Abstract
Purpose::
For many gene transfer applications it is often desirable to deliver more than one gene to the targeted cells. One way to accomplish this has been to create bicistronic vectors in which two transgenes are linked by an internal ribosomal entry site (IRES). The performance of these vectors has been inconsistent. Most notably expression levels of the gene downstream of the IRES have been below those required by many applications. In our current studies, we need to be able to reliably identify photoreceptors transduced by our therapeutic virus. The purpose of these experiments was to determine if high-level, photoreceptor-specific expression of reporter genes normally placed downstream of IRES elements can be obtained by replacing the IRES with a second independent promoter.
Methods::
We constructed two dual-promoter lentiviral vectors that included guanylate cyclase (GC1) under the regulation of either an EF1α or IRBP promoter followed by GFP under the regulation of a murine opsin promoter, mOP500 (mOP). Constructs were packaged into lentiviruses (~1010 TU/ml) and injected into the optic vesicles of stage 12 chicken embryos. The day before hatching, the retinas were removed and GFP expression was examined in fixed flatmount tissue and in frozen sections. Cells expressing GFP were identified using immunohistochemistry.
Results::
High levels of GFP expression were consistently observed in rod photoreceptors in retinas transduced with either EF1α.GC1-mOP.GFP or IRBP.GC1-mOP.GFP. Surprisingly, no GFP was detected in cone photoreceptors. While GFP expression was restricted to rod cells in retinas treated with IRBP.GC1-mOP.GFP, low levels of GFP were occasionally observed in inner retinal cells in retinas treated with EF1α.GC1-mOP.GFP.
Conclusions::
Replacement of IRES with mOP significantly increased GFP expression levels in transduced cells. Unlike the photoreceptor-specific expression pattern (rods and cones) exhibited by the mOP promoter in mouse retina, the expression pattern of mOP in chicken retina was limited to rod cells. The presence of GFP in non-photoreceptor cells in retinas transduced with EF1α.GC1-mOP.GFP shows that the expression of the individual promoters can be altered in the context of dual-promoter vectors, an issue that we are examining using dual-promoter vectors expressing dsRED2 and GFP under the control of EF1α, IRBP, GCAP1 and mOP promoters. Dual-promoter lentiviral vectors can solve IRES-related problems and may make it possible to target two different cell types in neural retina using one vector.
Keywords: gene transfer/gene therapy • photoreceptors • gene/expression