Engineered tRNA Restores Full-Length Proteins in Cystic Fibrosis Models, Offering a Common Strategy for Thousands of Genetic Diseases
核心洞察
University of Toronto researchers engineered transfer RNA (搜索) (tRNA) to suppress disease-causing nonsense mutations and restore full-length protein production across laboratory and preclinical models of cystic fibrosis (搜索).
A single chemical modification, N1-methyladenosine (m1A), increased suppressor tRNA readthrough activity by about 10.6-fold and extended its apparent half-life from roughly 12 to 29 days.
A custom lipid nanoparticle, TTP-3, delivered the tRNA to key airway cells, and combining the therapy with Trikafta restored CFTR (搜索) function in patient-derived organoids.
Researchers at the University of Toronto have developed a next-generation RNA therapeutic approach with the potential to treat a wide range of genetic diseases that share certain disease-causing mutations. The work, published in Science on Aug. 27, advances an emerging platform in genetic medicine centred on transfer RNA (搜索), or tRNA, by engineering tRNA to help cells read through premature stop signals and complete production of full-length proteins that would otherwise be truncated or absent.
Study lead Bowen Li, an associate professor in U of T's Leslie Dan Faculty of Pharmacy and an affiliate scientist at the University Health Network's Princess Margaret Cancer Centre, says the research could lay the foundation for a new class of drugs designed to treat a swath of genetic diseases through a common therapeutic strategy. "There are so many types of disease-causing mutations – many affecting only a small number of people – that developing a separate gene therapy for every individual mutation is extremely challenging," Li said. "With tRNA therapeutics, our goal is to develop a common therapeutic approach that could potentially address the same type of mutation across many different genes and diseases, including rare conditions that currently have few or no effective treatment options."
Targeting Nonsense Mutations
Li and his team homed in on "nonsense mutations," which introduce a premature stop signal into the genetic instructions for making a protein. The result is that cells may produce little or no full-length functional protein, disrupting vital functions in ways that are difficult to treat. Although nonsense mutations are estimated to cause only about 11 per cent of inherited genetic disorders, those number in the thousands, including subsets of cystic fibrosis (搜索) and certain muscular and neurological diseases.
While nonsense mutations occur in many different genes and cause many different diseases, they arise from only three possible premature stop signals. "The same type of premature stop signal can occur in many different genes, causing diseases that affect the lungs, brain, muscles and other tissues," Li said. "Our long-term goal is to develop tRNA medicines that recognize these shared stop signals, so that one therapeutic strategy could potentially be applied across many different genetic diseases."
Li described the mechanism with an analogy: "Think of tRNA as a little car. A nonsense mutation is like putting a stop sign in the middle of the road. The car has to slam on the brakes, and the protein never gets finished." He added that nonsense mutations are among the most damaging ways a gene can go wrong, because they "introduce a premature stop codon, which can prevent the full-length protein from being made or produce a shortened, usually non-functional protein. This leaves little or no functional protein for conventional small-molecule drugs to act on."
A Chemical Modification That Boosts Potency
While scientists have established tRNA's potential, turning it into a drug has proved difficult, with one hurdle being engineering a tRNA potent enough to restore meaningful levels of protein. Study co-lead Haissi Cui, assistant professor of chemistry in the Faculty of Arts & Science, pointed the team toward adding back the chemical tags found in natural tRNAs.
The team systematically tested modifications at different locations. One, called N1-methyladenosine, or m1A, produced particularly strong results when placed at position 57 or 58. In an arginine suppressor tRNA, the modification increased premature-stop readthrough by about 10.6-fold, improved attachment of the correct amino acid, prolonged the RNA's functional persistence and reduced innate immune activation.
"Interdisciplinary collaboration was key to this project," Cui said. "We used nature as our design guide and found that adding one specific modification made the engineered tRNA more active and longer-lasting. It shows what becomes possible when chemistry and RNA biology come together."
In cell experiments, one modified suppressor tRNA had an estimated apparent half-life of about 29 days, compared with roughly 12 days for its unmodified counterpart. Reporter activity from the modified RNA remained detectable after 30 days, and experiments in mice also showed activity lasting beyond 30 days.
