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Blood. 2019 May 23;133(21):2255-2262. doi: 10.1182/blood-2019-01-895094. Epub 2019 Jan 31.

Editing aberrant splice sites efficiently restores β-globin expression in β-thalassemia.

Xu S1,2,3,4,5,6, Luk K7, Yao Q1,2,3,4,5,8,9,10,11, Shen AH1,2,3,4,5, Zeng J1,2,3,4,5, Wu Y1,2,3,4,5,12,13,14, Luo HY15,16,17, Brendel C1,2,3,18, Pinello L4,8,9,10,11, Chui DHK15,16,17, Wolfe SA7,19, Bauer DE1,2,3,4,5.

Author information

1
Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA.
2
Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA.
3
Harvard Stem Cell Institute, Cambridge, MA.
4
Broad Institute, Cambridge, MA.
5
Department of Pediatrics, Harvard Medical School, Boston, MA.
6
Department of Haematology, Qilu Hospital, Shandong University, Jinan, Shandong, China.
7
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA.
8
Molecular Pathology Unit.
9
Center for Cancer Research, and.
10
Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA.
11
Department of Pathology, Harvard Medical School, Boston, MA.
12
Shanghai Key Laboratory of Regulatory Biology.
13
Institute of Biomedical Sciences, and.
14
School of Life Sciences, East China Normal University, Shanghai, China.
15
Department of Medicine and.
16
Department of Pathology & Laboratory Medicine, Boston University School of Medicine, Boston, MA.
17
Hemoglobin Diagnostic Reference Laboratory, Boston Medical Center, Boston, MA.
18
Gene Therapy Program, Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Boston, MA; and.
19
Li Weibo Institute for Rare Diseases Research, University of Massachusetts Medical School, Worcester, MA.

Abstract

The thalassemias are compelling targets for therapeutic genome editing in part because monoallelic correction of a subset of hematopoietic stem cells (HSCs) would be sufficient for enduring disease amelioration. A primary challenge is the development of efficient repair strategies that are effective in HSCs. Here, we demonstrate that allelic disruption of aberrant splice sites, one of the major classes of thalassemia mutations, is a robust approach to restore gene function. We target the IVS1-110G>A mutation using Cas9 ribonucleoprotein (RNP) and the IVS2-654C>T mutation by Cas12a/Cpf1 RNP in primary CD34+ hematopoietic stem and progenitor cells (HSPCs) from β-thalassemia patients. Each of these nuclease complexes achieves high efficiency and penetrance of therapeutic edits. Erythroid progeny of edited patient HSPCs show reversal of aberrant splicing and restoration of β-globin expression. This strategy could enable correction of a substantial fraction of transfusion-dependent β-thalassemia genotypes with currently available gene-editing technology.

PMID:
30704988
PMCID:
PMC6533605
[Available on 2020-05-23]
DOI:
10.1182/blood-2019-01-895094

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