EYE MICRORNAS · Study of the functional role of microRNAs in the eye

Study of the functional role of microRNAs in the eye

MicroRNAs (miRNAs) are small noncoding RNAs that have important roles in the regulation of gene expression. At the beginning of this project, the contribution of individual miRNAs in vertebrate eye development was largely unexplored. This study aimed to enhance our understanding of the functional role of miRNAs expressed in the non-retinal parts of the eye. The main results obtained in the course of the project are the following: 1.comprehensive analysis of miRNA expression in the eye. To define miRNA function in the eye, it is essential to determine a high-resolution profile of their spatial and temporal distribution. In this project, we performed the first comprehensive survey of miRNA expression in ocular tissues, using both microarray and RNA in situ hybridisation (ISH) procedures (Karali, M. BMC Genomics. 2010). We initially determined the expression profiles of miRNAs in the retina, lens, cornea and retinal pigment epithelium of the adult mouse eye by microarray. Each tissue exhibited notably distinct miRNA enrichment patterns and cluster analysis identified groups of miRNAs that showed predominant expression in specific ocular tissues or combinations of them. Next, we performed RNA ISH for over 220 miRNAs, including those showing the highest expression levels by microarray, and generated a high-resolution expression atlas of miRNAs in the developing and adult wild-type mouse eye, which is accessible in the form of a publicly-available web database (http://mirneye. tigem. it). We found that 122 miRNAs displayed restricted expression domains in the eye at different developmental stages, with the majority of them expressed in one or more cell layers of the neural retina. This analysis revealed miRNAs with differential expression in ocular tissues and provided a detailed atlas of their tissue-specific distribution during development of the murine eye. 2. Functional characterisation of miR-204 using in vitro and in vivo systems. We then showed that a single miRNA, miR-204, which is expressed in the lens and cornea as well as in the retina and retinal pigment epithelium, regulates multiple aspects of eye development in the medaka fish (Oryzias latipes) (Conte, I.. PNAS. 2010). Morpholino-mediated ablation of miR-204 expression resulted in an ocular phenotype that involved also the non-retinal parts of the eye (e. g. lens). In particular, the obtained phenotype was characterised by microphthalmia, abnormal lens formation, and altered dorsoventral (D-V) patterning of the retina, which is associated with optic fissure coloboma. Using a variety of in vivo and in vitro approaches, we identified the transcription factor Meis2 as one of the main targets of miR-204 function. We showed that, together with altered regulation of the Pax6 pathway, the abnormally elevated levels of Meis2 resulting from miR-204 inactivation are largely responsible for the observed phenotype. These data provided an example of how a specific miRNA can regulate multiple events in eye formation; at the same time, they uncovered an as yet unreported function of Meis2 in the specification of D-V patterning of the retina. 3. Use of eye-expressed miRNAs to restrict trangene expression. Finally, we used the knowledge obtained from the miRNA expression profiling to select candidate miRNAs that could be exploited to fine-tune transgene expression in the eye. Gene transfer using adeno-associated viral (AAV) vectors has been successfully applied in the retina for the treatment of inherited retinal dystrophies. As part of this project, we evaluated the ability of retinal-expressed miRNAs to restrict AAV-mediated transgene expression to specific retinal cell types that represent the main targets of common inherited blinding conditions. To 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 (Karali, M.. Plos One. 2011). These vectors were administered subretinally to adult C57BL/6 mice and Large White pigs. Our results demonstrated 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. We concluded that miRNA-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. Overall, the project was successful in addressing the initial objectives. Primarily, the work carried out under this funding has enriched the knowledge on the spatial and temporal distribution of eye-expressed miRNAs. Moreover, it provided an elegant in vivo example of how perturbation of a single eye-expressed miRNA can yield strong ocular phenotypes and by target validation established the developmental pathway through which miR-204 exerts this function. As a side scope, this study confirmed that eye-expressed miRNAs can be used as tools to fine-tune AAV-mediated transgene expression in the retina. In a long-term perspective, the combined knowledge generated during this project sets the foundations for understanding miRNA contribution to eye pathological conditions, with eventual implications in miRNA-based diagnostics and therapy.

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