Exploring the Benefits and Challenges of circRNA Therapy

Published on: July 25, 2024

The success of Pfizer/BioNTech and Moderna’s COVID-19 mRNA vaccines has accelerated the development of nucleic acid-based drugs. Currently, mRNA is primarily used in preventive vaccines for infectious diseases, therapeutic cancer vaccines, protein replacement therapies, and gene editing. However, mRNA has several limitations. It requires modification to resist nuclease degradation and avoid triggering the innate immune response. Additionally, mRNA encapsulation presents challenges related to cost and stability. All these challenges of mRNA, along with its short lifespan limit the sustained production of therapeutic proteins and efficacy of treatments.

Fortunately, in the rapidly emerging world of RNA therapeutics, circular RNA (circRNA), as a new and versatile therapeutic genetic transmitting vehicle, holds promise as a potential alternative to mRNA due to its superior stability. According to a 2022 survey, designing circRNA therapeutics became one of six academic spinouts of “Nature Biotechnology 2021”, and are a key focus for pharmaceutical companies. In 2022, Merck entered a $3.5 billion collaboration with Orna Therapeutics, a pioneer in the field of circRNA.

Given the similarities in the mechanisms of therapeutic protein expression between mRNA and circRNA, it is possible to expand the therapeutic applications of mRNA to circRNA. This article provides a brief overview of the potential applications of circRNA therapy and discusses its advantages and challenges as a therapeutic medium.

cirRNA in Protein Replacement Therapy

Protein replacement therapy aims to restore the function of proteins that are deficient or defective due to genetic mutations, which is accomplished by delivering mRNA that encodes functional endogenous proteins to specific cells and tissues to restore their biological functions.

Protein replacement therapy can offer a low-risk treatment option for various rare diseases characterized by gene loss of function. Conditions such as hemophilia, methylmalonic acidemia, and cystic fibrosis can potentially benefit from this approach.

1. Advantages of circRNA over mRNA in Protein Replacement Therapy

Greater stability: The circular structure of circRNA provides greater stability, leading to longer protein expression period compared to mRNA. As a result, compared to mRNA, circRNA-based therapies can achieve higher and more sustained levels of protein expression. These therapies can maintain the expression of therapeutic proteins for a longer duration, potentially reducing the frequency of dosing.

Lower immunogenicity: mRNA’s high immunogenicity comes from the innate immune system activation via pathogen recognition receptors, triggering the interferon production that hinder its therapeutic efficacy. In contrast, unmodified circRNA is less likely to be recognized by these receptors, resulting in reduced immunogenicity. Considering its lower immunogenicity and higher potential for repeated dosing, this makes circRNA more suitable for applications in protein replacement therapy.

2. Challenges of circRNA in Protein Replacement Therapy

RNA delivery system limitation: Lipid nanoparticles (LNPs) represent a significant advancement in delivering RNA to target organs. Since most LNPs tend to localize in the liver post systemic administration, most protein replacement therapies are limited to genes that can be expressed in the liver. Researchers are also considering inhalation delivery, but its efficiency is lower than systemic delivery. Researchers are also considering inhalation as a method of administration, but its delivery efficiency is lower than that of systemic administration. In the future, it will be necessary to innovate the formulation and targeted delivery of LNPs to expand the application of protein replacement therapies to non-hepatic tissues.

Lack of fine-tuning mechanisms for protein expression: circRNA and mRNA lack mechanisms for fine-tuning protein expression. The expression of therapeutic proteins needs to reach appropriate functional levels—effectively but without exceeding toxic limits. The expression of endogenous proteins is precisely controlled by regulatory mechanisms and feedback loops at the genetic level. However, for exogenous engineered RNA, only a few small molecule regulatory mechanisms are used in gene expression control. Further development of controllable engineered circRNA could achieve precise regulation of protein expression, thereby increasing the efficacy and safety of therapies.

