POL Scientific / JBM / Volume 1 / Issue 2 / DOI: 10.14440/jbm.2014.30
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Use of retroviral-mediated gene transfer to deliver and test function of chimeric antigen receptors in human T-cells

Ana C. Parente-Pereira1 Scott Wilkie2 Sjoukje J.C. van der Stegen1 David M. Davies3 John Maher1,4,5
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1 King’s College London, Department of Research Oncology, Guy’s Hospital Campus, Great Maze Pond, London SE1 9RT, UK
2 Drug Discovery Unit, James Black Centre, University of Dundee, College of Life Sciences, Dow Street, Dundee, DD1 5EH, UK
3 University College London Cancer Institute, London, WC1E 6DD, UK
4 Department of Immunology, Barnet and Chase Farm NHS Trust, Barnet, Hertfordshire, EN5 3DJ, UK
5 Department of Allergy and Clinical Immunology, King’s College Hospital NHS Foundation Trust, Denmark Hill, London SE5 9RS, UK
Published: 14 September 2014
© 2014 by the author. Licensee POL Scientific, USA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
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.

Keywords
Retroviral
transduction
T-cell
chimeric antigen receptor
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Journal of Biological Methods, Electronic ISSN: 2326-9901 Print ISSN: TBA, Published by POL Scientific