The potential and challenges of circular RNA in the development of vaccines and drugs for emerging infectious diseases

Abstract

Circular RNAs (circRNAs) are a class of RNA molecules with a covalent closed-loop structure that play important roles in the regulation of biological processes and disease genesis. In recent years, circRNAs have become a research hotspot due to their unique stability and biological functions. Compared with linear RNAs, synthetic circRNAs have higher stability and show great potential in therapeutic applications. Although circRNA technology is still at an early stage of development, breakthroughs in mRNA technology provide an important reference for its application. This review systematically explores the promising applications of circRNAs in vaccine development and drug research, evaluates their feasibility as vaccine components and drug carriers, and experimentally validates their efficacy in disease models. At the same time, this paper analyzes the advantages and challenges of circRNA application in depth and looks forward to the future research direction, which provides new ideas for the prevention and treatment of new outbreaks of infectious diseases.

Graphical abstract

This review explores the potential of circular RNA (circRNA) in vaccine and drug development for emerging infectious diseases (EIDs). It highlights circRNA’s stability, immunomodulatory properties, and therapeutic promise, offering innovative solutions to overcome the limitations of traditional vaccine and drug strategies in rapidly evolving disease contexts.

Introduction

Emerging infectious diseases (EIDs) are infectious diseases that are newly emerging or have significantly increased in incidence and geographic distribution, and pose a major threat to global public health, economic and social stability. In recent years, multiple outbreaks of EIDs have taken a heavy toll¹: the 2013–2016 West African Ebola Virus Disease epidemic resulted in more than 11,000 deaths²; the emergence of SARS-CoV in 2003, MERS-CoV in 2012, and SARS-CoV-2 in 2019 have all caused global outbreaks, with SARS-CoV-2 having caused billions of infections and millions of deaths.^3,4,5 Apart from respiratory diseases, arboviruses are also a major public health threat. Arboviruses are viruses transmitted by arthropods such as mosquitoes, ticks, and fleas.⁶ The spread of arboviruses continues to expand as a result of climate change, globalized trade, and population migration, among other factors, making the need for related vaccines and therapeutics increasingly urgent.⁷ Mosquito-borne viruses, especially dengue virus and neo-bunyavirus, of which neo-bunyavirus is more prevalent in China and dengue virus is more prevalent in the tropics.⁸ The development of vaccines and drugs has become a key part of the response to these diseases.

However, existing vaccines and drugs still have many limitations in responding to EIDs. Traditional vaccine development has a long track record of time-consuming processes, which limits the ability to respond promptly to infectious disease outbreaks. Inactivated and live attenuated vaccines may have certain safety risks during use; while mRNA-based vaccines, although developed at a faster pace, still need to be further evaluated for long-term safety and efficacy due to their high requirements for stability and storage conditions.⁹ In addition, the development of broad-spectrum vaccines against viral variants remains a challenge. On the drug front, antiviral drugs are insufficiently specific and prone to drug resistance, and the development of novel viruses is lagging behind the epidemic. These shortcomings highlight the urgent need to develop novel vaccines and drugs.¹⁰ In this, cyclic RNA technology offers a new solution for vaccine development for EIDs.¹¹ The ability of cyclic RNA technology to enhance immune responses through its unique structure, along with its low immunogenicity and precise molecular regulatory mechanisms that may help to reduce the risk of side effects, make it a key component of global defense strategies as it demonstrates unique advantages in dealing with rapidly changing pathogens.

circRNAs are a class of endogenous RNA molecules with a unique circular structure that differs significantly from linear RNAs.¹² Its structural features are characterized by the lack of a 5′-end cap structure and a 3′-end polyadenylate tail, but rather by the formation of a closed ring structure through covalent bonds.¹³ In recent years, circRNAs have received extensive attention in the life sciences. Studies have shown that circRNAs are generated mainly by two splicing mechanisms: co-transcriptional post-splicing and selective splicing, which occur in the messenger RNA precursors transcribed by RNA polymerase II.¹⁴ circRNAs are highly abundant, stable, evolutionarily conserved, and exhibit tissue-specific and developmental stage-specific properties.^15,16,17 These unique properties make circRNAs potential biomarkers and therapeutic targets for a variety of diseases. Current studies have demonstrated that circRNAs play key regulatory roles in a variety of pathological processes such as cardiovascular diseases,¹⁸ renal diseases,¹⁹ and cancer.^20,21 Compared with mRNAs, circRNAs have significant advantages in terms of stability, expression persistence and immunogenicity, providing a broad prospect for their application in disease diagnosis and therapy.

