The advantages of using circular RNAs—including increased durability, enhanced protein expression and substantially lower manufacturing costs compared to linear mRNAs—have driven a spate of investment in this technology.
When Thomas Hansen and I published our characterization of the first thoroughly studied endogenous human circular RNA in 2011, we had little idea of the importance this field would take on. Eleven years later, we launched Circio, a biotech devoted to harnessing the therapeutic potential of circRNAs, but by that time, we weren’t the only ones to recognize it, with other companies having also sprung up to develop these molecules into enhanced nucleic acid medicines. Now, a range of circRNA products are expected to enter the clinic in the next few years.
Multiple types of RNA have been in the clinic for therapeutics and vaccines since the turn of the millennium. Messenger RNA (mRNA) finally reached its first approval with the COVID-19 vaccines after over 30 years of development. However, linear mRNA has major limitations, and many in the industry are moving on to circRNA, whose benefits include increased durability (generating robust expression in animal models for 7 to 10 days, compared to just 2 to 3 days for the current gold-standard modified linear mRNA designs) and enhanced protein expression, as well as an expected 80–90% reduction in manufacturing cost vs. linear mRNA.
Most importantly, circRNA’s benefits can extend the therapeutic applications of RNA technology to novel areas such as gene and cell therapy. While only at the cusp of clinical entry, the data so far should interest all groups across the therapeutic development market. Let me explain.
What Is CircRNA?
CircRNA had been observed anecdotally in plants and vertebrates for decades and was detected in human cells more than 30 years ago. However, it was not until the early 2010s that circRNA garnered significant interest as a common, highly conserved, non-coding RNA species with a variety of important structural and regulatory functions. In the 13 years since Thomas Hansen’s and my initial human circRNA article, over 100,000 human circRNA species have been annotated, compared to only around 20,000 protein-coding genes in the human genome. This gives an idea of the biological importance of this class of RNA.
One of the most important reasons circRNA has potential as a therapeutic is its substantially increased durability compared to linear mRNA. In a process called exonucleolytic decay that occurs within all cells, ribonucleotides are chopped off one by one from the end of linear RNA. As a closed loop lacking a free end, circRNA is intrinsically resistant to this process, explaining why circRNAs tend to be some of the most abundant RNAs in human cells overall. One of the major drawbacks of mRNA as a therapeutic is its poor durability, and therefore the increased stability of engineered circRNAs has triggered substantial interest, with researchers working to design circRNAs that could express proteins. In 2019, a team of researchers at MIT succeeded in this pursuit, delivering a lipid nanoparticle (LNP)–packaged circular mRNA that led to robust protein expression in mouse models.
Based on this important milestone, oRNA Therapeutics of Cambridge, Massachusetts, was founded in 2021. Several high-profile biotechs have launched since then to capture this opportunity, including Sail Biomedicines, Orbital, Renegade, and, of course, Circio.
During the past three years, circRNA companies attracted around 40% of all VC capital that went into RNA-related concepts. This was reinforced in 2022 when Merck invested heavily to access the circRNA vaccine platform of oRNA Therapeutics, paying $150 million upfront and $100 million in equity. I, like most proponents of circRNA, believe that as the technology makes its way into the clinic and approved therapeutics, circRNA will rapidly make linear mRNA redundant as a therapeutic format.
CircRNA’s Potential for Gene Therapy
The simple replacement of linear by circular mRNA for infectious disease vaccines is an obvious first step in its therapeutic use, and is the aim of a number of development programs. However, companies are also now testing many ingenious ways to leverage the advantages of circular RNA in new settings. As one example, at Circio, we are exploring circular mRNA as a way to improve upon the durability and levels of protein expression from viral and DNA vectors. This has the potential to deliver a major, untapped opportunity for circRNA so far, bringing it into the space of genetic medicine.
Current gene therapy approaches, which are mainly based on the AAV vector, face major limitations due to low expression, which leads to high dosing levels and toxicity in patients. Circio has built a unique and powerful DNA-based genetic cassette, circVec, which codes for circular mRNA that is in turn translated into proteins inside cells. This has so far shown substantially improved protein output in vitro and prolonged durability in vivo compared to classic mRNA-based vector expression. We expect that circVec can boost protein output from an AAV by 10–100x over time. In addition to reducing the necessary dose, this could also lower the cost, which is a crucial step forward in widespread delivery of therapies to patients.
Furthermore, circRNA could potentially open up new disease targets where AAV gene therapy has not achieved sufficient expression levels, providing novel therapeutic options to patients for whom no treatments are currently available. Just as I expect that synthetic circRNA will replace linear mRNA for vaccines, I believe circRNA-based expression is likely to replace traditional mRNA vector systems in the future.
A New Type of CAR T Cell?
In addition to vaccines and gene therapies, circRNA can also be utilized for non-expression-related modes of action. These include antisense knockdown strategies and regulation of other RNA species such as microRNAs that are involved in many disease processes. For example, oRNA is developing a chimeric antigen receptor (CAR)–expressing circRNA that could engineer T cells directly in patients. If successful, this approach could circumvent the need for ex vivo processing of autologous cell therapy, potentially upending the CAR-T field as we know it today. oRNA intends to bring its lead CD19 circRNA candidate into clinical trials by 2026, and it is expected to be the first circRNA-based therapeutic to enter human studies.
While promising preclinical data have attracted significant interest from investors and the industry, circRNA will take time to demonstrate clinical results. Previous promising modalities such as mRNA and AAVs took decades to progress from promising research to approved therapies, and it’s uncertain how the preclinical results generated so far with circRNA will translate in patients. However, the biochemical advantages and potential breadth of applications for circRNA point to an exciting period ahead as several highly promising concepts are set to enter clinic development soon. While not all of these approaches will succeed, circRNA is poised to play an important role in both improving and shaping nucleic acid medicines of the future.
