The administration route and molecular architecture of nucleic acid vaccines are known to independently influence immunogenicity, yet how they interact to cooperatively shape in vivo immune polarization remains unexplored. This knowledge gap critically hinders the rational design of circular RNA (circRNA) vaccines for specific clinical applications. Here, through a systematic evaluation in murine models of two structurally distinct circRNAs delivered via four routes (intramuscular, subcutaneous, intradermal, and intravenous), we uncover a crucial synergy between delivery path and molecular design. The circRNAs comprised a compact, epitope-encoding "small" type and a full antigen-encoding "large" type. We demonstrate that the delivery route dictates the quality of the immune response: intravenous administration primes robust systemic CD8T cell immunity and durable memory, ideal for prophylaxis, whereas localized routes (intradermal) elicit superior antitumor efficacy, supporting their use in therapeutic oncology. Concurrently, we identify circRNA architecture as a key tunable lever for immunogenic potency, with small circRNAs driving heightened T cell activation. These results reveal a crucial synergy, demonstrating that route and structure are interdependent determinants. Our work provides a framework for tailoring circRNA vaccines by strategically pairing molecular design with administration route to achieve precise immunological goals, from prevention to cancer therapy. +
mRNA Technology