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Optimized Production of Lentiviral Vectors for CAR-T Cell

  • Pablo Diego Moço
  • Mário Soares de Abreu Neto
  • Daianne Maciely Carvalho Fantacini
  • Virgínia Picanço-CastroEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2086)

Abstract

Advances in the use of lentiviral vectors for gene therapy applications have created a need for large-scale manufacture of clinical-grade viral vectors for transfer of genetic materials. Lentiviral vectors can transduce a wide range of cell types and integrate into the host genome of dividing and nondividing cells, resulting in long-term expression of the transgene both in vitro and in vivo. In this chapter, we present a method to transfect human cells, creating an easy platform to produce lentiviral vectors for CAR-T cell application.

Key words

Chimeric antigen receptor Transfection Human cell line Lentiviral vectors Titration 

Notes

Acknowledgments

The authors acknowledge the financial support of: São Paulo Research Foundation—FAPESP (2016/08374-5); the National Council for Scientific and Technological Development—CNPq (381128/2018-0); Research, Innovation, and Dissemination Centers—RIDC (2013/08135-2); and the National Institute of Science and Technology in Stem Cell and Cell Therapy—INCTC (465539/2014-9). The authors also acknowledge financial support from Secretaria Executiva do Ministério da Saúde (SE/MS), Departamento de Economia da Saúde, Investimentos e Desenvolvimento (DESID/SE), Programa Nacional de Apoio à Atenção Oncológica (PRONON) Process 25000.189625/2016-16.

References

  1. 1.
    Brentjens RJ, Davila ML, Riviere I et al (2013) CD19-targeted t cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med 5:177ra38.  https://doi.org/10.1126/scitranslmed.3005930CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Kochenderfer JN, Dudley ME, Carpenter RO et al (2013) Donor-derived CD19-targeted T cells cause regression of malignancy persisting after allogeneic hematopoietic stem cell transplantation. Blood 122:151.  https://doi.org/10.1182/blood-2013-08-519413.R.E.GCrossRefGoogle Scholar
  3. 3.
    Kochenderfer JN, Dudley ME, Kassim SH et al (2015) Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol 33:540–549.  https://doi.org/10.1200/JCO.2014.56.2025CrossRefPubMedGoogle Scholar
  4. 4.
    Kalos M, Levine BL, Porter DL et al (2011) T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med 3:95ra73.  https://doi.org/10.1126/scitranslmed.3002842CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Beatty GL, Haas AR, Maus MV et al (2014) Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce antitumor activity in solid malignancies. Cancer Immunol Res 2:112–120.  https://doi.org/10.1158/2326-6066.CIR-13-0170CrossRefPubMedGoogle Scholar
  6. 6.
    Huls MH, Figliola MJ, Dawson MJ et al (2013) Clinical application of sleeping beauty and artificial antigen presenting cells to genetically modify T cells from peripheral and umbilical cord blood. J Vis Exp 72:e50070.  https://doi.org/10.3791/50070CrossRefGoogle Scholar
  7. 7.
    Singh H, Figliola MJ, Dawson MJ et al (2013) Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using sleeping beauty system and artificial antigen presenting cells. PLoS One 8:e64138.  https://doi.org/10.1371/journal.pone.0064138CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Morita D, Nishio N, Saito S et al (2018) Enhanced expression of anti-CD19 chimeric antigen receptor in piggyBac transposon-engineered T cells. Mol Ther Methods Clin Dev 8:131–140.  https://doi.org/10.1016/j.omtm.2017.12.003CrossRefPubMedGoogle Scholar
  9. 9.
    Jensen MC, Clarke P, Tan G et al (2000) Human T Lymphocyte Genetic Modification with Naked DNA. Mol Ther 1:49–55.  https://doi.org/10.1006/mthe.1999.0012CrossRefPubMedGoogle Scholar
  10. 10.
    Suerth JD, Schambach A, Baum C (2012) Genetic modification of lymphocytes by retrovirus-based vectors. Curr Opin Immunol 24:598–608.  https://doi.org/10.1016/j.coi.2012.08.007CrossRefPubMedGoogle Scholar
  11. 11.
    Merten O-W (2004) State-of-the-art of the production of retroviral vectors. J Gene Med 6:S105–S124.  https://doi.org/10.1002/jgm.499CrossRefPubMedGoogle Scholar
  12. 12.
    Merten O-W, Charrier S, Laroudie N et al (2011) Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application. Hum Gene Ther 22:343–356.  https://doi.org/10.1089/hum.2010.060CrossRefPubMedGoogle Scholar
  13. 13.
    van der Loo JCM, Wright JF (2016) Progress and challenges in viral vector manufacturing. Hum Mol Genet 25:R42–R52.  https://doi.org/10.1093/hmg/ddv451CrossRefPubMedGoogle Scholar
  14. 14.
    Picanço-Castro V, Fontes AM, Russo-Carbolante EMDS, Covas DT (2008) Lentiviral-mediated gene transfer – a patent review. Expert Opin Ther Pat 18:525–539.  https://doi.org/10.1517/13543776.18.5.525CrossRefGoogle Scholar
  15. 15.
    DuBridge RB, Tang P, Hsia HC et al (1987) Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol Cell Biol 7:379–387CrossRefGoogle Scholar
  16. 16.
    Gama-Norton L, Botezatu L, Herrmann S et al (2011) Lentivirus production is influenced by SV40 large T-antigen and chromosomal integration of the vector in HEK293 cells. Hum Gene Ther 22:1269–1279.  https://doi.org/10.1089/hum.2010.143CrossRefPubMedGoogle Scholar
  17. 17.
    Graessmann M, Menne J, Liebler M et al (1989) Helper activity for gene expression, a novel function of the SV40 enhancer. Nucleic Acids Res 17:6603–6612CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Pablo Diego Moço
    • 1
  • Mário Soares de Abreu Neto
    • 1
  • Daianne Maciely Carvalho Fantacini
    • 1
  • Virgínia Picanço-Castro
    • 1
    Email author
  1. 1.Center for Cell-Based Therapy CTC, Regional Blood Center of Ribeirão PretoUniversity of São PauloSão PauloBrazil

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