title: Evaluation of Tet-on system to avoid transgene down-regulation in ex vivo gene transfer to the CNS.
abstract: Ex vivo gene transfer to the CNS has so far been hampered by instability of transgene expression. To avoid the phenomenon of transgene down-regulation, we have employed strong, constitutive promoters and compared this expression system with the inducible Tet expression system incorporated in a single plasmid vector or in lentiviral vectors. Plasmid-based transgene expression directed by the constitutive, human ubiquitin promoter, UbC, was stable in transfected HiB5 cells in vitro and comparable in strength to the CMV promoter. However, after transplantation of UbC and CMV HiB5 clones to the rat striatum, silencing of the transgene occurred in most cells soon after implantation of transfected cells. The Tet-on elements were incorporated in a single plasmid vector and inducible HiB5 clones were generated. Inducible clones displayed varying basal expression activity, which could not be ascribed to an effect of cis-elements in the vector, but rather was due, at least in part, to intrinsic activity of the minimal promoter. Basal expression activity could be blocked in a majority of cells by stable expressing the transrepressor tTS. Fully induced expression levels were comparable to CMV and UbC promoters. Similar to the constitutive promoters transgene expression was down-regulated soon after grafting of inducible HiB5 clones to the rat striatum. Lentiviral vectors can direct long-term stable in vivo transgene expression. To take advantage of this quality of the lentiviral vector, the Tet-on elements were incorporated in two lentiviral transfer vectors followed by transduction of Hib5 cells. Interestingly, all HiB5 clones established by lentiviral transduction showed very similar expression patterns and tight regulatability that apparently was independent of transgene copy number and integration site. Nevertheless, transgene expression in all lentiviral HiB5 clones was down-regulated shortly after transplantation to the rat striatum. These results confirm the general phenomenon of transgene down-regulation. Moreover, the results suggest that the considerable advantages offered by lentiviral vectors for direct gene delivery cannot necessarily be transferred directly to ex vivo gene delivery. This emphasizes the need for alternative vector strategies for ex vivo gene transfer.
authors.affiliation: NsGene A/S, 97-Pederstrupvej, DK-2750 Ballerup, Denmark.
authors.forename: J
authors.initials: J
authors.lastname: Johansen
authors.forename: C
authors.initials: C
authors.lastname: Rosenblad
authors.forename: K
authors.initials: K
authors.lastname: Andsberg
authors.forename: A
authors.initials: A
authors.lastname: Møller
authors.forename: C
authors.initials: C
authors.lastname: Lundberg
authors.forename: A
authors.initials: A
authors.lastname: Björlund
authors.forename: T E
authors.initials: TE
authors.lastname: Johansen
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D003587:Cytomegalovirus / Q000235:genetics; D015536:Down-Regulation; D005260:Female; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D019076:Transgenes; D025801:Ubiquitin / Q000235:genetics
chemical_list: D012097:Repressor Proteins; D025801:Ubiquitin; C034811:tetracycline resistance-encoding transposon repressor protein