The Potential and Challenges of Circular RNA Vaccines: From

Mechanism to Clinical Use

Yuling Song

and Siyu Liu

Beijing Academy, Beijing, China

Tianjin Xinhua High School, Tianjin, China

Keywords: Circular RNA Vaccine, Stability, Immunotherapy.

Abstract: Circular RNA (circRNA) is a stable, covalently closed RNA that regulates gene expression by sponging

microRNAs and can sometimes translate into proteins. This makes circRNA a promising platform for

vaccines against infectious diseases and cancer immunotherapy. Compared to mRNA vaccines, circRNA

vaccines offer longer half-lives, enhanced stability, room-temperature storage, and reduced immunogenicity,

minimizing adverse reactions and logistical challenges. However, key challenges include inefficient delivery,

high production costs, and uncertain long-term safety. Ongoing research focuses on optimizing circRNA

design, improving delivery methods (e.g., lipid nanoparticles), and developing cost-effective production

techniques. This review discusses circRNA’s functions, its vaccine potential, and the obstacles to its clinical

application in infectious disease prevention and cancer therapy.

1 INTRODUCTION

Circular RNA (circRNA) is a category of RNA with

a covalently closed structure. Without a 5’ end or a 3’

end, circular RNAs have a higher stability than linear

RNAs. As for the functions, circRNA can regulate

many reactions in vivo. In terms of the mechanisms

functions in vivo. The majority of circRNA is formed

through a unique process called back-splicing. After

that, circRNA can bind to miRNAs, acting as RNA

sponge and regulate gene expression (Pan-da.2018).

In addition, some circRNA is able to be translated

into proteins, and induce immune response (Zhou et

al. 2023). Therefore, circRNA have high potential in

vaccine development. At pre-sent, circRNA vaccines

against SARS‐CoV‐2 and monkeypox virus are

already under development.

Compared to traditional mRNA vaccines,

circRNA vaccines have multiple distinctive ad-

vantages. Due to its closed-loop structure, circRNA is

less prone to degradation, providing a longer half-life

and sustained antigen presentation (Bu et al.2025).

This extended exposure enhances immune response

durability. In addition, traditional mRNA vaccines

need to be stored in an ultra-cold and sterile

environment, while circRNA vaccines have high

stability and can be stored at room temperature or low

temperatures, which is easy to transport. What's more,

the low immunogenicity of circRNA allows it to

alleviate the side effects of immune response, such as

inflammatory response, thus expanding the range of

applicable people. The purpose of this article is to

review the latest progress of circRNA in vaccine

development, and to explore its unique biological

characteristics, immune mechanism, and application

advantages. At the same time, this article will also

evaluate the current challenges faced by circRNA

vaccines, and by summarizing the existing research,

this article will provide theoretical guidance for the

future development of circRNA vaccines, and look

forward to their application prospects in the field of

immunotherapy.

2 circRNA AND ITS

BIOLOGICAL MECHANISM

2.1 Biosynthesis of circRNA

Most circRNAs in vivo are formed by back-splicing.

For circRNAs which have exons, if there are

complementary sequences or repeated Alu sequences

within the two introns flanking the exons, the

complementary parts will come close to each other

Song, Y. and Liu, S.

The Potential and Challenges of Circular RNA Vaccines: From Mechanism to Clinical Use.

DOI: 10.5220/0014488100004933

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 1st International Conference on Biomedical Engineer ing and Food Science (BEFS 2025), pages 335-339

ISBN: 978-989-758-789-4

335

due to base-pairing, thus trigger back-splicing,

forming a new circular pattern with the exons and the

remaining parts of the flanking introns involved.

(Hwang et al.2024) Additionally, RNA binding

proteins (RBPs) can regulate the back-splicing. They

can either facilitate the biosynthesis of exonic RNAs

(EcircR-NAs) and exonic-intrinsic RNAs

(ElciRNAs), or inhibit the splicing of circRNAs. The

unique process of back-splicing gives circRNAs its

special stability and functions.

2.2 Biological Function of Circ RNA

CircRNAs play a crucial role in biological processes.

