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Mol Ther. 2015 Jan;23(1):147-57. doi: 10.1038/mt.2014.195. Epub 2014 Oct 7.

An AAVS1-targeted minigene platform for correction of iPSCs from all five types of chronic granulomatous disease.

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Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA.
Neutrophil Monitoring Lab, Applied/Developmental Research Directorate, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.
Hematology Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland, USA.
Center for Regenerative Medicine, National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, Maryland, USA.
Laboratory of Clinical Infectious Diseases, NIAID, NIH, Bethesda, Maryland, USA.


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.

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