mRNA therapy utilizes the cell's own protein synthesis machinery. Compared with traditional protein drugs, mRNA drugs have shown a more streamlined R&D process and have become a cutting-edge and innovative treatment model. Against the backdrop of the COVID-19 global pandemic, vaccines based on mRNA technology have made breakthrough progress. This milestone has greatly promoted the popularity of mRNA therapy, especially in the research and development of viral and tumor vaccines. However, despite its broad prospects, mRNA still faces many technical challenges in terms of stability and immunogenicity.

circRNA is a type of RNA molecule that is a single-stranded closed ring formed by covalent bonds. First, because it lacks free ends, it is not easily degraded by nucleases and has stability. Secondly, the purified engineered circRNA shows low immunogenicity and side effects in vivo, reflecting its safety advantage as a drug. In addition, the synthesis of circRNA does not require structures such as capping and tailing, nor does it require the introduction of RNA modifications, which facilitates cost control. Compared with linear mRNA, circRNA with a closed-loop structure has become an attractive alternative due to its excellent stability, low immunogenicity and sustained expression.

The History of circRNA

In 1976, Hsu MT et al. discovered circRNA in eukaryotic cells for the first time through electron microscope observation. However, it was once considered to be a byproduct of intracellular mRNA splicing errors.

In 1993, Capel et al. found that the circRNA of the mouse Sry (sex-determining region Y) gene may play a specific function in the mouse testis. CircRNA has thus truly entered the field of scientific research and gradually become a research focus.

In 2012, Salzman et al. found that a large part of the spliced transcripts of hundreds of genes were circRNAs through deep sequencing of a variety of normal and malignant human cell RNAs. It proves that circRNA is not an error of abnormal RNA splicing, but a universal feature of gene expression programs in human cells. This discovery has rekindled people's great interest in circRNA research, and related research has grown exponentially.

July 2018 was a key turning point in the history of circRNA development. Daniel Anderson et al. of MIT first confirmed that engineered circRNA can stably and efficiently express proteins in eukaryotic cells. It has pioneered a new application of exogenous circRNA in expressing proteins in eukaryotic cells, and has also proved that circRNA is an effective substitute for linear mRNA. Many startups have emerged to focus on the research and development of circRNA.

Figure 1. Timeline of circular RNA. (Chen, Xinjie, and Yuan Lu. 2021)

Common Circularization Methods for Engineered circRNA

Using linear mRNA as a precursor, the common idea for synthesizing circRNA in vitro is to connect the two ends to form a covalently closed circular structure. This process can be achieved by chemical ligation, enzymatic ligation or ribozyme splicing.

Chemical ligation: Due to the problem that phosphate migration will form a 2'-5'-phosphate bond instead of a natural 3'-5' phosphodiester bond, it is not currently the mainstream in vitro cyclization method.

Enzymatic ligation: Enzymes commonly used for RNA cyclization include T4 DNA ligase 1 (T4 Dnl 1), T4 RNA ligase 1 (T4 Rnl 1) and T4 RNA ligase 2 (T4 Rnl 2), all of which are ATP-dependent. Through three nucleotide transfer steps, the connection of the 5'-phosphate and 3'-OH at the RNA end is catalyzed, and the two ends of the RNA molecule are connected to produce circRNA. The three enzymes have different optimal ligation substrate types and ligation characteristics, and can be selected according to the type of substrate to be connected and whether a splint is required.

Ribozyme splicing: The most commonly used ribozyme system for RNA cyclization is the PIE system (type I intron or type II intron). Based on the self-splicing function of type I or type II introns, in the presence of magnesium ions and free GTP, the splicing effect is achieved, leading to the cyclization of introns and the connection of intermediate sequences, thereby generating circRNA.

Figure 2. Strategies for ribozyme methods. (Chen, Xinjie, and Yuan Lu. 2021)

The use of T4 td gene or Anabaena tRNALeu precursor gene to design and arrange intron exon (PIE) structure belongs to the type I intron circularization system. This system, combined with reasonable circularization rate improvement strategies (such as adding homology arm sequences), can achieve circularization of sequences up to 5 kb. However, the disadvantage is that type I introns will retain auxiliary exon sequences. Compared with the type I intron PIE system, the use of type II introns does not require auxiliary exon sequences to produce the expected circRNA sequence. The wide applicability of this method still needs more research verification.

Application Areas of circRNA

Gene editing: Gene editing can achieve precise replacement of bases, and precise insertion, replacement and deletion of small fragments. At present, circRNA has been reported in the application of CRISPR guide editors. Using circRNA to express CRISPR-Cas9 protein for gene editing has considerable specificity. In addition, multiple crRNAs targeting multiple sites and RTT-PBS sequences targeting multiple sites are inserted in series in circRNA to guide the guide editor (CPE) system developed based on Cas12a to successfully edit up to four genes simultaneously in the T-rich genomic region of human cell lines.

Protein replacement: The continuous and long-term expression of functional proteins using circRNA is conducive to the accumulation of functional proteins and their efficacy. It has been reported that a single transfection of circRNA expressing NEUROD1 in human pluripotent stem cells (hPSCs) can induce neuronal differentiation. In addition, a single dose of U-LNP-delivered VEGF-AcircRNA preparation expressed and released VEGF-A for a long time, and almost complete healing of the wound surface of diabetic mice was observed on the 12th day of injection. The effect is significantly better than that of linear VEGF-A mRNA and rhVEGF protein.

Tumor immunity: In a spontaneous tumor model caused by KRAS G12D mutation, GSDMDENG circRNA improves the survival rate of diseased mice and can continuously induce KRAS G12D tumor antigen-specific cytotoxic T lymphocyte responses, and has significant effects in preventing pancreatic cancer, lung adenocarcinoma, and colon adenocarcinoma.

Vaccine development: The circRNA vaccine encoding SARS-CoV-2 Spike RBD can induce effective neutralizing antibodies and T cell immune responses, and provide strong protection against mutant SARS-CoV-2 in mice and rhesus monkeys. Other studies have reported that a single-dose optimized Zika virus circRNA vaccine can improve antigen expression and has effective and lasting protection in mice without inducing obvious dengue virus susceptibility effects.

Mechanism research: With the deepening of circRNA research, it has been confirmed that it is widely present in various organisms and plays an important role in various pathological processes such as cancer, cardiovascular disease, and Alzheimer's disease. Deciphering its mechanism of action under specific physiological or pathological conditions remains an important task at present.

Creative Biogene offers a series of premade circular RNA products with high stability and translation efficiency to satisfy your various downstream applications.

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