PROJECT SUMMARY/ABSTRACT The curative potential for hematopoietic stem cell (HSC) transplant has expanded with therapeutic evidence in diseases as far ranging as diabetes, autoimmune diseases such as multiple sclerosis and scleroderma, or non-malignant hematologic disorders such as sickle cell anemia and thalassemia. If HSC transplantation, and hematopoietic gene therapy, are going to be primary methods of treating, or curing disease, numerous deficiencies in the current approaches must be addressed. In the context of gene therapy, limitations in HSC number and/or reductions in engraftment efficiency and true multi-lineage reconstitution reduce the efficacy potential of many current clinical programs. There is a critical need to define the “rules” of HSC engraftment during transplantation, which will then provide targets for therapeutic intervention on HSCs, or at the very least define distinct release criteria for altered HSC products. This proposal will utilize a newly discovered subset of highly engraftable hematopoietic stem cells (heHSCs) and a novel method to mobilize them from the bone marrow niche. The first aim is to Identify the mechanism(s) governing high engraftment of HSCs in competitive environments. This aim will use functional assays to determine what features make an heHSC more competitive and molecular analysis to identify a target(s) that can then be exploited to enhance clinical transplant. The second aim is to evaluate non-toxic conditioning regimens that utilize stem cell trafficking and endothelial opening. These studies will explore non-toxic methods to achieve hematopoietic engraftment that do not rely on radiation or chemotherapeutics. The advances accomplished in this proposal will enhance our understanding of HSC heterogeneity, define potential therapeutic strategies to enhance stem cell engraftment, and will provide a proof of concept for trafficking-based conditioning regimens for non-malignant stem cell transplantation.