A significant obstacle in current hematopoietic stem cell (HSC) gene therapy studies has been the ability to achieve persisting high-level engraftment of gene-modified cells to provide long-term therapeutic efficacy. Subtherapeutic engraftment occurs even under full myeloablative conditioning and will likely become an even bigger issue with nonmyeloablative and nongenotoxic conditioning. The only corrective measure for subtherapeutic correction levels is an allogeneic HSC transplant, which subjects patients to the risks of graft- versus-host disease. Here we propose an alternative, minimally toxic approach to increase engraftment of gene-modified cells into the therapeutic range that avoids the significant side-effects associated with myeloablative conditioning and allogeneic HSC transplantation. Our approach will be applied to sickle cell disease (SCD) and ß-thalassemia, which represent the most common severe monogenic diseases worldwide. Gene replacement therapies for these disorders have shown promising early results but also in many cases subtherapeutic gene-correction levels. In this application, we propose an innovative strategy to enrich genome- edited and therapeutically relevant HSC post-transplantation in the context of reduced, nonmyeloablative conditioning. This strategy relies on three coordinated aims that collectively address current HSC gene therapy/genome editing transplantation. Specific Aim 1 will optimize safety and efficacy of novel base editors/prime editors to allow simultaneous modification of the therapeutic globin target and of the selection gene. Specific Aim 2 will apply these novel editing tools to the modification of HSCs derived from both healthy or sickle cell patients in order to support high levels of therapeutic globin production upon engraftment and drug selection in the mouse xenograft model. Specific Aim 3 will build upon these findings to evaluate engraftment and selection protocols for edited HSC in the clinically relevant rhesus macaque autologous transplant model in the setting of reduced-intensity conditioning. To accomplish these aims, we assembled a multidisciplinary team which includes investigators with complementary expertise in base editing/prime editing (Dr. Liu), hemoglobinopathies (Dr. Weiss) and HSC biology and transplantation (Dr. Kiem). Our findings should be applicable to other diseases in which genetically corrected cells do not have a natural selective advantage and also to other reduced-intensity, nongenotoxic conditioning regimens. Collectively, the proposed studies will define a safer and effective HSCs transplantation protocol that will serve as a foundation for clinical testing in patients suffering from hemoglobinopathies and from other genetic diseases.