Use of retroviral-mediated gene transfer to deliver and test function of chimeric antigen receptors in human T-cells

Authors

  • Ana C. Parente-Pereira King's College London
  • Scott Wilkie Dundee University
  • Sjoukje J.C. van der Stegen King's College London
  • David M. Davies King's College London
  • John Maher King's College London

DOI:

https://doi.org/10.14440/jbm.2014.30

Keywords:

Retroviral, transduction, T-cell, chimeric antigen receptor

Abstract

Chimeric antigen receptors (CARs) are genetically delivered fusion molecules that elicit T-cell activation upon binding of a native cell surface molecule. These molecules can be used to generate a large number of memory and effector T-cells that are capable of recognizing and attacking tumor cells. Most commonly, stable CAR expression is achieved in T-cells using retroviral vectors. In the method described here, retroviral vectors are packaged in a two-step procedure. First, H29D human retroviral packaging cells (a derivative of 293 cells) are transfected with the vector of interest, which is packaged transiently in vesicular stomatitis virus (VSV) G pseudotyped particles. These particles are used to deliver the vector to PG13 cells, which achieve stable packaging of gibbon ape leukaemia virus (GALV)-pseudotyped particles that are suitable for infection of human T-cells. The key advantage of the method reported here is that it robustly generates polyclonal PG13 cells that are 100% positive for the vector of interest. This means that efficient gene transfer may be repeatedly achieved without the need to clone individual PG13 cells for experimental pre-clinical testing. To achieve T-cell transduction, cells must first be activated using a non-specific mitogen. Phytohemagglutinin (PHA) provides an economic and robust stimulus to achieve this. After 48-72 h, activated T-cells and virus-conditioned medium are mixed in RetroNectin-coated plasticware, which enhances transduction efficiency. Transduced cells are analyzed for gene transfer efficiency by flow cytometry 48 h following transduction and may then be tested in several assays to evaluate CAR function, including target-dependent cytotoxicity, cytokine production and proliferation.

Author Biography

John Maher, King's College London

Senior Lecturer in Immunology,
NIHR Biomedical Research Centre at Guy

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Published

2014-09-14

How to Cite

1.
Parente-Pereira AC, Wilkie S, van der Stegen SJ, Davies DM, Maher J. Use of retroviral-mediated gene transfer to deliver and test function of chimeric antigen receptors in human T-cells. J Biol Methods [Internet]. 2014Sep.14 [cited 2021Jun.16];1(2):e7. Available from: https://jbmethods.org/jbm/article/view/30

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Protocols