Circio invited Alexander Wesselhoeft who is a pioneer in circular RNA research and a founder of ORNA Therapeutics, now doing consulting work for Circio. He gave an overview of the science and motivations behind developing circular RNA therapeutics. In short, the reason for using circular RNA is because it is more stable than linear RNA which leads to it having a potentially higher therapeutic index (i.e. a larger range of doses in which it is effective), so you could use much lower doses to obtain the same effect. This is particularly important for viral vector-based drugs, which have safety issues when administered in high doses. By incorporating circular RNA, lower non-toxic doses that are therapeutically active could be administered. This technology will likely supplant mRNA and DNA vector modalities in the future.

The first hurdle when creating circular RNA is the circularisation of the original straight RNA. The DNA (or template) from which RNA is produced is linear, so the resulting RNA has to fold back on itself. Furthermore, the design has to allow a large enough proteins to be encoded (at least several kB). Secondly, the expression of protein from the circularised mRNA has to be optimised. Circular mRNA is translated in the ribosomes through an IRES site. There are different IRES types with dramatically varying levels of protein expression, so the choice of the correct IRES is important. In fact, it can make the difference between a functioning drug and a non-functioning prototype. These problems have generally been solved, but research is ongoing to further optimise protein expression. Other problems not yet solved are purification and commercial-scale production of circular mRNA (which Circio can evade since it uses DNA as a vector) and targeted delivery to specific tissue types.

Alexander Wesselhoeft thinks that DNA based circular RNA therapeutics are likely superior to pure circular RNA in genetic diseases, due to it’s the potentially even longer period of protein production compared to circular RNA. Circular RNA, on the other hand, is likely superior for autoimmune diseases, while both technologies should work well in oncology and vaccines.

Circio has previously shown that circVec 1.0 has a 15 times longer half-life than single-stranded RNA in a bioinformatics model. In the webinar, results from an experiment with version 2.1 was shown. It showed an improvement in protein expression of 8.5 times over circVec 1.0 (in a luminescence model) after 192 hours (8 days).