• Yuxuan Wu, Jing Zeng, Benjamin P Roscoe, Pengpeng Liu, Qiuming Yao, Cicera R Lazzarotto, Kendell Clement, Mitchel A Cole, Kevin Luk, Cristina Baricordi, Anne H Shen, Chunyan Ren, Erica B Esrick, John P Manis, David M Dorfman, David A Williams, Alessandra Biffi, Carlo Brugnara, Luca Biasco, Christian Brendel, Luca Pinello, Shengdar Q Tsai, Scot A Wolfe, and Daniel E Bauer.
• Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.
• Nat. Med. 2019 May 1; 25 (5): 776-783.

AbstractRe-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, α2γ2)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 are dispensable in non-erythroid cells2-6. 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 non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with β-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.

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