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Sci Transl Med. 2016 Oct 12;8(360):360ra134.

Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells.

Author information

1
Innovative Genomics Initiative, University of California, Berkeley, Berkeley, CA 94720, USA. Department of Molecular and Cellular Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
2
Children's Hospital Oakland Research Institute, University of California San Francisco (UCSF) Benioff Children's Hospital, Oakland, CA 94609, USA.
3
Digital Biology Center, Bio-Rad Laboratories, Pleasanton, CA 94588, USA.
4
Departments of Microbiology, Immunology, and Molecular Genetics; Pediatrics; and Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
5
Department of Molecular and Cellular Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
6
Children's Hospital Oakland Research Institute, University of California San Francisco (UCSF) Benioff Children's Hospital, Oakland, CA 94609, USA. Blood and Marrow Transplant Program, Division of Hematology, UCSF Benioff Children's Hospital, Oakland, CA 94609, USA.
7
Innovative Genomics Initiative, University of California, Berkeley, Berkeley, CA 94720, USA. Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112, USA. jcorn@berkeley.edu dimartin@chori.org dana@biochem.utah.edu.
8
Children's Hospital Oakland Research Institute, University of California San Francisco (UCSF) Benioff Children's Hospital, Oakland, CA 94609, USA. jcorn@berkeley.edu dimartin@chori.org dana@biochem.utah.edu.
9
Innovative Genomics Initiative, University of California, Berkeley, Berkeley, CA 94720, USA. Department of Molecular and Cellular Biology, University of California, Berkeley, Berkeley, CA 94720, USA. jcorn@berkeley.edu dimartin@chori.org dana@biochem.utah.edu.

Abstract

Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs), and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the β-globin gene (HBB). Sickle hemoglobin damages erythrocytes, causing vasoocclusion, severe pain, progressive organ damage, and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA, together with a single-stranded DNA oligonucleotide donor (ssODN), to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice, ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing, enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells, and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases.

PMID:
27733558
PMCID:
PMC5500303
DOI:
10.1126/scitranslmed.aaf9336
[Indexed for MEDLINE]
Free PMC Article

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