Therapeutic base editing of human hematopoietic stem cells

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. Here we purified A3A (N57Q)-BE3 protein for ribonucleoprotein (RNP) electroporation of human peripheral blood (PB) mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11A +58 erythroid enhancer 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 (SCD) and B-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, to our knowledge for the first time, the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC-targeted therapeutic genome modification.