B16F10-OVA and MC38-OVA | circRNA Applications Beyond Vaccines
Circular RNAs (circRNAs) offer inherent advantages over linear mRNAs due to their covalently closed structure, which confers resistance to exonuclease degradation, leading to superior stability and prolonged expression in vivo. However, their clinical translation has been hindered by two major obstacles:1. Immunogenicity: Unmodified RNA triggers potent innate immune responses. While linear mRNA overcomes this via nucleoside modification (e.g., pseudouridine), applying such modifications to circRNAs often disrupts the complex secondary structures of internal ribosome entry sites (IRES) required for translation, creating a dilemma where modification prevents efficient circularization or protein production.
2. Limitations on Rolling Circle Translation (RCT): Theoretically, circRNAs without stop codons could undergo highly efficient RCT. However, conventional IRES elements (600-700 nt) frequently contain unavoidable stop codons, effectively blocking this process.
Consequently, circRNA technologies have struggled to advance beyond vaccines into protein replacement therapies.
On June 29, 2026, a team led by Professor Liang Qu and Assistant Researcher Qian Pan from Fudan University, in collaboration with other institutions, published a pivotal study in Nature Biomedical Engineering titled "Engineered circular RNA compatible with complete nucleoside modification and rolling circle translation through a Cap-independent translation enhancer." By moving beyond traditional IRES and screening a cap-independent translation enhancer (CITE) from the Black Beetle Virus (BBV) , they simultaneously addressed both immunogenicity and RCT limitations.
This work achieved the delivery of fully nucleoside-modified circRNAs with low immunogenicity and demonstrated in vivo proof-of-concept for RCT platform in three distinct therapeutic areas: cancer immunotherapy, metabolic disease, and autoimmune disease. This significantly expands the potential applications of circRNA beyond vaccines.
Bypassing IRES: A 39-nt Element Solves Both Modification and RCT Challenges
Instead of modifying traditional IRES, the team turned to cap-independent translation enhancers (CITEs) derived from plant viruses. CITEs are shorter and contain fewer stop codons, making them inherently more suitable for RCT. After screening numerous CITE candidates, they selected a short 39-nt sequence from the Black Beetle Virus (BBV) . This BBV element consistently drives translation of modified RNAs and supports RCT in engineered circRNAs.To build a versatile platform, the team established two parallel circularization systems compatible with the BBV element:
· In vitro: A "scarless" PICC system based on a rearranged Tetrahymena group I intron.
· In vivo/In vitro: Twister ribozyme and RzL ligase ribozyme systems.
They demonstrated that incorporating modifications like mo5U and hm5C significantly reduced pro-inflammatory cytokines (IL-6, TNF) while preserving RCT capability. This integrated engineering framework concurrently addresses translation efficiency, circularization precision, and immunogenicity.
Validation in Three Therapeutic Areas: Expanding Beyond the Vaccine Narrative
To validate the platform's potential, the team performed in vivo proof-of-concept studies in three disease models:1. Cancer Vaccine: A Natural Application for Short Peptide Expression
The platform's efficacy was tested using circRNAs encoding the OVA antigen peptide in B16F10-OVA melanoma and MC38-OVA models, benchmarked against N1-methylpseudouridine (m1ψ)-modified linear mRNA vaccines.· Superior Efficacy: The circRNA vaccine showed better tumor growth inhibition than the linear mRNA vaccine.
· Significant Dose Advantage: At equimolar (and lower mass) doses, the circRNA vaccine achieved comparable or superior anti-tumor effects to a higher dose of the mRNA vaccine. The circRNA group also showed enhanced antigen-presenting cell infiltration and stronger CD8+/CD4+ T cell responses in the tumor microenvironment.
These results highlight the advantage of RCT for sustained short-peptide antigen expression. However, it's important to note these are results from a model antigen in an animal model.
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Figure 2. Nucleoside-modified circRNA cancer vaccines demonstrate a dose advantage in mouse models.
2. GLP-1 Peptide: Expanding circRNA's Therapeutic Reach Beyond Vaccines
This study extended circRNA application to metabolic disease by engineering a nucleoside-modified circRNA encoding the GLP-1 peptide, a key hormone for glycemic control.· Glucose Control: In mice, circRNA-GLP-1 significantly reduced the area under the glucose curve, showing efficacy comparable to semaglutide.
· Therapeutic Effects: In high-fat diet-induced obese mice, repeated administration improved glucose tolerance, reduced fasting/random blood glucose, lowered serum leptin and ALT levels, and alleviated hepatic steatosis.
While not intended to replace existing GLP-1 receptor agonists, this experiment serves as a "proof-of-concept" demonstrating that low-immunogenicity circRNAs can produce functional therapeutic short peptides, extending their potential use to chronic disease management and peptide replacement therapies.
Figure 3. circRNA-GLP-1 extends the platform's application to metabolic disease.
3. Autoimmune Disease: Low Immunogenicity as a Therapeutic Mechanism
In autoimmune diseases, the goal is immune tolerance, making the "invisibility" of low-immunogenicity RNA an asset. The team tested modified circRNA-MOG₃₅₋₅₅ in an experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis.· Alleviated Disease: Modified circRNA significantly reduced disease severity compared to unmodified circRNA.
· Mechanistic Changes: It increased regulatory T cells (Treg) and PD-1⁺CD4⁺ T cells in the spleen, reduced immune cell infiltration in the CNS, and lessened myelin damage.
This positions low immunogenicity not just as a safety feature, but as a potential component of the therapeutic mechanism, enabling tolerance induction in autoimmune diseases.
Figure 4. Low-immunogenicity circRNA induces autoimmune tolerance.
Delivery and Technical Considerations: Promise and Constraints
All RNA drugs require efficient delivery. The team also developed an EASY extracellular vesicle (EV) delivery platform for packaging circRNAs. This system showed delivery activity in vitro and in vivo with lower inflammatory cytokine induction compared to some controls. However, this is early-stage proof-of-concept, and EV production faces challenges in uniformity, loading efficiency, quality control, and scalable manufacturing.Platform Boundaries:
· Translation Strength: The BBV element's initiation activity is moderate; high protein yields rely on the cumulative effect of RCT ("loop-reading").
· Sequence Length Constraints: The most compelling RCT data so far involve short peptides. Whether the BBV element can support efficient RCT for large functional proteins (hundreds/thousands of amino acids) remains unproven.
· Compatibility Constraints: There is a strict matching relationship between nucleoside modifications and circularization systems; not all modifications are compatible, and their impact varies. Development requires finding the precise balance among modification type, translation element, and circularization method.
Conclusion: The Birth of an Independent Variable
This study's key value lies in its integrated engineering framework that resolves the long-standing conflicts between nucleoside modification, circularization efficiency, and rolling circle translation for circRNA platforms.
While this research is at the cell and animal model stage, requiring further validation in complex disease models and larger animals, it provides a clear direction for the field. It shows that the future of circRNA lies not just in "being more stable than mRNA," but in establishing a designable, manufacturable, and repeatedly administrable drug system capable of leveraging low immunogenicity, prolonged expression, and short peptide/protein output.
Success in these areas will allow circRNA to truly evolve beyond the vaccine narrative, creating a distinct and important space for itself in protein replacement, chronic disease management, and autoimmune tolerance induction—becoming an independent and significant variable in the RNA therapeutic landscape.