The discovery and research history of circRNA can be traced back to 1976,²² when researchers first observed circRNA molecules formed by the processing of mRNA precursors while probing the tumor suppressor gene deleted in colorectal carcinoma and the human proto-oncogene Ets-1, and this discovery confirmed for the first time the eukaryotic cellular the existence of circRNAs in eukaryotic cells.²³ However, early studies generally assumed that these loop structures were byproducts of RNA splicing, and thus were not sufficiently emphasized.²⁴ The research turning point came in the early 2010s, when circRNA research entered a new era with the breakthrough of second-generation sequencing technology. In 2012, researchers systematically identified a large number of circRNAs in the mammalian transcriptome, some of which were expressed in abundance even more than their linear counterparts, suggesting that they may have important regulatory functions in life processes.²⁵ In recent years, significant progress has been made in the study of circRNA function, and it has been demonstrated that circRNAs are involved in the regulation of gene expression through a variety of mechanisms, including acting as microRNA molecular sponges, participating in protein translation, and interacting with host genes.²⁶ In 2018, circRNA research saw an important breakthrough as researchers successfully developed circRNAs with protein-coding functions, opening up new avenues for vaccine development.²⁷ In 2022, several research teams applied circRNA technology to the development of COVID-19 vaccine and therapeutic vaccines for oncology and genetic diseases, marking circRNA’s entry into the exploratory phase of clinical application.²⁸ As research continues, the important role of circRNAs in physiological and pathological processes is gradually being revealed, and their unique protein-coding ability opens up new possibilities for vaccine development. These breakthroughs have made circRNA research an important research direction in molecular biology and related fields, showing great potential in both basic research and clinical applications.

In recent years, circRNA research has made remarkable progress, and its application has expanded from basic research to the clinical translational stage.²⁹ In the field of vaccine development, circRNA vaccines have entered preclinical and clinical studies, and the preliminary data in Table 1 show that they have good immunogenicity and safety characteristics. Meanwhile, the potential of circRNAs in disease diagnosis and treatment is becoming more and more prominent, especially in the mechanism of colorectal cancer, diagnostic markers, and therapeutic targets.³³ However, the analysis of biological mechanisms of circRNAs, the exploration of clinical translation pathways, and the optimization of delivery systems are still the focus and difficulty of current research. With the rapid development of molecular biology technology, circRNA, as a new type of regulatory molecule, has demonstrated unique advantages in the fields of cell function regulation, disease mechanism analysis, and biomedical applications.³⁴ Studies have shown that circRNAs play key roles in a variety of physiopathological processes by participating in gene expression regulation, cell signaling, immune response regulation, and protein translation, and have become important targets for disease research and therapeutic intervention.³⁵ By combining the technical approaches and recent advances in circRNA research, we have not only deepened our understanding of its biological functions but also provided new strategies and tools for disease diagnosis and treatment.

Table 1.

Research on the use of circRNA as a vaccine

National Clinical Trial number, a unique identifier assigned to each clinical study registered on ClinicalTrials.gov	Study phase	Vaccine type	Participant number	Study year	Study status
NCT01799954	phase 1	HIV vaccine	96	2012	completed30
NCT02109354	phase 1	HIV vaccine	202	2013	completed
NCT02404311	phase 2	HIV vaccine	252	2015	completed
NCT02207920	phase 1	HIV vaccine	104	2014	completed
NCT02296541	phase 1	HIV vaccine	105	2014	completed
NCT03284710	phase 2	HIV vaccine	132	2017	completed
NCT02915016	phase 2	HIV vaccine	334	2016	completed
NCT02997969	phase 1	HIV vaccine	132	2016	completed
NCT03122223	phase 2	HIV vaccine	160	2018	completed
NCT03409276	phase 1	HIV vaccine	60	2018	completed
NCT02968849	phase 3	HIV vaccine	5404	2016	completed
NCT03060629	phase 2	HIV vaccine	2636	2017	completed
NCT00223080	phase3	HIV vaccine	16402	2003	completed
NCT04605159	phase 3	RSV vaccine	11194	2020	completed31
NCT04355351	–	SARS-CoV-2	303	2020	completed32
NCT04429594	–	SARS-CoV-2	1000	2020	not completed

This review focuses on the application of circRNAs in vaccine and drug discovery and development. By reviewing the research results in related fields, this paper comprehensively describes the application potential, technical challenges, and future development direction of circRNAs in these two key areas, and provides theoretical basis and research ideas for the clinical translational research of circRNAs.

