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Biomaterials. 2015 Nov;69:191-200. doi: 10.1016/j.biomaterials.2015.07.057. Epub 2015 Aug 3.

TALEN-mediated functional correction of X-linked chronic granulomatous disease in patient-derived induced pluripotent stem cells.

Author information

1
Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
2
Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany; RG Reprogramming and Gene Therapy, REBIRTH Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; JRG Translational Hematology of Congenital Diseases, REBIRTH Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
3
Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany; RG Reprogramming and Gene Therapy, REBIRTH Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany.
4
Institute of Cell and Molecular Pathology, Hannover Medical School, 30625 Hannover, Germany.
5
Institute for Cell and Gene Therapy, University Medical Center Freiburg, 79106 Freiburg, Germany; Center for Chronic Immunodeficiency, University Medical Center Freiburg, 79108 Freiburg, Germany.
6
Division of Immunology, University Children's Hospital, 8032 Zurich, Switzerland.
7
Division of Immunology, University Children's Hospital, 8032 Zurich, Switzerland; Center for Applied Biotechnology and Molecular Medicine, Swiss Center for Regenerative Medicine, Zurich Centre for Integrative Human Physiology, University of Zurich, 8091 Zurich, Switzerland.
8
Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, 60596 Frankfurt, Germany.
9
Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany; Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA. Electronic address: schambach.axel@mh-hannover.de.
10
Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany; Institute for Cell and Gene Therapy, University Medical Center Freiburg, 79106 Freiburg, Germany; Center for Chronic Immunodeficiency, University Medical Center Freiburg, 79108 Freiburg, Germany. Electronic address: toni.cathomen@uniklinik-freiburg.de.

Abstract

X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of the immune system. It is characterized by a defect in the production of reactive oxygen species (ROS) in phagocytic cells due to mutations in the NOX2 locus, which encodes gp91phox. Because the success of retroviral gene therapy for X-CGD has been hampered by insertional activation of proto-oncogenes, targeting the insertion of a gp91phox transgene into potential safe harbor sites, such as AAVS1, may represent a valid alternative. To conceptually evaluate this strategy, we generated X-CGD patient-derived induced pluripotent stem cells (iPSCs), which recapitulate the cellular disease phenotype upon granulocytic differentiation. We examined AAVS1-specific zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for their efficacy to target the insertion of a myelo-specific gp91phox cassette to AAVS1. Probably due to their lower cytotoxicity, TALENs were more efficient than ZFNs in generating correctly targeted iPSC colonies, but all corrected iPSC clones showed no signs of mutations at the top-ten predicted off-target sites of both nucleases. Upon differentiation of the corrected X-CGD iPSCs, gp91phox mRNA levels were highly up-regulated and the derived granulocytes exhibited restored ROS production that induced neutrophil extracellular trap (NET) formation. In conclusion, we demonstrate that TALEN-mediated integration of a myelo-specific gp91phox transgene into AAVS1 of patient-derived iPSCs represents a safe and efficient way to generate autologous, functionally corrected granulocytes.

KEYWORDS:

AAVS1 site; Designer nuclease; Gene editing; Genome engineering; Neutral integration site; X-linked chronic granulomatous disease

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