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Nat Biomed Eng. 2019 Dec;3(12):974-984. doi: 10.1038/s41551-019-0409-0. Epub 2019 Jun 10.

Orthotopic replacement of T-cell receptor α- and β-chains with preservation of near-physiological T-cell function.

Author information

1
Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany.
2
German Center for Infection Research (DZIF), Munich, Germany.
3
Juno Therapeutics GmbH, Munich, Germany.
4
Institute for Advanced Study, Technische Universität München, Munich, Germany.
5
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.
6
Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany. dirk.busch@tum.de.
7
German Center for Infection Research (DZIF), Munich, Germany. dirk.busch@tum.de.
8
Institute for Advanced Study, Technische Universität München, Munich, Germany. dirk.busch@tum.de.

Abstract

Therapeutic T cells with desired specificity can be engineered by introducing T-cell receptors (TCRs) specific for antigens of interest, such as those from pathogens or tumour cells. However, TCR engineering is challenging, owing to the complex heterodimeric structure of the receptor and to competition and mispairing between endogenous and transgenic receptors. Additionally, conventional TCR insertion disrupts the regulation of TCR dynamics, with consequences for T-cell function. Here, we report the outcomes and validation, using five different TCRs, of the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) with non-virally delivered template DNA for the elimination of endogenous TCR chains and for the orthotopic placement of TCRs in human T cells. We show that, whereas the editing of a single receptor chain results in chain mispairing, simultaneous editing of α- and β-chains combined with orthotopic TCR placement leads to accurate αβ-pairing and results in TCR regulation similar to that of physiological T cells.

PMID:
31182835
DOI:
10.1038/s41551-019-0409-0

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