C. C. Ren, Li Song, Ming Hu, Xiu Jin, Licong Liang, Fanfei Liu, Jing Su, Jiamei Fu, Yiliu Yang, Yiliu Yang, Man Liu, Xiaoyi Wu, Xiaoyi Wu, Min Luo, Qiqi Li, Qiqi Li, Yifang An, Manjun Li, Kaiqin She, Yuquan Wei, Fang Lü, Yang Yang, Yang Yang
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-tRNAArg 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-tRNAArg significantly restores retinal function in mice, with effects lasting for at least 36 weeks. Furthermore, scAAV8.sup-tRNAArg 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-tRNAArg is a potential therapeutic intervention for IRDs caused by nonsense mutations in RPE65 gene. Insufficient efficacy is one of the major impediments for further application of sup-tRNA therapies. Here, authors engineer the body sequence of sup-tRNAArg and restored retinal function and vision-guided behavior in a mouse model carrying the RPE65-R44X nonsense mutation.