title: Functional Restoration of gp91phox-Oxidase Activity by BAC Transgenesis and Gene Targeting in X-linked Chronic Granulomatous Disease iPSCs.
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.
authors: Department of Pediatrics, University Clinic Carl Gustav Carus, TU Dresden, Dresden, Germany.
authors: Magdalena
authors: M
authors: Laugsch
authors: Stem Cell Engineering, BIOTEC, TU Dresden, Dresden, Germany.
authors: Maria
authors: M
authors: Rostovskaya
authors: Stem Cell Engineering, BIOTEC, TU Dresden, Dresden, Germany.
authors: Sergiy
authors: S
authors: Velychko
authors: Department of Pediatrics, University Clinic Carl Gustav Carus, TU Dresden, Dresden, Germany.
authors: Cornelia
authors: C
authors: Richter
authors: Department of Pediatrics, University Clinic Carl Gustav Carus, TU Dresden, Dresden, Germany.
authors: Ariane
authors: A
authors: Zimmer
authors: Institute for Clinical Genetics, Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany.
authors: Barbara
authors: B
authors: Klink
authors: Institute for Clinical Genetics, Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany.
authors: Evelin
authors: E
authors: Schröck
authors: Department of Pediatric Surgery, University Clinic Carl Gustav Carus, TU Dresden, Dresden, Germany.
authors: Michael
authors: M
authors: Haase
authors: Stem Cell Engineering, BIOTEC, TU Dresden, Dresden, Germany.
authors: Katrin
authors: K
authors: Neumann
authors: Department of Pediatrics, University Clinic Carl Gustav Carus, TU Dresden, Dresden, Germany.
authors: Sebastian
authors: S
authors: Thieme
authors: Department of Pediatrics, University Clinic Carl Gustav Carus, TU Dresden, Dresden, Germany.
authors: Joachim
authors: J
authors: Roesler
authors: Department of Pediatrics, University Clinic Carl Gustav Carus, TU Dresden, Dresden, Germany.
authors: Sebastian
authors: S
authors: Brenner
authors: Stem Cell Engineering, BIOTEC, TU Dresden, Dresden, Germany.
authors: Konstantinos
authors: K
authors: Anastassiadis
mesh_terms: D002454:Cell Differentiation; D002478:Cells, Cultured; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006105:Granulomatous Disease, Chronic / Q000235:genetics / Q000473:pathology* / Q000628:therapy; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000201:enzymology*; D015603:Keratinocytes / Q000166:cytology; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism
chemical_list: D008562:Membrane Glycoproteins; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases