Journal
NATURE MEDICINE
Volume 25, Issue 5, Pages 776-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41591-019-0401-y
Keywords
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Funding
- Translational Research Program at BCH
- NHLBI [U01HL11772, R01HL137848, DP2OD022716, P01HL053749, P01HL032262]
- NHGRI [R00HG008399]
- NIAID [R01AI117839]
- NIGMS [R01GM115911]
- NIDDK [K08DK093705, R03DK109232]
- Harvard Stem Cell Institute Seed Grant
- St. Jude Children's Research Hospital Collaborative Research Consortium
- Burroughs Wellcome Fund
- American Society of Hematology
- Doris Duke Charitable Foundation
- Charles H. Hood Foundation
- Cooley's Anemia Foundation
- bluebird bio
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Re-expression of the paralogous gamma-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe beta-globin disorders sickle cell disease (SCD) and beta-thalassemia by induction of fetal hemoglobin (HbF, alpha(2)gamma(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 are 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 gamma-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 beta-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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