Highly efficient therapeutic gene editing of human hematopoietic stem cells

Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal hemoglobin (HbF, α(2)γ(2))(1). Previously we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but dispensable in non-erythroid cells(2–6). CRISPR-Cas9 mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP) mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo nonhomologous as compared to microhomology mediated end-joining repair. Erythroid progeny of edited engrafting sickle cell disease (SCD) HSCs express therapeutic levels of fetal hemoglobin (HbF) and resist sickling, while those from β-thalassemia patients show restored globin chain balance. NHEJ-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.