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: Shamim H Rahman, Johannes Kuehle, Christian Reimann, Tafadzwa Mlambo, Jamal Alzubi, Morgan L Maeder, Heimo Riedel, Paul Fisch, Tobias Cantz, Cornelia Rudolph, Claudio Mussolino, J Keith Joung, Axel Schambach, Toni Cathomen
• Journal: PLoS genetics (PLoS Genet) Vol. 11 Issue 5 Pg. e1005239 (May 2015) ISSN: 1553-7404 [Electronic] United States
• PMID: 26000857 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• DNA-Binding Proteins
• Nuclear Proteins
• Protein Kinases
• DNA-Activated Protein Kinase
• Prkdc protein, mouse

Topics

• Animals
• Cell Differentiation
• DNA-Activated Protein Kinase (deficiency, genetics, metabolism)
• DNA-Binding Proteins (deficiency, genetics, metabolism)
• Disease Models, Animal
• Fibroblasts (cytology, metabolism)
• Genome
• Genotyping Techniques
• HEK293 Cells
• Humans
• Induced Pluripotent Stem Cells (cytology)
• Male
• Mice
• NIH 3T3 Cells
• Nuclear Proteins (deficiency, genetics, metabolism)
• Phenotype
• Protein Kinases (genetics, metabolism)
• Signal Transduction
• T-Lymphocytes (cytology, metabolism)
• Zinc Fingers