title: An AAVS1-targeted minigene platform for correction of iPSCs from all five types of chronic granulomatous disease.
abstract: There are five genetic forms of chronic granulomatous disease (CGD), resulting from mutations in any of five subunits of phagocyte oxidase, an enzyme complex in neutrophils, monocytes, and macrophages that produces microbicidal reactive oxygen species. We generated induced pluripotent stem cells (iPSCs) from peripheral blood CD34(+) hematopoietic stem cells of patients with each of five CGD genotypes. We used zinc finger nuclease (ZFN) targeting the AAVS1 safe harbor site together with CGD genotype-specific minigene plasmids with flanking AAVS1 sequence to target correction of iPSC representing each form of CGD. We achieved targeted insertion with constitutive expression of desired oxidase subunit in 70-80% of selected iPSC clones. Neutrophils and macrophages differentiated from corrected CGD iPSCs demonstrated restored oxidase activity and antimicrobial function against CGD bacterial pathogens Staphylococcus aureus and Granulibacter bethesdensis. Using a standard platform that combines iPSC generation from peripheral blood CD34(+) cells and ZFN mediated AAVS1 safe harbor minigene targeting, we demonstrate efficient generation of genetically corrected iPSCs using an identical approach for all five genetic forms of CGD. This safe harbor minigene targeting platform is broadly applicable to a wide range of inherited single gene metabolic disorders.
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Randall K
authors.initials: RK
authors.lastname: Merling
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Colin L
authors.initials: CL
authors.lastname: Sweeney
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Chu
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Aaron
authors.initials: A
authors.lastname: Bodansky
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Uimook
authors.initials: U
authors.lastname: Choi
authors.affiliation: Neutrophil Monitoring Lab, Applied/Developmental Research Directorate, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.
authors.forename: Debra Long
authors.initials: DL
authors.lastname: Priel
authors.affiliation: Neutrophil Monitoring Lab, Applied/Developmental Research Directorate, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Kuhns
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Wang
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Sam
authors.initials: S
authors.lastname: Vasilevsky
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Suk See
authors.initials: SS
authors.lastname: De Ravin
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Winkler
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland, USA.
authors.forename: Cynthia E
authors.initials: CE
authors.lastname: Dunbar
authors.affiliation: Center for Regenerative Medicine, National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, Maryland, USA.
authors.forename: Jizhong
authors.initials: J
authors.lastname: Zou
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Kol A
authors.initials: KA
authors.lastname: Zarember
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: John I
authors.initials: JI
authors.lastname: Gallin
authors.affiliation: Laboratory of Clinical Infectious Diseases, NIAID, NIH, Bethesda, Maryland, USA.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Holland
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Harry L
authors.initials: HL
authors.lastname: Malech
mesh_terms: D016947:Acetobacteraceae / Q000254:growth & development / Q000276:immunology; D002454:Cell Differentiation; D000229:Dependovirus / Q000235:genetics*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D005838:Genotype; D006105:Granulomatous Disease, Chronic / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D006412:Hematopoietic Stem Cells / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism* / Q000473:pathology; D008264:Macrophages / Q000276:immunology / Q000382:microbiology / Q000473:pathology; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D009504:Neutrophils / Q000276:immunology / Q000382:microbiology / Q000473:pathology; D013211:Staphylococcus aureus / Q000254:growth & development / Q000276:immunology; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D019255:NADPH Oxidases