Capasso, Dalila (2024) RNA-based approaches to enhance diagnosis and therapy of Inherited Retinal Diseases. [Tesi di dottorato]

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Abstract

Inherited Retinal Diseases (IRDs) are a group of genetic disorders characterized by a high genetic heterogeneity and clinical overlap. Over 310 causative genes have been identified to date, with a vast repertoire of pathogenic variants, yet about 30-40% of patients still lack a conclusive molecular diagnosis preventing them from having a proper genetic counseling, or therapeutic indications, when available. A huge effort is then required to increase the knowledge about molecular basis of these diseases in order to ameliorate patients’ general conditions. In this thesis, I aimed at exploiting different RNA-based approaches to address important issues pertaining to IRDs, involving diagnosis, new insights into IRD pathogenic mechanisms and possible therapies. The first one being described consists in a new pipeline demonstrated to be effective in highlighting deleterious transcripts alterations previously missed by other currently used techniques contributing to the diagnosis of four previously unsolved patients from our cohort. This method could provide a complementary tool to improve diagnosis for IRDs and other additional diseases when transcript anomalies could be present. The second effort we made was directed towards the identification and further characterization of long non-coding RNAs (lncRNAs) with a putative role in retinal functions and/or pathogenic mechanisms. To this aim, we applied co-expression analysis on pre-existing RNA-seq data from human retina donors and a set of functional experiments on the lncRNAs identified. Finally, we focused on a new strategy to simultaneously screen more miRNA targets to develop a therapeutic approach not relying on the specific genetic alteration involved in the disease etiology. To do so, multicistronic plasmids were designed for miRNA overexpression and silencing to be screened firstly in vitro and then, in vivo in a mouse model of IRD. Before concluding the screening, a beneficial miRNA was already identified able to decrease the rate of retinal degeneration demonstrating the efficacy of the multicistronic approach.

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