For what indications are circRNA vaccine being investigated?

Overview of circRNA Vaccines

CircRNA vaccines represent an emerging class of RNA‐based vaccines distinguished by their covalently closed, circular structure. This unique feature not only makes them more resistant to exonuclease-mediated degradation but also confers a longer half-life and more sustained protein expression compared to conventional linear mRNA vaccines. Their mechanism of action is based on the delivery of synthetic circRNAs that encode therapeutic polypeptides such as antigenic proteins, functional proteins, receptor proteins, or targeting proteins. When delivered in vivo—often encapsulated in a lipid nanoparticle (LNP) formulation—the circRNA can be taken up by cells, where it undergoes cap-independent translation to produce antigenic proteins that trigger both innate and adaptive immune responses.

Definition and Mechanism of Action

CircRNAs are a class of single-stranded RNAs that are produced by back-splicing of pre-mRNA molecules. In contrast to linear mRNA, circRNAs form a covalently closed loop without free 5’ or 3’ ends. This structure is crucial for their exceptional stability and resistance to degradation by exonucleases, which in turn enhances their durability when used as a vaccine platform. Once delivered into host cells, circRNAs use internal ribosome entry sites (IRESs) or N6-methyladenosine (m6A) modifications to initiate translation in a cap-independent manner. The antigen encoded by a circRNA is expressed over an extended period, providing prolonged immune stimulation compared to conventional mRNA vaccines. In addition, circRNAs have been found to possess self-adjuvant properties that can activate innate immune receptors and enhance T cell and B cell activation.

Advantages over Traditional Vaccines

Traditional vaccines, such as inactivated, attenuated, protein subunit, and even conventional linear mRNA vaccines, often face challenges including rapid degradation, reduced expression duration, and sometimes the need for strict storage conditions. CircRNA vaccines overcome several of these limitations because:
- Enhanced Stability: The circular structure protects the RNA from degradation and renders it thermostable, which could simplify cold chain logistics and reduce manufacturing costs.
- Prolonged Protein Expression: Due to extended half-lives compared to linear mRNA, circRNAs sustain antigen expression for a longer duration, potentially leading to improved immune memory.
- Self-Adjuvanticity: CircRNA molecules can themselves trigger innate immune pathways indirectly, reducing or even eliminating the need for external adjuvants.
- Ease of Manufacture: Since they require minimal nucleotide modifications and are less prone to degradation, circRNAs might enable simpler and more scalable manufacturing processes.

Current Research on circRNA Vaccines

The investigation of circRNA vaccines spans multiple indications, ranging from infectious diseases to cancer immunotherapy, and even extends to veterinary applications. Research in this field is rapidly accelerating as circRNAs continue to show promising advantages over conventional vaccine platforms. The evidence comes from structured patent disclosures, preclinical animal studies, and early phase clinical evaluations.

Indications Under Investigation

CircRNA vaccines are being actively explored and developed for multiple indications, including but not limited to:
- Infectious Diseases – SARS-CoV-2 (COVID-19):
The global urgency in addressing the COVID-19 pandemic has accelerated research into RNA vaccine platforms. Several patents and studies have specifically disclosed circRNA vaccines encoding nucleic acid sequences for the coronavirus Spike (S) protein or its fragments. For instance, describe circRNA vaccines that target SARS-CoV-2 by encoding the S protein or portions thereof, providing a basis for effective immunization against COVID-19. Moreover, patent outlines the development of circRNA vaccines specifically designed against SARS-CoV-2 variants such as Delta and Omicron. The rationale is that the enhanced stability and persistent protein expression of circRNAs may lead to robust immune responses and long-lasting protection, an essential attribute in addressing rapidly mutating viruses.

- Veterinary Applications – Infectious Spleen and Kidney Necrosis Virus (ISKNV):
In addition to human infectious diseases, circRNA vaccines are also being developed for veterinary use. Patent discloses a circRNA vaccine specifically targeting the infectious spleen and kidney necrosis virus. This virus is a significant pathogen in aquaculture and veterinary medicine, and the circRNA vaccine platform offers a promising approach to reduce the incidence of the disease in affected animal populations.

- Cancer Immunotherapy:
Beyond infectious diseases, circRNA vaccines are also being investigated as therapeutic cancer vaccines. Preclinical studies have shown that circRNA-based vaccines can induce potent antigen-specific CD8 T cell responses and have demonstrated tumor inhibition in multiple mouse tumor models. The concept here involves encoding tumor-associated antigens in a circRNA format to stimulate a robust antitumor immune response. The potential to combine circRNA vaccines with adoptive cell transfer therapies has also been explored, which could further boost the immune response against hard-to-treat cancers.

