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.
Engineering bacteria to secrete gut therapeutics has been limited by their poor autonomous sensing of pathological cues and inability to sustain localized, long-term therapeutic activity. Here we engineer nonpathogenic Escherichia coli with a blood-inducible gene circuit that secretes the barnacle-derived adhesive protein CP43K and the therapeutic gut-barrier-healing factor TFF3 in response to gastrointestinal bleeding, an indicator of severe inflammatory bowel disease (IBD). Adhesive production enables sustained bacterial attachment to inflamed tissues for up to 10 days or 7 days following a single rectal or oral administration, respectively. This effect depends on bleeding-induced adhesion. Using two mouse models of IBD, the colitis model induced by dextran sulfate sodium and the interleukin-10-knockout mouse model, we demonstrate improved weight recovery, reversed colonic shortening and reduced intestinal bleeding. Additionally, the treatment decreases intestinal inflammation, promotes mucosal repair and restores gut barrier integrity, demonstrating comprehensive therapeutic efficacy.
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