Comparison of circRNA and mRNA

The research of mRNA technology started abroad, and the research progress was slow in the early stage due to the problems of poor stability, easy degradation, and low cellular uptake of mRNA molecules.³⁶ However, the outbreak of the New Crown epidemic led to a breakthrough in mRNA vaccines,³⁷ which have significant advantages: high expression efficiency, short development cycle, high immunogenicity, and large-scale production through in vitro transcription.^38,39 However, mRNA vaccines still have limitations, such as demanding storage and transportation conditions, high costs, and the possibility of triggering excessive immune responses in the body.⁴⁰

In recent years, circRNA has emerged as an emerging form of nucleic acid drugs. circRNAs have unique advantages over mRNAs: their covalently closed loop structure confers greater stability, extends half-life, reduces storage and transportation requirements, and promotes their accumulation in cells and tissues.⁴¹ In addition, the in vitro synthesis of circRNA does not require 5′-end-capping and 3′-end-poly(A) tailing as in linear mRNAs, nor does it require pseudouridine modification to reduce immunogenicity, and the production cost is more advantageous.⁴² By comparison, it can be seen that mRNA technology is more mature at this stage, especially in the field of vaccine development; whereas circRNA, with its stability and multifunctionality, shows great potential in the field of therapeutics and gene regulation, and provides a new direction for future drug development.

Application of circRNA in vaccines

Introduction to vaccines

The main types of vaccines include live attenuated, inactivated, subunit, nucleic acid, and vector vaccines. In order to more visually compare the characteristics of the different vaccine types, Table 2 summarizes the key differences between the aforementioned vaccines in a number of ways. The comparison provides a better understanding of the scope of application of each vaccine technology and its potential in disease prevention and control. Conventional vaccines rely on inactivated or attenuated technologies to stimulate innate, cellular, and humoral immune responses.⁹ However, there are clear limitations to these traditional vaccines: live-toxic vaccines may pose a risk of infection in immunocompromised individuals, and inactivated vaccines often require multiple vaccinations to maintain effective immunity. Among the types of vaccines available, the advantages and disadvantages of each make them suitable for different disease prevention and for specific populations. With the rapid development of biotechnology, the development of novel vaccine platforms has become a current hotspot. Among them, circRNAs have attracted much attention due to their unique molecular properties. Studies have shown that circRNAs have excellent stability, controlled immunogenicity, and persistent expression properties.⁵³ These features allow for extended antigen expression and enhanced immune response, demonstrating unique advantages in addressing EIDs and developing therapeutic vaccines.⁵⁴ The development of circRNA vaccine platforms is expected to overcome the limitations of traditional vaccines and provide new directions for vaccine development.⁵⁵

Table 2.

Comparison between different vaccines

	Attenuated live vaccine	Inactivated vaccine	Subunit vaccine	Vector vaccine	mRNA vaccine	circRNA vaccine
Structure	composed of attenuated live pathogens	composed of inactivated pathogens or parts of pathogens	composed of parts of the pathogen’s proteins or antigens	composed of viral vectors (such as adenovirus and adeno-associated virus) carrying exogenous genes	linear RNA, with a 5′-cap and a 3′-polyadenylated tail	circular structure, lacking a 5′-cap and a 3′-tail, with higher stability
Stability	poor, easily affected by temperature and light	good, convenient for storage and transportation	good, requires cold chain storage	good, but dependent on the stability of the vector43	lower, easily degraded by RNase	good, not easily degraded by RNase
Manufacturing process	more complex	simple	simple	relatively complex	relatively simple, with established in vitro transcription technology44	more complex, requires specialized circularization technology
Delivery method	injection	injection	injection, usually used with adjuvants43	injection, relies on the vector to enter cells	technologically mature, primarily delivered through lipid nanoparticle systems	still in the early stages, requires more research
Translation efficiency	high, it directly enters the body to produce antigens45	moderate, the vaccine requires cellular response in the body	low, usually requires adjuvants to enhance the immune response44	high, the vector virus transfects host cells to express the antigen46	high, can be directly used for protein synthesis47	low to moderate, some circRNAs have translation capability48
Immunogenicity	strong, capable of inducing a complete immune response49	moderate, immune response is mild and may require multiple doses50	weaker, requires adjuvants to enhance the immune response51	strong, capable of rapidly inducing a specific immune response	may cause an immune response	relatively low, may reduce immune side effects
Half-life	relatively long	relatively short	short	moderate	short, requires repeated dosing	relatively long, may enable long-term expression with a single dose
Precision targeting	poor	poor	moderate	high	weak, prone to degradation and interference	strong, with high stability and cell specificity15
Production cost	relatively high	relatively low	relatively low	moderate	mature technology, with gradually decreasing costs	not yet fully commercialized, with high costs
Application range	applicable to common viral diseases (such as measles, smallpox, etc.)	applicable to viral diseases and some bacterial infections	suitable for diseases that require protein immunity, such as influenza, HPV, etc	suitable for the rapid control of emerging diseases (such as Ebola, COVID-19, etc.)44	it has been widely applied in vaccine development, gene therapy, protein expression, and more	it has great potential in disease diagnosis, therapeutic targets, gene regulation, and other areas
Sideeffects	mild	relatively small, but with weaker immune effects	fewer, but with a weaker immune response52	fewer, but there may be immunogenic reactions	it may cause local or systemic immune responses	relatively small, but long-term safety still needs to be studied
Research maturity	both research and application are relatively mature, with widely used vaccines already available	the research maturity is high, and many inactivated vaccines have already been approved	in research, some vaccines have already entered the clinical trial stage	the research is relatively mature and has been applied in certain diseases (such as Ebola)	it has been widely applied in clinical research and treatments, such as the COVID-19 vaccine	it is primarily in the basic research stage, with clinical applications still in the exploration phase35

