CD117 Antibody Conditioning and Multiplex Base Editing Enable Rapid and Robust Fetal Hemoglobin Reactivation in a Rhesus Autologous Transplantation Model

Current genetic therapies for sickle cell disease (SCD) require busulfan or other myeloablative conditioning regimens prior to cell infusion. Busulfan leads to both acute and chronic toxicities including infertility, organ injury and the potential for increased rates of malignancy. Due to the significant adverse effects of myeloablative conditioning with busulfan or other conditioning agents, many patients will not be eligible or will not choose to receive a potentially transformative therapy.

To address this challenge, we developed a non-genotoxic conditioning strategy with our Engineered Stem Cell Antibody Paired Evasion (ESCAPE) approach where we epitope engineered the hematopoietic stem and progenitor cell (HSPC) surface protein CD117 (cKIT) with a base editor to retain normal CD117 receptor function, but escape recognition by a cognate monoclonal antibody (mAb) that recognizes the wildtype CD117 protein and can be used for conditioning and in vivo selection of edited cells. The ESCAPE CD117 edit is combined with a therapeutic target edit in the γ-globin gene (HBG1/2) promoters to induce high levels of fetal hemoglobin (HbF) to prevent polymerization of sickle hemoglobin and the associated pathophysiology of SCD. This ESCAPE approach enables elimination of the toxic effects of myeloablative conditioning and provides a selective advantage for edited cells as the mAb does not need to be cleared before or following infusion of edited cells.

We demonstrated the ESCAPE concept in humanized wild type (WT) NBSGW mice treated with CD117 mAb followed by transplantation with multiplex edited human HSPCs. This conditioning/transplant strategy resulted in >50% HbF in sorted bone marrow GlyA positive cells, indicating the potential for significant therapeutic benefit. Highlighting the exquisite dependency of erythropoiesis on CD117 signaling, we also observed that ex vivo CD117 mAb treatment of multiplex edited HSPCs led to rapid selection of multiplex edited erythroid cells in mixtures of unedited and edited cells.

To investigate whether ESCAPE, incorporating single agent CD117 mAb conditioning, could achieve rapid and robust induction of HbF in an immunocompetent host, we used a rhesus macaque (Macaca mulatta) autologous CD34+ HSCT model. First, we demonstrated that the CD117 mAb was cross-reactive and bound rhesus HSPCs to induce apoptosis ex vivo. Mobilized rhesus CD34+ HSPCs were then multiplex edited for CD117 and HBG1/2 and HbF levels of >50% were confirmed in the cell product. Successful engraftment was achieved in a busulfan-conditioned animal, resulting in peripheral blood F-cell levels up to 98% and γ-globin levels up to 88%. We next employed the ESCAPE multiplex-edited autologous CD34+ HSPC approach in two additional rhesus macaques conditioned with 10 mg/kg or 25 mg/kg of CD117 mAb prior to HSCT. Subsequently, these animals received additional mAb treatments post-transplantation to provide a competitive advantage for edited cells with WT CD117 receptor occupancy maintained at 80-90%. In contrast to the busulfan treated animal, the mAb was well tolerated at both doses and no supportive care was necessary for the mAb conditioned animals. We observed significant induction of HbF at early timepoints post-transplant. F-cell levels rose to 61% in the periphery as early as 8 weeks post-transplant in one of the non-human primates (NHPs), and both NHPs stabilized at ~85% F-cells at week 35. Concomitant early induction of γ-globin was observed in these NHPs with ~37% γ-globin levels achieved at 8 weeks post-transplant and reaching ~55% at 35 weeks. Two nontransplant control NHPs received 10 mg/kg mAb dosing and did not demonstrate HbF induction (< 1% total γ-globin).

Our findings suggest that the ESCAPE approach, enabled by multiplex base editing and using CD117 mAb conditioning and selection, effectively reactivates HbF production without the need for genotoxic conditioning or post-transplant supportive measures and hematological perturbations in NHPs. This work lays the groundwork for potential therapeutic advancements in SCD treatment that avoid busulfan or other myeloablative conditioning related morbidities, enable rapid induction of therapeutically relevant levels of HbF and brings us closer to the goal of advancing gene therapy to SCD patients across the full spectrum of disease severity, including in resource limited environments.