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.
Highlights
Studying the molecular pathology of human disease is often difficult due to the limited availability of particular primary cells, their limited lifespan, or because complex developmental differentiation procedures cannot be followed in vivo
Due to the limited availability and lifespan of some primary cells, in vitro disease modeling with induced pluripotent stem cells offers a valuable complementation to in vivo studies
The goal of our study was to establish an in vitro disease model for severe combined immunodeficiency (SCID), a group of inherited disorders of the immune system characterized by the lack of T-lymphocytes
Summary
Studying the molecular pathology of human disease is often difficult due to the limited availability of particular primary cells, their limited lifespan, or because complex developmental differentiation procedures cannot be followed in vivo. NHEJ is essential for physiological B- and T-lymphocyte development as it plays an important role in the B-cell receptor (BCR) and T-cell receptor (TCR) recombination process [4]. DNA-PK dependent signaling is a paradigmatic example of how a single molecule can be simultaneously involved in both, DNA repair and T- and B-cell development, and of how such a process can be disturbed by a single point mutation. These particularities make PRKDC an optimal target for novel site-specific gene therapy approaches, such as designer nuclease mediated genome editing
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