To demonstrate more clearly the mechanism of action of circRNA vaccines in vivo, Figure 1 summarizes the whole process of circRNA vaccines from delivery to immune activation.

Figure 1.

The mechanism of action of circRNA vaccines diagram

Advantages of circRNA vaccines

High stability

The unique covalent closed-loop structure of circRNA endows it with significantly better stability characteristics than linear mRNA. Unlike linear mRNAs with a cap at the 5′-end and a poly(A) tail at the 3′-end, the closed-loop structure of circRNAs allows them to effectively evade recognition and degradation by nucleic acid exonucleases.¹³ It has been shown that the intracellular half-life of exonic circRNAs generally exceeds 48 h, which is much higher than the average 10-h half-life of mRNAs.^15,56 This excellent stability allows circRNAs to show significant advantages in alternative therapies: not only can they achieve higher yields of protein expression, but they can also maintain longer-lasting therapeutic protein levels, thus potentially dose sparing.⁵⁷ Taking SARS-CoV-2 vaccine development as an example, circRNA vaccines encoding the receptor-binding domain of spiking proteins have demonstrated higher stability and longer half-life than linear mRNA vaccines in both in vitro and in vivo experiments.^58,59 This property not only improves the storage stability of circRNA vaccines but also significantly reduces their stringent requirements for cold-chain transportation conditions, which facilitates the widespread use of vaccines.

Enhanced immunogenicity

circRNAs exhibit unique advantages in regulating host immune responses. Studies have shown that circRNAs can effectively activate the host’s innate immune system, induce a strong immune response, and promote the formation of long-term immune memory, thus providing a durable protective effect for hosts.⁶⁰ As a novel immunostimulant, circRNA enhances the immune response through multiple pathways: on the one hand, it can regulate B cell and T cell functions, break the state of immune tolerance, and enhance the body’s ability to recognize pathogens⁶¹;on the other hand, circRNA significantly enhances the host’s antiviral immune defenses in viral infection models through mechanisms such as activating RIG-I-like receptors and initiating the interferon response.⁶² These properties give circRNAs significant advantages in the prevention and treatment of infectious diseases and provide new ideas for the development of novel vaccines and immunotherapies.

Safety

The unique molecular structure of circRNAs confers significant safety advantages. Unlike linear mRNAs, circRNAs lack a 5′-cap structure and a 3′-poly(A) tail and have reduced immunorecognition, a feature that not only reduces unwanted immune responses, but also significantly reduces unwanted inflammatory responses and other side effects.⁶³ More importantly, circRNAs do not integrate into the host genome and therefore do not pose a risk of mutation or genetic alteration.⁶⁴ In addition, the targeting and specificity of circRNAs in disease treatment indirectly reflect their safety advantages. For example, circPVT1 regulates miRNA-16 through the ceRNA (competing endogenous RNA) mechanism, thereby affecting the proliferation and survival of tumor cells.⁶⁵ This precise molecular regulatory mechanism enables circRNAs to directly target disease-related molecular pathways, which theoretically minimizes interference with normal tissues and reduces the risk of adverse effects during therapy.⁶⁶ This potential for targeted therapy provides a new dimension of consideration for the safety of circRNAs as vaccines and therapeutic agents.

Design flexibility

The design flexibility of circRNAs is mainly reflected in their programmability and versatility, which provides a wide scope for biomedical research and applications.⁶⁷ By precisely designing the sequence of circRNAs, molecules with specific functions can be constructed.⁶⁸ Researchers can introduce chemical modifications such as 2′-O-methylation and phosphorylation on circRNAs to enhance their stability and functionality.⁶⁹ In addition, circRNAs not only carry specific antigenic coding sequences for precise targeting of pathogens, but also integrate multiple immune adjuvants to enhance the immune effect.⁷⁰ This versatility allows circRNAs to exhibit remarkable flexibility in vaccine design, enabling the customization of vaccine formulations to specific needs. For example, circRNA-000203 has shown potential application in cancer therapy by designing multiple miR-7 binding sites to effectively inhibit miR-7 functions.⁷¹

Although circRNA vaccines offer many advantages and show great potential, their application still faces some challenges and limitations, and these disadvantages need to be overcome in further research and development.

