Astatine 211-based conditioning with a humanized CD45 antibody for autologous hematopoietic stem cell gene therapy

Successful transplantation of autologous gene-modified hematopoietic stem and progenitor cells (HSPCs) requires efficient ablation of resident hematopoietic stem cells. Considering that conventional myeloablative conditioning regimens are associated with nonhematologic toxicities, we evaluated CD45-directed radioimmunotherapy (RIT) using the α-emitter astatine 211 (211At) before the transplantation of ex vivo gene-edited autologous HSPCs in nonhuman primates as an alternative. We humanized the CD45 antibody, BC8, and labeled it with 211At. As a model, mobilized CD34+ HSPCs were multiplex gene-edited using an adenine base editor, modifying the HBG promoter to reactivate fetal hemoglobin and deleting CD33. Two animals each received 300 or 400 μCi/kg of 211At. In contrast to historic controls conditioned with total body irradiation, CD45-RIT animals did not show any noticeable nonhematopoietic toxicities and were almost entirely transfusion independent with rapid recovery of neutrophils and platelets. Dose-dependent engraftment of gene-edited cells was enabled by 211At. A new single-cell sequencing assay revealed up to 70% combined monoallelic and biallelic gene-editing efficiency in the blood, consistent with complete replacement of the bone marrow stem cell compartment. Assessment by bulk analysis underestimated the frequency of gene-edited cells, highlighting the importance of a single-cell readout. Single-cell sequencing further confirmed stable and unbiased contribution of multiplex-edited HSPCs to all mature lineages in the blood, providing high-resolution data assuring successful replacement upon autologous HSPC gene therapy. The levels of edited cells remained stable for the entire follow-up of >18 months. Collectively, these studies identify 211At-CD45 RIT as a targeted alternative for myeloablative conditioning for autologous gene therapy.