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	Jizhong Zou, Colin L Sweeney, Bin-Kuan Chou, Uimook Choi, Jason Pan, Hongmei Wang, Sarah N Dowey, Linzhao Cheng, Harry L Malech
Journal	Blood (Blood) Vol. 117 Issue 21 Pg. 5561-72 (May 26 2011) ISSN: 1528-0020 [Electronic] United States
PMID	21411759 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
Chemical References	Membrane Glycoproteins RNA, Messenger Reactive Oxygen Species CYBB protein, human NADPH Oxidase 2 NADPH Oxidases
Topics	Adult Animals Blotting, Southern Blotting, Western Bone Marrow (metabolism) Cell Differentiation DNA Fingerprinting Dependovirus (genetics) Flow Cytometry Granulomatous Disease, Chronic (genetics, pathology, therapy) Humans Induced Pluripotent Stem Cells (metabolism) Karyotyping Male Membrane Glycoproteins (physiology) Mesenchymal Stem Cells (metabolism) Mice Mice, Inbred NOD Mice, Nude NADPH Oxidase 2 NADPH Oxidases (deficiency, genetics, physiology) Neutrophils (enzymology) Phagocytosis RNA, Messenger (genetics) Reactive Oxygen Species (metabolism) Recombination, Genetic Reverse Transcriptase Polymerase Chain Reaction Zinc Fingers

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