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- Fisher, Linda, et al.
(författare)
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Cellular delivery of a double-stranded oligonucleotide NFκB decoy by hybridization to complementary PNA linked to a cell-penetrating peptide
- 2004
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Ingår i: Gene Therapy. - : Springer Science and Business Media LLC. - 0969-7128 .- 1476-5462. ; 11, s. 1264-1272
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Tidskriftsartikel (refereegranskat)abstract
- The activation of nuclear factor B (NFB) is a key event in immune and inflammatory responses. In this study, a cell-penetrating transport peptide, transportan (TP) or its shorter analogue TP 10, was used to facilitate the cellular uptake of an NFB decoy. Peptide nucleic acid (PNA) hexamer or nonamer was linked to the transport peptide by a disulfide bond. NFB decoy oligonucleotide consisted of a double-stranded consensus sequence corresponding to the B site localized in the IL-6 gene promoter, 5'-GGGACTTTCCC-3', with a single-stranded protruding 3'-terminal sequence complementary to the PNA sequence was hybridized to the transport peptide–PNA construct. The ability of the transport peptide–PNA–NFB decoy complex to block the effect of interleukin (IL)-1-induced NFB activation and IL-6 gene expression was analyzed by electrophoretic mobility shift assay and reverse transcriptase-polymerase chain reaction in rat Rinm5F insulinoma cells. Preincubation with transport peptide–PNA–NFB decoy (1 M, 1 h) blocked IL-1-induced NFB-binding activity and significantly reduced the IL-6 mRNA expression. The same concentration of NFB decoy in the absence of transport peptide–PNA had no effect even after longer incubations. Our results showed that binding of the oligonucleotide NFB decoy to the nonamer PNA sequence resulted in a stable complex that was efficiently translocated across the plasma membrane.
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- Johansen, J, et al.
(författare)
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Evaluation of Tet-on system to avoid transgene downregulation in ex vivo gene transfer to the CNS
- 2002
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Ingår i: Gene Therapy. - : Springer Science and Business Media LLC. - 0969-7128 .- 1476-5462. ; 9:19, s. 1291-1301
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Tidskriftsartikel (refereegranskat)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.
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