PROJECT SUMMARY Sickle cell disease (SCD) and transfusion-dependent b-thalassemia (TDT) are severe, prevalent blood disorders for which fetal hemoglobin (HbF) induction can bypass the fundamental hemoglobin defects and hematopoietic stem/progenitor cell (HSPC) transplantation (HSCT) offers curative potential. Allogeneic and autologous trans- plant approaches can succeed, nonetheless, the short- and long-term toxicities of genotoxic alkylating chemo- therapy-based conditioning regimens remain a substantial barrier to the widespread application of curative HSCT for SCD and TDT. Immunotherapies targeting HSPC antigens have been proposed as a safer conditioning strat- egy, however the pharmacokinetics of these agents currently hamper their clinical efficacy. The long-term goal of our proposal is to address this unmet medical need by developing an effective, novel strategy for the engraft- ment and progressive enrichment of autologous gene modified HSPCs in SCD and TDT by coupling non-geno- toxic immunotherapy-based myeloablation with epitope-engineering. Our central hypothesis is that precise multiplexed base editing of HbF determinants and targeted epitopes within HSPCs can endow hematopoietic lineages with both HbF induction capacity and selective resistance to monoclonal Abs or CAR-T cells without affecting protein function or regulation (so-called stealth status). We have identified defined minimal amino-acid changes within the extracellular domains (ECD) of the cytokine receptors KIT, FLT3 and IL3RA, each expressed in long-term repopulating HSCs, that abrogate recognition by therapeutic Abs while preserving physiologic responses to stimulation with their respective ligands. Here, we will capitalize on these results and further expand the reach of these innovative genetic engineering tools with the objectives to i) generate “stealth” g-globin derepressed HSPCs by multiplex CRISPR-Cas base-editing; ii) validate efficacy of this approach on suitable pre-clinical models of b-hemoglobinopathy, and iii) further optimize and scale the manufacturing process for production of efficiently and precisely engineered cellular products suitable to progress to the clinic. We aim: 1) to optimize multiplex base editing approaches to simultaneously derepress HbF and engineer stealth HSPC epitopes that will generate immunotherapy resistant hematopoietic stem cells capable of ameliorating sickling and globin chain imbalance in SCD and TDT patient erythroid cells; 2) to maximize the engraftment of edited HSCs by optimizing immunotherapy regimens to enrich multiplex edited HSPCs through modeling parameters for therapeutic selection of edited HSPCs, and thereby obtaining proof-of-concept chemotherapy-free engraft- ment and selection of edited patient HSPCs; and 3) to identify conditions that produce efficient on-target base edits without measurable off-targets at clinical scale. This project will provide fundamental advancement of a new chemotherapy-free gene therapy approach to HSCT for hemoglobinopathies that should additionally have broad applicability to other hematopoietic disorders.

PROJECT NARRATIVE Sickle cell disease (SCD) and transfusion-dependent b-thalassemia (TDT) are severe blood disorders for which the only currently available curative therapies require chemotherapy, which comes with substantial short-term and long-term drawbacks. We have developed a novel approach where we produce base edits in blood stem cells to both re-activate therapeutic fetal hemoglobin and create cells resistant to immune-based therapies. Our premise to be tested is that this approach could allow the development of curative therapies for SCD and TDT that do not require chemotherapy (or radiation therapy), and thus sidestep inherent risks such as nausea, hair loss, mucositis, life-threatening infections, infertility, DNA damage, and cancer.