injected SNpc, as compared to GFP treated animals. These results confirm that loss of functional α-syn is toxic to nigrostriatal dopaminergic neurons. Future studies will determine if differential aggregation profiles underlie the ability of α-synC6, but not WT α-syn, to rescue nigrostriatal neurons from loss of endogenous α-syn. Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy