Adenine Base Editing of the Sickle Allele in CD34⁺ Hematopoietic Stem and Progenitor Cells Eliminates Hemoglobin S

While there are several small molecule, gene therapy, and gene editing approaches for treating sickle cell disease (SCD), these strategies do not result in the direct elimination of the causative sickle β-globin (HbS) variant itself. The reduction or complete removal of this pathologic globin variant and expression of normal β-hemoglobin (HbB) or other non-polymerizing β-globin variant may increase the likelihood of beneficial outcomes for SCD patients. Adenine base editors (ABEs) can precisely convert the mutant A-T base pair responsible for SCD to a G-C base pair, thus generating the hemoglobin variant, Hb G-Makassar, a naturally occurring β-globin variant that is not associated with human disease.

Our studies have identified ABEs that can achieve highly efficient Makassar editing (>70%) of the sickle mutation in both sickle trait (HbAS) and homozygous sickle (HbSS) patient CD34⁺ cells with high cell viability and recovery and without perturbation of immunophenotypic hematopoietic stem and progenitor cell (HSPC) frequencies after ex vivo delivery of guide RNA and mRNA encoding the ABE. Furthermore, Makassar editing was retained throughout erythropoiesis in bulk in vitro erythroid differentiated cells (IVED) derived from edited CD34⁺ cells. To gain an understanding of allelic editing at a single clone resolution, we assessed editing frequencies of clones from both single cell expansion in erythroid differentiation media, as well as from single BFU-E colonies. We found that we could achieve >70% of colonies with bi-allelic Makassar editing and approximately 20% of colonies with mono-allelic Makassar editing, while <3% of colonies remained completely unedited.

Previously, conventional hemoglobin capillary electrophoresis and high-performance liquid chromatography (HPLC) were unable to distinguish between HbS and HbG-Makassar. Here, we developed an ultra-high-performance liquid chromatography (UPLC) method that resolves sickle globin (HbS) from Hb G-Makassar globin in IVED cells. The Makassar globin variant was further confirmed by liquid chromatography mass spectrometry (LC-MS). By developing this new method to resolve these two β-globin variants in edited HbSS cells, we were able to detect, in bulk IVED cultures, >80% abundance Hb G-Makassar of total β-globins, which corresponded to a concomitant reduction of HbS levels to <20%. Furthermore, we were also able to determine globin abundance as well as allelic editing at the level of single clones and found that HbS was completely eliminated in >70% of cells that had bi-allelic Makassar editing. Moreover, in the approximately 20% of colonies that were found to be mono-allelically edited for the Makassar variant, there was a 60:40 ratio of Hb G-Makassar:HbS globin abundance in individual clones, at levels remarkably similar to the HbA(wildtype HbB):HbS levels found in HbAS individuals, with minimal observable in vitro sickling when exposed to hypoxia. Thus, with our ABEs, we were able to reduce HbS to <40% on a per cell basis in >90% of IVED cells and found that in vitro sickling under hypoxia inversely correlated with the level of Hb G-Makassar globin variant installation and corresponding reduction in HbS levels.

By converting HbS to Hb G-Makassar, our direct and precise editing strategy replaces a pathogenic β-globin with one that has been shown to have normal hematologic parameters. Coupled with autologous stem cell transplant, this next generation gene editing strategy presents a promising new modality for treating patients with SCD.