The tetracycline (Tet)-dependent regulatory system has been widely used for controlling gene expression. The Tet-on version of the system, in which the reverse Tet-responsive transcriptional activator (rtTA) is positively regulated by Tet or its analogs, such as doxycycline (Dox), is of potential utility for gene therapy applications in humans. However, rtTA may display a high basal activity, especially when delivered in vivo by using episomal vectors such as plasmids. Two novel Dox-inducible activators, called rtTA2(S)-S2 and rtTA2(S)-M2, which have a significantly lower basal activity than rtTA in stably transfected cell lines, have been described. In this study we tested the capability of these trans-activators to control expression of mouse erythropoietin (mEpo) and to modulate hematocrit (Hct) increase in vivo on delivery of plasmids into quadriceps muscles of adult mice by DNA electroinjection. Both rtTA2(S)-M2 and rtTA2(S)-S2 displayed a considerably lower background activity and higher window of induction than rtTA in vivo. Moreover, a stringent control of mEpo gene expression and Hct levels in the absence of any background activity was maintained over a 10-month period by injecting as little as 1 microg of a single plasmid containing the rtTA2(S)-S2 expression cassette and the Tet-responsive mEpo cDNA. This constitutes the first report of a stringent ligand-dependent control of gene expression in vivo obtained by delivering a single plasmid encoding both the trans-activator and the regulated gene. Notably, the rtTA2(S)-S2-based system was induced by oral doses of doxycycline comparable to those normally used in clinical practice in humans.

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 tTS(kid) has proved capable of substantially reducing the baseline activity of rtTA. On the other, novel versions of the activator, namely rtTA2(s)-S2 and rtTA2(s)-M2, with a lower basal activity have been engineered. We have combined these two approaches by co-expressing TS(kid) with the novel transactivators. Bicistronic vectors were constructed that co-express TS(kid) with rtTA, rtTA2(s)-S2, or rtTA2(s) 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.