AAV-delivered engineered suppressor tRNA rescues visual function in mice with an inherited retinal disease

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 engineer the body sequence of sup-tRNA^Arg to enhance readthrough efficiency at clinically relevant PTCs in the RPE65 and ABCA4 genes. By using the self-complementary adeno-associated virus (scAAV), we achieve restoration of RPE65 protein expression in up to 50.2% retinal pigment epithelium (RPE) cells in a mouse model carrying the RPE65-R44X nonsense mutation. Notably, the engineered sup-tRNA^Arg significantly restores retinal function in mice, with effects lasting for at least 36 weeks. Furthermore, scAAV8.sup-tRNA^Arg exhibits minimal retinal toxicity and had negligible effects on global readthrough at native termination codons as revealed by ribosome profiling. Our findings demonstrate scAAV-delivered sup-tRNA^Arg is a potential therapeutic intervention for IRDs caused by nonsense mutations in RPE65 gene.