ABSTRACT Gene therapy with recombinant AAV vectors continues to show promise towards clinical treatment of rare genetic diseases. Despite several encouraging results from ongoing clinical trials, numerous challenges have been identified. Amongst these, cross-species variability in AAV tropism, vector dose-related hepatotoxicity and neutralizing antibodies (NAbs) against AAV capsids remain major unaddressed concerns facing effective clinical translation. The current renewal application hinges on the rationale that different aspects of the AAV capsid and the genome can be engineered to selectively enhance vector performance. In the previous funding cycle, we made significant progress towards understanding AAV biology and capsid interactions with glycan and integrin receptors. We successfully exploited this information to engineer new AAV strains with enhanced transduction efficiency and altered tissue tropisms. In the current proposal, we will build on our progress over the past two grant cycles. We propose to tackle the challenges associated with evading neutralizing antibodies and increasing vector potency by shifting our focus to as yet unexplored aspects of AAV capsid structure and the genome. The specific aims are focused on improving AAV transduction at the pre-entry and post-entry level rather than at the point of cellular entry. Our comprehensive approach is supported by exciting new preliminary data and led by a team with expertise in AAV biology and engineering and extensive experience handling large animal models. We expect the outcome of the proposed studies to reduce the burden associated with high costs of scale up and hepatotoxicity associated with high vector dose as well as expand the patient cohort eligible for gene therapy trials.