@article{c84a26b900e44ff0810788e66d641de2,
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.",
author = "Magdalena Laugsch and Maria Rostovskaya and Sergiy Velychko and Cornelia Richter and Ariane Zimmer and Barbara Klink and Evelin Schr{\"o}ck and Michael Haase and Katrin Neumann and Sebastian Thieme and Joachim Roesler and Sebastian Brenner and Konstantinos Anastassiadis",
note = "Funding Information: We would like to thank Mandy Obst, Isabell Kolbe, Katrin Navratiel, and Jenny Marzahn for excellent technical assistance. We would like to thank Martin Wermke and Xenia Lojewski for sharing methods and protocols. We are thankful to Dirk Hoffmann and Axel Schambach, Medizinische Hochschule Hannover, for advice and for kindly donating the LV-OKSM-Tomato reprogramming vector. Further, we would like to thank A. Francis Stewart for advice and support. This project was financially supported by the Bundesministerium f{\"u}r Bildung und Forschung (BMBF) grant number 01GN1007 to K.A. and S.B. The authors have no competing financial interests. M.L., M.R., K.N., A.Z., and C.R. derived and validated X-CGD iPSCs from patient keratinocytes; M.R., S.V., and K.A. designed and generated constructs and performed BAC transgenesis and targeting; M.H., M.L., C.R., A.Z., and S.T. performed teratoma assays; B.K. and E.S. performed karyotyping and FISH; J.R. provided patient samples and helped with functional analysis; M.L., M.R., and C.R. established the hematopoietic differentiation; M.L., M.R., S.B., and K.A. designed experiments and wrote the manuscript. Publisher Copyright: {\textcopyright} 2016 The American Society of Gene & Cell Therapy.",
year = "2016",
month = apr,
day = "1",
doi = "10.1038/mt.2015.154",
language = "English",
volume = "24",
pages = "812--822",
journal = "Molecular Therapy",
issn = "1525-0016",
publisher = "Cell Press",
number = "4",
}