One key function is their "sponging" effect, where

circRNAs bind to miRNAs, preventing them from

interacting with target mRNAs and inhibiting

miRNA-mediated gene suppression. In other words,

circRNAs counteract RNA inhibition. Depending on

the number of miRNAs bound to circRNAs, they can

have a bidirectional effect on gene expression. A

higher number of bound miRNAs suppresses mRNA

degradation, leading to increased gene expression.

Conversely, a lower number of bound miRNAs can

reduce gene expression (Pamudurti et al., 2020).

Additionally, circRNAs containing exons can be

translated into proteins. These proteins and peptides

can act as antigens, triggering immune responses. As

a result, synthetically engineered circRNAs can serve

as vaccines, providing immune protection and disease

prevention.

2.3 Mechanism of circRNA Vaccine

The circRNA undergoes three main processes in the

cell when it functions: endosome escape, antigen

encoding, and immune initiation. In APCs, lipid

nanoparticles (LNPs) containing circRNA vaccines

form endosomes which are membrane-encapsulated

vesicle structures in the cytoplasm. Then the

endosomes release the circRNA vaccine, which is

called endosome escape. In the next step, the

circRNA encoding sequence is translated by the

ribosome into an antigenic protein or peptide.

Endogenous antigens are degraded into peptides by

proteases and presented by MHC I molecules,

activating cytotoxic T cells which are CD8+ T cells.

In addition to cellular immunity, circRNA-induced

humoral immunity is also important. Endogenous

antigens in APCs can be secreted and presented by

MHC II molecules to helper T cells, helper T cells

that are CD4+ T cells, which further stimulate B cells

to produce neutralizing antibodies (Dun et al., 2023).

3 THE POTENTIAL OF RNA IN

VACCINE DEVELOPMENT

At present, the application of circRNA in vaccine

research and development has gradually at-tracted

attention, especially in the field of viral vaccines and

cancer immunotherapy. In terms of viral vaccine

research and development, circRNA vaccines have

shown potential in the preven-tion of novel

coronavirus, Mpox virus, Rabies virus and other

viruses. In terms of cancer immu-notherapy, in

cancers such as colorectal cancer, gastric carcinoma,

and liver cancer, the expres-sion changes of circRNA

and the relationship between these expression

changes and immune response have been confirmed.

Based on these differences, we can mobilize immune

cells to re-spond to cancer cells by controlling

circRNA expression, and then develop

immunotherapies against these cancers (Wan et al.,

2024).

There are currently circRNA vaccines that are

being studied against the novel coronavirus. The

research team synthesized circRNA using a linear

template containing RBD coding sequences using in

vitro transcription. Cyclization was performed using

a group I intron method and a T4 RNA ligase-based

approach. Experiments in mice have shown that

circRNA vaccines can significantly reduce the

infection of the SARS-CoV-2 beta variant, reduce the

viral load in the lungs, and there is no obvious disease

exacerbation (Qu et al., 2022). Through experiments

in animal models, the research team found that

circRNA vaccines performed well in preventing

tumor growth and inhibiting tumor growth.

Compared with linear mRNA vaccines, circRNA

vaccines induce a higher proportion of tumor-specific

T cells and have stronger immune activa-tion

capabilities (Yu et al., 2025).

CircRNA vaccines also have great potential for

the treatment of cancer. The research team has

experimentally found that circRAPGEF5 and

circMYH9 can translate distinctive peptides and

activate CD8+ T cells. The research team detected the

presence of circMYH9 in blood samples from

colorectal cancer patients, suggesting that circRNA

can be used as a biomarker in liquid biopsy for early

detection of cancer and vaccine development, which

is expected to improve treatment effect (Ren et al.,

2024).

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336

4 ADVANTAGES OF circRNA

VACCINES

4.1 Stability

Without a traditional 5’end or 3’ end, circRNAs are

immune to degradation led by RNase. At the same

time, CircRNAs have high stability due to the

covalently closed structure. As a result, compared

with mRNA vaccines, circRNAs have longer half-life

in vivo, which means that circRNAs vaccines can be

expressed at a longer time and be translated into more

antigen proteins than mRNA vaccines. Thus,

circRNA vaccines can induce immune response more

effectively. Moreover, many traditional mRNA

vaccines can only be stored and transported in highly

restricted environment such as refrigeration, while

circRNA vaccines can still maintain its functions in

room temperature, which reduces the transportation

and storage cost.

