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Generation of CAR-T Cells with Slee** Beauty Transposon Gene Transfer

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Gene Therapy of Cancer

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2521))

Abstract

Human T lymphocytes that transgenically express a chimeric antigen receptor (CAR) have proven efficacy and safety in gene- and cell-based immunotherapy of certain hematological cancers. Appropriate gene vectors and methods of genetic engineering are required for therapeutic cell products to be biologically potent and their manufacturing to be economically viable. Transposon-based gene transfer satisfies these needs, and is currently being evaluated in clinical trials. In this protocol we describe the basic Slee** Beauty (SB) transposon vector components required for stable gene integration in human cells, with special emphasis on minicircle DNA vectors and the use of synthetic mRNA. We provide a protocol for functional validation of the vector components in cultured human cell lines on the basis of fluorescent reporter gene expression. Finally, we provide a protocol for CAR-T cell engineering and describe assays that address transgene expression, biological potency and genomic vector copy numbers in polyclonal cell populations. Because transposons allow virus-free gene transfer with naked nucleic acids, the protocol can be adopted by any laboratory equipped with biological safety level S1 facilities.

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Acknowledgments

M.H. and Z.I. is receiving funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 754658 (CARAMBA). S.P. and M.H. are supported by the patient advocacy group ‘Hilfe im Kampf gegen den Krebs e.V.’, Würzburg, Germany and ‘Forschung hilft’—Stiftung zur Förderung der Krebsforschung an der Universität Würzburg. Further, the authors are supported by the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 853988 (imSAVAR to M.H.) and No. 945393 (T2EVOLVE to M.H. and Z.I.). Some of the figures were prepared with the help of Biorender.com.

Author information

Author notes

  1. Sabrina Prommersberger, Razieh Monjezi, Lacramioara Botezatu, Csaba Miskey and Maximilian Amberger contributed equally to this work.

    Michael Hudecek and Zoltán Ivics contributed equally to this work.

Authors and Affiliations

  1. Department of Internal Medicine II, University Hospital of Würzburg, Würzburg, Germany

    Sabrina Prommersberger, Razieh Monjezi, Katrin Mestermann & Michael Hudecek

  2. Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany

    Lacramioara Botezatu, Csaba Miskey, Maximilian Amberger & Zoltán Ivics

Authors
  1. Sabrina Prommersberger
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  2. Razieh Monjezi
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  3. Lacramioara Botezatu
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  4. Csaba Miskey
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  5. Maximilian Amberger
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  6. Katrin Mestermann
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  7. Michael Hudecek
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  8. Zoltán Ivics
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Corresponding author

Correspondence to Zoltán Ivics .

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Editors and Affiliations

  1. Max-Delbrück-Center for Molecular Medicine, Berlin, Berlin, Germany

    Wolfgang Walther

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Prommersberger, S. et al. (2022). Generation of CAR-T Cells with Slee** Beauty Transposon Gene Transfer. In: Walther, W. (eds) Gene Therapy of Cancer. Methods in Molecular Biology, vol 2521. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2441-8_3

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  • DOI: https://doi.org/10.1007/978-1-0716-2441-8_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2440-1

  • Online ISBN: 978-1-0716-2441-8

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