Why circular RNA could be the next revolution in RNA therapeutics

While RNA-based therapeutics are taking the biotechnology world by storm following the rapid development of the COVID-19 vaccines, the next generation of treatments could already be here.1 Despite being the lesser-known cousin of linear mRNA, circular RNA (circRNA) holds tremendous potential for emerging pharmaceutical applications by increasing the stability of the therapeutics and lowering manufacturing costs.

What is circular RNA? 

mRNA-based vaccinescircRNA is naturally produced by eukaryotic cells and consists of a single-stranded RNA with covalently linked 5ʹ and 3ʹ ends. There is still a lot to learn about the roles of these molecules in the cell, which have drawn interest as protein/gene sponges, cell activity modulators and disease biomarkers.1  
Although initially thought only to be non-coding, some natural circRNAs have since been discovered that code for proteins.1 This ability means that these intriguing structures have significant appeal as a new generation of mRNA therapeutics. Building on the success of modified linear mRNA, synthetic circRNA could help overcome some major challenges holding back the field. 

Advantages for developing therapeutics

The covalently-closed structure of circRNAs offers a significant stability advantage over linear mRNA for therapeutic applications. Without 5ʹ or 3ʹ ends, circular RNAs are more resistant to exonuclease degradation than linear RNA.2 This increased stability may promote pharmacokinetic accumulation in various cell or tissue types. This characteristic could mean that lower doses are required for therapeutic impact, which would reduce manufacturing costs and potentially improving patient safety.1 In addition, without a 5ʹ end, circRNA doesn’t require a capping agent, which accounts for about 40 percent of the spend on mRNA raw materials, making circRNA considerably less expensive to manufacture than linear mRNA.3  

The circular structure may also prolong product shelf life compared to current mRNA vaccines and relieve stringent storage and shipping conditions. Research has shown that LNP-encapsulated circRNA vaccines can be preserved for at least four weeks at 4 °C and two weeks at room temperature with little loss in expression.4 By comparison, linear mRNA resulted in lower levels of antigens produced at these temperatures.  
One challenge associated with circular RNA is that it lacks the 5ʹ end necessary for cap-dependent translation. However, circRNA can be modified to enable protein translation through an internal ribosome entry site or by incorporating m6A modifications upstream of the open reading frame.5 These modifications enable engineered circRNAs to express proteins similarly to modified mRNAs but for a longer duration.  
The protein-coding ability of circular RNAs, coupled with the stability, storage and pharmacokinetic improvements compared to linear mRNA, suggest that circRNA may be the next revolution in nucleic acid therapeutics.1   
Duchenne muscular dystrophy mRNA

Considerations for circRNA therapeutics developers

Engineered circRNA can be synthesised using one or multiple precursor linear RNAs that are circularised by chemical or enzymatic ligation.6 However, several obstacles must be overcome to use circular RNAs successfully in therapeutic applications: finding efficient circularisation methods for long in vitro transcribed (IVT) RNA, ensuring sufficient protein expression, and purifying circRNA adequately.5

Circularisation efficiency optimisation 
Long IVT RNAs pose challenges for typical enzymatic ligation circularisation. Typically, ribozymatic circularisation methods using self-splicing introns are more efficient for larger RNA fragments.5 Using this approach, researchers have been able to circularise RNAs up to 12 kb in length in vitro.6 
Translation efficiency optimisation 
For successful therapeutic applications, optimising the translation efficiency of circRNAs is critical.1 Research has shown that optimising five elements: vector topology, 5′ and 3′ untranslated regions, internal ribosome entry sites and synthetic aptamers recruiting translation initiation machinery can maximise circRNA translation.8 Together, these elements:  
  • May improve circRNA protein yields by several hundred-fold 
  • Provide increased translation compared to mRNA in vitro 
  • Deliver more durable translation in vivo 
  • Are generalisable across multiple transgenes
Cancer immunotherapy mRNA treatmentcircRNA purification 

Another aspect that may limit therapeutic utility is purity. Impure circRNA may induce strong immune responses, adding safety risks for patients.9 RNase R is a 3′ to 5′ exoribonuclease with helicase activity that digests most linear RNAs, while leaving circRNA untouched. This makes it invaluable for removing precursor linear RNA that remains after the circularisation process, helping to purify the therapeutic circRNA.8 

RNA-seq data from HeLa total RNA has shown that coupling poly(A)-tailing with RNase R in LiCl-containing buffer can enable more efficient degradation of linear RNAs, thereby resulting in more efficient enrichment of circular RNA transcripts.10 With engineered circRNA, high-performance liquid chromatography followed by RNase R treatment improves the quantity and stability of the protein produced.

