Enhancing Circular RNA Translation Efficiency Through Dual Internal Ribosome Entry Sites

Circular RNA (circRNA) has emerged as a promising vector for drug delivery because, unlike linear mRNA, it does not require costly chemical modifications and offers greater stability and sustained expression in cells. Lacking the canonical 5' cap structure, circRNA relies primarily on internal ribosome entry sites (IRES) to initiate translation, but IRES-mediated initiation is less efficient than cap-dependent translation. To overcome this limitation, we devised a dual-IRES strategy that introduces a second IRES to drive translation of the coding sequence (CDS). By testing several IRES elements known for high translational activity, this study shows that IRESs derived from the EMCV (Encephalomyocarditis virus) family can enhance expression when placed at the 3' of the CDS, in coordination with the 5' EMCV-derived IRES. The optimal dual-IRES combinations identified in this study display compatibility with two different coding sequences, offering a useful strategy to enhance circRNA translation.