title: TALEN-mediated functional correction of X-linked chronic granulomatous disease in patient-derived induced pluripotent stem cells.
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.
authors: Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
authors: Anne-Kathrin
authors: AK
authors: Dreyer
authors: Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany.
authors: Dirk
authors: D
authors: Hoffmann
authors: 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.
authors: Nico
authors: N
authors: Lachmann
authors: 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.
authors: Mania
authors: M
authors: Ackermann
authors: Institute of Cell and Molecular Pathology, Hannover Medical School, 30625 Hannover, Germany.
authors: Doris
authors: D
authors: Steinemann
authors: Institute for Cell and Gene Therapy, University Medical Center Freiburg, 79106 Freiburg, Germany; Center for Chronic Immunodeficiency, University Medical Center Freiburg, 79108 Freiburg, Germany.
authors: Barbara
authors: B
authors: Timm
authors: Division of Immunology, University Children's Hospital, 8032 Zurich, Switzerland.
authors: Ulrich
authors: U
authors: Siler
authors: 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.
authors: Janine
authors: J
authors: Reichenbach
authors: Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, 60596 Frankfurt, Germany.
authors: Manuel
authors: M
authors: Grez
authors: 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.
authors: Thomas
authors: T
authors: Moritz
authors: 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.
authors: Axel
authors: A
authors: Schambach
authors: 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.
authors: Toni
authors: T
authors: Cathomen
mesh_terms: D002454:Cell Differentiation; D002460:Cell Line; D003851:Deoxyribonucleases / Q000235:genetics; D005818:Genetic Engineering; D015316:Genetic Therapy; D006098:Granulocytes / Q000166:cytology / Q000378:metabolism*; D006105:Granulomatous Disease, Chronic / Q000235:genetics* / Q000628:therapy; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics*; D022423:Myeloid Cells / Q000166:cytology; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics*
chemical_list: D008562:Membrane Glycoproteins; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D003851:Deoxyribonucleases
keywords: AAVS1 site; Designer nuclease; Gene editing; Genome engineering; Neutral integration site; X-linked chronic granulomatous disease