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Abstract
BackgroundGene transfer using adeno-associated viral (AAV) vectors has been successfully applied in the retina for the treatment of inherited retinal dystrophies. Recently, microRNAs have been exploited to fine-tune transgene expression improving therapeutic outcomes. Here we evaluated the ability of retinal-expressed microRNAs to restrict AAV-mediated transgene expression to specific retinal cell types that represent the main targets of common inherited blinding conditions.Methodology/Principal FindingsTo this end, we generated AAV2/5 vectors expressing EGFP and containing four tandem copies of miR-124 or miR-204 complementary sequences in the 3′UTR of the transgene expression cassette. These vectors were administered subretinally to adult C57BL/6 mice and Large White pigs. Our results demonstrate that miR-124 and miR-204 target sequences can efficiently restrict AAV2/5-mediated transgene expression to retinal pigment epithelium and photoreceptors, respectively, in mice and pigs. Interestingly, transgene restriction was observed at low vector doses relevant to therapy.ConclusionsWe conclude that microRNA-mediated regulation of transgene expression can be applied in the retina to either restrict to a specific cell type the robust expression obtained using ubiquitous promoters or to provide an additional layer of gene expression regulation when using cell-specific promoters.
Highlights
MicroRNAs are a class of small 20–25-nucleotide long non-coding RNAs that negatively regulate expression of their target genes by binding to specific sequence elements in the 39 untranslated region (UTR) of their respective mRNAs [1]
We sought to assess whether post-transcriptional restriction of associated viral (AAV)-mediated transgene expression to PRs could be achieved by exploiting endogenous miRNAs
For this purpose we selected miR-204, a miRNA that is strongly expressed in the retinal pigment epithelium (RPE) from as early as E10.5 to adulthood [25,26] (Figure 1a). miR-204 expression was absent from the PRs and was detected by in situ hybridization (ISH) at low levels in the inner nuclear layer of the neural retina as well in the ganglion cell layer [26] (Figure 1a)
Summary
MicroRNAs (miRNAs) are a class of small 20–25-nucleotide long non-coding RNAs that negatively regulate expression of their target genes by binding to specific sequence elements in the 39 untranslated region (UTR) of their respective mRNAs [1]. Incorporation of target sites for a specific miRNA (miRTs) at the 39 end of a transgene cassette has been adapted to provide a means of restricting transgene expression domains to specific cell types, lineages or differentiation states [4,5,6,7,8]. This strategy is useful to further improve transgene specificity, when combined with the transcriptional targeting provided by tissuespecific promoters. We evaluated the ability of retinal-expressed microRNAs to restrict AAV-mediated transgene expression to specific retinal cell types that represent the main targets of common inherited blinding conditions
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