title: Oxidase-deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease-mediated safe harbor targeting.
abstract: We have developed induced pluripotent stem cells (iPSCs) from a patient with X-linked chronic granulomatous disease (X-CGD), a defect of neutrophil microbicidal reactive oxygen species (ROS) generation resulting from gp91(phox) deficiency. We demonstrated that mature neutrophils differentiated from X-CGD iPSCs lack ROS production, reproducing the pathognomonic CGD cellular phenotype. Targeted gene transfer into iPSCs, with subsequent selection and full characterization to ensure no off-target changes, holds promise for correction of monogenic diseases without the insertional mutagenesis caused by multisite integration of viral or plasmid vectors. Zinc finger nuclease-mediated gene targeting of a single-copy gp91(phox) therapeutic minigene into one allele of the "safe harbor" AAVS1 locus in X-CGD iPSCs without off-target inserts resulted in sustained expression of gp91(phox) and substantially restored neutrophil ROS production. Our findings demonstrate how precise gene targeting may be applied to correction of X-CGD using zinc finger nuclease and patient iPSCs.
authors.affiliation: Division of Hematology, Department of Medicine, and Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Jizhong
authors.initials: J
authors.lastname: Zou
authors.forename: Colin L
authors.initials: CL
authors.lastname: Sweeney
authors.forename: Bin-Kuan
authors.initials: BK
authors.lastname: Chou
authors.forename: Uimook
authors.initials: U
authors.lastname: Choi
authors.forename: Jason
authors.initials: J
authors.lastname: Pan
authors.forename: Hongmei
authors.initials: H
authors.lastname: Wang
authors.forename: Sarah N
authors.initials: SN
authors.lastname: Dowey
authors.forename: Linzhao
authors.initials: L
authors.lastname: Cheng
authors.forename: Harry L
authors.initials: HL
authors.lastname: Malech
mesh_terms: D000328:Adult; D000818:Animals; D015139:Blotting, Southern; D015153:Blotting, Western; D001853:Bone Marrow / Q000378:metabolism; D002454:Cell Differentiation; D016172:DNA Fingerprinting; D000229:Dependovirus / Q000235:genetics; D005434:Flow Cytometry; D006105:Granulomatous Disease, Chronic / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D007621:Karyotyping; D008297:Male; D008562:Membrane Glycoproteins / Q000502:physiology*; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D051379:Mice; D016688:Mice, Inbred NOD; D008819:Mice, Nude; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000172:deficiency* / Q000235:genetics / Q000502:physiology; D009504:Neutrophils / Q000201:enzymology*; D010587:Phagocytosis; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016335:Zinc Fingers
chemical_list: D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases