A team of researchers from the Centre of New Technologies at the University of Warsaw, the Medical University of Warsaw, and the International Institute of Molecular and Cell Biology has achieved an unprecedented milestone in the field of mRNA circularization – the process of creating circular molecules of messenger RNA (mRNA) that deliver therapeutic genes. The circular structure engineered by the scientists significantly extends the lifespan of RNA molecules, enabling longer production of therapeutic proteins. This marks a major breakthrough in the development of next-generation RNA-based drugs, which could in the future be used to treat rare genetic diseases, including cystic fibrosis.
The study, titled “Chemical circularization of in vitro transcribed RNA for exploring circular mRNA design”, was published on July 12 in Nature Communications. The paper describes several pioneering achievements in RNA circularization, including chemical circularization of full-length protein-coding mRNA, in vitro RNA, CAP-dependent translatable circular RNAs, and chemically modified RNA.
“Circular RNA is an unusual form of messenger RNA. It does occur in nature, but it was only recently discovered, and for a few years now its therapeutic potential has been under consideration,” explains Prof. Jacek Jemielity from the Centre of New Technologies at the University of Warsaw in an interview with Newseria. “Biochemical methods of synthesis already exist, but they have certain drawbacks. For example, circular RNA typically translates like viral RNA. For it to translate like human RNA inside our cells, it requires the addition of a CAP structure. What we achieved for the first time was chemically closing these very large, fragile RNA molecules into a loop while also incorporating the CAP structure responsible for proper translation in human cells.”
Translation is the process of decoding the information in mRNA to produce proteins. In linear RNA, a CAP (a chemical “cap” structure) is located at one end of the molecule and is essential for translation. The researchers succeeded in closing RNA into a circle and chemically attaching the CAP structure to it – something only achievable through chemistry.
“Circular RNA has several advantages. The most important is that it lacks ends, which are the weakest points in linear mRNA where degradation usually begins. By eliminating these ends and closing RNA into a circle, the molecule becomes much more stable inside cells, and as a result, protein production lasts much longer,” Prof. Jemielity adds.
As a result, RNA serves longer as a template for protein synthesis. In some sequences, the amount of protein produced from circular RNA compared to linear precursor RNA was up to 370 times greater.
The scientists used a gentle chemical reaction to create circular RNA – one mild enough not to damage the highly sensitive molecules. At first, they tested the method on very short RNA fragments of just a few nucleotides.
“To our surprise, it worked quite well. Of course, we expected much greater difficulty when moving to mRNAs that actually encode proteins – molecules 1,000 to 2,000 nucleotides long, and the longest we tested was 4,000 nucleotides, encoding a COVID-19 vaccine,” says Prof. Jemielity. “We were astonished that, despite the enormous size of the molecules and the challenge of bringing their two ends together, the reaction was extremely efficient – with yields exceeding 60%.”
The research demonstrates that even extremely fragile RNA molecules can undergo chemical reactions successfully.
“When we first began this project, the idea seemed almost crazy. But this opens up an entirely new way of thinking about RNA modifications for therapeutic applications,” the professor emphasizes.
Extending the lifespan of mRNA creates new potential applications for this technology in medicine – particularly in cases requiring long-term protein production, unlike in vaccines.
“These are rare genetic diseases, where therapeutic proteins must be supplied for the patient’s entire life. Linear RNA, which degrades relatively quickly, is not well suited for such applications,” says Prof. Jemielity.
One example is cystic fibrosis, a lung disease caused by mutations in the protein responsible for ion transport.
“A single building block in this protein is replaced with another, and that small change causes dysfunction. The therapeutic idea is to deliver RNA encoding the correct form of this protein,” explains the researcher.
The team stresses that studies on circular RNA are still at an early stage. The next goal is to increase the efficiency of circularization, potentially to above 80%.
“We’ll also work on improving biological activity. The first in vivo tests in mice show that our circular RNA is biologically active, but now we aim to optimize its structure so that protein production not only lasts longer but is also as efficient as possible,” Prof. Jemielity notes. “Ultimately, the final stage will be demonstrating the therapeutic use of circular RNA. We’re already considering which disease model would best show that this technology works better than existing solutions on the market.”
