Circular RNA-based Biomedical Applications

As research into the functions and mechanisms of circular RNA (circRNA) continues to deepen, the field has gradually expanded from basic scientific research to industrial applications, demonstrating significant potential particularly in drug development. In the process of drug R&D, circRNA is primarily categorized into two research directions: non-coding RNA and coding RNA.

In the field of non-coding RNA applications, circRNA serves not only as nucleic acid aptamers to regulate intracellular signal transduction but also as molecular sponges for miRNAs or proteins to modulate the functions of corresponding molecules. Furthermore, it plays a vital role in gene editing as guide RNA.

In the field of coding RNA applications, circRNA is capable of encoding proteins with various functions. Consequently, it possesses immense potential and broad application prospects in areas such as vaccine development, in vivo chimeric antigen receptor T-cell (CAR-T) immunotherapy, regenerative medicine, and protein replacement therapy.

Figure 1. Circular RNA-based biomedical applications. (Liu C X, Chen L L., 2022)

circRNA Aptamers

Aptamers are structures composed of short-chain DNA or RNA molecules expressed within cells, possessing the ability to bind specific target molecules and regulate intracellular signal transduction processes. However, a major challenge in the application of RNA aptamers is the difficulty of maintaining stable expression at sufficiently high concentrations to be effective. The high stability of circRNA provides a practical solution for RNA aptamer-based applications. For example, a circRNA fluorescent sensor expressed via the Tornado system exhibited fluorescence intensity nearly 200 times higher than that of linear RNA when binding small molecules in living cells. By constructing nuclear factor-kappa B (NF-κB) aptamers into circRNA, it proved more effective at inhibiting the NF-κB signaling pathway than linear RNA of the same structure. Additionally, researchers embedded S-adenosylmethionine (SAM) and Broccoli fluorescence aptamers into a circRNA structure to create a highly sensitive metabolite biosensor. This sensor can continuously and dynamically detect changes in intracellular SAM levels, showcasing the potential of circRNA aptamers in regulating intracellular signaling.

Sponge circRNA

circRNA can act as a sponge for intracellular miRNAs by adsorbing specific miRNAs to inhibit their regulatory effects on target mRNAs, a mechanism known as the competing endogenous RNA (ceRNA) regulatory model. By synthesizing circRNA in vitro or expressing stable circRNA in vivo containing binding sites partially complementary to miRNAs, the activity of disease-related miRNAs can be effectively reduced, showing therapeutic potential. For instance, a circRNA sponge designed for miRNA-122, which targets the Hepatitis C virus, inhibited viral protein production more effectively than existing locked nucleic acid (LNA) antisense oligonucleotides. circRNA can also be engineered with specific RNA binding sites to adsorb particular proteins and regulate their activity. For example, heterogeneous nuclear ribonucleoprotein L (hnRNPL) regulates splicing by binding to CA repeats in pre-mRNA; circRNA carrying these CA repeats can significantly reduce the distribution of hnRNPL within the nucleus, thereby affecting its splicing function. Long non-coding RNAs such as NORAD (non-coding RNA activated by DNA damage) prevent Pumilio family (PUM) proteins from binding to the mRNA of cell cycle regulators through multiple Pumilio response elements (PREs). Engineered circRNA can mimic NORAD's function by adsorbing PUM proteins to maintain genomic stability.

circRNA sponges can not only neutralize pathologically accumulated miRNAs but also regulate the activity of intracellular RNA-binding proteins, providing new directions for treating related diseases and demonstrating their potential as therapeutic tools.

Guide circRNA

In the field of gene editing, guide RNA serves as a sequence-positioning element and plays a crucial role in editing efficiency and precision. Transforming guide RNA into circRNA can significantly enhance both efficiency and accuracy. In 2022, two studies published concurrently in Biotechnology showed that by using guide circRNA to recruit adenosine deaminase acting on RNA 1 (ADAR1), efficient and programmable adenosine-to-inosine (A-I) RNA editing could be achieved both in vitro and in vivo. Due to its unique structure, guide circRNA can avoid cleavage by exonucleases, significantly improving editing efficiency and precision while largely eliminating off-target editing issues on target transcripts within double-stranded RNA regions.

Furthermore, guide circRNA can be applied to the CRISPR-Cas9 gene editing system. Research indicates that using guide circRNA in human embryonic kidney (HEK) 293 cell models reduces off-target effects compared to linear guide RNA, although this is accompanied by a slight decrease in editing efficiency. Notably, by introducing linker sequences into guide circRNA, the editing efficiency in Escherichia coli was increased two-fold compared to natural linear guide RNA. Therefore, by optimizing the structural design of guide circRNA, it is possible to improve editing efficiency while reducing off-target risks, providing a more precise "navigation system" for gene editing and offering broad application prospects in the treatment of hereditary diseases.

circRNA Vaccines

Because circRNA has the ability to encode antigenic proteins, it can be used to develop vaccines. In 2022, the journal Cell first reported research results on a novel circRNA coronavirus vaccine. Researchers used IRES sequences and sequences encoding the receptor-binding domain (RBD) of the spike protein to prepare circRNA, which was then encapsulated in lipid nanoparticles (LNPs) to develop a circRNA vaccine against COVID-19 strains. In mouse models, immunization with doses of 10 and 50 μg induced a higher proportion of neutralizing antibodies and a significant T helper cell 1 (Th1)-skewed immune response compared to mRNA vaccines. Further challenge experiments in monkey models showed that the viral load in the lungs, the degree of lung injury, and the number of pulmonary inflammatory infiltrating cells were significantly reduced in the immunized group. Additionally, circRNA vaccine experiments targeting the Delta and Omicron BA1 variants of COVID-19 further confirmed the broad-spectrum activity and high stability of the vaccine.

In Vivo circRNA CAR-T

As a novel cancer treatment, CAR-T cell therapy transforms a patient's own T cells to recognize and attack tumor cells, and is regarded as a revolutionary breakthrough in oncology. However, the therapy still faces challenges such as high production costs, clinical compliance constraints, and potential risks of secondary carcinogenesis. To improve the accessibility and safety of CAR-T cell therapy, researchers are exploring innovative technologies and methods. Among these, the strategy of generating CAR-T cells in vivo using mRNA technology is expected to reduce treatment costs and improve clinical acceptance. Notably, circRNA, with its unique biological characteristics, also shows great application potential in this field.

Researchers have developed a novel cardiolipin-mimetic phosphoramide (CAMP) LNP that demonstrates significant advantages in targeting T cells. The morphology, high rigidity, and core chemical structure (piperazine central group) of this LNP may be key to its preferential targeting of T cells. Using this specially designed LNP, the research team successfully delivered a CARcircRNA encoding for the urokinase-type plasminogen activator receptor (uPAR), an antigen associated with aging and inflammation. This effectively alleviated uPAR-related liver fibrosis and rheumatoid arthritis (RA) symptoms, demonstrating the potential of circRNA in the field of in vivo CAR-T therapy.