Among contemporary gene transfer vehicles, non-pathogenic recombinant adeno-associated viral vectors (rAAV) show exceptional promise for liver-targeted therapeutic approaches. The broad focus of studies described in this thesis was the development of rAAV-mediated gene therapy to treat Progressive Familial Intrahepatic Cholestasis type 3. This autosomal recessive condition, caused by mutations of ABCB4, results in deficient hepatocanalicular phosphatidylcholine translocation and leads to progressive cholestatic liver disease with approximately 50% of patients requiring liver transplantation before reaching adulthood. Using an Abcb4-knockout mouse model, in vivo liver transduction with rAAV2/8 vectors encoding hABCB4 led to increased biliary phosphatidylcholine in disease-free heterozygous, but not in homozygous adults with established liver disease, despite varying vector genome size and routes of administration. Maximal transduction was achieved prior to onset of liver disease, optimally in neonates. However, loss of transgene expression occurs following neonatal vector delivery, due to rAAV episomal degradation during rapid liver growth. A novel, hybrid rAAV-piggyBac transposon vector strategy was devised to sustain hABCB4 expression in neonatally-treated homozygotes. Successful correction of liver disease was demonstrated in Abcb4-/- mice up to 9 months post-inoculation, with preliminary results indicating reduction in disease-related hepatocarcinogenic risk. These results demonstrate that rAAV-mediated gene therapy has the potential to offer patients with this heritable cholestatic liver disease an effective alternative treatment to liver transplantation, but also illustrate the importance of addressing challenges, such as the impact of liver pathology on vector performance, which is vital before this potential can be realised for this and related conditions
