Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials in patients with diverse disorders. Two AAV based gene therapy drugs have been recently approved by the FDA. Luxturna has been valued at $850,000 for a one-time treatment for a rare form of blindness and Zolgensma priced at $2,100,000 for spinal muscle atrophy. As such, AAV vector based gene therapy is an increasingly attractive market. Although successful in clinical studies, two concerns restrict broader AAV vector applications for patients requiring liver targeted AAV gene therapy following systemic administration: low human hepatocyte transduction and neutralizing antibody (Nab)-mediated inhibition of AAV transduction. Several approaches have been explored for AAV transduction enhancement or capsid Nab evasion. Engineering of the AAV capsid presents a very powerful and popular technology that has been extensively studied to develop novel AAV vectors for enhanced transduction in animal models or Nab escape in vitro. However, it has been demonstrated that the results from mouse experiments do not recapitulate those of large animals such as primates and dogs. Thus, the data for AAV variants generated in animal cells and organs may not translate into successful human applications. Recently, a mouse xenograft model with human hepatocytes has been used to develop human liver targeted AAV vectors for gene therapy. In our previous studies, we have successfully isolated several AAV mutants from the liver of chimeric mice with human hepatocyte xenografts in the presence of human Nabs (IVIG) using the AAV shuffled capsid library approach. Specifically, BDRK001 (AAV mutant LP2-10) demonstrated a much higher ability to evade Nabs than any other AAV serotypes or mutants. However, BDRK001 was not enhanced for transduction in human hepatocytes when compared to the best natural serotype. In this application, we will use rational design strategy to generate novel AAV capsids by variable region I (VRI) domain swapping of BDRK001 using natural serotypes or mutants with high human liver tropism. This panel will then be evaluated in chimeric mice for human hepatocyte transduction (Aim 1) and Nab evasion (Aim 2). Bedrock's long-term goal of this approach is low dose AAV gene therapy for the successful treatment of a variety of liver diseases, independent of the patient's Nab prevalence.