Disadvantages of circRNA vaccines

Development and production complexity

Although circRNAs show many potential advantages, their development and production technologies are still in their infancy. The synthesis and purification of high-quality circRNAs require specific technical platforms and specialized equipment, a technological threshold that limits their widespread application to some extent.⁷² The production process of circRNA involves several key steps, including RNA synthesis, cyclization reactions, and product purification, each of which may introduce impurities or affect the quality of the final product.⁷³ To address these challenges, researchers are exploring more efficient production strategies, such as the use of plasmid DNA templates or gene synthesis techniques to prepare circRNAs, which can not only reduce production costs, but also significantly improve the yield and quality of circRNAs, laying the foundation for their large-scale application.⁷⁴

Difficulty in regulating the immune response

Despite the significant advantages of circRNAs in enhancing immunogenicity, the precise regulation of their immune responses still faces major challenges. Excessive or inappropriate immune activation may lead to strong inflammatory responses or autoimmune problems.⁶⁰ Animal studies have shown that some circRNA vaccines may induce strong local or systemic immune responses, resulting in adverse reactions such as pain, redness, and swelling at the injection site.^75,76 To address this challenge, researchers are exploring structural engineering strategies to modulate the immunogenicity of circRNAs. Specifically, the sequence or spatial structure of circRNAs can be rationally designed to reduce their excessive interactions with immune receptors, such as Toll-like receptors and RIG-I-like receptors, so as to achieve fine regulation of the intensity of immune responses.⁷⁷ This strategy based on structure optimization provides new ideas for the safe application of circRNA vaccines.

Limited clinical validation

Currently, research on circRNA vaccines is still mainly confined to the laboratory stage, with relatively limited clinical studies and validation data. circRNA vaccines still need to be systematically evaluated and validated for safety and efficacy in large-scale clinical trials.⁷⁸ To ensure the quality and safety of circRNA vaccines, regulatory agencies need to establish appropriate evaluation criteria and approval processes. For example, authorities such as the U.S. Food and Drug Administration and the European Medicines Agency are required to fully evaluate the manufacturing process, safety, and efficacy of circRNA vaccines to ensure that they meet the criteria for clinical use.⁷⁹

Although the development of circRNA vaccines still faces many challenges, it shows great potential for application in the field of vaccine development. circRNA’s unique molecular stability and immunomodulatory properties make it an important direction for the development of next-generation vaccines. With further research and technological development, circRNA vaccines are expected to provide new solutions for the prevention and control of infectious diseases.

Application of circRNA in drugs

With the in-depth research on the biological functions of circRNAs, their potential application in the therapeutic field has become increasingly prominent. In recent years, the study of circRNAs as novel therapeutic agents has made remarkable progress in the field of EIDs, especially in the prevention and treatment of viral infections, abnormal immune regulation, and other diseases, showing unique advantages.⁸⁰ The unique closed-loop structure and stable expression properties of circRNAs make them a potential therapeutic tool for coping with EIDs. With the deepening understanding of its molecular mechanism, researchers are actively exploring the innovative applications of circRNAs in disease treatment, providing an important direction for the development of novel therapeutic strategies.⁸¹

Drug carriers

The unique covalent closed-loop structure of circRNA confers excellent in vivo stability, enabling efficient RNase degradation and long-lasting activity in blood and other body fluids.⁸² Based on this property, researchers are developing circRNAs as novel drug delivery vectors: on the one hand, exploring the encapsulation of therapeutic molecules in circRNAs to improve drug stability and bioavailability⁸³; on the other hand, utilizing circRNAs as nanocarriers to achieve precise drug delivery. Studies have shown that circRNAs can serve as ideal vectors for siRNAs or miRNAs to achieve therapeutic effects by regulating the expression of specific genes.⁸⁴ For example, circRNA-0001946 is able to act as a molecular sponge for miR-138, upregulating the expression of its target genes by binding to miR-138, thus exerting an oncogenic role in breast cancer.⁸⁵ This circRNA can target tumor cells through delivery systems such as lipid nanoparticles, providing new strategies for cancer therapy.

The circRNA vector technology not only opens up new avenues for cancer treatment, but also provides innovative ideas for the prevention and treatment of EIDs. In antiviral therapy, circRNA can be used as an efficient carrier to deliver antiviral small molecules or immunomodulatory factors, which significantly improves the targeting and efficacy of treatment.⁸⁶ This circRNA-based delivery system is expected to overcome the limitations of conventional drugs and provide a powerful tool for dealing with EIDs.

