Efficient transduction and selection of human T-lymphocytes with bicistronic Thy1/HSV1-TK retroviral vector produced by a human packaging cell line
✍ Scribed by François M. Lemoine; Mariana Mesel-Lemoine; Mustapha Cherai; Géraldine Gallot; Henri Vié; Virginie Leclercq; Hélène Trébèden-Negre; Olivier Mammès; Olivier Boyer; Patricia Noguiez-Hellin; David Klatzmann
- Publisher
- John Wiley and Sons
- Year
- 2004
- Tongue
- English
- Weight
- 390 KB
- Volume
- 6
- Category
- Article
- ISSN
- 1099-498X
- DOI
- 10.1002/jgm.538
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✦ Synopsis
Abstract
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.