title: CRISPR-mediated genotypic and phenotypic correction of a chronic granulomatous disease mutation in human iPS cells.
abstract: Chronic granulomatous disease (CGD) is a rare genetic disease characterized by severe and persistent childhood infections. It is caused by the lack of an antipathogen oxidative burst, normally performed by phagocytic cells to contain and clear bacterial and fungal growth. Restoration of immune function can be achieved with heterologous bone marrow transplantation; however, autologous bone marrow transplantation would be a preferable option. Thus, a method is required to recapitulate the function of the diseased gene within the patient's own cells. Gene therapy approaches for CGD have employed randomly integrating viruses with concomitant issues of insertional mutagenesis, inaccurate gene dosage, and gene silencing. Here, we explore the potential of the recently described clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 site-specific nuclease system to encourage repair of the endogenous gene by enhancing the levels of homologous recombination. Using induced pluripotent stem cells derived from a CGD patient containing a single intronic mutation in the CYBB gene, we show that footprintless gene editing is a viable option to correct disease mutations. Gene correction results in restoration of oxidative burst function in iPS-derived phagocytes by reintroduction of a previously skipped exon in the cytochrome b-245 heavy chain (CYBB) protein. This study provides proof-of-principle for a gene therapy approach to CGD treatment using CRISPR-Cas9.
authors.affiliation: James Martin Stem Cell Facility, Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom; Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors.forename: Rowan
authors.initials: R
authors.lastname: Flynn
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors.forename: Alexander
authors.initials: A
authors.lastname: Grundmann
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors.forename: Peter
authors.initials: P
authors.lastname: Renz
authors.affiliation: James Martin Stem Cell Facility, Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom; Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors.forename: Walther
authors.initials: W
authors.lastname: Hänseler
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors.forename: William S
authors.initials: WS
authors.lastname: James
authors.affiliation: James Martin Stem Cell Facility, Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom; Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors.forename: Sally A
authors.initials: SA
authors.lastname: Cowley
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom. Electronic address: kenny.moore@path.ox.ac.uk.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Moore
mesh_terms: D064113:CRISPR-Cas Systems; D015316:Genetic Therapy; D006105:Granulomatous Disease, Chronic / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000628:therapy; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000201:enzymology* / Q000473:pathology; D007438:Introns; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D009154:Mutation; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000096:biosynthesis* / Q000235:genetics; D016897:Respiratory Burst / Q000235:genetics
chemical_list: D008562:Membrane Glycoproteins; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases