Adeno-associated virus (AAV) vectors have become the leading biologic for the long-term correction of monogenic diseases. Success in numerous clinical trials led in the approval of currently six AAV-based biologics. However, significant challenges for their wide-spread utilization remain including pre-existing immunities in large portions of the human population and toxicities at high doses. Neutralizing antibodies originate from prior exposure to naturally circulating AAVs that primarily target the capsid leading to vector inactivation and loss of treatment efficacy. Patients having these antibodies are usually excluded from receiving these therapeutics. Additionally, other aspects of the immune system pose significant barriers for effective gene delivery especially following high dose AAV vector administration. These high doses are required to counteract the low transduction efficiencies of many natural AAV variants to achieve sufficient transgene expression for therapeutic success. The ability of AAV vectors to deliver their payload to the target cells is determined by a series of interactions of the capsid to the cell surface receptors as well as intracellular trafficking factors. Hence, the overall objective of this project is to characterize these AAV capsids interactions structurally and functionally. Specific questions our proposal will address are: “How do mAbs from clinical trial participants or donors with natural AAV exposure bind and neutralize AAV capsids compared to the previously described mouse mAbs?” (Aim 1) and “How do proteins of the innate immune system, receptors, and other intracellular factors interact to the AAV capsids?” (Aim 2). We will use cryo-electron microscopy to determine high-resolution structures of the complexes to ≤3 Å resolution and apply the obtained information to engineer vectors that retain their cell binding properties but evade recognition by human antibodies or defensins. Additionally, we will characterize non-primate AAVs and evaluate their potential as gene delivery vectors (Aim 3). Due to highly diverse capsid amino acid sequence compared to the commonly utilized AAV serotypes most pre-existing antibodies will be prevented from binding. Thus, this aim will ask the question: “Can we design new AAV capsids utilizing structural information from non-primate AAV, while retaining/inserting known receptor-binding interfaces of the primate AAVs on the capsid surfaces to allow for transduction of human cells and prevention of antibody neutralization and/or complement activation?” Overall, this project will culminate in an expanded “pillbox” of engineered vectors with the ability to overcome the challenges of the host immune responses and efficient gene transfer thereby improving clinical efficacy and increase the cohort of patients eligible for treatment.