Efficient transduction and selection of human T-lymphocytes with bicistronic Thy1/HSV1-TK retroviral vector produced by a human packaging cell line.
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 (approximately 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.
Lemoine
François M
FM
UPMC/CNRS UMR 7087, CERVI, Hôpital Pitié-Salpêtrière, 83 bd de l'hôpital, 75651 Paris cedex 13, France. francois.lemoine@chups.jussieu.fr
Mesel-Lemoine
Mariana
M
Cherai
Mustapha
M
Gallot
Géraldine
G
Vié
Henri
H
Leclercq
Virginie
V
Trébèden-Negre
Hélène
H
Mammès
Olivier
O
Boyer
Olivier
O
Noguiez-Hellin
Patricia
P
Klatzmann
David
D
D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D015774:Ganciclovir / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D012190:Retroviridae / Q000235:genetics*; D013601:T-Lymphocytes / Q000187:drug effects / Q000502:physiology*; D018800:Thy-1 Antigens / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*; D014776:Virus Cultivation / Q000379:methods
D018800:Thy-1 Antigens; D013937:Thymidine Kinase; D015774:Ganciclovir