4.2 High Translation Efficiency

circRNA has a unique translation mechanism, leading

to high translation efficiency. The translation

initiation of circRNA is dependent on Internal

Ribosome Entry Site (IRES) or m6A modification

instead of conventional 5’ cap, increasing the

translation efficiency. In addition, when one round of

translation is over, the ribosome can directly pass

over the circRNA even if after reaching a stop codon

and start the next round of translation right away (Xie

et al., 2023). This kind of translation is called rolling

circle translation, which may seem slow but actually

effective. As a result, more stable and efficient

antigen production can be achieved, enabling stronger

immune protection.

4.3 Immunogenicity and

Self-Adjuvanted Vaccination

CircRNA itself has immunogenicity and has the

ability to activate immune cells like natural killer

cells and antigen presenting cells, and it can promote

the presentation of antigen translated by itself.

Subsequently, circRNAs are able to enhance the

immune response against the antigen produced by

themselves and be made into self-adjuvanted

vaccines. Compared with mRNA vaccines, circRNA

vaccines induces higher antibody production and

effective immune protection (Das et al., 2024).

5 PREPARATION PROCESS

5.1 Sequence Adjustment and

Modification

To use circRNA in vaccine development, the first step

is to synthesize linear precursor RNA. Since there is

no 5' end, circRNA cannot initiate translation in a

conventional 5' end-determined manner. Therefore,

sequence optimization of circRNAs is required for

successful initiation of translation. The first manner

is the addition of an untranslated region (UTR)

containing an RNA-binding protein (RBP) motifs

upstream or downstream of the IRES-ORF gene

cassette of the linear RNA. IRES can recruit

ribosomes through IRES-transacting factors (ITAFs)

to initiate translation without the involvement of

traditional translation initiation factors. The most

efficient IRES currently known is EV-A IRES (Yu et

al., 2025). Another approach is to introduce m6A

modification upstream of circRNA and use it to

mediate translation initiation. This modification may

reduce the immunogenicity of circRNA (Niu et al.,

2023).

5.2 Linear RNA Precursor for

Circularization

Next, the translated precursor RNA needs to be

cyclized to become circRNA and be used as a

vaccine. At present, there are three cyclization

methods for circRNA: chemical synthesis, T4 ligase

method and ribozyme ligation. The chemical

synthesis method uses chemicals to induce the

formation of phosphodiester bonds between the 5' end

and 3' end of linear RNA to form circRNA. The T4

ligase method involves the use of T4 DNA ligase, T4

RNA ligase I, and T4 RNA ligase II. These ligases

can directly form phosphodiester bonds between the

5' end phosphate group and the 3' end hydroxyl group

of linear RNA through nucleotide transfer reactions,

thus forming circRNA. Ribozymes are bioactive

RNA molecules with catalytic functions. There are

several ribozyme-based methods, the most prominent

of which is the arrangement of introns and exons

(PIE) methods. The group I intron self-splicing

system is more commonly used than the group II

intron self-splicing system. In this method, two

continuous transesterification reactions are achieved

with the help of type I intron self-splicing system or

type II intron self-splicing system to form circRNA

(Niu et al., 2023).

The Potential and Challenges of Circular RNA Vaccines: From Mechanism to Clinical Use

337

5.3 Vaccine Delivery System

The circRNA vaccines need to be delivered into cells

to function. The most common is that using lipid

nanoparticles (LNP) delivery systems, which are a

new generation of liposomes with high

biocompatibility and biodegradability. LNP delivery

system can improve the stability and permeability of

circRNA vaccines, which is a typical and widely used

delivery system with significant advantages. In

addition to LNP delivery system, using viral vectors,

electroporation techniques and exosomes can also

deliver circRNA vaccines. Viral vectors are rarely

used for circRNA delivery. When using viral vector

delivery, the viral vector integrates DNA as an

intermediate into the host genome, then transcribes it

into RNA, and then cyclize it into circRNA in cells.

