An engineered UGA suppressor tRNA gene for disease-agnostic AAV delivery

Suppressor transfer RNAs (sup-tRNAs) have the potential to rescue nonsense mutations in a disease-agnostic manner and are an alternative therapeutic approach for many rare and ultrarare disorders. Among all human pathogenic nonsense variants, approximately 20% arise from C-to-T transitions that convert the CGA arginine codon into a UGA stop codon. While recombinant adeno-associated virus (rAAV) has been successfully used to deliver a UAG-targeting sup-tRNA gene in vivo, extending this approach to UGA-targeting sup-tRNA genes has posed unique challenges related to rAAV vector production. Here, we demonstrate that an engineered UGA-sup-tRNA gene, designed with transcriptional regulatory elements, can be efficiently packaged into rAAV for in vivo delivery. A single administration in mouse models of two distinct lysosomal storage disorders restores enzymatic activity to approximately 10% of normal levels. Comparative analysis reveals differential sup-tRNA expression and aminoacylation patterns across tissue types, which correlate with enhanced therapeutic effects. The applied rAAV-based agents and engineering strategies expand the potential therapeutic scope of sup-tRNA therapies.