Harnessing the Therapeutic Potential of Circular RNA

The rapid development and widespread use of mRNA vaccines during the COVID-19 pandemic brought RNA therapeutics and their potential into the spotlight. However, issues with mRNA durability and immunogenicity present ongoing challenges, driving interest in next-generation RNA therapeutics.

Technology Networks recently spoke with Erik Digman Wiklund, CEO of Circio, to learn about circular RNA (circRNA), a previously overlooked alternative RNA that could overcome some of mRNA’s limitations. As well as developing a cancer vaccine targeting KRAS mutations, Circio has established a circRNA platform, circVec, to produce multifunctional circRNA from DNA and viral vectors.

In this interview, Wiklund tells us about the discovery of circRNA and discusses some of the challenges of harnessing its full potential for therapeutic interventions.

Anna MacDonald (AM): What makes circRNA a major advance for RNA therapeutics?

Erik Wiklund (EW): circRNA is a significant advance towards next-generation mRNA. One of the major reasons for this is its ability to overcome the durability issue of mRNA, which is chemically unstable and therefore has a short half-life. circRNA has a closed loop structure with no free ends, and is resistant to exonuclease degradation, which is the major biological mechanism for RNA degradation inside of cells.

In addition, circRNA is less immunogenic than mRNA, avoiding RNA sensing mechanisms that normally detect and destabilize foreign RNA. The major result of this is that circRNA has intrinsic features that make it a more suitable and superior RNA format for therapeutic applications than mRNA.

Adding to these major advantages, circRNA is also expected to be cheaper to manufacture than mRNA. Because of the durability and immunogenicity issues described above, mRNA requires heavy modifications to be therapeutically active. This includes 5' end cap protection and utilization of nucleotide analogs to extend half-life and reduce immunogenicity – the latter discovery led to the mRNA Nobel Prize in Medicine in 2023.

By being circular, circRNA avoids the need for 5' end cap protection and the reduced immunogenicity omits the need to use nucleotide analogs. As these two process steps substantially drive up cost in mRNA manufacturing, it has been estimated that circRNA manufacturing at scale will be 80% lower than for current mRNA vaccine technology. 

AM: Why is it widely expected to replace mRNA as the RNA format of choice? 

EW: The opportunity to achieve extended durability and increased potency, combined with reduced manufacturing cost, suggests that circRNA will outcompete mRNA as the future RNA format of choice for vaccines and any other therapeutic strategy depending on efficient expression of proteins or peptides.

The expected improvement in shelf life and storage also has the potential to make circRNA vaccines more easily implemented and distributed in developing countries where healthcare logistics and infrastructure are more challenging.

However, the field is still in its infancy and, as with any next-generation technology, it will take time to be validated in the clinic and on a larger manufacturing scale. 

AM: Are there any challenges associated with circRNA therapeutics?

EW: One current challenge is that circRNA manufacturing is not yet available on a general basis from CDMOs, and therefore cannot easily be sourced for in vivo experiments, primate toxicology studies and clinical trials.

To date, the major circRNA players have invested in in-house CMC development and manufacturing systems, which drives up the investment need and limits entrants into the space. Once this bottleneck is resolved and circRNA production becomes more broadly available, we can expect that the field will rapidly expand with a large number of programs moving into the clinic.

As with any new and novel class of medicine, circRNA has yet to be tested in the clinic, and it remains unknown how the human body will react to a circRNA therapeutic. However, linear mRNA is now well understood clinically and nothing suggests that circRNA will pose a safety risk beyond the manageable issues known for mRNA. Human physiology is complex, though, and safety will need to be broadly tested and evaluated in patients.

AM: Why is DNA-format circRNA the key to unlocking the potential of circular RNA in genetic medicine?

EW: Synthetic circRNA or mRNA does not have sufficient durability to be relevant in a gene therapy context, where expression of a missing or dysfunctional protein is required on a permanent basis. Modified mRNA can typically achieve expression for 2–3 days in the human body. However, circRNA is estimated to extend this short expression window up to 7–10 days. This is very valuable for many applications, such as vaccines, but it is nowhere near the durability needed for gene therapy, where the aim is to achieve durable expression for months or years.

In addition, a major limitation of current gold-standard AAV-based gene therapy is the need for very high dosing levels. This increases the toxicity in patients and drives up manufacturing cost.

