Epitope Editing Enables In vivo Co-Selection of Multiplex Genome-Engineered Hematopoietic Stem Cells and Non-Genotoxic Conditioning

Epitope Editing Enables In vivo Co-Selection of Multiplex Genome-Engineered Hematopoietic Stem Cells and Non-Genotoxic Conditioning Hematopoietic stem cell transplantation (HSCT) is a curative therapy for various hematological disorders. However, traditional myeloablative conditioning regimens pose significant toxicity, limiting broader clinical applications. HSCT risk/benefit assessment is particularly important when HSCT is proposed as curative approach for non-malignant diseases by enabling the replacement of endogenous hematopoiesis with gene-corrected cells. However, the short- and long-term toxicities of genotoxic conditioning remain a major barrier to the wider adoption of HSCT and gene therapy. Monoclonal antibody (mAb)-based conditioning, particularly targeting CD117 (c-KIT), offers a promising non-genotoxic alternative but pharmacokinetic limitations can lead to unintended on-target depletion of transplanted hematopoietic stem/progenitor cell (HSPCs), increasing the risk of graft failure and incomplete myeloablation. To overcome these limitations, we exploited precise epitope editing of the targeted c-KIT epitope in HSPCs to confer selective resistance to SR1, an anti-CD117 monoclonal antibody currently in clinical development, without impairing stem cell functionality. This approach enables the selective engraftment of gene-modified HSPCs while preserving normal hematopoiesis and reducing the risks associated with mAb-based conditioning. First, we discovered and characterized two specific mutations in the c-KIT receptor which can be induced to generate HSPCs resistant to SR1, without affecting surface expression, ligand affinity and proliferation capacity. We exploited adenine base-editing (ABE) and prime editing (PE) to efficiently introduce S123P and D121L mutations, respectively, in human HSPCs (~ 80% ABE efficiency and ~ 40% PE efficiency) and combined it with the disruption of the BCL11A erythroid enhancer motifs to promote fetal hemoglobin (HbF) expression. Importantly, we optimized HSPCs in vitro culture and stimulation to allow an efficient multiplex PE protocol that results in a preservation of the CD90+ compartment. This strategy can provide therapeutic benefits for sickle cell disease and beta thalassemia patients allowing for an in vivo co-selection of gene-engineered cells to reach their therapeutic threshold. Indeed, we observed a progressive enrichment of KIT/BCL11A enhancer multiplex-edited hematopoiesis under SR1 selective pressure, both in vitro by performing HSPCs liquid culture and colony forming assays in presence of various antibody doses and in vivo by: i) performing a competitive transplant with co-injection of multiplex KIT/BCL11A BE and AAVS1 BE CD34+ cells in NBSGW mice and ii) selectively enriching the HSPCs for KIT/BCL11A PE cells following SR1 intraperitoneal administrations. In both experiments we observed progressive enrichment of both KIT and BCL11A edits (up to 4x). Lastly, we demonstrated the efficacy of SR1-based conditioning in a transplant NBSGW model, where SR1 successfully eradicated pre-established human hematopoiesis, enabling efficient multiplex epitope edited HSPCs engraftment. Our results suggest that SR1-based conditioning, combined with epitope-edited HSCs, could allow safer gene-corrected cells transplantation, by reducing toxicity and improving donor cell selection. This approach may allow to expand the indication of genetic therapies and HSCT to benign/congenital hematologic diseases.