Development of a modified RNA circularization system to improve circRNA-based protein expression in mammalian cells

Circular RNA (circRNA) is emerging as a highly promising technology in various biomedical applications, offering advantages over traditional linear RNA. The Twister-optimized RNA for the durable overexpression (Tornado) system has been widely investigated for generating circRNAs in mammalian cells; however, the use of the Tornado system for large RNA inserts, especially those containing the internal ribosome entry site (IRES) sequences, is hindered by low circularization efficiency and limited circRNA abundance. Therefore, developing novel strategies to enhance RNA circularization in cells is of critical importance. In this study, we present a modified Tornado system that significantly improves circRNA-based protein expression by incorporating an optimal distance between the IRES and the upstream CMV promoter. Furthermore, we elucidate the dual roles of HRV-B3 IRES in mammalian cells, demonstrating its negative regulatory effect on RNA abundance and its positive contribution to RNA circularization. Additionally, the integration of a truncated 5′ long terminal repeat (LTR) from HIV-1 upstream of the HRV-B3 IRES, combined with the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), further enhances transcriptional efficiency in the Tornado system. This modified system holds great potential for advancing circRNA-based therapeutics and vaccines, and these findings provide valuable insights for refining the Tornado system and designing regulatory elements in synthetic biology applications.