379. Intravenous AAV9 Based Gene Therapy Corrects a Mouse Model of Sandhoff Disease

NEUROLOGIC GENE & CELL THERAPY I with robust MFP-inducible target transgene expression and minimal background expression. Switch and Target vectors were delivered to cells in a stepwise manner. Murine and human cells were infected with Switch vectors first and selected by antibiotic resistance or FACS. SWITCH expression was robust and was downregulated upon NPC differentiation. Switch-expressing cells were then infected with Target vectors containing selectable markers. Dual-infected cells were exposed to MFP or vehicle. Initially, the fidelity and inducibility of the system was evaluated using fluorescent marker target transgenes. In the absence of MFP there was no expression of these transgenes; while MFP exposure induced robust expression. Other Target loci with transgenes encoding IGF-1, GDNF or luciferase linked to slectable markers also showed very tight control of transgene expression in vitro. In vivo analysis of the MFP-inducible NPC system is ongoing in rodents with CNS transplantation with NPCs capable of inducible expression of target transgenes. Initial analysis will look at NPCs inducibly expressing fluorescent markers and/or luciferase. Rodents will be treated with placebo or MFP for set periods of time and expression of target transgenes will be evaluated by fluorescent microscopy, immunohistochemistry, or bioluminescence.

379. Intravenous AAV9 Based Gene Therapy Corrects a Mouse Model of Sandhoff Disease

Jagdeep S. Walia,1 Alexander Bello,2 Naderah Altaleb,3 Christa Kruck,3 Richard Hemming,3 Gary P. Kobinger,2 Barbara TriggsRaine.3 1 Medical Genetics, Queen’s University, Kingston, ON, Canada; 2 Special Pathogens Program, Public Health Agency of Canada, National Microbiology Laboratory, Winnipeg, MB, Canada; 3 Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB, Canada. The G M2 gangliosidoses are neurodegenerative disorders characterized by GM2 ganglioside accumulation, particularly in the central nervous system (CNS). These disorders are caused by a deficiency in the activity of the lysosomal enzyme, -hexosaminidase A (HexA). One such disorder, Sandhoff disease, results from mutations in HEXB, which encodes the -subunit of HexA. To correct Sandhoff disease in a mouse model, we decided to use systemic gene therapy with an AAV9 viral vector expressing the -subunit of HexA. AAV9 is known for its ability to cross the blood brain barrier and has been successfully used for systemic gene therapy of CNS disorders. We intravenously injected AAV9 engineered to express murine HEXB (rAAV9-CMV-muHEXB) into Sandhoff (HexB -/-) or unaffected (HexB+/+ or +/-) mice. The same injections were also performed with a control vector expressing LacZ, rAAV9-CMV-LacZ. The dose used was 3 x 1011 vector genomes (vg) for neonatal (day 0 or 1) and 7 x 1011 vg for adult (6 weeks) mice. Mice were monitored for survival, serum HexA  B activity and motor strength until 10 months unless a humane end point was reached earlier. Sandhoff mice treated with the lacZ vector reached a humane end point at 15-17 weeks of age, as expected based on previous literature. All (5/5) neonatally treated Sandhoff mice survived till the end point of the study at 10 months (p<0.001). The total serum hexosaminidase activity was found to be approximately 10-fold of LacZ treated Sandhoff mice at 16 weeks of age. We noticed a decrease in enzyme activity over time to approximately 2-fold at the end point of the study at 10 months. Out of the 5 mice, 3 didn’t show any signs of weakness/tremors, but slight weakness of hind limbs/tremors were found in 2 of 5 mice at the 10 month end point. Of the Sandhoff mice treated at 6 weeks of age, most died at18-19 weeks, and 1 mouse survived to each of 25 and 36 weeks. This study shows very long term correction of Sandhoff disease after one time systemic treatment with rAAV9-CMV-muHEXB with sustained enzyme expression in peripheral blood. The final analysis will include measurement of enzyme activity and GM2 ganglioside levels in the brain, viral copy number analysis and histopathology of S146

different tissues. This study demonstrates the potential of long-term/ permanent correction of Sandhoff disease/ GM2 gangliosidosis through AAV9 based systemic gene therapy.

380. A Specific Cleavage of Amyloid-β by a Viral Protease NIa Prevents the Amyloid-β-Mediated Mitochondrial Dysfunction Woo Jin Park,1 Bae-Hyun Shin,1 Hye-Eun Han,1 Sang Min Park.1 1 Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea.

Alzheimer’s disease (AD) is a neurodegenerative disease marked by extracellular deposition of amyloid- and progressive cognitive dysfunction. Amyloid- levels in the brain are delicately determined by the production and clearance of amyloid-. Several endogenous proteases including neprilysin (NEP) are known to play a role in the clearance of amyloid-. Although these proteases reduced the extracellular amyloid- plaques, they failed to reduce the toxic oligomeric amyloid- levels and to improve cognitive deficits when overexpressed in the brain. Besides, they can cleave a variety of other peptides and cytokines present in the extracellular matrix due to their broad substrate specificity. This property poses a serious drawback as a modality for clearance of amyloid-. Therefore, a novel strategy that permits the specific cleavage of amyloid- is sought after. The nuclear inclusion a protease (Nia) of the turnip mosaic virus has a strict substrate specificity for the consensus sequence of Val-Xaa-His-Gln. We previously showed that Nia specifically cleaved amyloid- which contains the sequence of Val-His-His-Gln near the putative -secretase cleavage site. In addition, we showed that lentivirus-mediated expression of Nia in the brain of AD mice significantly reduced plaque deposition levels and improved cognitive deficits. However, the detailed molecular mechanism underlying the neuro-protective effects of Nia was unknown. In this study, we show that Nia cleaves oligomeric as well as monomeric amyloid-, which is an essential feature that is not shared by other known amyloid- cleaving enzymes such as NEP. The progression of AD is reportedly associated with mitochondrial dysfunction. We found that Nia restored mitochondrial function in neuronal cells damaged by exogenously added oligomeric amyloid-. We further traced the fate of the fluorescence-labeled oligomeric amyloid- within cells under confocal microscope. At earlier times of treatment, the fluorescence was observed mostly in endosomes and lysosomes. However, at 48 hours after the treatment, about 70-80% of the fluorescence was colocalized with lyso-tracker, whereas about 20-30% was co-localized with mito-tracker. This finding supports the previous hypothesis that the endocytosed amyloid- leaks out of lysosomes and enters mitochondria. This transit of amyloid- to mitochondria was perfectly prevented by Nia. Therefore, it appears that Nia cleaves the amyloid- that leaks out of lysosomes and thus prevents the accumulation of amyloid- in mitochondria. With its capability of cleaving amyloid- in a highly specific manner, Nia provides a unique opportunity for gene therapy of AD.

381. Viral Vector Mediated Overexpression of Pleiotrophin for Disease Modification in Parkinson’s Disease

S. E. Gombash,3,1 F. P. Manfredsson,1 C. J. Kemp,1 S. L. Wohlgenant,1 N. C Kuhn,1 S. M. Fleming,3 R. J. Mandel,2 T. H. Collier,1 C. E. Sortwell.1 1 Michigan State University, Cincinnati, OH; 2University of Florida, Gainesville, FL; 3University of Cincinnati, Cincinnati, OH. Trophic factor gene therapy to slow nigral degeneration in Parkinson’s disease (PD) has been extensively tested in preclinical animal models. Despite encouraging results in toxin based PD models, no neurotrophic factor therapy to date has yielded clinical Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy