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TALEN-mediated editing of endogenous T-cell receptors facilitates efficient reprogramming of T lymphocytes by lentiviral gene transfer

Gene Therapy volume 21, pages 539–548 (2014)Cite this article

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Abstract

Adoptive immunotherapy with T lymphocytes expressing transgenic T-cell receptors (TCRs) has shown significant clinical efficacy in various malignant diseases. However, concurrent expression of endogenous and transgenic TCRs in one and the same T cell may impair efficacy and cause safety problems owing to mispairings. The most elegant approach to address these issues is the complete shutoff of the endogenous receptor chains by genome editing. To this end, we designed TCR-α and TCR-β-specific pairs of transcription activator-like effector nucleases (TALENs). TALENs were delivered into T cells using an optimized messenger RNA-electroporation protocol. Based thereon, we obtained precise and highly efficient knockout (KO) in Jurkat (TCR-α: 59.7±4.0%, TCR-β: 37.4±7.3%) as well as primary T cells (TCR-α: 58.0±15.0%, TCR-β: 41.0±17.6%). Moreover, a successive KO strategy for the endogenous TCR chains combined with subsequent transduction of the respective chains of an Influenza virus-specific model TCR led to complete reprogramming of T cells with strongly improved expression and functionality of transgenic TCRs. In conclusion, we have developed novel means for the efficient genome editing in primary T lymphocytes.

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Abbreviations

TCR:

T-cell receptor

Flu:

Influenza

Flu-M:

Influenza matrix protein

TALEN:

TAL effector nuclease

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Acknowledgements

This work has been part of the PhD thesis of BB at the MIN faculty of the University of Hamburg. We wish to thank Johannes Polke, Tanja Sonntag, Tim Aranyossy and the FACS Core Facility of the University Medical Center Hamburg-Eppendorf for expert technical assistance and the Department of Transfusion Medicine of the UMC Hamburg-Eppendorf for kind help. Our work was supported by a grant of the Deutsche Krebshilfe (to BF and DA) and, in part, the SFB841/Z2 and a post-doc grant to UM within the Forschungsförderung Medizin (FFM) program of the Medical faculty of the UMC Hamburg-Eppendorf.

Author contributions

BB designed and performed experiments; UM helped with design and application of TALENs; DA with T-cell assays; BF designed the study and wrote the manuscript. All authors have read the manuscript and confirmed their authorship.

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Authors and Affiliations

  1. Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, UCCH, University Medical Center (UMC) Hamburg-Eppendorf, Hamburg, Germany

    B Berdien, U Mock & B Fehse

  2. Department of Oncology/Hematology, UMC Hamburg-Eppendorf, Hamburg, Germany

    D Atanackovic

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  1. B Berdien
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  2. U Mock
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Correspondence to B Fehse.

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Berdien, B., Mock, U., Atanackovic, D. et al. TALEN-mediated editing of endogenous T-cell receptors facilitates efficient reprogramming of T lymphocytes by lentiviral gene transfer. Gene Ther 21, 539–548 (2014). https://doi.org/10.1038/gt.2014.26

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