An AAVS1-targeted minigene platform for correction of iPSCs from all five types of chronic granulomatous disease.
Mol Ther
; 23(1): 147-57, 2015 Jan.
Article
en En
| MEDLINE
| ID: mdl-25288370
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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Células Madre Hematopoyéticas
/
Dependovirus
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NADPH Oxidasas
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Células Madre Pluripotentes Inducidas
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Enfermedad Granulomatosa Crónica
Límite:
Humans
Idioma:
En
Revista:
Mol Ther
Asunto de la revista:
BIOLOGIA MOLECULAR
/
TERAPEUTICA
Año:
2015
Tipo del documento:
Article
País de afiliación:
Estados Unidos