Johansen J, Rosenblad C, Andsberg K, Møller A, Lundberg C, Björlund A, Johansen T E
NsGene A/S, 97-Pederstrupvej, DK-2750 Ballerup, Denmark.
Gene Ther. 2002 Oct;9(19):1291-301. doi: 10.1038/sj.gt.3301778.
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.
迄今为止，转基因表达的不稳定性一直阻碍着中枢神经系统的离体基因转移。为避免转基因下调现象，我们采用了强组成型启动子，并将该表达系统与整合在单个质粒载体或慢病毒载体中的可诱导Tet表达系统进行了比较。由组成型人泛素启动子UbC指导的基于质粒的转基因表达在体外转染的HiB5细胞中稳定，且强度与CMV启动子相当。然而，将UbC和CMV HiB5克隆移植到大鼠纹状体后，转基因在大多数细胞中于转染细胞植入后不久就发生了沉默。将Tet-on元件整合到单个质粒载体中，并产生了可诱导的HiB5克隆。可诱导克隆表现出不同的基础表达活性，这不能归因于载体中顺式元件的作用，而至少部分是由于最小启动子的内在活性。在大多数细胞中，通过稳定表达反式阻遏物tTS可阻断基础表达活性。完全诱导的表达水平与CMV和UbC启动子相当。与组成型启动子类似，可诱导HiB5克隆移植到大鼠纹状体后不久，转基因表达就下调了。慢病毒载体可指导体内转基因的长期稳定表达。为利用慢病毒载体的这一特性，将Tet-on元件整合到两个慢病毒转移载体中，随后转导Hib5细胞。有趣的是，通过慢病毒转导建立的所有HiB5克隆都显示出非常相似的表达模式和严格的调控性，这显然与转基因拷贝数和整合位点无关。然而，所有慢病毒HiB5克隆中的转基因表达在移植到大鼠纹状体后不久就下调了。这些结果证实了转基因下调这一普遍现象。此外，结果表明慢病毒载体在直接基因递送方面所提供的显著优势不一定能直接转移到离体基因递送中。这强调了需要用于离体基因转移的替代载体策略。
