Multiplex base editing of BCL11A regulatory elements to treat sickle cell disease

Sickle cell disease (SCD) is a genetic anemia caused by the production of an abnormal adult hemoglobin. The clinical severity is lessened by elevated fetal hemoglobin (HbF) production in adulthood. A promising therapy is the transplantation of autologous, hematopoietic stem/progenitor cells (HSPCs) treated with CRISPR/Cas9 to downregulate the HbF repressor BCL11A via generation of double strand breaks (DSBs). Here, to further enhance HbF production without increasing the mutagenic load, we targeted both BCL11A erythroid-specific enhancers using base editors. We systematically dissected DNA motifs recognized by the key transcriptional activator within these regions and identified the critical nucleotides required for activator binding. Multiplex base editing of these residues was efficient and safe, and generated no or little DSBs and genomic rearrangements. We observed substantial HbF reactivation, exceeding the levels achieved using the FDA-approved CRISPR/Cas9 nuclease-based strategy, thus efficiently rescuing the sickling phenotype. Multiplex base editing was efficient in long-term repopulating HSPCs and resulted in potent HbF reactivation in vivo. In summary, these results show that multiplex base editing of BCL11A erythroid-specific enhancers is a safe and potent strategy for treating sickle cell disease.