What circRNA vaccine are being developed?

Introduction to circRNA Vaccines

Definition and Basic Concepts
Circular RNA (circRNA) vaccines are an emerging class of RNA vaccine platforms that utilize covalently closed circular RNA molecules as a template for protein translation rather than the traditional linear messenger RNA (mRNA). Unlike linear mRNA molecules, circRNAs lack the free 5′- and 3′-end structures, which renders them inherently resistant to exonuclease activity and thereby significantly enhances their stability in vivo. The basic concept is to engineer these circRNAs to encode antigenic proteins or fragments that stimulate an adaptive immune response when delivered into host cells. The designed circRNA includes essential regulatory elements, such as internal ribosome entry sites (IRES) or modifications (e.g., m6A) to facilitate cap-independent translation. This inherent stability coupled with persistent protein expression gives circRNA vaccines an advantageous profile over conventional RNA vaccines.

Historical Development and Significance
Although circRNAs were first discovered several decades ago in viroids and other systems, their significance in vaccine development has only recently been appreciated. Initial observations that circRNAs existed in various eukaryotic cells led to further investigation into their biological roles. However, it was the evolution of high-throughput sequencing and synthetic biology that opened the door for designing engineered circRNAs as vaccine candidates. Over the past few years, researchers have successfully synthesized circRNAs and demonstrated their utility in in vitro and in vivo models for triggering immune responses. For instance, early patents described circRNA vaccines based on group I intron elements such as the microcystis aeruginosa I-type intron for preventing infections like varicella-zoster virus. More recently, development efforts have focused on circRNA vaccines against SARS-CoV-2 and other emerging pathogens, highlighting the rapid evolution from concept to application. Their robust and prolonged antigen expression, reduced innate immunogenicity, and simplified manufacturing process make circRNA vaccines particularly significant in controlling rapid outbreaks and expanding therapeutic applications.

Current circRNA Vaccine Developments

Types of circRNA Vaccines in Development
Multiple circRNA vaccine approaches are under development, targeting various pathogens and disease indications. The major types include:

1. SARS-CoV-2 circRNA Vaccines:
- Several groups have developed circRNA vaccines that encode the receptor binding domain (RBD) or trimeric spike protein of SARS-CoV-2. These vaccines have been shown to generate high titers of neutralizing antibodies with a Th1-skewed immune response. Some designs incorporate modifications like IRES elements to drive translation activity in immune cells, enhancing the secretion of protective antigens.
- Notably, patents have described circRNA vaccines that encode therapeutic polypeptides based on coronavirus antigens, such as spike protein fragments, to prevent and treat COVID-19. These circRNA vaccines display advantages in stability and sustained antigen production compared to conventional mRNA vaccines.

2. Multivalent and Variant-Specific circRNA Vaccines:
- In response to the evolving variants of SARS-CoV-2, some circRNA vaccine candidates are being engineered to be multivalent. For example, circRNA vaccines that include sequences tailored for both Delta and Omicron variants have been developed. These designs can be used either as standalone vaccines or boosters to broaden the neutralization spectrum against emerging variants.

3. Cancer circRNA Vaccines:
- Beyond infectious diseases, circRNA vaccines are being examined for therapeutic cancer applications. Preclinical studies have demonstrated that circRNA vaccines encoding tumor antigens can stimulate potent CD8+ T cell responses and achieve tumor inhibition in multiple mouse tumor models. These vaccines are designed to harness the immune system to target antigens specific to malignancies, thereby serving as a next-generation cancer immunotherapy platform.

4. Lyophilized and Lymph Node-targeting circRNA Vaccines:
- Another development focuses on improving the storage and delivery of circRNA vaccines. One promising strategy involves formulating circRNA vaccines with mannose-modified lipid nanoparticles (LNPs) to target dendritic cells in lymph nodes, while also allowing lyophilization for enhanced long-term stability at 4°C. This design overcomes common logistical challenges associated with RNA vaccine cold chain requirements, making them more suitable for global distribution.

