Rescue of DNA-PK Signaling and T-Cell Differentiation by Targeted Genome Editing in a prkdc Deficient iPSC Disease Model.

Rahman SH, Kuehle J, Reimann C, Mlambo T, Alzubi J, Maeder ML, Riedel H, Fisch P, Cantz T, Rudolph C, Mussolino C, Joung JK, Schambach A, Cathomen T
Source: PLoS Genet
Publication Date: (2015)
Issue: 11(5): e1005239
Research Area:
Stem Cells
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Nucleofector® I/II/2b
For targeted genome editing, iPSC clone iPS.6 cells were nucleofected with donor and ZFN expression plasmids. After selection and expansion, inside-out PCR was used to detect correct genomic targeting. Gene targeting in RS-SCID cells iPSCs were grown feeder-free before and after transfection. 3x106 cells were nucleofected with 10 µg of pJET.SAE8586Neo and 5 µg of each ZFN expression plasmid using the Mouse ES Cell Nucleofector Kit (LONZA) and Nucleofector II with program A-030. After 5 days of recovery, G418 selection was applied for 7 days at a concentration of 400 µg/ml. After 1 week, iPSC clones were isolated and cultivated on feeders. Lonza Summary: The authors developed a protocol that allowed them to model T-cell differentiation in vitro. They also sowed that iPSCs can be differentiated into hematopoetic progenitors and beyond into various stages of thymocyte development. Overall, they feel their results provide proof of concept that iPSC-based disease modeling is able to reflect thymocyte maturation in vivo and that modeling strategies such as this will be useful in developing gene therapy strategies.
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.