Improving the Circularization Efficiency, Stability and Translatability of Circular RNA by circDesign

Abstract Synthetic circular RNA (circRNA) has recently emerged as the next generation RNA technology for vaccine and drug development, featuring its uniqueness in a closed-loop structure and cap-independent translation mechanism. However, rational design and optimization of circRNA to improve its stability and protein coding potential remains challenging due to lack of structure prediction tool and sequence design algorithm for endless circular-shaped RNA. In this study, we presented circDesign, a computational approach to achieve optimal design of the circRNA with enhanced circularization efficiency, stability, and translatability. Using rabies virus glycoprotein (RABV-G) and varicella-zoster virus (VZV) glycoprotein gE as the exemplified antigens, we demonstrated that optimized circRNAs encoding corresponding antigens exhibited higher stability and protein translation efficiency in vitro . Formulated by proprietary delivery system lipopolyplex (LPP), the optimized circRNA vaccines encoding RABV-G or VZV gE antigen further elicited enhanced humoral and cellular in vivo immune responses in mice. Taken together, this circular RNA design approach provided a general strategy to leverage the capability of circRNA as next generation vaccines or therapeutics.