Highly efficient therapeutic gene editing of human hematopoietic stem cells.

Autor: Wu Y; Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.; Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA.; Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China., Zeng J; Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.; Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA., Roscoe BP; Department of Molecular, Cell and Cancer Biology, Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, USA., Liu P; Department of Molecular, Cell and Cancer Biology, Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, USA., Yao Q; Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.; Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA.; Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA, USA.; Department of Pathology, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA., Lazzarotto CR; Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA., Clement K; Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA, USA.; Department of Pathology, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA., Cole MA; Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.; Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA., Luk K; Department of Molecular, Cell and Cancer Biology, Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, USA., Baricordi C; Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.; Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA.; Gene Therapy Program, Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Boston, MA, USA., Shen AH; Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.; Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA., Ren C; Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.; Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA., Esrick EB; Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.; Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA., Manis JP; Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.; Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA., Dorfman DM; Department of Pathology, Brigham and Women's Hospital , Boston, MA, USA.; Department of Pathology, Harvard Medical School, Boston, MA, USA., Williams DA; Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.; Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA., Biffi A; Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.; Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA.; Gene Therapy Program, Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Boston, MA, USA., Brugnara C; Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.; Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA., Biasco L; Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.; Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA.; Gene Therapy Program, Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Boston, MA, USA.; University College of London, Great Ormond Street Institute of Child Health, Faculty of Population Health Sciences, London, UK., Brendel C; Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA.; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.; Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA.; Gene Therapy Program, Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Boston, MA, USA., Pinello L; Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA, USA.; Department of Pathology, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA., Tsai SQ; Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA., Wolfe SA; Department of Molecular, Cell and Cancer Biology, Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA, USA., Bauer DE; Division of Hematology/Oncology, Boston Children's Hospital , Boston, MA, USA. daniel.bauer@childrens.harvard.edu.; Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. daniel.bauer@childrens.harvard.edu.; Department of Pediatrics, Harvard Stem Cell Institute, Broad Institute of Harvard and MIT, Harvard Medical School, Boston, MA, USA. daniel.bauer@childrens.harvard.edu.
Jazyk: angličtina
Zdroj: Nature medicine [Nat Med] 2019 May; Vol. 25 (5), pp. 776-783. Date of Electronic Publication: 2019 Mar 25.
DOI: 10.1038/s41591-019-0401-y
Abstrakt: 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 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 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.
Databáze: MEDLINE
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