Exciting applications of circRNA in therapeutics 



Figure 1. Examples of the types of therapeutic applications for circular RNA currently being developed

The molecular characteristics of circular RNA make it a useful candidate for various therapeutic applications, including protein and peptide replacement, vaccines, and biosensors.1 Many academic groups and biotechnology companies are currently investigating circRNA therapeutics for a range of indications, including:

  • Orna™, a therapeutics company specialising in circRNA products, uses the technology to create modified immune cells within a patient (in situ CAR therapy) and deliver improved gene therapy for Duchenne Muscular Dystrophy. 

  • Circio, a therapeutics company specialising in circRNA medications for cancer and rare diseases, has created an immunotherapy program that uses a patient’s immune system to fight cancer cells.  

  • A joint effort between Ginkgo Bioworks and Esperovax aims to develop circRNAs that specifically target colorectal cancer cells to induce the expression of a toxic payload that causes cell death. The novel treatment reduces toxicity and the resulting side effects from the death of non-cancerous cells. 

  • Researchers have developed a circRNA vaccine that encodes the SARS-CoV-2 receptor-binding domain. The team successfully induced potent and sustained neutralising antibodies via the LNP-encapsulated vaccine, which was reported to be highly heat-stable and could help to reduce cold-chain limitations.
It’s clear that circRNA could be the next revolution in nucleic acid therapeutics. More work needs to be done to develop efficient, cost-effective production systems that support scalable, commercial circRNA development.11 However, as more institutions devote time and research to circRNA therapeutics, many of the associated production and biological challenges will be overcome.  
LGC Biosearch Technologies™ supplies a range of enzymes for circRNA development and is the leading supplier of RNase R (Epicentre/Lucigen RNase R). 
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Related content

  1. Liu X, Zhang Y, Zhou S et al. Circular RNA: An emerging frontier in RNA therapeutic targets, RNA therapeutics, and mRNA vaccines. J Control Release. 2022. 348:84-94. doi: 10.1016/j.jconrel.2022.05.043 
  2. Abe H, Abe N, Uda M et al. Synthetic nanocircular RNA for controlling of gene expression. Nucleic Acids Symp. Ser. Oxf. 2009. 65–66. doi: 10.1093/nass/nrp033 
  3. Grand View Research. mRNA Synthesis Raw Materials Market Size, Share & Trends Analysis Report By Type (Capping Agents, Nucleotides, Plasmid DNA), By Application, By End-user, By Region, And Segment Forecasts, 2023 - 2030. https://www.grandviewresearch.com/industry-analysis/mrna-synthesis-raw-materials-market-report  
  4. Qu L, Yi Z, Shen Y et al. Circular RNA vaccines against SARS-CoV-2 and emerging variants. Cell. 2022. 185(10):1728-1744.e16. doi: 10.1016/j.cell.2022.03.044 
  5. Wesselhoeft RA, Kowalski PS and Anderson DG. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat. Commun. 2018. 9:2629. doi: 10.1038/s41467-018-05096-6 
  6. Dolgin E. Why rings of RNA could be the next blockbuster drug. Nature. 2023. 622, 22-24 doi: 10.1038/d41586-023-03058-7 
  7. Petkovic S and Muller S. Synthesis and engineering of circular RNAs. Methods Mol. Biol. 2018. 1724:167–180. doi: 10.1007/978-1-4939-7562-4_14 
  8. Chen R, Wang SK, Belk JA et al. Engineering circular RNA for enhanced protein production. Nat Biotechnol. 2023. 41: 262–272. doi: 10.1038/s41587-022-01393-0 
  9. Wesselhoeft RA, Kowalski PS, Parker-Hale FC et al. RNA circularization diminishes immunogenicity and can extend translation duration in vivo. Mol. Cell. 2019. 74(3):508–520 e4. doi: 10.1016/j.molcel.2019.02.015 
  10. Xiao MS and Wilusz JE. An improved method for circular RNA purification using RNase R that efficiently removes linear RNAs containing G-quadruplexes or structured 3' ends. Nucleic Acids Res. 2019. 47(16):8755-8769. doi: 10.1093/nar/gkz576 
  11. Lee KH, Kim S and Lee S-W. Pros and cons of in vitro methods for circular RNA preparation. Intl J of Mol Sci. 2022. 23(21):13247. doi: 10.3390/ijms232113247 

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