Optimized circular RNA vaccines for superior cancer immunotherapy

<b>Rationale:</b> Circular RNA (circRNA) has gained attention as a promising platform for mRNA vaccines due to its stability, sustained protein expression, and intrinsic immunostimulatory properties. This study aimed to design and optimize a circRNA cancer vaccine platform by screening for efficient internal ribosome entry sites (IRES) and enhancing circRNA translation efficiency for improved cancer immunotherapy. <b>Methods:</b> We screened 29 IRES elements to identify the most efficient one for immune cell translation, ultimately discovering the <i>Enterovirus</i> A (EV-A) IRES. Using SHAPE-MaP technology, we analyzed the secondary structure of circRNA and introduced targeted mutations and deletions to optimize translation efficiency. Additionally, we investigated the regulatory roles of spacer sequences and microRNA recognition sites in circRNA design and examined the mechanisms behind IRES-mediated translation initiation. <b>Results:</b> The EV-A IRES was identified as the most efficient for immune cell translation. Structural modifications and optimization of spacer sequences enhanced the translation efficiency of circRNA. Comparative studies demonstrated that circRNA vaccines induced stronger T cell immune responses and exhibited superior tumor prevention and therapeutic efficacy compared to traditional linear mRNA vaccines. <b>Conclusion:</b> The optimized tumor antigen circRNA vaccine platform offers a stable, efficient alternative to conventional mRNA vaccines for cancer immunotherapy, with enhanced immune responses and improved therapeutic outcomes. This work lays the foundation for developing circRNA-based vaccines as a novel strategy for cancer treatment.