Innovative Transformer Base Editor (tBE)-Mediated Editing at BCL11A Binding Motif Triggers High Level of γ-Globin Expression with No Observable Off-Target Mutations

Activating the silent γ-globin expression is a universal therapeutic strategy for treating β-hemoglobinopathies including β-thalassemia and sickle cell disease. Several therapeutic target sites have been identified and the editing at these sites can reactivate γ-globin expression. In this study, we used a recently developed cytosine base editor, transformer base editor (tBE), which triggers no observable off-target mutations, to edit the therapeutic target sites for triggering γ-globin expression. Through editing six therapeutic sites, we found that the editing at BCL11A binding motif in the HBG1/2 promoters induced the highest γ-globin expression level. By comparing to other approaches such as Cas9 nuclease or other base editors, tBE-meditated editing at BCL11A binding motif induced higher fetal hemoglobin (HbF) expression and meanwhile did not trigger any detected DNA or RNA off-target mutations. Of note, tBE-mediated editing at the HBG1/2 promoters induced more robust HbF expression in the erythroid progeny from β ⁰/β ⁰ thalassemia patient-derived hematopoietic stem and progenitor cells (HSPCs) than CRISPR/Cas9 approach. Moreover, persistent base editing by tBE was found in long-term repopulating hematopoietic stem cells. Taken together, this study demonstrates that tBE-mediated therapeutic base editing at BCL11A binding motif in HBG1/2 promoters is a safe and highly effective strategy for treating β-hemoglobinopathies.