Genetic determinants of AAV liver transduction and toxicity - Six adeno-associated virus (AAV) vector based biodrugs have been approved by the FDA to treat genetic disorders. The great therapeutic success of AAV gene transfer has been tempered by the often-associated liver toxicity and, with liver-directed therapies, the concomitant fall in the transgene expression. There are marked interpatient differences in susceptibility to AAV liver toxicity and transgene expression, and we have demonstrated similar inter-donor variability in cultured human hepatocytes and in various strains of mice, strongly implicating the role of genetic background. We are proposing to study AAV liver interactions in Collaborative Cross (CC) mice, a relatively new genetic reference population engineered to maximize genetic diversity. We have successfully used CC mice to identify candidate genes which may underlie susceptibility to liver toxicity from several drugs. We have also observed marked differences between CC lines in the extent and kinetics of transgene expression after AAV liver targeting as well as liver toxicity, and this susceptibility was reproduced in hepatocyte spheroids. Our central hypothesis is that CC inter-strain susceptibility to AAV hepatotoxicity and transgene expression will be mirrored in cultured hepatocyte spheroids prepared from the strains and that combining in vivo and hepatocyte spheroid data will provide new insights that will improve the safety and efficacy of AAV gene therapy. To this end, we will pursue 3 specific aims: Aim 1. Elucidate the role of genetic factors underlying susceptibility of AAV liver toxicity and variation in transduction efficiency. After systemic administration of AAV vectors to the fully inbred CC populations (63 lines), we will identify the associations between host genetic background and protein/metabolite biomarkers and susceptibility to liver toxicity and to the long-term, stable transgene expression. Aim 2. Identify non-genetic factors influencing susceptibility to AAV liver toxicity and transduction efficiency. The effect of different parameters (AAV serotypes/variants, dose, transgene, dsAAV vs ssAAV, steroids, empty AAV virions) on AAV liver toxicity and transduction efficiency will be studied in CC strains identified in Specific Aim 1 as susceptible to liver toxicity and/or reduced transgene expression. Aim 3. Explore a novel model culture system to study AAV liver toxicity and transduction. We have demonstrated that primary hepatocytes cultured as spheroids in 386 well plates maintain a mature hepatocyte phenotype and are transduced by AAV. We propose to expand these studies to determine correlations with results from our live CC studies, and to further pursue the underlying mechanisms. Our proposed studies should provide mechanistic insight that may result in the design of safer AAV vectors, as well as identify promising biomarkers to guide safer dosing of existing AAV vectors. Moreover, our studies may support future susceptibility testing of iPSC-derived hepatocytes obtained from patients who are candidates for AAV gene therapy.
