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Nat Med. 2019 Mar 25. doi: 10.1038/s41591-019-0401-y. [Epub ahead of print]

Highly efficient therapeutic gene editing of human hematopoietic stem cells.

Wu Y1,2,3,4, Zeng J1,2,3, Roscoe BP5, Liu P5, Yao Q1,2,3,6,7, Lazzarotto CR8, Clement K6,7, Cole MA1,2,3, Luk K5, Baricordi C1,2,3,9, Shen AH1,2,3, Ren C1,2,3, Esrick EB1,2,3, Manis JP1,2,3, Dorfman DM10,11, Williams DA1,2,3, Biffi A1,2,3,9, Brugnara C1,2,3, Biasco L1,2,3,9,12, Brendel C1,2,3,9, Pinello L6,7, Tsai SQ8, Wolfe SA5, Bauer DE13,14,15.

Author information

1
Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.
2
Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.
3
Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA.
4
Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
5
Department of Molecular, Cell and Cancer Biology, Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, USA.
6
Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA, USA.
7
Department of Pathology, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA.
8
Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA.
9
Gene Therapy Program, Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Boston, MA, USA.
10
Department of Pathology, Brigham and Women's Hospital , Boston, MA, USA.
11
Department of Pathology, Harvard Medical School, Boston, MA, USA.
12
University College of London, Great Ormond Street Institute of Child Health, Faculty of Population Health Sciences, London, UK.
13
Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA. daniel.bauer@childrens.harvard.edu.
14
Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. daniel.bauer@childrens.harvard.edu.
15
Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA. daniel.bauer@childrens.harvard.edu.

Abstract

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, 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.

PMID:
30911135
DOI:
10.1038/s41591-019-0401-y

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