Characterising the use of microRNA as therapeutics for retinal degenerations

Age-related macular degeneration (AMD) is the leading cause of blindness in the western world, with a global prevalence expected to reach 280 million people by 2040. The most prevalent form of the disease, known as dry/atrophic AMD, currently has no cure or therapy. Genetic and molecular studies are beginning to untangle the intricate mechanisms underlying this complex disease, which is a required first step to develop better treatment strategies for reducing the incidence and progression of this disease. MicroRNA (miRNA) are short, non-coding RNAs that are pivotal in post-transcriptional regulation in almost every biological system. Furthermore, they have gained significant attention since the turn of the century for their crucial regulatory roles in various neurodegenerative diseases. Despite their importance in gene regulation in the central nervous system, the role miRNA play in the degenerating retina has been largely unstudied. The studies presented in this thesis help further elucidate the active role that miRNA play in retinal degenerations and investigate their potential for use as therapeutics in the retina. In this thesis, I have characterised miRNA involved in the degenerating retina using a sterile model of retinal inflammation, the photo-oxidative damage model, and use this model for investigating the use of miRNA as a therapeutic. First, through a novel technique in identifying functional miRNA involving high-throughput sequencing of retinas immunoprecipitated with a key protein required in miRNA activity (argonaute), an analysis of the active miRNA and their targets in retinal degenerations is provided. In particular, the importance and abundance of a particular miRNA, miR-124, is highlighted as potentially a key player in the maintenance of retinal homeostasis. Secondly, we examine the efficacy of a lipid-based transfection reagent, Invivofectamine 3.0, as a vehicle to allow for the alteration manipulation of miRNA expression levels in the retina through intravitreal injections. It is demonstrated that Invivofectamine 3.0 is a safe and efficient tool for the delivery of small nucleic acids, including miRNA, into the retina allowing for the further study of the downstream effects of such on the molecular biology and phenotype of the rodent retina. Finally, we demonstrate that miR-124 is strongly depleted in the degenerating retina in both human AMD and rodent retina. We demonstrate that miR-124 is present in the Muller glia and that it directly regulates CCL2, a pro-inflammatory chemokine highly expressed in AMD donor retinas. Further, intravitreal supplementation of miR-124 using Invivofectamine 3.0, ameliorated retinal function, reduced photoreceptor cell death and retinal inflammation, demonstrating the potential of miRNAs as therapeutics in the retina. This thesis allows for a deeper understanding of the role that miRNA play in the degenerating retina and will further unravel the molecular complexities of neurodegenerative diseases such as AMD, which may elucidate novel therapeutic strategies