- Other Potential Indications – Respiratory Diseases and Beyond:
Some patents and literature suggest the investigation of circRNA vaccines for potentially treating or preventing acute lung injury and other inflammatory conditions. Although not as extensively documented as the work on SARS-CoV-2, this direction indicates that the inherent immunogenic and stable nature of circRNAs might be leveraged to deliver therapeutic proteins for non-viral, inflammatory, or immune‐mediated conditions.
In addition, there is expert commentary predicting that in the future, circRNA platforms might be adapted for mRNA vaccines addressing a variety of tissue-targeted therapies, such as in liver, lung, muscle, and even central nervous system (CNS) disorders. These investigations are still in early preclinical stages but highlight the expansive potential of circRNA vaccines.

- Multicistronic and Polycistronic RNA Vaccines:
Recent patents describe multicistronic and polycistronic RNA vaccines which include circRNA configurations as well as conventional and self-replicating RNA variants. Although these patents address a broader category of RNA vaccine platforms, they underline the feasibility of using circRNA's structure to encode multiple antigens simultaneously, thereby expanding the scope of indications, potentially including multi-pathogen protection or multi-antigen cancer vaccines.

Preclinical and Clinical Trial Data

The current body of evidence is primarily preclinical, involving in vitro experiments and animal models that showcase the promising immunogenicity and protective efficacy of circRNA vaccines:
- In Vivo Mouse Models:
Studies have demonstrated that circRNA vaccines can elicit robust anti-tumor immunity in mouse models. These studies reported that vaccination with circRNA encoding a tumor antigen can drive both innate and adaptive immune responses, leading to significant tumor growth inhibition in multiple cancer models. Similarly, circRNA vaccines targeting SARS-CoV-2 have been shown in preclinical mouse studies to produce potent neutralizing antibodies and T cell responses.

- Comparative Studies with mRNA Vaccines:
Additional studies have compared the performance of circRNA vaccines with self-amplifying mRNA vaccines. For example, presents data indicating that circRNA vaccines not only induce comparable antibody titers but also achieve significantly higher memory T cell responses and prolonged antigen expression. These preclinical comparisons underscore the potential of circRNA vaccines to improve upon the efficacy limitations sometimes observed in conventional mRNA vaccines.

- Patent Disclosures and Early-Stage Clinical Considerations:
While the majority of research is still in the preclinical phase, several patent applications reflect concerted efforts to transition circRNA vaccine candidates toward clinical evaluation. These patents emphasize the use of circRNAs in prophylactic settings against viral infections like COVID-19 and suggest that further clinical trials may soon assess safety, immunogenicity, and protective efficacy in human subjects.

- Potential in Veterinary Medicine:
The investigation of circRNA vaccines is not limited to human medicine. As described, a circRNA vaccine targeting ISKNV has been developed with the aim of reducing infection in susceptible animal populations. Although clinical trials in veterinary settings follow different regulatory pathways, the preclinical data provide critical insights into dosing, immunogenicity, and protective outcomes.

Potential Impact on Disease Treatment

CircRNA vaccines hold considerable promise for reshaping disease treatment paradigms. Their enhanced stability, prolonged antigen expression, and self-adjuvant properties offer advantages that could lead to more effective vaccines for both infectious diseases and cancers.

Effectiveness in Comparison to Existing Treatments

CircRNA vaccines, by virtue of their intrinsic molecular stability and robust translation efficiency, are positioned to provide several improvements over existing therapeutic approaches:
- Sustained Immune Activation:
Because circRNAs can sustain protein expression for extended periods, the immune system is likely to be exposed to the antigen for a much longer duration, which can result in stronger humoral and cellular immune responses. Compared with conventional mRNA vaccines that require cold-chain storage and have relatively short half-lives, circRNA vaccines may offer more durable protection. This sustained expression could be crucial in inducing long-lasting memory T cell responses, a key component in preventive as well as therapeutic settings.

- Lower Immunogenicity of the RNA Molecule Itself:
Unlike conventional mRNAs that may require chemical modifications to reduce innate immune recognition, naked circRNAs often possess a natural capacity to evade certain innate sensors, thereby potentially reducing reactogenicity. At the same time, the self-adjuvanting nature of circRNA formulations is beneficial for effective immune training without excessive inflammation.

- Broad-Spectrum Application:
The versatility of circRNA vaccine design—enabling multicistronic constructs—could allow for addressing multiple strains or even multiple pathogens within a single formulation. In the context of rapidly mutating viruses like SARS-CoV-2, a circRNA vaccine can be designed to encode antigenic variants. This broad-spectrum potential represents a significant improvement over vaccines that target a single antigenic variant, thereby mitigating the risk of immune escape.

