Project Summary Adeno-associated viruses (AAVs) are non-pathogenic parvoviruses that are currently the foremost gene therapy vectors. As gene therapy is increasingly employed to treat a variety of indications, AAVs serve a critical role in delivering therapeutic DNA. However, effective packaging of recombinant transgenes in AAV vectors remains a paramount challenge to successful gene therapy. First, the packaging capacity of the AAV capsid restricts transgene size to under 4.7 kb, precluding delivery of many transgenes and gene editing cassettes. Relatives of AAV within Parvoviridae contain larger genomes, including Densovirus (DNV), a subfamily of parvoviruses that naturally infect invertebrates. I propose to engineer chimeric DNV/AAV vectors that support robust transduction of mammalian cells and enhanced packaging capacity relative to traditional AAV vectors. I then plan to investigate DNV/AAV transduction, and leverage this knowledge to perform directed evolution on DNV/AAV capsids to confer robust transduction of mammalian cells. Second, immune recognition and clearance of AAVs following high dose vector administration remains a key issue in gene therapy delivery, as high doses are often indispensable to achieve transgene expression at therapeutic levels. One reason for the necessity of high dose treatments is the lacking potency of recombinant AAV vector preparations, which have been demonstrated to have lower infectious-to-physical particle ratios relative to AAVs packaging their endogenous genome. Previous studies have shown that these differences in AAV production may be due to uncharacterized cis-acting packaging signals within the AAV genome. Therefore, I aim to perform an unbiased screen for novel motifs within the AAV genome that confer enhanced genome packaging, and to investigate how incorporation of additional cis-acting sequences affects recombinant vector production. Together, these studies aim to address core issues in AAV mediated gene therapy by augmenting both the size of transgenes that can be delivered and the potency of recombinant vector production.