Comparative analysis of expression, immunogenicity, and safety profiles between linear and circular RNA vaccine...

mRNA vaccines have revolutionized development and continue to advance through clinical and research applications. Circular RNA is emerging as a promising alternative to linear RNA. However, no direct comparison has identified the optimal RNA platform for mRNA-based drug development. This study compares linear and circular RNA platforms in vitro and in vivo, focusing on expression dynamics, immune responses, and safety. In terms of expression efficiency, the circular RNA showed prolonged expression compared to linear RNA. The immunogenicity was evaluated using influenza HA as an antigen. In humoral immune response, linear RNA with modified nucleosides (Lin-m1Ψ) and circular RNA with unmodified nucleosides (Circ-WT) showed higher neutralizing antibody titers than linear RNA with unmodified nucleosides (Lin-WT). Lin-m1Ψ and Lin-WT induced slightly higher cell-mediated immune responses than Circ-WT. In mice, all RNA types showed a decrease in reticulocytes, monocytes, and alkaline phosphatase on day 2 following high-dose intramuscular injection. Furthermore, Lin-WT and Circ-WT showed greater decreases in platelets than Lin-m1Ψ. However, these changes recovered to normal by day 14 post-vaccination in all groups. These findings provide insights into developing RNA-based immunotherapies and selecting suitable RNA platforms for various applications, including preventive and therapeutic vaccines as well as protein-replacement therapies.