In general, AAV8-Fah displayed a linear dose response over the range of doses administered (Fig. 4) where the highest doses administered produced the greatest gene repair. The difference in repair frequencies between the highest dose and all other doses administered was significant. In contrast, AAV2-Fah had no significant change in repair frequency over the entire range of doses administered. Overall, results indicate

that AAV8-mediated gene repair is superior to that with AAV2. The adult liver has considerably less cellular turnover than neonatal liver undergoing rapid growth and proliferation. Thus, gene repair frequencies are predicted to be lower in adults as homologous recombination is most prevalent during mitotic S-phase.37 AAV8 was chosen to RAD001 supplier test the feasibility of gene repair in the nearly quiescent adult liver as it had now been demonstrated to be both faster and more efficient at gene repair than AAV2. Adult Fah5981SB mice (8-12 weeks old) were injected with 1 × 1011 vg of AAV8-Fah (n = 6), whereas age-matched littermate controls were injected with isotonic NaCl (n = 8). Mice were withdrawn from NTBC to allow selection of corrected hepatocytes. Serum for liver function tests and liver tissue were harvested >12 weeks after treatment. Mice treated with AAV8-Fah showed clinical improvement and repopulation with FAH+ hepatocytes (Fig. 5A), whereas all mice Dasatinib order in the control

group had

to be euthanized and showed no hepatic repopulation. Surprisingly, the initial correction frequency of FAH+ nodules was comparable to that seen with neonatal administration. The clonal expansion of corrected hepatocytes was able to reverse the tyrosinemic phenotype and was highly reproducible. Liver function tests for AST and bilirubin demonstrated near complete correction when compared to controls (Fig. 5B). Although phenotypic reversion of Fah5981SB mice indicates successful site-specific gene repair, random integration could also occur.38 To assess random integration frequencies, d3 Fah5981SB neonates were coinjected with 4 × 1010 vg of AAV8-Fah and an irrelevant serotype-matched control vector AAV8-hAAT. Post-weaning, mice were MCE subjected to NTBC withdrawal to select for corrected hepatocytes. To ensure no episomes remained, 5 × 105 random hepatocytes were then serially transplanted into eight secondary Fah5981SB recipients. After >12 weeks off NTBC, serum and liver tissue were collected at harvest. qPCR was used to determine Fah and hAAT copy numbers in each mouse (Table 1). The frequency of randomly integrated hAAT ranged from 0 (undetectable) to 0.06/dGE and averaged 0.005/dGE. Only half the hepatocytes in repopulated livers were donor-derived, thus frequencies were corrected by a factor of two, resulting in an average random integration frequency of 0.01/dGE (1%) in corrected hepatocytes.