- Implications for Cancer Treatment:
For cancer immunotherapy, the ability of circRNA vaccines to induce potent T cell responses is of particular interest. As seen in preclinical models, circRNA vaccines can stimulate robust cytotoxic T lymphocyte (CTL) responses that are critical for combating tumors. Compared to conventional therapeutic cancer vaccines, which sometimes suffer from poor immunogenicity or insufficient antigen persistence, circRNA vaccines may offer a means to overcome these barriers. This holds special promise for the treatment of hard-to-treat malignancies, where conventional immunotherapeutic strategies have not yielded sufficient clinical benefit.

Case Studies and Examples

Several examples from the literature and patent disclosures underscore the potential of circRNA vaccines across diverse indications:
- COVID-19 Vaccines:
In patents, circRNA vaccines encoding the SARS-CoV-2 Spike protein or its fragment have been designed. These constructs are aimed at eliciting robust immune responses against the virus. The early preclinical data suggest that such vaccines may induce strong neutralizing antibody responses and T cell responses comparable to, or even surpassing, those achieved with linear mRNA vaccines. This is particularly critical given the challenges posed by emerging viral variants that partially evade existing immunity.

- Veterinary Vaccines for ISKNV:
Patent exemplifies the veterinary application of circRNA vaccines. Here, a circRNA vaccine targeting the infectious spleen and kidney necrosis virus is developed to reduce disease incidence in affected animal populations. This application not only broadens the market for circRNA vaccines but also highlights the versatile manufacturing platforms that can be adapted across species.

- Cancer Immunotherapy:
Preclinical studies have demonstrated that circRNA vaccines encoding tumor antigens can drive potent immune responses and exhibit significant anti-tumor efficacy in mouse models. For instance, studies have shown that circRNAs can be formulated in LNPs for in vivo delivery, leading to robust antigen-specific T cell responses and reduction in tumor growth. These findings are a critical proof-of-concept for circRNA vaccines as a next-generation option for cancer immunotherapy, particularly in malignancies that have been refractory to conventional treatments.

- Potential Applications in Acute Lung Injury and Other Inflammatory Diseases:
Although not as extensively developed as the COVID-19 or cancer vaccine indications, some reports propose the use of circRNA-based approaches for conditions such as acute lung injury. The rationale is that the exceptional stability and translational capacity of circRNAs may be harnessed to deliver therapeutic proteins that modulate inflammation and tissue repair, opening potential avenues for non-infectious diseases.

Challenges and Future Directions

Despite their promise, circRNA vaccines face several challenges related to their design, manufacturing, delivery, and clinical evaluation. In addition, future research directions must address these limitations to fully realize the clinical potential of this novel vaccine platform.

Current Research Challenges

Several obstacles remain to be overcome before circRNA vaccines can be widely applied:
- Manufacturing and Cyclization Efficiency:
One crucial challenge is the efficient synthesis and cyclization of circRNAs. Although circRNAs are naturally more stable than linear RNAs, current in vitro methods for circularization often suffer from low yields due to gene sequence length and sequence-dependent efficiency issues. Improving these methods is imperative for scalable production.

- Delivery Systems:
The optimal delivery of circRNAs remains an area of active research. Lipid nanoparticles (LNPs) have proven effective in delivering RNA vaccines, but there are still questions related to tissue-specific targeting, biodistribution, and achieving high transfection efficiency with minimal off-target effects. Alternative delivery vectors (such as exosomes, virus-like particles, or advanced nanoparticle formulations) are under evaluation to optimize the therapeutic outcomes.

- Immunogenicity and Safety:
Although circRNAs can potentially evoke a balanced immune response, excessive or misdirected activation of innate immunity could reduce vaccine efficacy or cause adverse effects. The dual role of circRNA immunogenicity—as a self-adjuvant and as a potential inducer of unwanted inflammatory responses—represents a delicate balance that must be carefully tuned. In addition, while early preclinical studies are encouraging, long-term safety data remain limited.

- Clinical Validation:
The transition from preclinical studies to clinical trials is complex, and circRNA vaccines must be subjected to rigorous clinical evaluation. There is currently a paucity of data from human trials, and issues such as dose optimization, immunization scheduling, and evaluation of correlates of protection still need to be defined in clinical settings.

- Regulatory Hurdles:
As an emerging technology, circRNA vaccines do not yet have well-established regulatory guidelines. This situation creates uncertainty regarding the clinical trial design and approval pathways, especially when compared with conventional vaccine platforms that have decades of accumulated experience.

Future Prospects and Research Directions

Given the promising attributes of circRNA vaccines, long-term research is geared toward overcoming current challenges and broadening the spectrum of clinical indications:
- Advanced Cyclization and Manufacturing Techniques:
Future research initiatives should focus on optimizing in vitro circRNA synthesis. The development of more efficient cyclization protocols, possibly involving novel enzymatic or chemical ligation techniques, may significantly boost production yields and reduce costs. Patents are already paving the way for multicistronic and polycistronic constructs, which could encode multiple antigens or epitopes for broad-spectrum protection or combination therapies.

