Optimizing Functional‐Domain Integrity of Circular Ribonucleic Acid With Improved Vaccine Immunogenicity by circ Design Algorithm

ABSTRACT Synthetic circular mRNA (hereafter referred to as circRNA) reduces susceptibility to exonuclease‐mediated degradation by its covalently closed circular structure, enabling prolonged protein expression for therapeutic applications. In this circular format, protein expression from engineered circRNAs is achieved mainly through cap‐independent translation initiation, commonly mediated by internal ribosome entry site (IRES) elements whose activity is influenced by RNA structure. Consequently, the coding sequence (CDS) and other elements should be designed with consideration of inter‐region base pairing that can shift IRES folding, a constraint not explicitly addressed by existing linear mRNA CDS optimization algorithms. Here, we present circ Design, an algorithm that explicitly incorporates IRES structural deviation into circRNA sequence design while jointly optimizing codon adaptation and thermodynamic stability. In a rabies virus glycoprotein (RABV‐G) vaccine model, circ Design‐generated circRNAs showed improved stability, translation efficiency, and vaccine immunogenicity compared with benchmark sequences optimized using conventional linear mRNA CDS design strategies, with CR3 achieving a 3.5‐fold increase in neutralizing antibody titers. Polysome profiling and targeted IRES‐disruption experiments support IRES structural integrity as a critical determinant of circRNA translation performance. Together, these results establish IRES structural preservation as a mechanistic design principle for circRNA engineering and position circ Design as a rational framework for therapeutic circRNA development.