Why the RNA world needed a new shape

The success of COVID-19 mRNA vaccines proved that synthetic RNA can be a safe, scalable way to turn a patient’s own cells into drug factories. Yet cracks soon appeared: linear mRNAs, which are produced through linear splicing, a canonical splicing process that joins exons in sequence to form messenger RNA, degrade within hours, demand deep-freeze storage, and can activate innate immune sensors that blunt repeat dosing, serious drawbacks for chronic-disease settings such as cancer¹. Researchers began asking whether the molecule’s chemistry could be re-engineered rather than endlessly tweaking caps, modified nucleotides, and lipid nanoparticles. The answer arrived in a deceptively simple geometry: a circle. Many circular RNAs (circRNAs) were initially classified as non-coding RNA, but recent research shows some circRNAs can encode proteins.

The structure of a circular RNA

Circular RNAs (circRNAs) are covalently closed, single-stranded RNA loops produced naturally by “back-splicing” at canonical splice sites; snipping off the vulnerable 5’ and 3’ ends makes them highly resistant to exonucleases due to their increased stability compared to linear RNAs, extending intracellular half-life from mere hours into multi-day territory². As nucleic acids, circRNAs are a focus of molecular cell biology research. In vitro, properly engineered circRNAs can drive 3- to 10-fold higher cumulative protein output and enhanced protein production compared to length-matched mRNAs, thanks to a phenomenon called rolling-circle translation that re-uses the same template repeatedly³. The efficiency of circRNA translation is influenced by translation initiation, and engineered circular RNAs can improve translation initiation to encode proteins more effectively. Equally important, circles appear less visible to pattern-recognition receptors such as RIG-I and MDA-5, reducing the need for heavy pseudouridine or cap analog decoration that complicates matters⁴, and potentially modulating the immune response to circRNAs. The RNA structure of circRNAs also plays a key role in their translation and stability.

Behind the scenes, new production tricks have made these loops practical at scale. In vitro transcription (IVT) is commonly used to generate linear RNA precursors for subsequent RNA circularization, which is a key step in circRNA synthesis. The use of permuted intron-exon (PIE) systems further enhances the efficiency of RNA circularization. Trans-splicing and enzymatic ligation can now stitch together kilobase-length constructs free of bacterial scars, while purification with RNase R removes any linear contaminants that might reignite innate^5-7. Advances in circRNA synthesis and engineering circular RNA have enabled the development of therapeutic circRNAs. Companies are also testing lyophilisation protocols that keep circRNAs stable at standard refrigerator temperatures⁸, and the importance of RNA delivery systems for therapeutic circRNAs is increasingly recognized. Exon intron circRNAs (ElciRNAs) represent a type of circRNA with regulatory roles in gene expression.

From nature to the lab: circRNA expression and biogenesis

CircRNAs have emerged as a fascinating player in the world of RNA therapeutics, thanks to their unique structure and promising therapeutic potential. Unlike linear RNA, which features exposed ends vulnerable to degradation, circular RNA forms a covalently closed loop, making it remarkably stable within cells. The journey from a precursor linear RNA to a mature circRNA involves a process known as back-splicing, where the 3’ end of an exon is joined to the 5’ end of the same or an upstream exon. This circularization is orchestrated by a network of RNA-binding proteins and spliceosomal components, which guide the enzymatic ligation that seals the RNA into a loop.

The expression of circRNA is not a random event; it is tightly regulated by the cell, influenced by factors such as cell type, developmental stage, and environmental signals. This precise control over circRNA expression allows cells to fine-tune protein production and translation efficiency, making circRNAs attractive candidates for therapeutic applications. By harnessing the natural biogenesis pathways of circRNA, researchers can design RNA therapeutics that deliver enhanced protein expression and improved translation efficiency compared to traditional linear RNAs. As our understanding of circRNA biogenesis deepens, so does the potential to engineer next-generation therapies that leverage the stability and versatility of circular RNA for a wide range of diseases.

Endogenous vs. synthetic circRNAs: Nature’s blueprint and human design

Endogenous circRNAs play diverse roles in gene expression, protein translation, and even disease progression, acting as regulators and sometimes as disease biomarkers. Their presence in various tissues and their involvement in key cellular processes make them intriguing targets for RNA therapeutics.

Synthetic circRNAs, on the other hand, represent the cutting edge of genetic medicine. By leveraging the principles of natural circRNA formation, scientists can design synthetic circRNAs with tailored properties, such as enhanced stability, improved translation efficiency, and reduced immunogenicity. This design flexibility opens the door to a new generation of RNA-based therapeutics, from vaccines that express specific antigens to protein replacement therapies for genetic disorders. Synthetic circRNAs can be optimized to highly efficient express proteins or peptides, making them ideal for applications where durable and robust protein production is essential. As research advances, the ability to engineer synthetic circRNAs with precise functions is poised to revolutionize the field of RNA therapeutics, offering hope for treating a wide spectrum of human diseases.

