Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. In this study, we purified A3A (N57Q)-BE3 base editor for ribonucleoprotein (RNP) electroporation of human-peripheral-blood-mobilized CD34⁺ hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11A erythroid enhancer at +58 with few indels. Fetal hemoglobin (HbF) induction in erythroid progeny after base editing or nuclease editing was similar. A single therapeutic base edit of the BCL11A enhancer prevented sickling and ameliorated globin chain imbalance in erythroid progeny from sickle cell disease and β-thalassemia patient-derived HSPCs, respectively. Moreover, efficient multiplex editing could be achieved with combined disruption of the BCL11A erythroid enhancer and correction of the HBB −28A>G promoter mutation. Finally, base edits could be produced in multilineage-repopulating self-renewing human HSCs with high frequency as assayed in primary and secondary recipient animals resulting in potent HbF induction in vivo. Together, these results demonstrate the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC-targeted therapeutic genome modification.

通过与可编程 DNA 结合蛋白连接的核苷酸脱氨酶进行碱基编辑代表了一种有希望的方法来永久治疗血液疾病，尽管其在移植造血干细胞 (HSC) 中的应用仍未得到探索。在这项研究中，我们纯化了 A3A (N57Q)-BE3 碱基编辑器，用于人外周血动员 CD34 ⁺造血干细胞和祖细胞 (HSPC) 的核糖核蛋白 (RNP) 电穿孔。我们在 +58 处观察到BCL11A红系增强子频繁的靶向胞嘧啶碱基编辑，几乎没有插入缺失。碱基编辑或核酸酶编辑后红系后代的胎儿血红蛋白 (HbF) 诱导相似。BCL11A的单一治疗基础编辑增强剂分别防止镰状细胞病和β-地中海贫血患者衍生的HSPCs的红系后代中的镰状和改善珠蛋白链失衡。此外，可以通过组合破坏BCL11A红细胞增强子和纠正HBB -28A> G 启动子突变来实现有效的多重编辑。最后，如在初级和次级受体动物中测定的那样，可以在多谱系重新增殖的自我更新人类 HSC 中产生碱基编辑，从而在体内产生有效的 HbF 诱导。总之，这些结果证明了人类 HSPC 的 RNP 碱基编辑作为 HSC 靶向治疗性基因组修饰的核酸酶编辑的可行替代方案的潜力。