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A retroviral vector for the enhanced expression of the herpes simplex virus thymidine kinase (HSV tk) gene was developed by using a tetracycline-responsive expression system (TRES). The two components of the TRES, the chimeric transactivator (tTA) and the corresponding tTA-binding cis element (tetO), were both incorporated into a retroviral vector and resulted in high levels of tk gene expression from tetO in target cells. Amphotropic virus supernatants from stable producer cells, generated by the retroviral vector containing the TRES, gave titers of 10(4) to 10(5) G418-resistant CFU/ml on murine NIH 3T3 cells. The retroviral vector (G1tTA-[tetOTkINa]R), in which tetO was used in the opposite orientation relative to viral transcription, was capable of transducing tk and neo genes into murine NIH 3T3 cells to yield a high level of tk gene expression. TK enzyme activity in NIH 3T3 cells transduced by this vector was 417-fold higher than in control cells. This increased TK activity was returned to basal levels in the presence of tetracycline. The level of tk gene expression driven by tetO from G1tTA-[tetOTkINa]R vector in NIH 3T3 cells was fourfold higher at both the mRNA level and the TK enzyme level than that produced by the long terminal repeat of G1Tk1SvNa, the vector being used in the ongoing brain tumor gene therapy trial. Retroviral vectors containing the TRES may be useful therefore in achieving higher levels of tk gene expression, which should facilitate gene therapy approaches in the treatment of cancer.

journal of virology

Novel retroviral vector transferring a suicide gene and a selectable marker gene with enhanced gene expression by using a tetracycline-responsive expression system