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The tetracycline (Tc)-dependent system in its “on” version (rtTA system) displays a baseline activity in the uninduced state, severely limiting its potential applicability in human gene therapy. So far, two different strategies to circumvent this limitation have been described. On one side, co-expression of the tetracycline regulated repressor tTSkid has proved capable of substantially reducing the baseline activity of rtTA. On the other, novel versions of the activator, namely rtTA2s-S2 and rtTA2s-M2, with a lower basal activity have been engineered. We have combined these two approaches by co-expressing TSkid with the novel transactivators. Bicistronic vectors were constructed that co-express TSkid with rtTA, rtTA2s-S2, or rtTA2s M2, through an internal ribosome entry site (plasmids IRES-A, IRES-S2, and IRES-M2, respectively). IRES-M2 proved to be the most effective construct ex vivo: it displayed a negligible basal activity, > 1000 fold inducibility, and high responsiveness to doxycycline (Dox). Upon delivery as plasmid DNA in mouse muscles, IRES-M2 facilitated 1000-fold induction of serum alkaline phosphatase (SEAP) gene expression and long-term, stringent, and strictly Dox-dose-dependent regulation of erythropoietin (Epo) gene expression. Tight regulation of the gene encoding SEAP was demonstrated also in non-human primates. Notably, the system was induced in animals by Dox-dosing regimens comparable to those used in humans. [Copyright &y& Elsevier]