PROJECT SUMMARY Recombinant adeno-associated virus (rAAV) vectors have demonstrated efficient gene transduction in preclinical gene therapy studies and are approved for clinical use in the US. Although we know the relevant viral genes that facilitate rAAV replication and packaging, we have very little understanding of the cellular proteins required to facilitate AAV replication. We also know very few of the host cell interactions with the vector genome which impact the efficiency of rAAV transduction in target cells. Recruitment of cellular proteins to AAV genomes promotes replication in producer cells, whereas host antiviral factors limit rAAV transduction in target cells. Production of rAAV vectors and their use for transduction and gene therapy will benefit from a deeper understanding of the cellular factors that interact with AAV genomes to facilitate gene expression, DNA replication, genome encapsidation, and vector transduction. In this proposal we aim to identify cellular factors that associate with AAV genomes. Knowledge of proteins that interact with AAV genomes will be harnessed to improve both rAAV production and effective gene delivery. We will employ a novel proteomics technology that identifies proteins on replicating DNA by coupling Isolation of Proteins on Nascent DNA (iPOND) with Mass Spectrometry (MS). Our lab has extensive experience using iPOND-MS to identify cellular factors on replicating viral DNA genomes, with existing experimental and bioinformatic pipelines for acquiring these proteomics data and analyzing results. We are the first to employ iPOND-MS for AAV, and our preliminary data demonstrate feasibility with an innovative approach to label AAV genomes. In Aim 1 we will adapt the iPOND-MS technique to identify cellular factors associated with replicating wild-type AAV genomes. We will optimize labeling of AAV genomes and identification of associated proteins, and determine their impact on AAV replication. In Aim 2 we will identify cellular proteins associated specifically with DNA genomes of rAAV vectors. We will employ iPOND- MS to define proteins on replicating rAAV genomes in producer cells. We will also label rAAV vector genomes and define factors associated with labeled genomes during rAAV transduction of target cells. We will use cellular assays to determine the impact of these host proteins on rAAV transduction. In this way we anticipate that our results will identify the cellular factors associated with rAAV genomes that regulate both vector production and efficient transduction of target cells. This knowledge will suggest ways that production and transduction can be improved. This application is based on innovative proteomics methodology we have developed to identify the repertoire of proteins associated with DNA of viral genomes. Our combined expertise and reagents make us ideally suited for this project which has the potential to provide insights into cellular factors that can be harnessed to improve AAV re...