Leading Research Institutions and Companies
Research and development in the circRNA vaccine domain is being pursued both in academic settings and by biopharmaceutical companies. Leading contributors include:

- Academic Initiatives: Several peer-reviewed studies have emerged from prominent research institutions that focus on circRNA design, synthesis, and preclinical evaluation. For instance, early patents and academic reports have described the generation of circRNA vaccines based on various intronic self-splicing elements.
- Biopharmaceutical Companies and Startups:
- Companies that had previously focused on mRNA vaccine innovation are now venturing into circRNA vaccine development in light of its potential benefits over conventional linear mRNA vaccines. Collaborations involving circRNA vaccine formulations, LNP encapsulation techniques, and clinical development strategies have been reported.
- Furthermore, startups working on next-generation RNA technologies are actively engaged in advancing circRNA vaccine platforms, often supported by significant funding rounds as indicated in broader reports on RNA-based drug development.
- Patent Filings: A number of patents have been filed internationally concerning circRNA-based vaccines. For instance, patents describe various circRNA vaccine designs, preparation methods, and applications for infectious diseases and cancer, indicating a competitive and innovative landscape in this field.

Mechanism of Action

How circRNA Vaccines Work
CircRNA vaccines operate by delivering engineered circular RNA molecules into host cells, where they are translated into antigenic proteins. The lack of free RNA ends confers resistance to exonuclease degradation, resulting in prolonged persistence and sustained protein production. Key mechanistic components include:

- Translation Initiation via IRES Elements:
Since circRNAs lack a 5′ cap structure critical for canonical mRNA translation, translation is initiated using internal ribosome entry sites (IRES). Studies have demonstrated that selecting efficient IRES elements, such as the Enterovirus A (EV-A) IRES, can significantly enhance protein translation in immune cells.
- CircRNA Stability and Prolonged Expression:
The circular nature of circRNA provides remarkable stability under cellular conditions, enabling continuous antigen production over an extended period. This sustained expression is vital for maintaining an effective level of antigen and inducing a robust adaptive immune response over time.
- Delivery and Cellular Uptake:
Delivery modalities, such as charge-altering releasable transporters or LNPs, facilitate the efficient cellular uptake of circRNA molecules. In some designs, LNPs are modified, for instance with mannose residues, to target dendritic cells in the lymph nodes, further enhancing the immune response by promoting antigen presentation.
- Immune Activation:
CircRNA vaccines not only serve as templates for antigen production but also act as intrinsic adjuvants. This is evidenced by the transient cytokine release and activation of myeloid cells observed upon circRNA vaccine administration. The direct uptake by antigen-presenting cells leads to strong T cell priming and robust adaptive immune responses.

Comparison with Traditional RNA Vaccines
CircRNA vaccines offer several mechanistic and practical distinctions compared to traditional linear mRNA vaccines:

- Stability and Durability:
While mRNA vaccines inherently suffer from stability issues due to exonuclease degradation, circRNA vaccines maintain a covalently closed structure, offering an extended half-life and more durable protein expression.
- Translation Efficiency and Safety:
CircRNA vaccines use alternative translation initiation mechanisms (IRES-mediated), which can be optimized by structure-function analyses such as SHAPE-MaP to further enhance translational efficiency. Moreover, circRNAs tend to trigger lower innate immune responses because they avoid detection by certain RNA sensors, potentially leading to fewer side effects.
- Manufacturing and Scale-Up:
The simplified design and enhanced stability of circRNAs can translate into easier manufacturing and storage conditions. For example, the ability to lyophilize circRNA vaccines without compromising the targeting modifications improves their logistical feasibility compared to traditional mRNA vaccines that require stringent cold chain management.
- Adjuvant Characteristics:
In contrast to some mRNA vaccines that require additional adjuvants to elicit a strong immune response, circRNAs may provide intrinsic adjuvant properties due to their RNA nature and potential to interact with innate immune sensors transiently, thereby enhancing overall efficacy.

