78. An IND-Enabling GLP-Toxicology and Biodistribution Study Assessing Systemic rAAV9-hNAGLU Gene Delivery for Treating MPS IIIB: Genotype- and Sex-Specific Dose-Limiting Acute Liver Toxicity in Male Wild Type C57BL/6 Mice

Abstract

No treatment is currently available for mucopolysaccharidosis (MPS) IIIB, a neuropathic lysosomal storage disease due to autosomal recessive defect in α-N-acetylglucosaminidase (NAGLU). In preparation for a clinical trial, we performed an IND-enabling GLP-toxicology study to assess systemic rAAV9-CMV-hNAGLU administration in wt C57BL/6 mice (6-8wk-old), at 1e14 vg/kg or 2e14 vg/kg (n=30/group, M:F=1:1). These were 2 and 4-fold higher, respectively, than our proposed clinical high dose. No adverse clinical signs were observed during the entire 6mo study duration. NAGLU activity above wt levels and vector genomes were detected in all tested CNS and somatic tissues in rAAV-treated mice through 6mo. However, acute mortality was observed in 5 male mice at 2e14 vg/kg dose on Days 6 and 8 pi. All 5 animals exhibited liver discoloration correlating with severe hepatic necrosis and vacuolation, without detectable leukocyte infiltration. At 6, 12 and 24wk pi, minimal single cell hepatocyte necrosis and/or vacuolation, and minimal cardiomyopathy were noted in a dose-dependent manner, with slight transient elevation of ALT or AST at 6wk pi. T-cell responses to AAV9 capsid and rNAGLU were detected at 6wk pi, but partially and completely diminished at 12wk pi, respectively. ELISA showed antibody responses to both AAV9 and rNAGLU in treated animals. In response to the acute mortality in the GLP-tox test, we performed a 6-wk non-GLP study mimicking the GLP-tox study conditions in male MPS IIIB and wt littermates, to further assess the approach and obtain data prior to early mortality. We treated the mice with the same batch of rAAV9-CMV-hNAGLU at the high dose, 2e14 vg/kg, and performed necropsy at 5 days and 6wk pi (n=4/group). No mortality or clinical signs were observed in either rAAV9-treated MPS IIIB or wt mice. At Day 5 pi necropsy, of 4 rAAV9-treated male wt mice, 1 exhibited liver discoloration, correlating with multifocal, periportal and midzonal hepatocyte necrosis, and 2 showed minimal single cell hepatocyte necrosis. Minimal liver damage and elevation of ALT/AST in wt mice persisted to 6wk pi. However, no abnormal changes attributed to vector treatment were detected in the liver in MPS IIIB mice. Therefore, we have identified genotype- and sex-specific dose-limiting toxicity of systemic rAAV9-hNAGLU delivery, supporting a safe profile for our proposed approach for treating MPS IIIB in patients.