Construction of an rtTA2s-m2/ttskid-Based transcription regulatory switch that displays no basal activity, good inducibility, and high responsiveness to doxycycline in mice and Non-Human primates

Abstract

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.

Inducible expression of phospholipid transfer protein (PLTP) in transgenic mice: acute effects of PLTP on lipoprotein metabolism

“…The non-induced double transgenic mouse line (Tre-PLTPtg//rtTAtg without doxycycline) did not show any significant differences in RNA expression compared with control mice, so our inducible mouse model does not show any intrinsic background activity (''leakage'') in the non-induced state. The improved version of the tetracycline-controlled transactivator we used (rtTA2S-M2) has been reported to have a low background activity (Lamartina et al , 2003Salucci et al 2002). Our inducible mouse model shows a broad pattern of human PLTP expression (liver, kidney > lung > spleen, heart, adrenal, adipose tissue).…”

Gene therapy progress and prospects: transcription regulatory systems

“…tTS consists in the KRAB (Kruppel-Associated Box) transrepressing domain of human Kid-1 protein fused to the wt TetR. 6,9 Coexpressing rTA with a tTS unable to heterodimerize with rtTA results in very tight regulation. 1 In the absence of Dox, tTS binds to tetO and inhibits basal transcription; as Dox is added, tTS dissociates from the target DNA while rtTA becomes active and triggers transcription (Figure 1c).…”

“…7,8 Finally, the latest and most improved versions combine tTS with rtTA2 S -S2 and rtTA2 S -M2. 6,9,10 Various bicistronic cassettes coexpressing via internal ribosome entry site (IRES) tTS and rtTA or rtTA2 S -S2 or rtTA2 S -M2 (IRES-A, IRES-S2 and IRES-M2 systems, respectively) have been constructed. Both IRES-S2 and IRES-M2 displayed a negligible baseline activity and a 10-fold expanded dynamic range of induction than IRES-A: IRES-M2 also displayed an enhanced Dox-sensitivity.…”

“…Both IRES-S2 and IRES-M2 displayed a negligible baseline activity and a 10-fold expanded dynamic range of induction than IRES-A: IRES-M2 also displayed an enhanced Dox-sensitivity. 9 When delivered into mouse muscles in the context of plasmid or gutless-Ad vectors, the IRES-M2 expression cassette mediated very tight and strictly Dox-dose-dependent modulation of expression for more than 1 year. 6,9 In agreement with these findings, Ad-vectors containing distinct expression cassettes for rtTA2 S -S2, tTS and the transgene of interest display greatly improved Dox-inducible gene expression in terms of basal activity and drug responsiveness.…”

“…9 When delivered into mouse muscles in the context of plasmid or gutless-Ad vectors, the IRES-M2 expression cassette mediated very tight and strictly Dox-dose-dependent modulation of expression for more than 1 year. 6,9 In agreement with these findings, Ad-vectors containing distinct expression cassettes for rtTA2 S -S2, tTS and the transgene of interest display greatly improved Dox-inducible gene expression in terms of basal activity and drug responsiveness. 10 The Tet-system is apparently not immunogenic in several mouse strains; in contrast, recent studies indicate that intramuscularly delivered Tet-ON activators may elicit a cellular and humoral response in non-human primates.…”