Researchers at the University of Toronto have developed a next-generation RNA therapeutic strategy that could have the potential to treat a wide range of genetic diseases that share certain types of disease-causing mutations. The team showed that chemically enhanced suppressor transfer RNAs (sup-tRNAs) combined with a lung-targeted delivery system can restore production of a critical protein in models of cystic fibrosis (CF) caused by “nonsense mutations.”
These mutations 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.
The researchers, led by Bowen Li, PhD, an associate professor in U of T’s Leslie Dan Faculty of Pharmacy, engineered sup-tRNAs to help cells read through these premature stop signals introduced by nonsense mutations into the mRNAs, and complete production of full-length proteins that would otherwise be truncated or absent. They tested the approach in bronchial epithelial cells, mouse models, and patient-derived cystic fibrosis organoids and found that the chemical modifications increased the readthrough of premature termination codons and tRNA aminoacylation, prolonged the tRNAs’ functional activity, and reduced innate immune activation. Moreover, the approach restored CFTR protein production and function across cell, animal, and patient-derived organoid models. The researchers also found that the approach can be combined with existing cystic fibrosis drugs, suggesting the potential for combination therapy.
Li, who is also an affiliate scientist at the University Health Network’s Princess Margaret Cancer Centre, suggests that 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. 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.”
Senior and co-corresponding author Li, and colleagues reported on their study in Science, in a paper titled “Nonviral delivery of chemically modified tRNA rescues nonsense mutations in cystic fibrosis,” concluding “These findings provide broadly applicable engineering insights for the future development of tRNA-based medicines.”
Nonsense mutations introduce premature termination codons (PTCs) in messenger RNA, causing protein production in cells to halt early, often resulting in truncated, nonfunctional proteins. Because these mutations account for ~11% of human genetic disorders, there is considerable interest in developing therapies that can restore production of full-length proteins. However, the authors wrote, “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 tRNAs (sup-tRNAs) offer a promising approach. By modifying their anticodons to recognize premature stop codons, engineered sup-tRNAs can insert the appropriate amino acid and allow the cell to resume translation of full-length, functional proteins. Yet the clinical potential of sup-tRNAs has been constrained by inefficient readthrough, immunogenicity, and difficulty in delivering them into the body.
To address this, the researchers chemically modified sup-tRNAs and engineered lipid nanoparticles (LNPs) for delivery into the lungs via inhalation, looking to see if they could repair the nonsense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene responsible for cystic fibrosis. “Interdisciplinary collaboration was key to this project,” says co-senior author Haissi Cui, PhD, assistant professor of chemistry in the Faculty of Arts & Science. “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.”
Jingan (Charles) Chen, a researcher in Li’s lab and co-lead author of the study, noted that another challenge was getting the tRNAs to the cells that needed them. The team settled on lipid nanoparticles, which they designed to deliver tRNA. “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.”
In recent years, cystic fibrosis care has been transformed by CFTR modulators, such as Trikafta. But such drugs are not effective for the roughly one in 10 patients whose disease stems from a nonsense mutation. Cystic fibrosis leaves cells unable to move salt and water, so mucus clogs the airways and gut. Modulators repair and activate the misshapen CFTR protein that controls this flow. But they can’t fix what was never built.
The U of T researchers set out to test if the sup-tRNAs could change that. “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,” they stated. The question was not just whether the CFTR protein reappeared, but whether it was active. Through their studies the team showed that in human airway cells with two common nonsense mutations, the protein came back and worked as it should, sticking around for more than 40 days. Further preclinical tests pointed in the same direction.
Research team members Jim Hu, PhD, and Tanja Gonska, PhD, both SickKids scientists with appointments in U of T’s Temerty Faculty of Medicine, provided access to tissue from a cystic fibrosis patient with a complex CFTR genotype containing four mutations—two of them nonsense—that left the the patient unresponsive to existing drugs. These samples were grown into miniature organoid models. Test showed that while neither the modified sup-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.
“Notably, in a patient-derived organoid model with a complex genotype, cotreatment with modified sup-tRNAs and Trikafta enabled functional rescue of CFTR,” they noted in summary. “In this setting, sup-tRNAs suppressed the nonsense mutations to restore full-length CFTR synthesis, whereas Trikafta supported the folding, trafficking, and activity of CFTR … This finding showcased a promising complementary therapeutic approach and highlighted the importance of combining therapies to rescue complex CFTR genotypes.”
The researchers say the study represents a major step forward in demonstrating tRNA’s therapeutic promise. Li’s lab is looking to expand the approach to other organs, each of which will need a specialized delivery system. For the lungs, the team has shown its particles can survive being turned into a fine mist—a first step toward a treatment that patients could inhale at home.
“The same type of premature stop signal can occur in many different genes, causing diseases that affect the lungs, brain, muscles and other tissues,” said Li. “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.”
In a related perspective, Jacob W. Myerson, PhD, and Drew Weissman, PhD, at Perelman School of Medicine, University of Pennsylvania, commented, “The results of Chen et al. have implications for thousands of cystic fibrosis patients.” Myerson and Weissman note that translating the effects reported in the researchers’ preclinical studies to amelioration of respiratory function in cystic fibrosis patients will further work, including characterizing side effects. They suggest that “… a likely therapeutic regimen could include both tRNA to rescue aberrant CFTR expression and drugs to enhance normal CFTR function, a combination that might have an additional side effect profile. Navigating the therapeutic potential of the tRNA, drugs, and LNPs may still be a matter of rethinking both the cargo and the carrier.”
