Sickle cell disease and β-thalassemia affect the production of the adult β-hemoglobin chain. The clinical severity is lessened by mutations that cause fetal γ-globin expression in adult life (i.e., the hereditary persistence of fetal hemoglobin). Mutations clustering ~200 nucleotides upstream of the HBG transcriptional start sites either reduce binding of the LRF repressor or recruit the KLF1 activator. Here, we use base editing to generate a variety of mutations in the −200 region of the HBG promoters, including potent combinations of four to eight γ-globin-inducing mutations. Editing of patient hematopoietic stem/progenitor cells is safe, leads to fetal hemoglobin reactivation and rescues the pathological phenotype. Creation of a KLF1 activator binding site is the most potent strategy – even in long-term repopulating hematopoietic stem/progenitor cells. Compared with a Cas9-nuclease approach, base editing avoids the generation of insertions, deletions and large genomic rearrangements and results in higher γ-globin levels. Our results demonstrate that base editing of HBG promoters is a safe, universal strategy for treating β-hemoglobinopathies.

β-thalassemia is one of the most common genetic diseases worldwide and is caused by mutations affecting β-globin production. The only curative treatment is allogenic hematopoietic stem/progenitor cells (HSPCs) transplantation, an approach limited by compatible donor availability and immunological complications. Therefore, transplantation of autologous, genetically modified HSPCs is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors are not equally effective in all the patients and CRISPR/Cas9 nuclease-based strategies raise safety concerns. Thus, base editing strategies aiming to correct the genetic defect in patients HSPCs could provide a safe and effective treatment. Here, we developed a strategy to correct one of the most prevalent β-thalassemic mutations [IVS1-110 (G>A)] using the SpRY-ABE8e base editor. RNA delivery of the base editing system was safe and led to ~80% of gene correction in β-thalassemic patients' HSPCs without causing dangerous double-strand DNA breaks. In HSPC-derived erythroid populations, this strategy was able to restore β-globin production and correct inefficient erythropoiesis typically observed in β-thalassemia both in vitro and in vivo. In conclusion, this proof-of-concept study paves the way for the development of a safe and effective autologous gene therapy approach for β-thalassemia.