Researchers at the University of Toronto have developed an RNA therapeutic approach that could treat genetic diseases linked to certain disease-causing mutations. The work advances an emerging platform in genetic medicine centred on transfer RNA, or tRNA. The team engineered tRNA to help cells read through premature stop signals and complete the production of full-length proteins that would otherwise be truncated or absent.
Study lead Bowen Li, associate professor at U of T’s Leslie Dan Faculty of Pharmacy and affiliate scientist at the University Health Network’s Princess Margaret Cancer Centre, said the research could lay the foundation for a class of drugs designed to treat 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,” says Li, who is also an affiliate scientist at the University Health Network’s Princess Margaret Cancer Centre.
“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.”
Li and his team focused on “nonsense mutations”. These mutations introduce a premature stop signal into the genetic instructions for making a protein. As a result, cells may produce little or no full-length functional protein, disrupting vital functions.
Although nonsense mutations are estimated to cause about 11 per cent of inherited genetic disorders, they number in the thousands, including subsets of cystic fibrosis and certain muscular and neurological diseases.
The study, published in Science on Aug. 27, shows that tRNA can be engineered to suppress disease-causing nonsense mutations and restore full-length protein production across a series of laboratory and preclinical models of cystic fibrosis. The researchers also found that the approach can be combined with existing cystic fibrosis drugs, suggesting the potential for combination therapy.
“The same type of premature stop signal can occur in many different genes, causing diseases that affect the lungs, brain, muscles and other tissues”, says 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.”
Study co-lead Haissi Cui, assistant professor of chemistry in the Faculty of Arts & Science, pointed the team towards an approach involving the addition of chemical tags found in natural tRNAs. One modification increased the activity and duration of the engineered tRNA.
“Interdisciplinary collaboration was key to this project,” says Cui. “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, said the next challenge was delivering the tRNAs to the cells that needed them. The team used lipid nanoparticles, the fatty bubbles that carried mRNA in the COVID-19 vaccines, with a redesign for tRNA delivery.
“No matter how powerful you make those tRNAs, without delivery, they cannot be a drug,” says 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 drugs called CFTR modulators, such as Trikafta. However, they 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, causing mucus to clog the airways and gut. Modulators repair and activate the misshapen CFTR protein that controls this flow. However, they cannot fix what was never built.
The U of T researchers tested whether tRNA could change this. The question was whether the CFTR protein would reappear and whether it would function. In human airway cells with two common nonsense mutations, the protein returned and functioned, remaining for more than 40 days. Further preclinical tests pointed in the same direction.
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 patient with a complex CFTR genotype containing four mutations, two of them nonsense, that left them unresponsive to existing drugs.
The samples were grown into miniature models called organoids. Neither the modified tRNA nor Trikafta had much effect on its own, but 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.
Lisa Dolovich, dean of the Leslie Dan Faculty of Pharmacy, said the study forms part of U of T’s tradition of drawing from the body’s own biology to develop treatments, from insulin to the GLP-1 discoveries behind drugs such as Ozempic.
“This is the kind of foundational research that medical breakthroughs are built on,” Dolovich says. “By tackling the science and the delivery together, we’re closer to turning a discovery into a drug.”
The researchers say the study demonstrates tRNA’s therapeutic potential. Li’s lab is looking to expand the approach to other organs, each of which will need a specialised delivery system. For the lungs, the team has shown that its particles can survive being turned into a fine mist, a first step towards a treatment that patients could inhale at home.
The research was supported by the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council, Cystic Fibrosis 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.