A Delivery Vehicle Built Specifically for tRNA
The next challenge was getting the tRNAs to the cells that needed them. Jingan (Charles) Chen, a researcher in Li's lab and co-lead author of the study, said the team settled on lipid nanoparticles, the fatty bubbles that carried mRNA in the COVID-19 vaccines, but these needed a redesign to deliver tRNA.
The researchers used combinatorial chemistry to synthesize about 1,000 structurally diverse lipids in a single day, then screened them to find the right candidate. Their search produced TTP-3, short for tRNA-tailored pulmonary delivery–3. "No matter how powerful you make those tRNAs, without delivery, they cannot be a drug," said Chen, a PhD candidate in the Leslie Dan Faculty of Pharmacy and the Institute of Biomedical Engineering. "That cargo-specific delivery system is one of the major advances of our study. We used a tailored lipid nanoparticle delivery system that is specifically developed for tRNA."
After delivery into mouse lungs, about 60% of cells containing the tracked tRNA were epithelial cells. TTP-3 also reached several airway populations relevant to cystic fibrosis (搜索), including 22% of ciliated cells, 19% of club cells and 32% of basal cells, as well as about 15% of rare ionocytes.
Restoring CFTR Protein and Function
Cystic fibrosis (搜索) gave the researchers a practical test because about one in 10 patients has a nonsense mutation. In recent years, cystic fibrosis care has been transformed by a wave of drugs called CFTR (搜索) modulators, such as Trikafta, but these are not effective for the roughly one in 10 patients whose disease stems from a nonsense mutation. Modulators repair and activate the misshapen CFTR protein that controls salt and water flow, but they cannot fix what was never built.
In human bronchial epithelial cells carrying CFTR (搜索) nonsense mutations, modified suppressor tRNAs restored CFTR protein and its chloride-channel activity. In cells carrying the R1162X mutation, substantial CFTR levels remained for more than 40 days.
Next, research team members Jim Hu and Tanja Gonska, both SickKids scientists with appointments in U of T's Temerty Faculty of Medicine, provided access to tissue from a cystic fibrosis (搜索) patient with a complex CFTR (搜索) genotype containing four mutations – two of them nonsense – that left them unresponsive to existing drugs. These samples were grown into miniature models called organoids. Although neither the modified tRNA nor Trikafta did much on its own, the patient's cells responded when the two were used together. The tRNA restored production of the full-length protein and gave Trikafta something to work with.
"That was a great moment for us, where we saw the potential of the therapy," Chen said.
Selectivity, Safety, and Remaining Hurdles
The researchers found encouraging signs of selectivity. Ribosome profiling detected no global increase in readthrough at normal stop codons. Mouse safety experiments showed transient, dose-dependent lung inflammation at higher doses, while lower doses remained closer to controls, and no detectable liver toxicity appeared in the measurements reported.
Clinical development still presents major challenges. Different organs will require specialized delivery systems, repeated dosing must remain safe, and additional chemical modifications and tRNA designs need testing. The current experiments remain preclinical, and the patient-derived experiment involved only one participant, making it a proof of principle rather than evidence that the combination will work broadly.
For lung delivery, however, the team has already taken an early step toward a potentially more practical treatment. TTP-3 nanoparticles retained some activity after nebulization, although performance fell and further optimization is needed. That raises the possibility that a future version could be inhaled rather than injected.
Li's lab is looking to expand the approach to other organs, each of which will need a specialized delivery system. "We hope this is just the start of a much bigger future for this platform," Li said.
Lisa Dolovich, dean of the Leslie Dan Faculty of Pharmacy, sees the study as part of U of T's tradition of drawing from the body's own biology to unlock new treatments, from insulin to the GLP-1 discoveries behind drugs like Ozempic. "This is the kind of foundational research that medical breakthroughs are built on," Dolovich said. "By tackling the science and the delivery together, we're closer to turning a discovery into a drug."
The research was supported by the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council, Cystic Fibrosis (搜索) Canada, the Cystic Fibrosis Foundation, the New Frontiers in Research Fund, the Canada Research Chairs Program, the Connaught Fund, the Harrington Discovery Institute and the National Institutes of Health.