Applications and Potential Uses

Disease Targets
CircRNA vaccines are being developed with applications in a broad spectrum of diseases and conditions:

- Infectious Diseases:
- COVID-19: The most prominent target is SARS-CoV-2, for which circRNA vaccines encoding the spike protein RBD or trimeric spike structures have elicited potent neutralizing antibodies and T cell responses.
- Other Viral Pathogens: Beyond SARS-CoV-2, circRNA vaccines are also being conceptualized for other viral infections such as influenza, Ebola, and potentially emerging pathogens. The stable nature of circRNAs makes them attractive candidates for rapid vaccine development in response to new epidemics.

- Cancer:
- Cancer immunotherapy using circRNA vaccines is a novel approach where circRNAs are engineered to encode tumor-associated antigens. Preclinical studies have demonstrated that circRNA cancer vaccines can generate robust cellular immunity, leading to significant antitumor efficacy in various tumor models.
- These vaccines can be tailored to target specific cancer neoantigens, enabling personalized treatment strategies that induce a specific immune attack against cancer cells.

- Other Diseases:
- With further research, the application of circRNA vaccines could expand to include therapeutic vaccines for chronic infections, autoimmune modulatory vaccines, or even vaccines that prevent the development of severe disease forms, as observed with COVID-19.

Advantages Over Other Vaccine Types
CircRNA vaccines offer multiple advantages when compared with conventional vaccine platforms:

- Enhanced Stability:
The circular configuration of circRNA renders it resistant to enzymatic degradation, leading to a prolonged antigen presentation and sustained immune stimulation which is crucial for long-lasting immunity.

- Efficient and Prolonged Protein Expression:
Owing to the resistance to exonucleases and optimized IRES elements, circRNA vaccines can enable continuous and higher-level protein expression compared to linear mRNA vaccines. This property improves the immunogenicity and efficacy of the vaccine.

- Simplified Manufacturing and Storage:
The ability to lyophilize circRNA formulations without degradation or loss of function significantly eases the manufacturing, transportation, and storage challenges associated with mRNA vaccines, enabling broader global distribution.

- Reduced Innate Immunogenicity and Improved Safety Profile:
Due to their unique structure, circRNAs may circumvent excessive activation of innate immune pathways, reducing the risk of adverse inflammatory responses while maintaining the capacity to stimulate an adaptive immune response.

- Versatility and Adaptability:
CircRNA vaccine platforms can be rapidly adapted to incorporate sequences encoding different antigens, making them highly suitable for dealing with emerging variants or tailoring personalized cancer vaccines. The structural and functional modularity of circRNAs allows researchers to modify spacer sequences, microRNA recognition sites, and other elements to fine-tune the vaccine’s performance.

Challenges and Future Directions

Current Challenges in Development
Despite their promising benefits, circRNA vaccines face several challenges that need to be addressed for successful clinical translation:

- Optimization of Translation Efficiency:
Although the lack of free ends provides stability, achieving robust translation from circRNAs requires optimal selection of IRES elements and other regulatory sequences. Studies have shown that the choice of IRES, such as the EV-A IRES, is critical, but further improvements and standardizations are required.

- Manufacturing and Scalability:
While circRNAs are inherently more stable, the current in vitro synthesis and cyclization processes can be less efficient for producing lengthy circRNAs with high circularization rates. Advances in enzymatic and ribozyme methods are needed to scale up production while preserving high fidelity and minimizing the production of linear RNA contaminants.

- Delivery Systems and Formulation:
Ensuring efficient delivery into target cells remains a significant obstacle. Although lipid nanoparticles (LNPs) with targeting modifications (e.g., mannose modification) have shown promise, formulation stability post-lyophilization and targeted delivery are still under active investigation.

- Regulatory and Safety Assessments:
As circRNA vaccines represent a novel class of therapeutics, regulatory pathways are not yet fully established. Extensive preclinical and clinical studies are required to evaluate their long-term safety, immunogenicity, and potential off-target effects. Their ability to consistently induce the desired immune response without unintended activation of innate immunity remains a subject of ongoing research.

