Functional restoration of gp91phox-oxidase activity by BAC transgenesis and gene targeting in X-linked chronic granulomatous disease iPSCs

Magdalena Laugsch; Maria Rostovskaya; Sergiy Velychko; Cornelia Richter; Ariane Zimmer; Barbara Klink; Evelin Schröck; Michael Haase; Katrin Neumann; Sebastian Thieme; Joachim Roesler; Sebastian Brenner; Konstantinos Anastassiadis

doi:10.1038/mt.2015.154

Functional restoration of gp91phox-oxidase activity by BAC transgenesis and gene targeting in X-linked chronic granulomatous disease iPSCs

Magdalena Laugsch, Maria Rostovskaya, Sergiy Velychko, Cornelia Richter, Ariane Zimmer, Barbara Klink, Evelin Schröck, Michael Haase, Katrin Neumann, Sebastian Thieme, Joachim Roesler, Sebastian Brenner^*, Konstantinos Anastassiadis

^*Corresponding author for this work

Research output: Contribution to journal › Article › Research › peer-review

18 Citations (Scopus)

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.

Original language	English
Pages (from-to)	812-822
Number of pages	11
Journal	Molecular Therapy
Volume	24
Issue number	4
DOIs	https://doi.org/10.1038/mt.2015.154
Publication status	Published - 1 Apr 2016
Externally published	Yes

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10.1038/mt.2015.154

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Laugsch, M., Rostovskaya, M., Velychko, S., Richter, C., Zimmer, A., Klink, B., Schröck, E., Haase, M., Neumann, K., Thieme, S., Roesler, J., Brenner, S., & Anastassiadis, K. (2016). Functional restoration of gp91phox-oxidase activity by BAC transgenesis and gene targeting in X-linked chronic granulomatous disease iPSCs. Molecular Therapy, 24(4), 812-822. https://doi.org/10.1038/mt.2015.154

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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",

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Laugsch, M, Rostovskaya, M, Velychko, S, Richter, C, Zimmer, A, Klink, B, Schröck, E, Haase, M, Neumann, K, Thieme, S, Roesler, J, Brenner, S & Anastassiadis, K 2016, 'Functional restoration of gp91phox-oxidase activity by BAC transgenesis and gene targeting in X-linked chronic granulomatous disease iPSCs', Molecular Therapy, vol. 24, no. 4, pp. 812-822. https://doi.org/10.1038/mt.2015.154

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T1 - Functional restoration of gp91phox-oxidase activity by BAC transgenesis and gene targeting in X-linked chronic granulomatous disease iPSCs

AU - Laugsch, Magdalena

AU - Rostovskaya, Maria

AU - Velychko, Sergiy

AU - Richter, Cornelia

AU - Zimmer, Ariane

AU - Klink, Barbara

AU - Schröck, Evelin

AU - Haase, Michael

AU - Neumann, Katrin

AU - Thieme, Sebastian

AU - Roesler, Joachim

AU - Brenner, Sebastian

AU - Anastassiadis, Konstantinos

PY - 2016/4/1

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N2 - 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.

AB - 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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Functional restoration of gp91phox-oxidase activity by BAC transgenesis and gene targeting in X-linked chronic granulomatous disease iPSCs

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