Potential target applications in multi-disease therapy

In addition to serving as drug carriers, circRNAs are potential drug targets due to their multiple biological functions within the cell. circRNAs are involved in the regulation of gene expression through various mechanisms such as miRNA sponging and protein translation templates, which provide new intervention strategies for disease treatment.⁸⁷ Studies have shown that aberrant expression of circRNAs is closely related to the development of many diseases. In the field of oncology, circPVT1 is significantly overexpressed in a variety of cancers and promotes tumor cell proliferation and survival through adsorption of miR-16. Drug design targeting circPVT1 restores the function of miR-16, thereby inhibiting tumor growth.⁸⁸ In neurological disorders, circ-UBE2K is involved in the pathological process of depression by modulating neurotransmitter synthesis and neuronal plasticity, making it a potential therapeutic target.⁸⁹

Notably, circRNAs have also demonstrated significant value in the treatment of EIDs. Studies have shown that circRNAs can affect viral replication and immune escape processes by regulating specific immune signaling pathways.⁸⁶ For example, it was found that circRNAs may regulate the host immune response in neocoronavirus (SARS-CoV-2) infection by modulating the interferon pathway, nuclear factor κB signaling pathway, and so on, which in turn affects the replication of the virus and the immune escape of the host cells.⁹⁰ In addition, circRNAs have also been found to be involved in the regulation of macrophage activation and inflammatory responses, and are able to modulate host immune escape mechanisms, which are critical for the viral infection process.⁹¹ By precisely regulating the expression or function of these circRNAs, the host immune response can be effectively regulated, providing innovative therapeutic strategies for dealing with new outbreaks of infectious diseases.⁹² These findings not only expand the scope of clinical applications of circRNAs, but also provide new research directions for disease treatment.

As a biomarker

circRNA shows great potential as a non-invasive biomarker in the early detection of EIDs. By detecting changes in circRNA expression profiles in patients’ blood or other body fluids, early warning, precise diagnosis, and dynamic monitoring of the disease can be realized, which is of great significance for the prevention and control of epidemics.⁹³

circRNAs are closely associated with a variety of physiological processes and cell biological properties, including stem cell stemness and pluripotency, and their aberrant expression is closely related to cancer development.⁹⁴ Studies have shown that circRNA can be used as a potential biomarker for early detection of cancer, clinical diagnosis, prognostic assessment, and monitoring of treatment response.^95,96,97 Thanks to its unique expression pattern, molecular stability and tissue specificity, circRNA can be detected non-invasively in body fluids such as blood, sputum, and urine, and this liquid biopsy technique has the advantage of being less invasive and more reproducible than traditional tissue biopsies.^20,98

In disease monitoring, changes in circRNA expression are widely used in the diagnosis of various diseases and the evaluation of treatment response. In brain tissue, circRNA expression can reflect disease progression, such as the downregulation of ciRS-7 expression in Alzheimer’s disease, which provides important clues as a potential diagnostic marker.^48,99 The source of brain tissue can be obtained by surgical resection, puncture biopsy, or autopsy, with the choice depending on the type of disease and the purpose of the study.¹⁰⁰ For neurodegenerative diseases such as Alzheimer’s disease (AD), although biopsies are uncommon, they still provide important biological data for research.¹⁰¹

In order to summarize the application cases of circRNA in different diseases more systematically, Table3 lists the key research results of circRNA in different fields. These cases cover the application of circRNAs as biomarkers, therapeutic targets, and vaccine components, demonstrating their broad clinical applications.

Table 3.

Examples of circRNA applications

Areas of application	Application cases	Reference
Vaccine development	circRNA as a vaccine vector: using circRNA to express antigen and trigger an immune response	Niu et al.9; Amaya et al.102
Drug delivery	circRNAs as drug carriers: circRNAs can be designed with multifunctional structures to deliver drugs and enhance therapeutic effects	Guo et al.85
Immune regulation	circRNA modulation of immune response: circRNAs are able to modulate the response of the immune system, enhancing or inhibiting specific immune responses	Qu et al.60
Biomarker	circRNA as a disease biomarker: circRNA is a potential early diagnostic marker, especially in the fields of cancer, cardiovascular diseases and viral infections, due to its stability in body fluids such as blood and urine	Vo et al.20; Nanishi et al.97; Bahn et al.98
Cancer treatment	application of circRNAs in tumor immunotherapy: circRNAs are used in cancer therapy by modulating immune escape mechanisms and promoting anti-tumor immune responses	Wang et al.17; Kristensen et al.103
Neurodegenerative disease	relationship between circRNAs and neurological diseases: circRNAs are involved in the onset and progression of neurodegenerative diseases and may become new therapeutic targets	Su et al.104; Shen et al.105; Mo et al106
New outbreaks of disease	application of circRNAs in novel emerging diseases: circRNAs show potential as vaccine vectors or drug targets in responding to novel infectious diseases such as COVID-19	Wu et al.107

With their unique functions, circRNAs show a broad application prospect in the prevention and treatment of EIDs. As drug carriers, therapeutic targets and biomarkers, circRNAs provide new strategies for disease diagnosis and treatment. With the in-depth study of its biological mechanism and the continuous advancement of technology, circRNA is expected to become an important tool for the prevention and treatment of EIDs, and to promote the further development of precision medicine in clinical applications.

