Gene editing of hematopoietic stem and progenitor cells (HSPCs) has the potential to cure many genetic diseases of the blood and immune system. Using targeted nucleases followed by delivery of a DNA donor template for homology directed repair (HDR) using an adeno-associated viral (AAV) vector, we and others have achieved high efficiency gene correction at multiple therapeutically relevant genetic loci. Human HSPCs can undergo high efficiency correction and reconstitute the blood and immune system in vivo after engraftment into immune-compromised NSG mice. However, transduction by the AAV vector impairs cell survival, proliferation, and engraftment efficiency. This proposal aims to understand the innate immune cellular responses to the AAV vector at the bulk population and single-cell level in HSPCs in order to obtain high efficiency gene correction without impairment of HSPC survival or self-renewal capacity. Although HSPCs can undergo high efficiency HDR, these cells are drastically (4-10 fold) impaired for engraftment compared to untreated controls, which we have determined is due to the AAV vector. In dose response experiments, high copy number transduction demonstrated decreased cell viability and proliferation as well as decreased progenitor cell survival as measured by Colony Forming Unit assay, toxicity which correlates with increased nuclear accumulation of AAV genomes as measured by digital droplet PCR. We therefore propose to investigate two antiviral pathways, NF-kB (Aim 1) and cGAS (Aim 2), across a range of AAV copy numbers to determine their effect on HSPC survival by measuring activation of these pathways as well as inhibition and knock-out experiments using transduction of GFP expressing vectors and CFU assays for progenitor cell survival. After initial studies on progenitor cell survival we will determine whether transient NF-kB and cGAS inhibition improves self-renewal of long-term hematopoietic stem cells through engraftment in NSG mice. We will simultaneously use an unbiased hypothesis generating approach in Aim 3 to determine cellular responses to AAV occurring in HSPC subpopulations through single-cell RNA-seq experiments which may be missed in Aim 1 and 2 experiments using the whole population or large subpopulations. My scientific environment and training plan are ideal for successful completion of this research proposal. The Stanford Stem Cell Institute has many talented stem cell researchers which are highly collaborative as demonstrated by our RNA-seq collaboration with Dr. Irv Weissman. The wealth of scientific resources including our dedicated FACS core and career resources offered through the Office of Post-doctoral Affairs, as well as scientific and career training from my mentor Dr. Porteus will allow me to achieve my scientific and career goals. Successful completion of this proposal will both further our understanding of HSPC biology and self-renewal in the context of antiviral responses, as well as improve the...