- Immunogenicity Fine-Tuning:
While circRNAs can provide intrinsic adjuvant effects, there is a delicate balance between sufficient immune activation and adverse inflammatory responses. Detailed studies are needed to understand the systemic and local immune responses elicited by these vaccines to ensure they meet the desired safety profiles.

Future Prospects and Research Directions
The field of circRNA vaccines is rapidly evolving, and several future directions are being explored:

- Structural and Functional Optimization:
Future research is likely to focus on detailed structure–function analyses using techniques such as SHAPE-MaP, to map and optimize the secondary structures of circRNAs. This will help in refining IRES elements, spacer sequences, and miRNA recognition sites to further boost translation efficiency and antigen expression.

- Next-Generation Delivery Systems:
Continued development of advanced delivery vehicles such as novel LNP formulations or charge-altering releasable transporters will enhance cellular uptake and tissue-specific distribution. Emphasis on targeting antigen-presenting cells through receptor-mediated endocytosis, for example via mannose modifications, is expected to improve vaccine efficacy and reduce dose requirements.

- Combination Approaches:
Integrative approaches that combine circRNA vaccines with other immunomodulatory therapies may offer superior outcomes, particularly in cancer applications. For instance, combination strategies involving circRNA vaccines and adoptive cell therapy, or immune checkpoint inhibitors, are being investigated to overcome the immunosuppressive tumor microenvironment.

- Clinical Trials and Translational Studies:
As circRNA vaccine candidates advance from preclinical studies, well-designed clinical trials will be crucial to establish efficacy, dosing regimens, and safety profiles in humans. Future research will provide insights into the long-term persistence of immune responses and may even pave the way for personalized circRNA vaccines based on patient-specific tumor neoantigens or rapidly evolving pathogens.

- Regulatory Framework Establishment:
The establishment of robust regulatory guidelines specifically tailored for circRNA products is vital. Collaborative efforts between regulatory agencies, academic institutions, and industry players will be critical in standardizing production processes, quality control measures, and safety evaluation protocols.

- Broader Disease Applications:
In addition to infectious diseases and cancer, circRNA vaccines are being evaluated for potential roles in combating emerging viral threats and even for therapeutic vaccines to modulate autoimmune diseases and other chronic conditions. The versatility of the circRNA platform suggests that, as research deepens, its applicability may widen to include a broad array of therapeutic areas.

Conclusion
In summary, circRNA vaccines are being developed as a next-generation vaccine platform with significant potential for enhanced stability, prolonged protein expression, and intrinsic adjuvant properties compared to conventional mRNA vaccines. The development landscape includes various designs addressing SARS-CoV-2—including multivalent formulations to target emerging variants—as well as cancer vaccines targeting tumor-specific antigens. Research from leading academic institutions and active biotech companies has resulted in multiple patent filings and preclinical studies that outline the design, synthesis, and delivery of circRNAs using effective IRES elements and LNP-based formulations.

Mechanistically, circRNA vaccines work by leveraging their closed-loop structure to achieve sustained antigen production via cap-independent translation, thereby triggering robust adaptive immune responses. Compared to traditional mRNA vaccines, they offer enhanced stability, improved manufacturing feasibility, and potentially reduced adverse effects due to lower innate immune activation.

Their applications span from combating infectious diseases like COVID-19—with circRNA vaccines encoding the SARS-CoV-2 spike protein components—to personalized cancer immunotherapies that induce potent cytotoxic T cell responses. Despite these promising advantages, challenges remain in optimizing translation efficiency, scaling up manufacturing processes, and ensuring targeted delivery and regulatory compliance. Ongoing research is directed towards structural optimization, advanced delivery mechanisms, combination therapies, and rigorous clinical testing to establish circRNA vaccines as a reliable and versatile technology in both prophylactic and therapeutic contexts.

Ultimately, circRNA vaccines represent a transformative approach in vaccinology with the potential to address critical public health challenges across diverse diseases. As research progresses and clinical trials validate their efficacy and safety, circRNA vaccine platforms are poised to become a cornerstone of next-generation vaccine technology, offering rapid, stable, and adaptable immunization strategies for the future.