Challenges and future prospects for circRNAs

Although circRNAs have shown great therapeutic potential in basic research, their development as therapeutic agents still faces many challenges. Currently, the development of circRNA therapeutics is limited by three key aspects: the design and optimization of circRNA molecules, the improvement of cyclization efficiency, and the development of chemical manufacturing and control (CMC) processes.⁷² These technical bottlenecks have constrained the translation of circRNA from laboratory research to clinical applications. However, with the continuous advancement of molecular biology techniques and production processes, researchers are actively breaking through these technical barriers. By optimizing circRNA design strategies, improving the cyclization process, and establishing a standardized production process, circRNA is expected to overcome existing limitations and lay a solid foundation for clinical applications. These efforts will pave the way for a wide range of applications of circRNAs in the field of disease treatment.¹⁰⁸

circRNA design and optimization

With their unique functions in gene expression regulation, circRNAs have become an emerging tool in the field of disease treatment. However, the clinical application of circRNAs first needs to address the critical issue of their molecular design and optimization.¹⁰⁹ Especially when targeting disease-associated genes, how to precisely design circRNAs to maximize their therapeutic potential is the focus of current research.

In recent years, researchers have developed a variety of circRNA overexpression vector systems (e.g., pLCDH-ciR and pCD-ciR), which carry optimized flanking cyclization frameworks and significantly improve the cyclization efficiency and expression level of circRNAs.¹¹⁰ By introducing RNA-binding protein modification sites (e.g., ALU repeats [arthrobacter luteus repeats, short interspersed nuclear elements widely present in the human genome that can facilitate RNA circularization] and QKI binding sites [quaking protein binding sites, recognized by the RNA-binding protein QKI that promotes RNA circularization]) as well as novel cyclization-mediated sequences, these systems enable efficient and accurate cyclization of circRNAs.¹¹¹

Although significant progress has been made in circRNA design and optimization strategies, there are still many challenges to be faced in practical applications. Stable intracellular expression of circRNAs, target specificity, and large-scale production processes still need to be further resolved.⁷² To address these challenges, researchers are exploring a variety of strategies: enhancing the stability of the circularized structure by introducing stable linkage sequences (e.g., short-stranded inverted repeat sequences), reducing dependence on exogenous enzymes, and thus improving circRNA yield and function¹¹²;Tissue specificity and targeting of circRNAs are enhanced by designing specific targeting sequences (e.g., miRNA binding sites) in them.¹¹³ These innovative design strategies have laid an important foundation for the clinical application of circRNAs.

Cyclization efficiency of circRNAs

The unique covalent closed-loop structure of circRNAs confers excellent stability and resistance to enzymatic degradation, but also poses technical challenges in terms of cyclization efficiency. circRNA formation relies on specific cyclization mechanisms and sequence features, and its efficiency is often limited by a variety of factors, including complex secondary structures, sequence specificity requirements, and competitive reactions during the cyclization process.⁷² These limitations have led to difficulties in meeting the cyclization efficiency of circRNAs for clinical applications.

In recent years, artificial regulatory techniques have made significant progress in improving circRNA cyclization efficiency.¹¹⁴ Researchers have significantly improved the biosynthetic efficiency of exogenous and endogenous circRNAs by introducing an RNA-binding structural domain (PUF structural domain) specific to the human Pumilio1 sequence and combining it with an engineered circRNA regulatory factor.¹¹⁵ These innovative strategies not only enhanced the circRNA cyclization efficiency, but also laid an important foundation for its therapeutic applications.

Despite the remarkable progress, the cyclization efficiency of circRNAs remains a key bottleneck for their clinical applications. Future research should focus on the following aspects: optimizing the existing cyclization mechanism, developing novel cyclization regulators, and exploring strategies to overcome cyclization barriers in specific environments. These research directions will help to further improve the cyclization efficiency of circRNAs and promote their application in therapeutic areas.¹¹⁶

CMC process development

Significant challenges remain for efficient synthesis and purification during CMC of circRNAs.¹¹⁷ circRNA synthesis relies on ligases to catalyze the formation of ring structures from RNA molecules, but this process is often accompanied by the production of polymerization by-products, which reduces the efficiency of synthesis and increases the difficulty of purification and recycling.⁷² Therefore, improving the efficiency of the cyclase, reducing byproduct generation, and obtaining high-purity circRNA products are key issues that need to be addressed at present.

