Base-editing-mediated dissection of a γ-globin cis-regulatory element for the therapeutic reactivation of fetal hemoglobin expression

Sickle cell disease and beta-thalassemia affect the production of the adult beta-hemoglobin chain. The clinical severity is lessened by mutations that cause fetal gamma-globin expression in adult life (i.e., the hereditary persistence of fetal hemoglobin). Mutations clustering similar to 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 gamma-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 gamma-globin levels. Our results demonstrate that base editing of HBG promoters is a safe, universal strategy for treating beta-hemoglobinopathies.