Adeno-associated viral vectors (rAAV) have shown promise in some liver-based clinical trials including hemophilia B. However, one of the limitations is the loss of the vector genomes during tissue growth and cell division limiting the duration of expression when treatment is initiated early in life. For success in treating a number of genetic diseases treatment early in life is required. We previously developed a non-nuclease mediated AAV-mediated homologous recombination (AAV- HR) approach by using genomic homology arms to insert a protein coding sequence onto the end of the endogenous Albumin gene such that after homologous recombination, the modified locus would make a chimeric mRNA and both the endogenous albumin protein and a second protein, in our case human factor IX and treated the bleeding diathesis in a murine model of hemophilia B. Subsequently, this approach has been used to treat other murine models of human hepatodeficiency disorders and a Phase I/II clinical trial for methylmalonic acidemia was initiated by LogicBio therapeutics. The limitation is the efficiency of the process remains low and we have recently established that off-target integration likely can produce a proportion of the therapeutic protein. Our goal is to further study the genomic sources of the AAV-HR produced therapeutic product as well as parameters that influence the efficiency of AAV-HR in cells and in vivo. To do this, we will use high-throughput sequencing approaches to molecularly characterize the origin of off-target transcribed RNAs produced after AAV-HR mediated transduction. We will establish if they arise from vectors that integrate into regions of microhomologies or via non-homologous end joining as well as identify which sets of these RNAs are translated. Our preliminary data suggests that regions of the host genome that are more robust in R-loop formation will likely be more efficient at AAV-HR. We will directly test this hypothesis and use the information to design improved homology arms for AAV-HR. We will also do an unbiased in vivo genetic screen in mice to establish which genes when expression is reduced results in higher AAV-HR. We will confirm that parameters that influence non-nuclease mediated AAV-HR also effects nuclease mediated AAV-HR. The results from these studies will not only provide new mechanistic insights into AAV- HR but provide new approaches for enhanced genome editing, which would broaden the application for treating human genetic diseases.