Immunogenicity: Although engineered circRNA has lower immunogenicity than mRNA, it can still activate the innate immune system. Chen et al. (2017) first showed that transfecting engineered circRNA can stimulate the expression of several immune genes, notably retinoic acid-inducible gene I (RIG-I). Factors contributing to the immunogenicity of engineered circRNA include:

circRNAs derived from endogenous human ZKSCAN1 introns lack immunogenicity but type I introns from T4 bacteriophage thymidylate synthase (td) genes are immunogenic (see Figure 1A).
m6A modification promotes cycularization and reduces immunogenicity (see Figure 1A).
Small amounts of linear RNA impurities can trigger strong cellular immune responses.
Residual exons can form double-stranded secondary structures, activating PKR (see Figure 1C).

Figure 1: Immunogenicity of circRNA. (A) Engineered circRNA induces a strong immune response via RIG-I, but m6A-modified circRNA eliminates the immune response. (B) Engineered circRNA has low immunogenicity, but incomplete purification can lead to the presence of 5′ triphosphate linear RNA contaminants that stimulate cytokine release. (C) Residual exons produced using PIE self-catalytic splicing can activate PKR. circRNA without residual fragments does not activate PKR. (doi/10.1093/jmcb/mjad002/7036773)

circRNA in Vaccine Applications

The concept of mRNA vaccines was proposed as early as the 1990s. mRNA vaccine technology involves delivering mRNA molecules carrying the genetic information of viral antigens (specific viral information) to immune cells, which then express the corresponding proteins (viral antigens). These protein molecules can induce the desired adaptive immune response, thereby providing precise and rapid protection for the body.

mRNA vaccines have stronger immunogenicity, do not require adjuvants, have a shorter development and production cycle, and involve relatively simple manufacturing processes. This gives them significant advantages when dealing with rapidly mutating pathogens (such as influenza viruses) and emerging viruses (such as the coronavirus, Ebola virus, Zika virus, etc.). However, the application of mRNA vaccines is limited by their instability, low efficiency, and innate immunogenicity. In contrast, circRNA vaccines that combine IRESs and ORFs offer an improved RNA vaccine approach that is safe, stable, easy to manufacture, and scalable.

circRNA vaccines offer several advantages (see Table 1).

Greater stability and storage: mRNA vaccines exhibit extreme instability because they are easily degraded by RNases during transportation, storage, and inside the cells. Although nucleotide modifications in the mRNA backbone and UTR regions enhance its stability, they also complicate the manufacturing process and increase production costs. Due to poor thermal stability, mRNA vaccines still require low-temperature cold chain storage. On the other hand, unmodified circRNAs demonstrate high stability and better resistance to RNase degradation, allowing them to be stored at room temperature or under repeated freeze-thaw conditions.

Fewer side effects: The cellular toxicity and side effects associated with mRNA vaccines are partly due to their high immunogenicity. However，unmodified circRNA has lower immunogenicity and cellular toxicity. This reduced immunogenicity can be beneficial in minimizing unwanted immune responses and associated side effects.

Extended protein expression duration: circRNAs vaccines can prolong the expression of proteins, which helps antigens remain in antigen-presenting cells (APCs) longer and enhances antigen presentation. This contributes to a more robust adaptive response and increased production of neutralizing antibodies.

Table 1: Comparison of mRNA and circRNA vaccines (doi.org/10.1038/s41392-023-01561-x）

	mRNA vaccine	circRNA vaccine
Preparation	Codon optimizations UTR optimizations Cap and Poly (A) tail design Nucleotide modifications In vitro transcription (IVT) Purification	Translation optimizations Circularization optimizations Nucleotide modifications IVT Circularization Purification
Storage	Refrigeration in ultra-cold and RNase-free and sterile conditions	Non-cryopreservation and preservation under fewer freezing and thawing cycles
Biosecurity	High immunogenicity of unmodified mRNA Low immunogenicity of modified mRNA	Low immunogenicity of unmodified circRNA Lower and even no immunogenicity of modified circRNA
Delivery	LNP Polymetric nanoparticles Cationic nanoemulsion Exosomes Solid lipid nanoparticles nanostructured lipid carriers Naked delivery	Compatible with mRNA delivery options
Antigens encoding	Low antigen-encoding endurance due to biodegradation of mRNA	Prolonged antigen coding tolerances due to the high longevity of circRNA
Application	Vaccines, encoding adjuvant, encoding antibody, gene editing, and protein replacement	Compatible with mRNA application. In addition, circRNA can also be applied to sponges and endogenous circRNA mimics

Table 1: Comparison of mRNA and circRNA vaccines (doi.org/10.1038/s41392-023-01561-x）

Although cirRNA vaccines share many similarities with mRNA vaccines, circRNA can address multiple shortcomings of mRNA vaccines. Although circRNA vaccines are still in the early stages of design, synthesis, purification, delivery, and application, circRNA holds promise as a next generation RNA-based vaccine platform.

circRNA in Tumor Vaccine Applications

mRNA has a broad range of applications in cancer vaccines, employing various strategies to inhibit or even eliminate tumors (see Figure 2).

