Expression characteristics of dual-promoter lentiviral vectors targeting retinal photoreceptors and Müller cells

[摘要] Purpose: Growing evidence suggests thatsuccessful treatment of many inherited photoreceptor diseases willrequire multi-protein therapies that not only correct the geneticdefects linked to these diseases but also slow or halt the relateddegenerative phenotypes. To be effective, it is likely that therapeuticprotein expression will need to be targeted to specific cell types. Thepurpose of this study was to develop dual-promoter lentiviral vectorsthat target expression of two proteins to retinal cones and rods, rodsonly, or Müller cells. Methods: Dual-promoter lentivectors wereconstructed using the following promoters: Xenopus opsinpromoter (XOPS)1.3, murine opsin promoter (MOPS), interphotoreceptorretinoid binding protein promoter (IRBP156), rhodopsin kinase (RK),neural retina leucine zipper (NRLL), vimentin (VIM), clusterdifferentiation (CD44), and glial fibrillary acidic protein (GFAP).Vectors were packaged and injected into the neural tubes of chickenembryos. The activities of the promoters alone, in duplicate, or whenpaired with a different promoter were analyzed in transduced,fully-developed retinas, using direct fluorescent and immunofluorescentmicroscopy. Results: IRBP156, NRLL, and RK wereactive in cones and rods while XOPS1.3 was active only in rods. Of theglial promoters, only GFAP activity was restricted to Müller cells;both VIM and CD44 were active in Müller and neural cells. Dual-promotervectors carrying IRBP156 and RK or XOPS1.3 and MOPS, in the orderlisted, exhibited robust expression of both reporter transgenes incones and rods or rods only, respectively. Expression of the upstreamtransgene was much lower than the downstream transgene in dual-promotervectors constructed using two copies of either RK or IRBP156. Analysesof the expression of a dual-promoter vector carrying CD44 and VIM inthe order listed showed that the activity of the VIM promoter was morerestricted to glial cells when paired with the CD44 promoter, while theactivity of the CD44 promoter was inhibited to the extent that noCD44-driven reporter protein was detected in transduced cells. Conclusions: We have identified twodual-promoter vectors, one that targets cones and rods and one thattargets rods alone. Both vectors reliably express the two proteinsencoded by the transgenes they carry. When two well matched promotersare not available, we found that it is possible to target expression oftwo proteins to single cells using dual-promoter vectors carrying twocopies of the same promoter. These vectors should be useful in studiesof retina when co-delivery of a reporter protein with an experimentalprotein is desired or when expression of two exogenous proteins intargeted cells is required.

[学科分类] 生物化学/生物物理