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Published Online: 1 May 2018

Efficient Non-Viral T-Cell Engineering by Sleeping Beauty Minicircles Diminishing DNA Toxicity and miRNAs Silencing the Endogenous T-Cell Receptors

Publication: Human Gene Therapy
Volume 29, Issue Number 5

Abstract

Transposon-based vectors have entered clinical trials as an alternative to viral vectors for genetic engineering of T cells. However, transposon vectors require DNA transfection into T cells, which were found to cause adverse effects. T-cell viability was decreased in a dose-dependent manner, and DNA-transfected T cells showed a delayed response upon T-cell receptor (TCR) stimulation with regard to blast formation, proliferation, and surface expression of CD25 and CD28. Gene expression analysis demonstrated a DNA-dependent induction of a type I interferon response and interferon-β upregulation. By combining Sleeping Beauty transposon minicircle vectors with SB100X transposase-encoding RNA, it was possible to reduce the amount of total DNA required, and stable expression of therapeutic TCRs was achieved in >50% of human T cells without enrichment. The TCR-engineered T cells mediated effective tumor cell killing and cytokine secretion upon antigen-specific stimulation. Additionally, the Sleeping Beauty transposon system was further improved by miRNAs silencing the endogenous TCR chains. These miRNAs increased the surface expression of the transgenic TCR, diminished mispairing with endogenous TCR chains, and enhanced antigen-specific T-cell functionality. This approach facilitates the rapid non-viral generation of highly functional, engineered T cells for immunotherapy.

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Information & Authors

Information

Published In

cover image Human Gene Therapy
Human Gene Therapy
Volume 29Issue Number 5May 2018
Pages: 569 - 584
PubMed: 29562762

History

Published in print: May 2018
Published online: 1 May 2018
Published ahead of production: 21 March 2018
Accepted: 15 March 2018
Received: 28 July 2017

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Topics

    Authors

    Affiliations

    Julian Clauss
    Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany.
    Matthias Obenaus
    Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany.
    Charité Universitätsmedizin Berlin, Campus Virchow-Klinikum, Berlin, Germany.
    Csaba Miskey
    Division of Medical Biotechnology, Paul Ehrlich-Institut, Langen, Germany.
    Zoltán Ivics
    Division of Medical Biotechnology, Paul Ehrlich-Institut, Langen, Germany.
    Zsuzsanna Izsvák
    Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany.
    Berlin Institute of Health, Berlin, Germany.
    Wolfgang Uckert,* [email protected]
    Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany.
    Berlin Institute of Health, Berlin, Germany.
    Institute of Biology, Humboldt-Universität zu Berlin, Berlin, Germany.
    Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany.

    Notes

    These authors contributed equally to this work.
    *
    Correspondence: Dr. Wolfgang Uckert or Dr. Mario Bunse, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Straße 10, 13092 Berlin, Germany. E-mail: [email protected] or [email protected]

    Author Disclosure

    The Max Delbrück Center for Molecular Medicine applied for a patent on the method for the generation of gene-engineered T cells using transposon minicircles (J.C., M.B., W.U., Zs.Iz.) and for the MAGE-A1-specific TCR T1367 (M.O.). W.U. is on the Scientific Advisory Board of Medigene and PACT Pharma.

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