Production of Lentivirus for the Establishment of CAR-T Cells

  • Marlous G. Lana
  • Bryan E. StraussEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2086)


One of the most versatile gene transfer methods involves the use of recombinant lentiviral vectors since they can transduce both dividing and nondividing cells, are considered to be safe and provide long-term transgene expression since the integrated viral genome, the provirus, is passed on to daughter cells. These characteristics are highly desirable when a modified cell must continue to express the transgene even after multiple cell divisions. Lentiviral vectors are often used to introduce protein encoding cDNAs, such as reporter genes, or for noncoding sequences, such as mediators of RNA interference or genome editing, including shRNA or gRNA, respectively. In the gene therapy setting, lentiviral vectors have been used successfully for the modification of hematopoietic stem cells, resulting in restored immune function or correction of defects in hemoglobin, to name but a few examples. The success of chimeric antigen receptor (CAR) T cells for the treatment of B cell leukemias and lymphomas has been particularly striking and this approach has relied heavily on lentivirus-mediated gene transfer. Here we present a typical protocol for the production of lentivirus, concentration by ultracentrifugation and determination of virus titer. The resulting virus can then be used in laboratory assays of gene transfer, including the establishment of CAR T cells.

Key words

Lentivirus production Packaging vectors Transfection Titration Ultracentrifugation Biosafety 


  1. 1.
    Riviere I, Sadelain M (2017) Chimeric antigen receptors: a cell and gene therapy perspective. Mol Ther 25(5):1117–1124. Scholar
  2. 2.
    Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, Trono D (1998) Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72(12):9873–9880PubMedPubMedCentralGoogle Scholar
  3. 3.
    Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L (1998) A third-generation lentivirus vector with a conditional packaging system. J Virol 72(11):8463–8471PubMedPubMedCentralGoogle Scholar
  4. 4.
    Gandara C, Affleck V, Stoll EA (2018) Manufacture of third-generation Lentivirus for preclinical use, with process development considerations for translation to good manufacturing practice. Hum Gene Ther Methods 29(1):1–15. Scholar
  5. 5.
    White M, Whittaker R, Gandara C, Stoll EA (2017) A guide to approaching regulatory considerations for Lentiviral-mediated gene therapies. Hum Gene Ther Methods 28(4):163–176. Scholar
  6. 6.
    Merten OW, Charrier S, Laroudie N, Fauchille S, Dugue C, Jenny C, Audit M, Zanta-Boussif MA, Chautard H, Radrizzani M, Vallanti G, Naldini L, Noguiez-Hellin P, Galy A (2011) Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application. Hum Gene Ther 22(3):343–356. Scholar
  7. 7.
    Debyser Z (2003) Biosafety of lentiviral vectors. Curr Gene Ther 3(6):517–525CrossRefGoogle Scholar
  8. 8.
    Debyser Z (2003) A short course on virology/vectorology/gene therapy. Curr Gene Ther 3(6):495–499CrossRefGoogle Scholar
  9. 9.
    Geraerts M, Willems S, Baekelandt V, Debyser Z, Gijsbers R (2006) Comparison of lentiviral vector titration methods. BMC Biotechnol 6:34. Scholar
  10. 10.
    Sena-Esteves M, Gao G (2018) Titration of lentivirus vectors. Cold Spring Harb Protoc 2018(4):pdb prot095695. Scholar
  11. 11.
    Naviaux RK, Costanzi E, Haas M, Verma IM (1996) The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J Virol 70(8):5701–5705PubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Viral Vector Laboratory, Faculdade de Medicina, Centro de Investigação Translacional em Oncologia/LIM24, Instituto do Câncer do Estado de São PauloUniversidade de São PauloSão PauloBrazil

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