Originally regarded as a genetic anomaly or an experimental artifact, circular RNA (circRNA) garnered attention after scientists discovered it had a functional role in human cells. In 2011, Erik Digman Wiklund, then a molecular biology graduate student at Aarhus University and now Circio’s Chief Executive Officer, published the first study describing a functional human circRNA (1). 

“I forgot about circRNA after that,” he said, recalling his transition from academia to a business career. A decade later, as circRNA emerged as a promising therapeutic avenue, Wiklund revisited his earlier work, spurred by its newfound popularity. After persuading a former laboratory colleague to join him, they founded a company aimed at developing innovative circRNA medicines for rare diseases, vaccines, and cancer. While circRNA therapeutics are still in early stages, Wiklund's team has demonstrated circRNA’s superior and enduring protein expression compared to traditional mRNA vectors, hinting at its potential to redefine DNA and virus-based therapies in the future. 

What is circRNA, and why is it important? 

CircRNA is a form of intracellular RNA that performs structural and regulatory functions. It is noncoding, which means it doesn't express proteins. Scientists had observed circRNA mostly in non-human cells. After our original discovery 15 years ago, more than 100,000 circRNA have been described in humans. By comparison, there are only 20,000 protein coding genes, so circRNA is a common molecule and must be important. We have only scratched the surface of what circRNA does biologically. As a researcher, it's a goldmine because it’s an unexplored area, and few scientists are working on it. From a therapeutic context, circRNA is exciting because of its chemical advantages. Linear RNA is chemically unstable, and it tends to get chopped off from the ends, either via hydrolysis or by enzymes. Since circRNA has no ends, it is naturally resistant to these processes, and therefore it hangs around in the cell longer.

What are the therapeutic applications of circRNA? 

An obvious development area would be vaccines where mRNA has been successful, but there’s so much more potential. We saw an opportunity to use circRNA for gene therapy, which typically starts at a higher level than mRNA vaccines, using DNA or viruses to deliver a gene that stays in a patient permanently. This is fundamentally different from vaccines, which aim for a quick burst of the immune response. The current gold standard of gene therapy, adeno-associated viruses (AAV), suffers from poor potency. It's hard to get it to express enough protein and express it for a sufficiently long time. Patients also need high doses, which is costly, drives toxicity, and can be lethal in some cases. But it's the only available platform that works. A gene therapy that expresses its protein via circRNA instead of mRNA will be presumably more durable, and it can accumulate at a much higher concentration inside the cell. Switching to circRNA expression could boost the protein output by tenfold compared to an AAV, which would reduce dosing and make it cheaper for patients. Our first goal is to use circRNA technology to improve the current gold standard of gene therapy.

Why did you choose alpha-1 antitrypsin deficiency (AATD) as a lead target for circRNA gene therapy?

AATD is a genetic disorder where patients fail to express alpha-1 antitrypsin, an enzyme produced in the liver that helps break down mucus in the lungs. This condition causes breathing problems, affects about 100,000 people in the U.S., and has a high unmet medical need. Scientists have tried AAV gene therapy for AATD, but this has failed to produce sufficient alpha-1 antitrypsin protein. We saw an immediate opportunity using circRNA to increase protein output. We can kill two birds with one stone by using an approach we call “silence and replace.” AATD patients suffer from both lung and liver pathologies due to toxic aggregates of the mutant protein accumulating in the liver. Our technology can address both issues by knocking down the mutant protein and simultaneously expressing the functional protein. This would be difficult to achieve with other approaches. 

How does circRNA compare with mRNA for vaccine development?

The immune system does not view circRNA as genetic material, so it doesn’t trigger the alarm bells of the immune system in the same way that mRNA does. Scientists can modify mRNA to increase its durability and reduce its immunogenicity, but making those changes is complicated and adds a lot of manufacturing costs. CircRNA is naturally more durable compared to mRNA, which will make it cheaper.

In 10 to 20 years, all RNA vaccines will likely be circular. 
- Erik Digman Wiklund, Circio

Unlike mRNA, which requires a protective cap at one end and a poly-A tail at the other, circRNA does not need either. COVID-19 vaccines require a cold chain. In contrast, circRNA can be at higher temperatures for longer, which would simplify supply chain logistics and improve vaccine access.

What other diseases are you targeting with circRNA?

We picked AATD as a lead disease target as proof-of-concept. For our secondary programs, we're focusing on tissues rather than specific genetic rare diseases. We are exploring tissues in which circRNA performs best. The liver is the easiest organ to target, so we are currently investigating delivery to the liver, but also lung and muscle, and we hope to start a central nervous system (CNS) program soon. Our plan is to evaluate in vivo performance in these tissues to identify where we achieve the best expression and then determine which diseases are relevant in those settings. For instance, we might focus on a group of diseases affecting the muscle or CNS based on our findings. Gradually, we could expand our work by developing initial programs in-house while partnering with others for broader applications.

In what other therapeutic areas do you think circRNA will have the most influence?

In 10 to 20 years, all RNA vaccines will likely be circular. We must go through clinical trials and get the manufacturing in place, but it’s a promising direction. Another exciting area for circRNA is in vivo chimeric antigen receptor (CAR) T cell therapy to transduce T cells or other immune cells with T cell receptors or CAR without the need for ex vivo processing. CAR T cell therapy is effective but cumbersome and expensive. It's likely circRNA will play an important role as in vivo CAR T cell therapy evolves. Gene therapy is moving to a DNA format, and in the future, we're not going to use viruses. Future gene therapies could use next-generation, redosable synthetic DNA systems expressing from circRNA. This shift will take time as there are delivery issues to solve, but circRNA could play a significant role in therapeutic contexts. CircRNA will also be relevant in protein and antibody manufacturing because a longer-lasting RNA will produce more protein faster. We're not a manufacturing company, but we're exploring licensing our technology to manufacturing companies.

Right now, our focus is on getting the first programs into the clinic and seeing some early clinical validation that it works in patients. I look forward to that data coming up. We are still two to three years away from that.

This interview has been condensed and edited for clarity.

Reference

1. Hansen, T.B. et al. miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA. EMBO J  30, 4414–4422 (2011).