title: Construction of an rtTA2(s)-m2/tts(kid)-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.
authors.affiliation: IRBM-P. Angeletti, Via Pontina Km. 30,600, 00040 Pomezia (Rome), Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Lamartina
authors.forename: Luisa
authors.initials: L
authors.lastname: Silvi
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Roscilli
authors.forename: Danilo
authors.initials: D
authors.lastname: Casimiro
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Simon
authors.forename: Mary-Ellen
authors.initials: ME
authors.lastname: Davies
authors.forename: John W
authors.initials: JW
authors.lastname: Shiver
authors.forename: Cira Daniela
authors.initials: CD
authors.lastname: Rinaudo
authors.forename: Immacolata
authors.initials: I
authors.lastname: Zampaglione
authors.forename: Elena
authors.initials: E
authors.lastname: Fattori
authors.forename: Stefano
authors.initials: S
authors.lastname: Colloca
authors.forename: Odalys
authors.initials: O
authors.lastname: Gonzalez Paz
authors.forename: Ralph
authors.initials: R
authors.lastname: Laufer
authors.forename: Hermann
authors.initials: H
authors.lastname: Bujard
authors.forename: Riccardo
authors.initials: R
authors.lastname: Cortese
authors.forename: Gennaro
authors.initials: G
authors.lastname: Ciliberto
authors.forename: Carlo
authors.initials: C
authors.lastname: Toniatti
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004318:Doxycycline / Q000494:pharmacology*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008253:Macaca mulatta; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D019076:Transgenes; D014407:Tumor Cells, Cultured
chemical_list: D000900:Anti-Bacterial Agents; D013752:Tetracycline; D004318:Doxycycline