Antigen presentation: mRNA vaccines deliver cancer antigens to antigen-presenting cells (APCs) for presentation via major histocompatibility complex (MHC) class I and II molecules.

Adjuvant functions: mRNA stimulates immune activation by binding to pattern recognition receptors expressed on APCs.

Antigen receptors: mRNA expresses chimeric antigen receptors (CARs) or T-cell receptors.

Protein production: mRNA enables the expression of immune-modulatory proteins, including toll-like receptors, chemokine receptors, co-stimulatory ligands, cytokines, chemokines, and various monoclonal antibody formats in different cell subsets.

Figure 2: Overview of mRNA Tumor Vaccines Mechanisms (https://doi.org/10.1186/s12943-021-01348-0）

Current mRNA cancer vaccines have shown poor performance in immune induction, whereas the inherent advantages of circRNA make it a promising candidate in the field of cancer vaccines.

Stable therapeutic protein expression is achievable by the stability of circRNA. Like preventive pathogen vaccines, cancer vaccines could benefit from extended antigen presentation to elicit stronger immune responses. In cell therapies, the sustained transient expression of CARs can lead to more robust immune responses and enhanced safety.

Enhanced immunogenicity：The use of circRNA could enhance the immunogenicity of cancer vaccines, allowing immune-stimulating proteins to more effectively activate the immune system in cancer immunotherapy. The prolonged expression of small effector proteins, such as cytokines and small antibody fragments, by circRNA can better recruit immune cells to the tumor site.

Customizable circRNA can be tailored to individual cancers for precise cancer treatment. Multiple antigens can also be encoded on different circRNAs, and vaccinating with multiple neoantigens can reduce the risk of cancer escape and recurrence.

Lower immunogenicity: circRNA’s low inherent immunogenicity is advantageous for expressing immune activating proteins within tumors. In contrast, unmodified mRNA can activate innate immune responses, which may reduce antibody production and impede the recruitment of adaptive immune cells to tumors.

Cap-independent translation initiation: The cap-independent translation initiation mechanism of circRNA allows it to be unaffected by the state of cancer cells, enabling the reliable translation of immune-activating proteins (such as cytokines and antibodies). Even in quiescent cancer stem cell subpopulations, circRNA can maintain robust protein translation.

circRNA in Gene Editing

The stability of the circRNA holds great potential in the field of gene editing. Both gRNA and template RNA in gene editing systems can achieve greater stability and improved editing efficiency when circularized. Compared to mRNA, circRNA Cas9 can express more robustly, leading to more persistent expression of Cas9 protein, leading to higher editing efficiency.

However, there are still many challenges in appling circRNA in the field of gene editing.

Delivery to target tissues: Efficient delivery of circRNA-based gene editing tools into target cells remains a significant challenge. Therefore, finding suitable delivery systems to more target organs are future challenges for both circRNA and mRNA.

Off-target effects: Like other gene editing tools, circRNA-based gene editing systems may have off-target effects, due to its prolonged expression. Currently, there is no report on the off-target effect by Cas9 delivered by mRNA and circRNA.

GOI size limit with circRNA. The rate of circularization is inversely related to the size of the circRNA. Researchers are exploring solutions like using smaller Cas9 variants or splitting Cas9 into fragments for separate expression and reassembly.

Immunogenicity remains a concern for gene editing therapies. Both circRNA and Cas9 may provoke immune responses, potentially leading to the elimination of edited cells. Currently, up to 78% of the population is already immune to the Cas9 protein, which further increases the challenges of gene editing therapies. Although circRNA has relatively low immunogenicity, more research is needed to determine whether its low immunogenicity is sufficient to avoid adverse immune responses during the editing process..