The detailed process production flow is shown in Figure 2. To address these challenges, researchers are working to develop more efficient and specific cyclases. The ideal cyclase should be highly specific and able to catalyze the cyclization reaction precisely, minimizing byproduct generation and thus increasing circRNA yield and purity.¹⁵ Meanwhile, innovative production methods and optimization of existing purification technologies are also key strategies to enhance circRNA production efficiency. These efforts will help establish a standardized and scaled-up circRNA production process and provide a reliable guarantee for its clinical application.

Figure 2.

circRNA process production flow

Discussion

As a new member of the RNA family, circRNA has attracted much attention in the biomedical field in recent years.¹¹⁸ Although initially thought to be a byproduct of splicing errors, its biological functions have been gradually revealed since its discovery in the late 1970s.⁹ Advances in high-throughput sequencing technology and the development of bioinformatics tools such as CircBase and circRNADb have greatly facilitated the systematic study of circRNAs.²⁷ Studies have shown that circRNAs play multiple roles in gene regulatory networks: acting as miRNA sponges to regulate the expression of target genes,¹¹⁹ participating in protein synthesis through the internal ribosome entry site, and interacting with RNA-binding proteins to regulate transcription and mRNA processing, which in turn affects cellular function.⁷⁷

In the field of disease research, circRNAs show important scientific value and application potential. In cancer, circRNAs are potential biomarkers for early diagnosis and prognosis assessment by regulating the proliferation, migration, invasion, and apoptosis of cancer cells.¹⁰³ For example, changes in the expression profiles of circRNAs in lung and breast cancers have been shown to be of diagnostic value,^120,121and their involvement in tumorigenesis and progression through the regulation of oncogenes or oncogenes suppressors has made them a new target for cancer therapy.¹⁷ Important breakthroughs have also been made in the study of circRNAs in neurodegenerative diseases. For example, downregulation of circHIPK2 in AD may be involved in disease onset,¹⁰⁴whereas upregulation of circPABPN1 is associated with neuronal apoptosis and inflammatory responses.¹⁰⁵ In addition, circRNA expression changes in blood samples provide potential markers for early diagnosis of AD.¹²² In Parkinson’s disease (PD), changes in the expression of circSLC8A1 and circHIPK3 are closely associated with disease progression, suggesting that they may be involved in the pathogenesis of PD through multiple signaling pathways.^104,123

In the field of cardiovascular diseases, the regulatory role of circRNAs has received increasing attention.¹²⁴ Studies have shown that circRNAs play important roles in diseases such as atherosclerosis, myocardial infarction, heart failure, and valvular calcification,¹²¹ and the mechanism of their interaction with miRNAs has become a hot research topic.^125,126 Although the study of circRNAs in vascular calcification is still in its infancy, their expression changes suggest that they may be involved in this pathological process.¹²⁷ In addition, changes in the expression profile of circRNAs in myocardial infarction and their effects on cardiac function have become a focus of research, providing an important basis for their use as biomarkers and therapeutic targets for cardiovascular disease.¹²⁸

In the defense and control of EIDs, circRNAs show great potential by virtue of their unique loop structure and immunoregulatory function.¹²⁹ As a key component of vaccines, circRNAs not only serve as antigenic carriers to induce specific immune responses, but also achieve broad-spectrum immune protection against a wide range of pathogens by designing multi-antigenic epitope structures.¹³⁰ This property allows circRNA vaccines to potentially provide broader protection than conventional vaccines against novel infectious diseases such as influenza and SARS-CoV-2.¹³¹

Conclusions

In summary, circRNAs play important roles in the regulation of gene expression and in a variety of diseases (e.g., cancer, neurodegenerative diseases, cardiovascular diseases, and new outbreaks of infectious diseases), demonstrating significant diagnostic and therapeutic potential. Its unique circular structure and extensive intracellular expression give it an advantage in biomarker discovery, drug target screening, and vaccine design. Although circRNAs face challenges in clinical applications, such as biosynthesis mechanism, in vivo stability, delivery efficiency, and safety assessment, with in-depth research and technological advances, circRNAs are expected to become an important tool for early diagnosis of diseases, prognostic assessment and therapeutic interventions, and to promote the development of precision medicine.

Acknowledgments

This study was funded by the National Natural Science Foundation of China (U20A20410), the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2022-I2M-CoV19-006), the Provincial Industry-University Cooperation Collaborative Education Project (no. 318 2022 of the Zhejiang Development Reform Society) and the University Level Scientific Research Project of Zhejiang Shuren University (grant no. 2024R057).

Author contributions

Writing – original draft, K.C., Y.X., and Jiaxuan Li; writing – review & editing, K.C., Y.X., Jiaxuan Li, S.G., and Z.W.; project administration, Jianhua Li and Y.Z.; funding acquisition, K.C.

Declaration of interests

The authors declare no competing interests.