AAV-Delivered Engineered Suppressor tRNA Rescues Visual Function in Mice with an Inherited Retinal Disease (PRJCA038624)
Nonsense mutations change a sense codon into a premature termination codon (PTC) in mRNA and account for approximately 18.5% of human inherited retinal diseases (IRDs)-related mutation. Nonsense suppression therapies by small molecular drugs or suppressor tRNAs (sup-tRNAs) can introduce an amino acid at PTC, thereby, promoting the production of full-length proteins. While sup-tRNA-based therapies have shown promising results in cell culture models, challenges remain for in vivo delivery, particularly regarding efficacy, stability, and safety. In this study, we engineered the body sequence of sup-tRNAArg to enhance readthrough efficiency at clinically relevant PTCs in the RPE65 and ABCA4 genes, as well as at PTCs with +4 nucleotide shifts across all four types. By using the self-complementary adeno-associated virus (scAAV), we achieved restoration of RPE65 protein expression in up to 50.2% RPE cells in a mouse model carrying the RPE65-R44X nonsense mutation. Notably, the engineered sup-tRNAArg significantly restored retinal function and visual-guided behavior in mice, with effects lasting for at least 12 weeks. Furthermore, scAAV8.sup-tRNAArg exhibited minimal retinal toxicity and had negligible effects on the activation of unfolded protein response pathways, which is related to readthrough at normal stop codons. Our findings demonstrate the potential of scAAV-delivered sup-tRNAArg as a translatable therapeutic intervention for inherited retinal diseases harboring nonsense mutations.