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.

Therapeutic gene editing in hematopoietic progenitor cells from a mouse model of Fanconi anemia

“…Once the expression of TALEN monomers in HEK293T cells was confirmed (EV1B), we established proof of principle for gene editing in FA-A MEFs. Using a stepwise experimental approach as followed in previous gene-targeting studies (Bednarski et al, 2016;Diez et al, 2017;Rahman et al, 2015), we observed relatively high transfection efficiencies (mean value of 30%) and viabilities (ranging from 47% to 78%), indicating that in this cell type cytotoxicity was not a limiting factor to perform gene editing. We also observed that when a donor template was used together with the TALENs, the percentage of repair by NHEJ was significantly reduced; suggesting that a proportion of NHEJ repair was replaced by HDR in the presence of a donor template.…”

TALEN mediated gene editing in a mouse model of Fanconi anemia

“…1b), we established proof of principle for gene editing in FA-A MEFs. Using a stepwise experimental approach as followed in previous gene-targeting studies 25,68,69 , we observed relatively high transfection efficiencies (mean value of 30%) and viabilities (ranging from 47% to 78%), indicating that in this cell type cytotoxicity was not a limiting factor to perform gene editing. We also observed that when a donor template was used together with the TALENs, the percentage of repair by NHEJ was significantly reduced; suggesting that a proportion of NHEJ repair was replaced by HDR in the presence of a donor template.…”

Genome-editing Technologies for Gene and Cell Therapy

“… 57 Gene-editing tools have also been developed to correct gene mutations associated with ADA-SCID 258 and radiosensitive SCID, caused by impaired DNA-dependent protein kinase (DNA-PK) activity. 259 Thus, the ability to efficiently alter gene sequences in T cells, CD34+ HSCs, and human pluripotent cells can provide therapeutic gene-editing strategies for a broad range of different human immunodeficiencies.…”