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Evaluation of Tet-on system to avoid transgene downregulation in ex vivo gene transfer to the CNS

Johansen, J; Rosenblad, C; Andsberg, Kim LU ; Moller, A; Lundberg, Cecilia LU ; Björklund, Anders LU and Johansen, TE (2002) In Gene Therapy 9(19). p.1291-1301
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
vectors, lentiviral, transgene down-regulation, ex vivo gene transfer, Tet-on
in
Gene Therapy
volume
9
issue
19
pages
1291 - 1301
publisher
Nature Publishing Group
external identifiers
  • pmid:12224012
  • wos:000178129800004
  • scopus:0036792499
ISSN
0969-7128
DOI
10.1038/sj.gt.3301778
language
English
LU publication?
yes
id
49037222-3a73-49ef-9cd9-6e949963adbb (old id 328380)
date added to LUP
2007-10-29 11:59:26
date last changed
2017-08-06 03:35:47
@article{49037222-3a73-49ef-9cd9-6e949963adbb,
  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.},
  author       = {Johansen, J and Rosenblad, C and Andsberg, Kim and Moller, A and Lundberg, Cecilia and Björklund, Anders and Johansen, TE},
  issn         = {0969-7128},
  keyword      = {vectors,lentiviral,transgene down-regulation,ex vivo gene transfer,Tet-on},
  language     = {eng},
  number       = {19},
  pages        = {1291--1301},
  publisher    = {Nature Publishing Group},
  series       = {Gene Therapy},
  title        = {Evaluation of Tet-on system to avoid transgene downregulation in ex vivo gene transfer to the CNS},
  url          = {http://dx.doi.org/10.1038/sj.gt.3301778},
  volume       = {9},
  year         = {2002},
}