AAV-delivered suppressor tRNA overcomes a nonsense mutation in mice

Gene therapy is a potentially curative medicine for many currently untreatable diseases, and recombinant adeno-associated virus (rAAV) isthe most successful gene delivery vehicle for in vivo applications(1-)(3). However, rAAV-based gene therapy suffers from several limitations, such as constrained DNA cargo size and toxicities caused by non-physiological expression of a transgene(4-)(6). Here we show that rAAV delivery of a suppressor tRNA (rAAV.sup-tRNA) safely and efficiently rescued a genetic disease in a mouse model carrying a nonsense mutation, and effects lasted for more than 6 months after a single treatment. Mechanistically, this was achieved through a synergistic effect of premature stop codon readthrough and inhibition of nonsense-mediated mRNA decay. rAAV.sup-tRNA had a limited effect on global readthrough at normal stop codons and did not perturb endogenoustRNA homeostasis, as determined by ribosome profiling and tRNA sequencing, respectively. By optimizing the AAV capsid and the route of administration, therapeutic efficacy in various target tissues was achieved, including liver, heart, skeletal muscle and brain. This study demonstratesthe feasibility of developing a toolbox of AAV-delivered nonsense suppressor tRNAs operating on premature termination codons (AAV-NoSTOP) to rescue pathogenic nonsense mutations and restore gene function under endogenous regulation. As nonsense mutations account for 11% of pathogenic mutations, AAV-NoSTOP can benefit a large number of patients. AAV-NoSTOP obviates the need to deliver a full-length protein-coding gene that may exceed the rAAV packaging limit, elicit adverse immune responses or cause transgene-related toxicities. It therefore represents a valuable addition to gene therapeutics.

Automated summary

Gene therapy is a potential cure for currently untreatable diseases. This study demonstrates the successful use of recombinant adeno-associated virus (rAAV) to deliver suppressor tRNA (rAAV.sup-tRNA) and effectively treat a genetic disease in a mouse model with a nonsense mutation. The treatment lasted for more than 6 months and showed promising results for various target tissues. This research highlights the feasibility and potential benefits of using rAAV-delivered nonsense suppressor tRNAs to rescue pathogenic nonsense mutations.