Base Editing-Mediated Dissection of the -200 Region of the γ-Globin Promoters to Induce Fetal Hemoglobin and Rescue Sickle Cell Disease and β-Thalassemia

β-hemoglobinopathies are caused by mutations affecting the adult hemoglobin production. In sickle cell disease (SCD), the β6 Glu→Val substitution leads to sickle hemoglobin (HbS) polymerization and red blood cell (RBC) sickling. In β-thalassemia, reduced β-globin production leads to precipitation of uncoupled α-chains causing ineffective erythropoiesis and the production of poorly hemoglobinized RBCs. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option. The clinical severity of β-hemoglobinopathies is alleviated by the co-inheritance of mutations causing hereditary persistence of fetal Hb (HPFH). HPFH mutations clustering 200 nucleotides upstream of the TSS of the fetal γ-globin (HBG) genes either disrupt the binding site (BS) of the fetal Hb (HbF) repressor LRF or generate a de novo BS for the KLF1 activator. To reactivate γ-globin expression, nuclease-based approaches have been explored. However, nucleases generate double-strand breaks (DSBs), raising safety concerns for clinical applications. Base editing (BE) allows the introduction of point mutations without generating DSBs. In this study, we designed BE systems to introduce a variety of HPFH or HPFH-like mutations in the -200 region of the HBG promoters. First, we screened in erythroid cell lines known and novel BEs, and we selected combinations of BEs and guide RNAs that edit alternative bases of the -200 region. We then developed a clinically-relevant protocol based on RNA-transfection to deliver the BE system to HSPCs. The expression profile of genes activated by RNA stimuli revealed no immune response in HSPCs. A progenitor assay indicated no alteration in the growth and multilineage differentiation of edited HSPCs. We applied this protocol to SCD and β-thalassemia HSPCs, achieving editing efficiencies up to ~70% of the HBG promoters. In RBCs differentiated from edited SCD HSPCs, RT-qPCR, HPLC and flow cytometry showed a potent γ-globin reactivation with a high frequency of HbF + cells and a concomitant decrease in the HbS content/cell. Importantly, the pathological RBC sickling phenotype was corrected in the samples derived from edited HSPCs. Similarly, in β-thalassemia samples, RT-qPCR and HPLC analyses showed strong γ-globin induction and decrease of the α-globin precipitates. HbF expression rescued the delay in erythroid differentiation and ineffective erythropoiesis characterizing β-thalassemia, as demonstrated b