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

A tetracycline conditional expression retroviral vector was validated for its efficient expression of a therapeutic gene in various appropriate cell lines. We designed an inducible retroviral vector carrying a therapeutic transgene; the cytocine deaminase (CD). As the room for exogenous DNA sequences is limited in retroviral vectors, we had to choose short sequences and thus used the GTX IRES sequence and the CD suicide gene (shorter than for instance HSV1-TK). Also, the rtTA transactivator was fused to CD, further restricting the size of exogenous DNA to be added to the retroviral vector.

In a preliminary experiment, the new GTX-rtTA-CD cassette was assayed in a defective retroviral vector, propagated together with a replicative retroviral vector holding the GFP reporter transgene. Induction of GFP expression was fully reversible upon addition or withdrawal of doxycycline, as measured by mean fluorescence intensity. Furthermore, when 5FC (cytotoxic prodrug of the CD suicide gene) was added in the induced state it was toxic to the cells, but not in the uninduced state. This experiment further demonstrates the functionality of tet-inducible replicative retroviral vectors, and proves that the CD suicide gene fused to the rtTA is functional. 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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