title: Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells.
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.
authors.affiliation: 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.
authors.forename: Mark A
authors.initials: MA
authors.lastname: DeWitt
authors.affiliation: Children's Hospital Oakland Research Institute, University of California San Francisco (UCSF) Benioff Children's Hospital, Oakland, CA 94609, USA.
authors.forename: Wendy
authors.initials: W
authors.lastname: Magis
authors.affiliation: 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.
authors.forename: Nicolas L
authors.initials: NL
authors.lastname: Bray
authors.affiliation: 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.
authors.forename: Tianjiao
authors.initials: T
authors.lastname: Wang
authors.affiliation: Digital Biology Center, Bio-Rad Laboratories, Pleasanton, CA 94588, USA.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Berman
authors.affiliation: Departments of Microbiology, Immunology, and Molecular Genetics; Pediatrics; and Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Fabrizia
authors.initials: F
authors.lastname: Urbinati
authors.affiliation: Children's Hospital Oakland Research Institute, University of California San Francisco (UCSF) Benioff Children's Hospital, Oakland, CA 94609, USA.
authors.forename: Seok-Jin
authors.initials: SJ
authors.lastname: Heo
authors.affiliation: Department of Molecular and Cellular Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Therese
authors.initials: T
authors.lastname: Mitros
authors.affiliation: Children's Hospital Oakland Research Institute, University of California San Francisco (UCSF) Benioff Children's Hospital, Oakland, CA 94609, USA.
authors.forename: Denise P
authors.initials: DP
authors.lastname: Muñoz
authors.affiliation: Children's Hospital Oakland Research Institute, University of California San Francisco (UCSF) Benioff Children's Hospital, Oakland, CA 94609, USA.
authors.forename: Dario
authors.initials: D
authors.lastname: Boffelli
authors.affiliation: Departments of Microbiology, Immunology, and Molecular Genetics; Pediatrics; and Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Donald B
authors.initials: DB
authors.lastname: Kohn
authors.affiliation: 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.
authors.forename: Mark C
authors.initials: MC
authors.lastname: Walters
authors.affiliation: 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.
authors.forename: Dana
authors.initials: D
authors.lastname: Carroll
authors.affiliation: 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.
authors.forename: David I K
authors.initials: DI
authors.lastname: Martin
authors.affiliation: 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.
authors.forename: Jacob E
authors.initials: JE
authors.lastname: Corn
mesh_terms: D000328:Adult; D053687:Adult Stem Cells / Q000378:metabolism*; D000755:Anemia, Sickle Cell / Q000235:genetics* / Q000628:therapy*; D000818:Animals; D064113:CRISPR-Cas Systems; D002460:Cell Line; D000072669:Gene Editing / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006451:Hemoglobin, Sickle / Q000235:genetics*; D064593:Heterografts; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D018345:Mice, Knockout; D016513:Mice, SCID; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D057170:Translational Research, Biomedical
chemical_list: D006451:Hemoglobin, Sickle