August 27, 2026
Published in Science Magazine featuring Bowen Li, PhD, University of Toronto, 2024 Oxford-Harrington Rare Disease Scholar
Nonsense mutations cause many genetic diseases by inserting premature stop codons (PTCs) that prevent full-length proteins from being made. Suppressor transfer RNAs (tRNAs) can bypass these faulty stop signals, but their current therapeutic use has been limited by inefficient PTC readthrough, immunogenicity, and delivery challenges. Chen et al. improved suppressor tRNAs through chemical modifications that enhanced therapeutic efficacy and persistence while reducing innate immune activation (see the Perspective by Myerson and Weissman). They also developed nonviral lipid nanoparticles tailored for pulmonary tRNA delivery. In models of cystic fibrosis ranging from cells to mice to patient-derived organoids, this nonviral approach restored the missing CFTR (cystic fibrosis transmembrane conductance regulator) protein production and function. Thus, nonviral-delivered suppressor tRNAs represent a promising therapeutic platform for treating diseases caused by nonsense mutations. —Stella M. Hurtley
Nonsense mutations introduce premature termination codons (PTCs) into messenger RNAs (mRNAs), preventing full-length protein production and accounting for ~11% of human genetic diseases. Current therapeutic strategies remain limited: Gene-editing approaches can face challenges related to delivery, immunogenicity, and off-target effects, whereas pharmacological readthrough agents have shown limited efficacy or considerable toxicity. Suppressor transfer RNAs (sup-tRNAs), whose anticodons are engineered to read through PTCs, offer an RNA-level strategy for restoring endogenous protein synthesis without altering the genome. This reversibility, transcript-level action, limited detectable off-target activity, and potential applicability across diseases sharing the same nonsense codon make sup-tRNAs attractive therapeutic candidates. However, their therapeutic translation has been limited by suboptimal readthrough, immune activation, short functional persistence, and inefficient delivery to disease-relevant tissues.
RNA modifications have emerged as key regulators of RNA stability, translation, immunogenicity, and therapeutic performance. Mature endogenous tRNAs are extensively modified for shaping tRNA folding, stability, aminoacylation, decoding, and interactions with the translational machinery. We thus hypothesized that installing defined chemical modifications into engineered sup-tRNAs could improve their activity and suitability as medicines. In parallel, effective translation of tRNA therapeutics requires cargo-tailored delivery vehicles capable of efficiently transporting structured tRNA molecules to disease-relevant tissues. We combined site-specific tRNA engineering with a large lipid nanoparticle (LNP) screen to develop a nonviral delivery platform optimized for sup-tRNA cargo. We used cystic fibrosis (CF) as a disease model to evaluate the therapeutic efficacy of this sup-tRNA platform because nonsense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene represent an unmet clinical need.
We synthesized various chemically modified sup-tRNAs and systematically tested the effects of site-specific modifications. Incorporation of N1-methyladenosine (m1A) at position 57 or 58 improved sup-tRNA function, increasing PTC readthrough by up to 10.6-fold for an arginine sup-tRNA. This modification also improved aminoacylation, increased functional persistence, reduced innate immune activation, and benefited sup-tRNAs charged with different amino acids. Screening more than 1000 ionizable lipids identified TTP-3 (tRNA-tailored pulmonary delivery–3), an LNP optimized for sup-tRNA cargo. After intratracheal administration in mice, TTP-3 LNPs efficiently delivered sup-tRNAs to airway epithelial and progenitor cells and showed preliminary aerosolization compatibility. Mango II aptamer insertion enabled in vivo tracking of sup-tRNA biodistribution, revealing delivery to epithelial progenitor cells relevant to CFTR expression and airway repair. In CFTR-mutant bronchial epithelial cells, modified sup-tRNAs restored CFTR protein expression and channel activity. In a CFTR R553X mouse model, LNP–sup-tRNA treatment improved CFTR-dependent swelling in intestinal organoids. In a CF patient–derived organoid model carrying a complex CFTR genotype, cotreatment with sup-tRNAs and Trikafta restored CFTR function, consistent with complementary restoration of protein production and modulator-responsive CFTR activity.
This work establishes a chemically modified, nonvirally delivered sup-tRNA platform for rescuing disease-causing nonsense mutations. Site-specific m1A57/58 modification provides a chemical strategy for enhancing sup-tRNA activity, persistence, and tolerability, whereas TTP-3 LNPs illustrate the importance of cargo-specific delivery design for structured tRNA therapeutics. The aptamer-tagging approach further provides a general method for tracking tRNA biodistribution and cell type targeting in vivo. These findings provide broadly applicable engineering insights for the future development of tRNA-based medicines.
Rare/Orphan, Respiratory
University of Toronto
Oxford-Harrington Rare Disease Scholar Award
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