Therapeutic base editing of HBG promoter to reactivate high level of gamma-globin expression with no detectable off-target mutations

Reactivating the silenced gamma-globin expression is a therapeutic strategy for treating beta-thalassemia and sickle cell disease. Genetic studies have identified several transcription factor binding motifs and their disruption could reactivate gamma-globin expression, the level of which correlates positively with therapeutic outcome. Here, we used transformer base editor (tBE), a recently developed cytosine base editor with no detectable off-target mutations, to disrupt transcription factor binding motifs in hematopoietic stem cells. By performing functional screening of six motifs with tBE, we found that directly disrupting BCL11A binding motif in the HBG1/2 promoters triggered higher gamma-globin expression than disrupting other regulatory motifs. Via side-by-side comparison with other clinical and preclinical strategies using Cas9 nuclease or conventional base editors (ABE8e and hA3A-BE3), we found tBE-meditated disruption of BCL11A binding motif triggered the highest fetal hemoglobin in human hematopoietic stem/progenitor cells, meanwhile exhibiting no detectable DNA or RNA off-target mutations. Durable therapeutic editing by tBE persisted in long-term repopulating hematopoietic stem cells, demonstrating that tBE-mediated editing of BCL11A binding motif in HBG1/2 promoters is a safe and effective strategy for treating beta-hemoglobinopathies.