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Efficient transduction and selection of human T‐lymphocytes with bicistronic Thy1/HSV1‐TK retroviral vector produced by a human packaging cell line
Author(s) -
Lemoine François M.,
MeselLemoine Mariana,
Cherai Mustapha,
Gallot Géraldine,
Vié Henri,
Leclercq Virginie,
TrébèdenNegre Hélène,
Mammès Olivier,
Boyer Olivier,
NoguiezHellin Patricia,
Klatzmann David
Publication year - 2004
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.538
Subject(s) - transduction (biophysics) , biology , virology , cell culture , viral vector , stem cell , cd90 , microbiology and biotechnology , transplantation , t cell , genetic enhancement , gene , immunology , recombinant dna , immune system , genetics , medicine , biochemistry , surgery , cd34
Background T‐cells expressing the HSV1‐TK suicide gene can be used for the control of graft‐versus‐host disease following allogeneic stem cell transplantation. To develop clinical trials based on such a strategy, we have generated under good manufacturing procedures a novel ‘split genome’ human packaging cell line (1704 cells). Methods To minimize the risk of generating replication‐competent retroviruses, pol was truncated to remove sequences overlapping with env . To improve retroviral infection and selection of transduced T‐cells, high titers of GALV‐pseudotyped retroviral particles harboring a bicistronic Thy1‐IRES‐TK vector coding for the CD90 GPI‐anchored membrane molecule were produced by 1704 cells. Results Using 1704 cell supernatant and an optimized transduction protocol, approximately 50% of primary T‐cells were transduced and could then be purified (∼95%) using clinical‐grade immunomagnetic beads directed against CD90. Over 96% of these OKT3/IL‐2‐activated CD90 + ‐selected T‐cells were killed by ganciclovir. Cell proliferation and cytokine production of transduced T‐cells and HLA‐restricted cytotoxicity of transduced T‐cell clones were identical to those of their non‐transduced counterparts cultured under the same conditions. Conclusions GALV‐pseudotyped retroviral particles harboring a bicistronic Thy1‐IRES‐TK vector allow efficient transduction and rapid selection of human T‐cells under conditions applicable for clinical trials using the new human 1704 packaging cell line. Copyright © 2004 John Wiley & Sons, Ltd.