Tetracycline-controlled expression but not toxicity of an attenuated diphtheria toxin mutant

Tight transcriptional regulation of transferred bacterial toxin genes represents a potential approach for gene therapy of cancer. We have previously shown that the gene for wild type diphtheria toxin A chain (DT-A) placed under transcriptional control of a tetracycline-responsive promoter cannot be silenced due to its extreme toxicity. We now have explored a tetracycline-regulated DT-A mutant involving the histidine-21 catalytic domain (H21A) which shows 120-fold reduced ADP-ribosylation activity. Cellular toxicity was determined in NIH 3T3 fibroblasts and C6 glioma cells after triple transfections with the DT-A construct, the Tet transactivator gene and a luciferase plasmid as the reporter. Marked toxicity, i.e. reduced luciferase expression by more than 98%, was observed both in the absence and in the presence of tetracycline, suggesting leakiness of the Tet system, and absence of regulation, possibly due to inhibition of DT-A synthesis by activated DT-A itself. In contrast, the lacZ gene which was driven by the same promoter could be regulated by up to 49-fold. We conclude that (1) expression but not toxicity of the DT-A mutant can be sufficiently controlled by a tetracycline-responsive promoter, and (2) tight regulation of transferred genes encoding toxins remains a challenge for gene therapy of cancer.