ABSTRACT Although hematopoietic stem and progenitor cell (HSPC) transplantation now underlies two clinical cases of HIV- 1 remission/functional cure, a means to apply this approach to a wider array of patients has not yet been identified. In this project, we will address a key limitation for HSPC-based anti-HIV strategies: the engraftment and potency of gene-edited HSPC and their progeny. Although our previous findings demonstrate that gene edited HSPCs engraft long-term in vivo, only a limited proportion persist over time, and are incapable of supporting antiretroviral therapy (ART)-free virus remission. To address this, we have i) adapted a more advanced strategy to edit our locus of interest, CCR5, ii) identified an approach to not only disrupt the CCR5 gene, but simultaneously insert therapeutic anti-HIV transgenes, and iii) designed experiments to evaluate this strategy in our robust nonhuman primate (NHP) model of suppressed HIV infection. We will target two rationally designed, highly potent anti-HIV transgenes to the gene-edited CCR5 locus: the virus-specific chimeric antigen receptor CD4CAR, and the broadly neutralizing antibody-like molecule eCD4-Ig. Our preliminary data demonstrate our ability to insert defined genetic sequences at up to 50% of targeted CCR5 alleles in primary NHP HSPCs. Here, we will optimize our approach to insert CD4CAR or eCD4-Ig, and safely engraft an autologous HSPC product containing both CD4CAR∆CCR5 and eCD4-Ig∆CCR5 HSPCs into the same animal. As we are introducing two therapeutic transgenes and simultaneously disrupting the CCR5 coreceptor, we refer to this as a “three for one” approach. In addition to generating a potent and efficiently modified HSPC product, we will work closely with each project in our U19 consortium. We will coordinate with Project 3/Cannon to identify the most efficient means to modify HSPCs, prior to in vivo studies in our respective animal models. With Project 1/Scadden, we will evaluate a bone marrow cryogel (BMC) scaffold designed to enhance the differentiation of HSPC-derived T-cells, namely CD4CAR∆CCR5 T-cells. Finally, we will investigate the impact of safer, nongenotoxic conditioning (NGC) regimens characterized by Project 2/Magenta on infection with simian/human immunodeficiency virus (SHIV) and suppression by ART. We believe that safe and efficacious engraftment of gene-modified, virus-specific HSPCs and their progeny will enable robust protection against de novo SHIV challenge, and significantly impact viral reservoirs in infected, suppressed animals.