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


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.

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Data availability

The authors declare that all data generated or analysed for this study are available within the paper and its Supplementary Information. The sequences of HDR DNA templates are provided in the Supplementary Dataset. Additional raw data are available from the corresponding author upon reasonable request.


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We thank members of the Busch laboratory for experimental help and critical discussion, particularly M. Hammel, F. Mohr, S. Dötsch, E. d’Ippolito and V. R. Buchholz. We also thank L. Germeroth (Juno Therapeutics) for critical discussion. This work was mainly supported by the German Centre for Infection Research (DZIF).

Author information

K.Schober and D.H.B. conceived the study. K.Schober, T.R.M. and D.H.B. designed and analysed experiments. K.Schober, T.R.M. and F.G. performed CRISPR editing and transductions. F.G. performed double-stranded DNA production and gDNA analysis. S.G. performed FACS. T.R.M. and M.E. identified TCRs. M.E. produced pMHC reagents. K.Schober and T.R.M. performed flow cytometric analyses and functional assays. M.P., C.S., K.Schumann and A.M. advised on CRISPR–Cas9 RNP editing of T cells. T.L.R. and A.M. developed and advised on non-viral CRISPR–Cas9 large gene KI via HDR. K.Schober, T.R.M. and T.L.R. designed HDR DNA templates. K.Schober, T.R.M. and D.H.B. wrote the manuscript. All authors read and reviewed the manuscript.

Correspondence to Dirk H. Busch.

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Competing interests

D.H.B. is co-founder of STAGE Cell Therapeutics GmbH (now Juno Therapeutics/Celgene) and T Cell Factory B.V. (now Kite/Gilead). D.H.B. has a consulting contract with and receives sponsored research support from Juno Therapeutics/Celgene. M.P. and C.S. are employees of Juno Therapeutics/Celgene. A.M. is on the scientific advisory board of PACT Pharma, serves as an advisor to Sonoma Biotherapeutics and previously served as an advisor to Juno Therapeutics and is a co-founder of Spotlight Therapeutics and Arsenal Biosciences. T.L.R. is a co-founder of Arsenal Biosciences. The Marson lab has received sponsored research support from Epinomics, Juno Therapeutics and Sanofi, and a gift from Gilead. A.M. and T.L.R. have previously filed related patent applications.

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Reporting Summary

Supplementary Dataset

Sequences of HDR DNA templates.

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Fig. 1: Advanced T-cell engineering through non-viral CRISPR–Cas9-mediated KI of TCRs into the endogenous TCR gene locus.
Fig. 2: Antigen-specific maximum cytokine release and functional avidity of retrovirally transduced (with or without TRAC KO) and TRAC-KI T cells.
Fig. 3: Mispairing with endogenous β-chain occurs in transduced or TRAC-only edited T cells.
Fig. 4: α and β editing eliminates mispairing and enhances TCR surface expression of weak TCRs.
Fig. 5: Orthotopic TCR α- and β-chain replacement enables most physiological T-cell engineering.
Fig. 6: Physiological TCR downregulation after orthotopic TCR α- and β-chain replacement.