Viral vector-mediated RNA interference in the retina

RNA interference (RNAi) is a highly conserved post-transcriptional gene silencing process triggered by double-stranded RNA (dsRNA) in eukaryotic cells. Elucidation of the RNAi regulatory pathway and its components has led to the identification of endogenous dsRNA molecules, termed microRNAs (miRNAs), which are transcribed as a single hairpin molecule prior to their maturation into a cytoplasmic dsRNA. The efficient gene silencing achieved by these short hairpin RNA (shRNA) molecules and the cumulative understanding of the RNAi pathway has prompted the development of hairpin expression vectors capable of mediating stable gene silencing in vitro and in vivo. The aim of this thesis is to evaluate the efficacy of viral vector-mediated RNAi in the retina using recombinant adeno-associated viruses (AAV) and lentiviruses that contain silencing hairpin cassettes to target four genes in murine photoreceptors and the retinal pigment epithelium (RPE). A detailed assessment of the utility and extend of RNAi in the retina using different viral vectors and hairpin designs is presented in this thesis. Lentiviral and AAV vectors were firstly used to silence GFP in vitro and in vivo as a proof of concept for vector mediated RNAi in the retina. Subsequently, we used lentivirally-mediated RNAi to study disease processes in the retina concentrating on tight junction (TJ) modulators ZO-1 and ZONAB and their role in RPE homeostasis, cell-cycle progression and epithelial-mesenchymal transition (EMT). Here we demonstrated how TJ misregulation can lead to RPE loss, proliferation or dedifferentiation; processes involved in pathological conditions such as atrophic age-related macular degeneration (AMD) and proliferative vitroretinopathy (PVR). Whilst lentivirally-mediated RNAi was used to elucidate aspects of retinal function and disease, AAV-mediated RNAi was used to probe the therapeutic potential of shRNAs by silencing Peripherin-2 (Prph2), the second most abundant retinal protein, using a miRNA-based hairpin. AAV2/8 particles were used to target endogenous Prph2 and evasion of silencing was demonstrated using an engineered Prph2 cDNA that could be used in a suppression and replacement approach for the treatment of dominant retinal disorders