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The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies

Gene Therapy volume 22, pages 209–215 (2015)Cite this article

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Abstract

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3+ T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19+ human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2−/−γc−/− immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

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Acknowledgements

We thank Miyoko Mitsu and Satomi Fujiwara for the AAV preparations. This work was supported in part by a Grant-in-Aid for Scientific Research (Nos. 22700923 and 24390247), a JMU Graduate Student Start-Up Grant for Young Investigators to N Iwase and the MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2013–2017. We thank Dr M Ito (Central Institute for Experimental Animals) for providing Balb/c Rag2−/−γc−/− mice. We also thank JMU Core Center of Research Apparatus for assistance with flow cytometry. This publication was subsidized by JKA through its promotion funds from KEIRIN RACE.

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Authors and Affiliations

  1. Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan

    T Tsukahara, N Iwase, C Yamamoto, R Uchibori, Y Saga, M Urabe, H Mizukami, A Kume & K Ozawa

  2. Division of Immuno-Gene and Cell Therapy (Takara Bio), Jichi Medical University, Shimotsuke, Tochigi, Japan

    T Tsukahara, K Ohmine, R Uchibori, T Teruya, H Ido & K Ozawa

  3. Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka, Japan

    K Kawakami & M Iwasaki

  4. Division of Hematology, Department of Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan

    C Yamamoto, K Ohmine & K Ozawa

  5. Department of Obstetrics and Gynecology, Jichi Medical University, Shimotsuke, Tochigi, Japan

    Y Saga

  6. Human Gene Sciences Center, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan

    M Nakamura

  7. Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    R Brentjens

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  1. T Tsukahara
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  2. N Iwase
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Correspondence to T Tsukahara.

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Tsukahara, T., Iwase, N., Kawakami, K. et al. The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies. Gene Ther 22, 209–215 (2015). https://doi.org/10.1038/gt.2014.104

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