Building better cancer vaccines

Stability alone would not justify a platform switch; what excites oncologists is how that stability shifts the kinetics of antigen presentation. Mouse studies show that circRNA vaccine platforms, as part of the broader class of circular RNA vaccines, enable durable protein production, sustaining neoantigen expression for a week. This prolonged antigen presence leads to extended and robust immune responses, allowing dendritic cells to undergo multiple rounds of cross-presentation and prime higher-avidity  T-cell pools⁹. Because the backbone elicits weaker type-I interferonbursts, booster doses can be given without antagonistic innate noise, opening a path for multi-dose regimens that track tumour evolution¹⁰. Compared to mRNA vaccines and other mRNA therapeutics, circRNA vaccines may offer advantages in stability and the duration of protein expression, further enhancing their therapeutic potential.

Bench to bedside: RXRG001 may break the regulatory ice

All of this remained academic until recently, when RiboX Therapeutics announced the first patient dosed in a Phase I/IIa Clinical Trial of RXRG001, the first synthetic circRNA therapeutic for the treatment of radiation-induced Xerostomia and hyposalivation. The candidate encodes human aquaporin-1 (AQP1), a water-channel membrane protein whose expression is lost in salivary glands damaged by head-and-neck radiotherapy¹¹.

What regulators are watching

Regulators will scrutinize three issues:

Persistence vs reversibility. Greater durability is an advantage only if off switches exist.

Immunogenicity of circular junctions. The back-splice site creates a neo-epitope not present in native proteins.

Manufacturing comparability. Because rolling-circle constructs often exceed 5 kb and carry internal ribosome entry sites, any process change (eg, ligase source) could alter expression kinetics. The FDA’s RNA CMC guidance now demands circRNA-specific release assays for topology confirmation¹². Efficient RNA delivery systems, such as lipid nanoparticles, are also critical for the clinical success of circRNA therapeutics.

A pipeline taking shape

RXRG001 is merely the beachhead. At least half a dozen start-ups have disclosed circRNA pipelines spanning personalized melanoma vaccines, in vivo CAR-T priming, and liver-directed protein replacement. Over 40 pre-clinical circRNA assets were being developed in 2024, up from just six two years prior¹³.

Meanwhile, academic labs are deconstructing the rules of circular design: placing N6-methyladenosine at precise intervals boosts cap-independent initiation; nested coding cassettes can make multimeric antigens from a single loop; and self-cleaving ribozymes streamline “one-pot” IVT-ligation workflows^3,14. As these tools mature, the field is converging on a Lego™-like kit where payload, promoter, and chemical tweaks are swappable.

Outlook: From proof-of-concept to platform

If RXRG001 can show even modest clinical benefit without safety surprises, confidence in circRNA’s translatability will spike. Cancer vaccines stand to gain the most: they require long-lived antigen expression, repeated boosting, and clean safety in often-immunocompromised patients, exactly the attributes circRNA promises. CircRNAs are now recognized as promising RNA therapeutic targets, with potential to advance RNA-based therapeutics and mRNA vaccines. But avenues are possible: self-replicating circRNAs for gene editing, synergistic circRNA + mRNA regimens that layer short and long signal pulses, and all-RNA cell reprogramming where durability is mission-critical.

The tissue specificity of circRNAs, with over 10% of human circRNAs exhibiting tissue-specific expression patterns, opens new possibilities for targeted therapies tailored to specific tissues or organs. In addition, circRNAs are being investigated for their role in mesenchymal tumor progression, as certain circRNAs can influence tumor growth and angiogenesis by modulating gene expression in mesenchymal tumors.

Five years ago, the idea of an FDA-sanctioned circular RNA drug sounded like theory. Today, patients are receiving one in a Phase I study. As the pipeline widens, expect regulatory playbooks, analytical standards, and delivery technologies to co-evolve.

References

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6. Kim, Y. S.  et al.  The RNA ligation method using modified splint DNAs significantly improves the efficiency of circular RNA synthesis.  Anim. Cells Syst.  27, 208–218 (2023).

7. Du, Y.  et al.  Efficient circular RNA synthesis for potent rolling circle translation.  Nat. Biomed. Eng.  https://doi.org/10.1038/s41551-024-01306-3 (2024).

8. Wan, J.  et al.  Circular RNA vaccines with long-term lymph node-targeting delivery stability after lyophilization induce potent and persistent immune responses.  mBio  15, e0177523 (2024).

9. Seephetdee, C.  et al.  A circular mRNA vaccine prototype producing VFLIP-X spike confers a broad neutralization of SARS-CoV-2 variants by mouse sera.  Antiviral Res.  204, 105370 (2022).

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11. Xu, Y.  et al.  One-pot ligation of multiple mRNA fragments on dsDNA splint advancing regional modification and translation.  Nucleic Acids Res.  53, gkae1280 (2025).