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Generation of CAR+ T Lymphocytes Using the Sleeping Beauty Transposon System

  • Leonardo Chicaybam
  • Luiza Abdo
  • Martín H. BonaminoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2086)

Abstract

Adoptive immunotherapy of cancer using T cells expressing chimeric antigen receptors (CARs) is now an approved treatment for non-Hodgkin lymphoma (NHL) and B cell acute lymphoblastic leukemia (B-ALL), inducing high response rates in patients. The infusion products are generated by using retro- or lentiviral transduction to induce CAR expression in T cells followed by an in vitro expansion protocol. However, use of viral vectors is cumbersome and is associated with increased costs due to the required high titers, replication-competent retrovirus (RCR) detection and production/use in a biosafety level 2 culture rooms, and additional quality control tests. Nonviral methods, like the Sleeping Beauty transposon system, can stably integrate in the genome of target cells and can be delivered using straightforward methods like electroporation. This chapter describes a protocol for T cell genetic modification using Sleeping Beauty transposon system and electroporation with the Lonza Nucleofector II device for the stable expression of CAR molecules in T lymphocytes.

Key words

Chimeric antigen receptor Sleeping beauty transposon T lymphocyte CD19 Electroporation 

References

  1. 1.
    Maude SL, Laetsch TW, Buechner J et al (2018) Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 378(5):439–448CrossRefGoogle Scholar
  2. 2.
    Park JH, Riviére I, Gonen M et al (2018) Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N Engl J Med 378(5):449–459CrossRefGoogle Scholar
  3. 3.
    Neelapu SS, Locke FL, Bartlett NL et al (2017) Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 377(26):2531–2544CrossRefGoogle Scholar
  4. 4.
    June CH, Sadelain M (2018) Chimeric antigen receptor therapy. N Engl J Med 379(1):64–73CrossRefGoogle Scholar
  5. 5.
    Levine BL, Miskin J, Wonnacott K et al (2017) Global manufacturing of CAR T cell therapy. Mol Ther Methods Clin Dev 4:92–101CrossRefGoogle Scholar
  6. 6.
    Wang X, Rivière I (2016) Clinical manufacturing of CAR T cells: foundation of a promising therapy. Mol Ther Oncolytics 3:16015CrossRefGoogle Scholar
  7. 7.
    Ivics Z, Hackett PB, Plasterk RH et al (1997) Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91(4):501–510CrossRefGoogle Scholar
  8. 8.
    Mátés L, Chuah MK, Belay E et al (2009) Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet 41(6):753–761CrossRefGoogle Scholar
  9. 9.
    Chicaybam L, Sodre AL, Curzio BA et al (2013) An efficient low cost method for gene transfer to T lymphocytes. PLoS One 8(3):e60298CrossRefGoogle Scholar
  10. 10.
    Peng PD, Cohen CJ, Yang S et al (2009) Efficient nonviral Sleeping Beauty transposon-based TCR gene transfer to peripheral blood lymphocytes confers antigen-specific antitumor reactivity. Gene Ther 16(8):1042–1049CrossRefGoogle Scholar
  11. 11.
    Huang X, Guo H, Tammana S et al (2010) Gene transfer efficiency and genome-wide integration profiling of Sleeping Beauty, Tol2, and PiggyBac transposons in human primary T cells. Mol Ther 18(10):1803–1813CrossRefGoogle Scholar
  12. 12.
    Moldt B, Miskey C, Staunstrup NH et al (2011) Comparative genomic integration profiling of Sleeping Beauty transposons mobilized with high efficacy from integrase-defective lentiviral vectors in primary human cells. Mol Ther J Am Soc Gene Ther 19(8):1499–1510CrossRefGoogle Scholar
  13. 13.
    Field AC, Vink C, Gabriel R et al (2013) comparison of lentiviral and Sleeping Beauty mediated αβ T cell receptor gene transfer. PLoS One 8(6):e68201CrossRefGoogle Scholar
  14. 14.
    Kebriaei P, Singh H, Huls MH et al (2016) Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells. J Clin Invest 126(9):3363–3376CrossRefGoogle Scholar
  15. 15.
    Neumann E, Schaefer-Ridder M, Wang Y et al (1982) Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1(7):841–845CrossRefGoogle Scholar
  16. 16.
    Tsong TY (1991) Electroporation of cell membranes. Biophys J 60(2):297–306CrossRefGoogle Scholar
  17. 17.
    Imai C, Mihara K, Andreansky M et al (2004) Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia. Leukemia 18(4):676–684CrossRefGoogle Scholar
  18. 18.
    Siddappa NB, Avinash A, Venkatramanan M et al (2007) Regeneration of commercial nucleic acid extraction columns without the risk of carryover contamination. BioTechniques 42(2):186CrossRefGoogle Scholar
  19. 19.
    Ghassemi S, Nunez-Cruz S, O'Connor RS et al (2018) Reducing ex vivo culture improves the antileukemic activity of chimeric antigen receptor (CAR) T cells. Cancer Immunol Res 6(9):1100–1109CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Leonardo Chicaybam
    • 1
    • 2
  • Luiza Abdo
    • 1
  • Martín H. Bonamino
    • 1
    • 2
    Email author
  1. 1.Programa de Carcinogênese Molecular—Coordenação de PesquisaInstituto Nacional de CâncerRio de JaneiroBrazil
  2. 2.Vice-Presidência de Pesquisa e Coleções Biológicas (VPPCB)Fundação Instituto Oswaldo Cruz (FIOCRUZ)Rio de JaneiroBrazil

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