- Circular RNA (circRNA) vector expression technology refers to a set of molecular tools and strategies designed to artificially synthesize and express circular RNAs in living cells or organisms. This technology is instrumental for studying the functions of circRNAs, investigating their roles in gene regulation and disease, and harnessing their potential for therapeutic and vaccine development. Since natural circRNAs are generated by back-splicing events, recreating this in a controlled, efficient, and scalable manner has required the development of specialized vector systems.
- The core challenge in expressing circRNAs from a plasmid vector lies in facilitating the back-splicing mechanism. To address this, researchers typically design expression constructs in which exons flanked by complementary intronic sequences or inverted repeat elements promote the circularization of the pre-mRNA transcript. These repeats, often derived from Alu elements in humans or other synthetic sequences, facilitate the formation of RNA secondary structures (such as stem-loops), which bring the 3′ splice donor and 5′ splice acceptor sites into close proximity, promoting back-splicing. Commonly used vector backbones include those based on cytomegalovirus (CMV) promoters for robust transcription, sometimes coupled with self-splicing ribozyme systems or splicing-enhancing elements to increase circularization efficiency.
- There are two major types of synthetic circRNA expression approaches: plasmid-based vectors and in vitro transcription (IVT)-based synthetic circRNAs. In plasmid-based expression, circularization occurs inside the cell, allowing for sustained circRNA production under a strong promoter. This approach is especially useful for in vivo studies and long-term expression. In contrast, IVT-generated circRNAs are synthesized in vitro, circularized enzymatically (e.g., by ligases or ribozymes), purified, and then introduced into cells via transfection or injection. This latter method is more suitable for therapeutic applications where transient expression is desired and the use of DNA vectors is undesirable.
- To ensure high purity and efficacy, synthetic circRNAs are often engineered with internal ribosome entry sites (IRES) or N6-methyladenosine (m6A) modifications to enable cap-independent translation, which is critical if the circRNA is intended to express a functional peptide or antigen. This has led to growing interest in circRNA as a platform for protein expression, particularly in areas such as mRNA vaccines, where circRNAs may offer advantages over linear mRNA due to their enhanced stability and prolonged translation in cells. For example, several studies have shown that circRNA-based vaccines can elicit strong immune responses against viral or tumor antigens while being less susceptible to degradation.
- Despite these advances, circRNA vector expression technology still faces technical hurdles. These include optimizing circularization efficiency, preventing the formation of unwanted linear or concatemeric RNA species, ensuring nuclear export of synthetic circRNAs, and avoiding innate immune activation. Ongoing improvements in vector design, splicing motifs, and purification methods are helping to address these challenges.
- In summary, circRNA vector expression technology is a rapidly evolving field that enables precise functional studies of circular RNAs and opens new avenues for biomedical applications, including therapeutic protein production, gene regulation, and next-generation vaccines. As the technology matures, it holds great promise for both basic research and clinical translation.
