An AAVS1-Targeted Minigene Platform for Correction of iPSCs From All Five Types of Chronic Granulomatous Disease
| Autor: | John I. Gallin, Hongmei Wang, Jizhong Zou, Aaron Bodansky, Harry L. Malech, Jessica Chu, Colin L. Sweeney, Thomas Winkler, Uimook Choi, Debra A. Long Priel, Kol A. Zarember, Sam Vasilevsky, Douglas B. Kuhns, Randall K. Merling, Steven M. Holland, Cynthia E. Dunbar, Suk See De Ravin |
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| Rok vydání: | 2015 |
| Předmět: |
Staphylococcus aureus
Enzyme complex Genotype Neutrophils Cellular differentiation Genetic Vectors Induced Pluripotent Stem Cells Gene Expression Biology Granulomatous Disease Chronic 03 medical and health sciences 0302 clinical medicine Chronic granulomatous disease Drug Discovery medicine Genetics Humans Induced pluripotent stem cell Molecular Biology 030304 developmental biology Pharmacology 0303 health sciences Macrophages NADPH Oxidases Cell Differentiation Zinc Fingers Genetic Therapy Dependovirus Hematopoietic Stem Cells medicine.disease Molecular biology Zinc finger nuclease 3. Good health Haematopoiesis 030220 oncology & carcinogenesis Acetobacteraceae Molecular Medicine Original Article Stem cell Minigene |
| Zdroj: | Molecular Therapy. 23(1):147-157 |
| ISSN: | 1525-0016 |
| DOI: | 10.1038/mt.2014.195 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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