title: Rescue of DNA-PK Signaling and T-Cell Differentiation by Targeted Genome Editing in a prkdc Deficient iPSC Disease Model.
abstract: In vitro disease modeling based on induced pluripotent stem cells (iPSCs) provides a powerful system to study cellular pathophysiology, especially in combination with targeted genome editing and protocols to differentiate iPSCs into affected cell types. In this study, we established zinc-finger nuclease-mediated genome editing in primary fibroblasts and iPSCs generated from a mouse model for radiosensitive severe combined immunodeficiency (RS-SCID), a rare disorder characterized by cellular sensitivity to radiation and the absence of lymphocytes due to impaired DNA-dependent protein kinase (DNA-PK) activity. Our results demonstrate that gene editing in RS-SCID fibroblasts rescued DNA-PK dependent signaling to overcome radiosensitivity. Furthermore, in vitro T-cell differentiation from iPSCs was employed to model the stage-specific T-cell maturation block induced by the disease causing mutation. Genetic correction of the RS-SCID iPSCs restored T-lymphocyte maturation, polyclonal V(D)J recombination of the T-cell receptor followed by successful beta-selection. In conclusion, we provide proof that iPSC-based in vitro T-cell differentiation is a valuable paradigm for SCID disease modeling, which can be utilized to investigate disorders of T-cell development and to validate gene therapy strategies for T-cell deficiencies. Moreover, this study emphasizes the significance of designer nucleases as a tool for generating isogenic disease models and their future role in producing autologous, genetically corrected transplants for various clinical applications.
authors.affiliation: Institute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg, Germany; Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany; Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Shamim H
authors.initials: SH
authors.lastname: Rahman
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Kuehle
authors.affiliation: Institute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg, Germany; Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Reimann
authors.affiliation: Institute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg, Germany; Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany; Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany.
authors.forename: Tafadzwa
authors.initials: T
authors.lastname: Mlambo
authors.affiliation: Institute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg, Germany; Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany; Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Jamal
authors.initials: J
authors.lastname: Alzubi
authors.affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America; Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Morgan L
authors.initials: ML
authors.lastname: Maeder
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; Department of Biochemistry and Mary Babb Randolph Cancer Center, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia, United States of America.
authors.forename: Heimo
authors.initials: H
authors.lastname: Riedel
authors.affiliation: Institute of Pathology, University Medical Center Freiburg, Freiburg, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Fisch
authors.affiliation: Translational Hepatology and Stem Cell Biology, REBIRTH cluster of excellence, Hannover Medical School, Hannover, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Cantz
authors.affiliation: Institute for Cellular and Molecular Pathology, Hannover Medical School, Hannover, Germany.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Rudolph
authors.affiliation: Institute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg, Germany; Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany.
authors.forename: Claudio
authors.initials: C
authors.lastname: Mussolino
authors.affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America; Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
authors.affiliation: Institute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg, Germany; Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany; Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D051747:DNA-Activated Protein Kinase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004195:Disease Models, Animal; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D016678:Genome; D060005:Genotyping Techniques; D057809:HEK293 Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology; D008297:Male; D051379:Mice; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology* / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011494:Protein Kinases; D051747:DNA-Activated Protein Kinase; C497264:Prkdc protein, mouse