Electroporation technology uses short high-voltage

pulses to form temporary holes in the cell membrane

to allow RNA molecules to enter the cell.

Electroporation is a versatile and efficient method for

non-viral RNA delivery. Exosomes are natural lipid

bilayer vesicles with good biocompatibility and low

immunogenicity (Cai et al., 2024).

6 CHALLENGES AND

IMPROVEMENT STRATEGIES

FOR circRNA VACCINES

At present, the industrial production of circRNA

vaccines is not yet mature, and its production process

mainly includes three stages: linear RNA molecules

are synthesized through in vitro transcription using

plasmids as templates, RNA circularization and

vaccine encapsulation. High-purity plasmids are

critical to vaccine quality, and the industrial

production of plasmids goes through processes such

as fermentation, bacterial harvest, lysis, clarification,

ultrafiltration and concentration, and

chromatographic purification. During

chromatographic purification, RNA, trace impurities,

and endomycin can be removed effectively by adding

different types of filler, thereby obtaining high-purity

plasmids (Bai et al., 2023). In the process of RNA

synthesis, intron self-splicing is more suitable for

linear RNA synthesis with larger molecular weight

than other methods. The purity of circRNA directly

affects its low immunogenicity and protein

expression levels. The main purification methods are

RNase R treatment, high-performance liquid

chromatography (HPLC) and electrophoresis. These

methods are effective in separating linear RNA from

circRNA (Zhang et al., 2024). In terms of delivery

systems, LNP delivery system is currently widely

recognized and applied RNA vaccine delivery

methods, but they have poor heat tolerance, resulting

in extremely low temperatures for vaccine

transportation and storage. Meanwhile, using LNP

may cause side effects such as inflammation. As an

emerging technology, exosome delivery has gained

more and more attention, and its advantage is that it

can effectively reduce side effects, but large-scale

industrial production still faces great challenges and

needs to be further studied and optimized (Zhao et al.,

2022).

Additionally, as a kind of vaccine in clinical

examination stage, there are still uncertainty about the

immune response and side effects. Humans generate

endogenous circRNAs, and those endogenous

circRNAs participate in the regulation of many

essential chemical effects. it is still unknown whether

exogenous circRNAs will interfere the normal

regulation effects. As a result, to be sure about the

side effects, more clinical tests are needed. At the

same time, up to now, the preparation process and

generation equipment are not mature enough, so the

generation procedure is complicated and the scale of

production is low, not able to satisfy the massive

requirements in clinic use. Besides, due to the

complex equipment and high expense, the number of

patients who may use the product might be low. In

order to make breakthroughs, more equipment that

can be used in massive production of circRNA

vaccines needs to be developed to reduce

manufacture costs and difficulty.

7 CONCLUSION

With its unique covalent closed-loop structure,

circRNA vaccines demonstrate the advantages of

long lifespan, high stability, low immunogenicity and

translatability. However, its development still faces

many challenges, including improving the cyclization

efficiency of linear RNA pre-cursors, improving

purification methods to obtain high-purity circRNA

and establishing an efficient and safe delivery system.

The self-adjuvanting effect of circRNA vaccines

enhances the intensity and duration of the immune

response, which is crucial for improving vaccine

effectiveness. In animal models, circRNA vaccines

have been proven to activate cellular immune

response effectively, especially CD8+ T cell-

mediated immune response, thus circRNA vaccines

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338

have good anti-tumor effects. Moreover, some

circRNA have been found to be potential tumor

biomarkers, but a large number of research is required

to validate that in aspect of clinical applications. In

summary, circRNA vaccines have broad

development prospect in the field of infectious

disease prevention and immunotherapy for tumor,

and may be used as an effective treatment for major

infectious diseases or common viral diseases in the

future, and may also be developed into therapeutic

tumor vaccines.

AUTHORS CONTRIBUTION

All the authors contributed equally and their names

were listed in alphabetical order.

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