- Tailored Delivery Approaches:
Innovations in delivery strategies will be central to maximizing the efficacy of circRNA vaccines. The research community is actively exploring advanced LNP formulations and alternative carriers that can target specific tissues (e.g., lung, liver, muscle, or even the central nervous system) to deliver circRNA vaccines efficiently. In cancer immunotherapy, for example, precise targeting of the tumor microenvironment could significantly enhance the antitumor response while minimizing systemic side effects.

- Expanding Indications Through Multicistronic Platforms:
The ability to encode multiple antigens in a single circRNA molecule opens the door to broad-spectrum vaccines that can target multiple strains or even different pathogens simultaneously. In the context of SARS-CoV-2 variants, a bivalent vaccine that targets both Delta and Omicron variants has already shown potent neutralizing effects. Similar principles could be applied to develop vaccines against other rapidly mutating viruses or even composite vaccines that tackle both infectious diseases and cancer.

- Exploration into Cancer and Other Chronic Diseases:
The early preclinical evidence supporting circRNA vaccines in cancer immunotherapy is likely to spur further investigations into using circRNAs for the treatment of hard-to-treat malignancies. As our understanding of tumor immunology deepens, circRNA vaccines may become a key component of combination therapies, working synergistically with checkpoint inhibitors, adoptive cell therapies, or other immunomodulatory approaches. Additionally, the inherent advantages of circRNAs in terms of stability and protein translation hold promise for addressing chronic diseases beyond cancer and infectious diseases, including metabolic disorders and inflammatory conditions.

- Optimizing Safety Profiles and Immune Responses:
The dual nature of circRNA immunogenicity—providing adjuvant activity while avoiding excessive inflammatory responses—is a field ripe for innovation. Future studies may involve the precise engineering of circRNA sequences or the use of innovative modifications to fine-tune the immune activation profile. This will allow researchers to design circRNA vaccines that elicit the desired immune responses without triggering adverse effects, thereby improving their overall safety and effectiveness.

- Clinical Trial Design and Regulatory Pathways:
As circRNA vaccines progress from bench to bedside, comprehensive clinical trials will be essential. Early-phase trials should focus on safety, tolerability, and immunogenicity, gradually expanding to efficacy studies in larger populations. Collaborative efforts between academic institutions, industry players, and regulatory bodies will be key to establishing standardized protocols and guidelines for circRNA vaccine clinical evaluation. This collaborative approach is expected to streamline regulatory processes and hasten the clinical translation of circRNA vaccine candidates.

Conclusion

In summary, current research on circRNA vaccines is exploring a diverse range of indications. The most thoroughly investigated application is for infectious diseases—most notably COVID-19—where circRNA vaccines encoding the SARS-CoV-2 Spike protein have shown promising preclinical data and are included in multiple patent disclosures. Beyond COVID-19, circRNA vaccines are being developed for veterinary applications, such as those targeting the infectious spleen and kidney necrosis virus. Furthermore, pioneering preclinical studies demonstrate that circRNA vaccines hold potential in cancer immunotherapy, with evidence of robust antigen-specific CD8 T cell responses and tumor inhibition in animal models. Researchers are also beginning to consider the applicability of circRNA vaccines in conditions like acute lung injury and other inflammatory diseases, while expert opinion anticipates that circRNA platforms may soon be adapted for a wide range of tissue-targeted therapeutics across the liver, lung, muscle, and central nervous system.

From a general perspective, circRNA vaccines offer enhanced stability, prolonged protein expression, and self-adjuvant properties that can be pivotal in advancing next-generation vaccines. Specific preclinical data and robust patent filings underscore their potential for tackling urgent public health challenges such as the COVID-19 pandemic and emerging viral variants. At the same time, the versatility of circRNA vaccine design paves the way for applications in veterinary medicine and cancer immunotherapy, among other fields. However, challenges such as efficient manufacturing, optimized delivery, precise immunomodulation, and clinical validation remain and must be addressed through concerted research efforts.

Overall, the investigation into circRNA vaccines spans a broad spectrum of indications—from infectious diseases and viral variants to cancer treatment and beyond—with both human and veterinary applications being actively pursued. Future research aimed at refining circRNA production methods, tailoring delivery systems, and establishing robust clinical trial protocols will be critical for fully realizing the therapeutic potential of circRNA vaccines. If these challenges are met with innovative solutions, circRNA vaccines are poised to have a transformative impact on disease prevention and treatment, offering more durable, effective, and versatile vaccine platforms than current technologies.