Project Summary/Abstract This proposal outlines the five-year research and career development plans for the PI, Stephen P. Persaud, MD, PhD, who is preparing for a career as an academic physician-scientist. Dr. Persaud completed his MD and PhD in immunology in 2015 at Washington University School of Medicine (WUSM), then completed his residency in Clinical Pathology in 2018 at Barnes-Jewish Hospital as part of the Pathology Physician Scientist Training Program. During his residency elective time, he began postdoctoral research in the lab of John DiPersio, MD, PhD, in the Oncology Division of the Department of Medicine at WUSM. Dr. DiPersio is an expert in allogeneic hematopoietic stem cell transplantation (allo-HSCT), normal and malignant hematopoiesis, and cellular immunotherapy, and has an outstanding track record of training successful physician-scientists. The exemplary scientific resources and environment provided by the DiPersio lab and WUSM, combined with the mentoring and training plans described herein, will enhance Dr. Persaud’s research program and advance his progress towards becoming an independent investigator. The long-term goal of the proposed research is to optimize HSCT conditioning, stem cell mobilization, and therapeutic gene editing to safely enable transplantation for non-malignant hematologic diseases, particularly sickle cell disease (SCD). Dr. Persaud published the first study showing that CD45- or cKit-targeted antibody-drug conjugates (ADCs) combined with Janus kinase 1/2 (JAK1/2) inhibitors could enable fully MHC-mismatched HSCT in mice. This work was the DiPersio lab’s first contribution to the antibody-based conditioning field and gave rise to several successful projects, including 1) development of fully myeloablative ADCs suitable for HSCT conditioning in the context of leukemia therapy, 2) the combination of anti-CD47/cKit antibodies with JAK1/2 inhibitors for toxin- free allo-HSCT conditioning, and 3) development of a novel streptavidin-based platform for rapid ADC production and screening. Dr. Persaud will build on this body of work in this proposal via two Specific Aims, which focus on overcoming the major hurdles to autologous gene therapy for SCD. In Aim 1, he will evaluate novel stem cell mobilization regimens combining VLA-4 and CXCR4 inhibition and assess their ability to generate a sufficient quantity and quality of stem cells for autologous gene therapy compared to standard-of- care regimens. In Aim 2, Dr. Persaud will optimize toxin-free conditioning with anti-CD47/cKit antibodies for transplantation and cure of SCD, including the development of novel bispecific CD47 x cKit antibodies. Collectively, these studies will explore the basic biology and translational potential of several novel approaches for improving the safety and efficacy of HSCT. Although this proposal focuses on autologous gene therapy for SCD, the work has important implications for both autologous and allogeneic transplantation for mal...