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Obtention of Tetracycline conditional expression retroviral vectors efficiently expressing a marker gene in hepatoma and glioblastoma cell lines

Tetracycline conditional expression retroviral vectors were validated for their efficient expression of a marker gene in various appropriate cell lines. The feasibility to control retroviral replication with the tetracycline (tet) inducible system was previously described in the context of a lentiviral virus. We choose to adapt this system to our Moloney Murine Leukemia Virus (Mo-MLV) based vectors and designed hybrid MoMLV tet-inducible LTRs by replacing viral enhancer sequences with tet-responsive elements. The tet-inducible transactivator (tTA/tetOFF or rtTA/tetON) was trans-complemented either by the cell line, a replication-defective vector, or the RRV itself.

In vitro experiments, in H293 Tet-off or Hela Tet-on human cell lines, were performed to validate these new regulatable replicative vectors. Doxycycline (dox) was given or not at the time of the transfection or 10 days thereafter. Without dox at the time of transfection, the RRV-GFP propagated, but dox treatment prevented its propagation. When dox was added at day 10, the level of GFP expression was reduced in the uninduced state. Also propagation started when dox was withdrawn at day 10. These results demonstrate the functionality of the hybrid LTRs to control RRV-GFP propagation by modulating its level of expression, in a reversible manner.

We then aimed to carry the whole tet-inducible system within RRVs: one RRV holding the GFP reporter transgene and another one with the rtTA (tetON) transactivator. NIH3T3 cells were co-transfected with these two RRVs, and, in the dox induced state, propagation was observed as in the previous experiment. The supernatant of these induced cells was then passed onto NIH3T3 cells with or without dox. In the dox- induced state, GFP was clearly propagated with a high level of expression. Without dox, no propagation occurred. These results demonstrate that the whole tet-inducible system could be adapted to a pair of RRVs to achieve their inducible propagation.

These regulatable replicative retroviral vectors harbour great potential for gene therapy application and will be further analysed for efficacy and safety in vivo.

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UNiversite Pierre et Marie Curie - UMR7087
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