Functional Restoration of gp91phox-Oxidase Activity by BAC Transgenesis and Gene Targeting in X-linked Chronic Granulomatous Disease iPSCs

Molecular Therapy : the Journal of the American Society of Gene Therapy
Magdalena LaugschKonstantinos Anastassiadis

Abstract

Chronic granulomatous disease (CGD) is an inherited immunodeficiency, caused by the inability of neutrophils to produce functional NADPH oxidase required for fighting microbial infections. The X-linked form of CGD (X-CGD), which is due to mutations in the CYBB (gp91phox) gene, a component of NADPH oxidase, accounts for about two-thirds of CGD cases. We derived induced pluripotent stem cells (iPSCs) from X-CGD patient keratinocytes using a Flp recombinase excisable lentiviral reprogramming vector. For restoring gp91phox function, we applied two strategies: transposon-mediated bacterial artificial chromosome (BAC) transgenesis and gene targeting using vectors with a fixed 5' homology arm (HA) of 8 kb and 3'HA varying in size from 30 to 80 kb. High efficiency of homologous recombination (up to 22%) was observed with increased size of the 3'HA. Both, BAC transgenesis and gene targeting resulted in functional restoration of the gp91phox measured by an oxidase activity assay in X-CGD iPSCs differentiated into the myeloid lineage. In conclusion, we delivered an important milestone towards the use of genetically corrected autologous cells for the treatment of X-CGD and monogenic diseases in general.

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Citations

Feb 13, 2016·Indian Journal of Pediatrics·Amit RawatSurjit Singh
May 11, 2016·Chemical Reviews·Gretchen MeinkeFrank Buchholz
Jan 23, 2017·Molecular Therapy : the Journal of the American Society of Gene Therapy·Giorgia Santilli, Adrian J Thrasher
Feb 6, 2017·Molecular Therapy : the Journal of the American Society of Gene Therapy·Colin L SweeneyHarry L Malech
Jun 7, 2019·Disease Models & Mechanisms·Maria Georgomanoli, Eirini P Papapetrou

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Methods Mentioned

BETA
PCR
transgenic
flow cytometry
biopsies
transfection

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