By switching from AAV-mRNA to more durable AAV-circRNA-based expression, it is possible to substantially increase the gene therapy output of each AAV vector. It is estimated that Circio’s DNA-format circRNA can reduce the dosing of AAV-based gene therapy by 10- to 100-fold, which would be a massive advantage over the classic AAV-mRNA strategy. Therefore, just as synthetic IVT circRNA is expected to replace synthetic mRNA for vaccines, DNA-format circRNA is expected to replace DNA-format mRNA for genetic medicine and beyond. 

AM: How was circRNA and its biological function discovered?

EW: CircRNA was overlooked for a long time by the wider scientific community. This was because RNA sequencing techniques selected against circular species and only picked up linear RNA. There were anecdotal reports in the scientific literature of the presence of circRNAs, but these were largely disregarded as being by-products of incorrect splicing without any biological function.

It was only in the late 2000s that Circio’s CTO, Dr. Thomas Hansen, and myself stumbled across a circular RNA species by chance whilst trying to elucidate an RNA-driven gene regulatory pathway. Further investigation demonstrated that this circRNA was highly abundant, actively regulated by the cell and conserved across species. We then explored further, and after adapting RNA sequencing technology, thousands of circRNA species were identified in human cells.

Today the total number of known human circRNA species stands at over 100,000. There are only around 20,000 protein-coding genes in the human genome, so this speaks to the abundance and biological importance of circRNA.

The function of most circRNAs remains unknown; however, it is clear that they are naturally non-coding (i.e., they do not express proteins) and mainly have regulatory and structural functions in the cell. The first, and still most well described, circRNA function is microRNA sponging. In this case, the circRNA acts as an anti-microRNA by sequestering a specific microRNA species in the cytoplasm, thus impacting the regulation of the microRNA´s downstream targets. 

In the last five years, the MIT-based scientist Alex Wesselhoeft in the Dan Anderson group demonstrated that circRNA could be manufactured synthetically and engineered to express protein in mice, and the concept of an IVT circular mRNA was born. Furthermore, they demonstrated that the circular mRNA could be delivered and achieve sustained expression in vivo in mice, which was the key discovery that really kick-started the field. This has led to some of the massive fundraising rounds and BD transactions we have seen in the circRNA space. 

AM: Which diseases will the differentiated circVec platform target?

EW: Circio has built a unique remove-and-replace concept with its circVec technology. This is applicable to genetic diseases where it is necessary to both remove a toxic mutant form and replace the functional protein. We use an RNAi-based strategy to remove the incorrect transcript, and the circRNA is used to replace the wild-type protein in a durable manner. Thus, we kill two birds with one stone. Alpha-1-antitrypsin deficiency (AATD) is an example of such a disease that Circio is targeting with its circVec remove-and-replace design, where both problems need to be solved.

AM: Can you tell us more about your cancer vaccine, TG01?

EW: Circio’s cancer vaccine TG01 is being used to target KRAS mutations, which are the most commonly occurring neoantigens and present in over 30% of all cancers. KRAS is one of the few clinically validated shared neoantigens, which means KRAS mutations can be targeted with an off-the-shelf product, rather than patient-specific vaccines that are tailored to each individual, which is a much more complex and costly approach.

TG01 targets the seven most commonly occurring KRAS mutations in one product, which cover 99% of KRAS-mutated pancreatic cancer and > 90% of lung and colorectal cancers that are KRAS positive. As such, TG01 can deal with a major unmet medical need and address a large patient population.

There were very promising data published at ASCO 2023 for a similar approach, so the concept is gaining strong momentum. However, there are only a couple of other KRAS-targeting cancer vaccines in early clinical development, so the opportunity remains largely untapped, despite the target being highly attractive commercially, and scientifically and clinically validated.

TG01 is currently being tested in lung cancer, pancreatic cancer and multiple myeloma, both in early and late settings and in different immunotherapy combinations. As this program reads out, Circio plans to select the most promising patient population and treatment combination to move into registrational development.

TG01 additionally plays a major advantage to Circio in having a more advanced clinical program with the potential to generate future revenues to invest into the circRNA platform development, which is the main priority for us due to its unique features and broad potential. 

Erik Wiklund was speaking to Anna MacDonald, Senior Science Editor for Technology Networks.

About the interviewee:

Dr. Wiklund is CEO of Circio. He has deep scientific knowledge in RNA and cancer biology, and 13 years of pharma and biotech industry experience in a variety of functions including R&D, finance and business development. Previous employment includes the radiopharmaceutical company Algeta, which was acquired by Bayer in 2014, Aker BioMarine, and management consulting experience from the Pharma & Health Care practice of McKinsey & Company. Dr. Wiklund holds a PhD in Molecular Biology from Aarhus University, Denmark, and the Garvan Institute of Medical Research, Sydney, Australia.