Technology Overview
Circular RNA and cirRNA expression vector
Circular RNA (circRNA) is a class of single-stranded RNA molecules with a closed loop structure, formed by reverse splicing of the 5‘ and 3’ ends of linear RNA. Unlike linear RNAs, circular RNAs do not have 5′ and 3′ free ends, and therefore have higher stability and are less susceptible to degradation by exonucleases. Circular RNAs have a variety of important biological functions within the cell. For example, they can act as miRNA sponges, adsorbing miRNAs and thus regulating their activity; they may also be involved in processes such as regulating gene expression and influencing cell proliferation and differentiation. Although most natural circRNAs are non-coding RNAs and cannot be translated into proteins, it has been shown that circular RNAs with IRES sequences can be translated into proteins in vivo and in vitro (Hwang, H.J., Kim, Y.K., 2024). Due to the absence of a cap structure at the 5′ end and a poly(A) tail at the 3′ end, circular mRNAs are resistant to degradation by nucleic acid exonucleases and exhibit higher stability than normal linear mRNAs. This unique structure enables circular mRNAs to express proteins more persistently in cells, providing new possibilities in areas such as gene therapy and vaccine development. GeneCopoeia has integrated Gateway cloning technology with a circRNA vector to construct circRNA optimised for gene expression. We offer over 35,000 human and mouse sequence-verified ORFs in pShuttle plasmids, which allow rapidly transfer into a circRNA vector. This technology reduces the time required for clone construction, eliminating the need for a tedious, time-consuming, multi-step sub-cloning processes involving enzyme digestions, ligations, gel electrophoresis and purification.RNA in vitro circularization strategy
The broad occurrence of circRNAs in vivo and the study of their structural and functional properties have caused demand for methods that allow efficient preparation of circular RNAs in vitro. There are several methods have be documented such as chemical ligation / synthesis, enzymatic ligation by using T7 RNA in vitro transcription, T4 DNA ligase and T4 RNA ligases as well as ribozyme method. Genecopoeia applied group I intron self-splicing system-coupled with Gateway cloning technology, high quality human and mice shuttle ORF Entry clones and T7 RNA polymerase to produce circular mRNA.Application of circular mRNA
- Gene therapy: CircRNAs acts as a stable gene expression vector that can provide long-lasting therapeutic effects without integrating into the host genome. Circular mRNA has a longer half-life than linear mRNA, meaning that it can provide gene expression in cells for longer periods of time, reducing the frequency of treatment. Compared to other gene vectors (e.g. some viral vectors), circular mRNA triggers a lower immune response, reducing the risk of side effects during treatment. By designing specific promoters and regulatory elements, precise regulation of circular mRNA expression can be achieved, improving therapeutic targeting.
- Vaccine Development: The high stability of circular mRNAs allows them to maintain their structural and functional integrity during vaccine production, storage and transport, increasing the utility of vaccines. Circular mRNAs are capable of sustained expression of antigenic proteins, which provides a long-lasting immune stimulus and enhances the immunogenicity of the vaccine. Compared to vaccines using live or attenuated viruses, circular mRNA-based vaccines do not involve live pathogens, reducing the risk of potential infection.
- Protein Production: In the biopharmaceutical and biomaterials industries, circular mRNA can be used as an efficient and stable method to produce recombinant proteins. The stability and long-term expression properties of circular mRNA allow it to produce large amounts of proteins consistently in cell factories or bioreactors.
