Engineered circular RNA compatible with complete nucleoside modification and rolling circle translation through a Cap-independent translation enhancer

Circular RNA (circRNA) exhibits extended stability and enhanced protein expression, and its translational efficiency and immunogenicity can be improved through nucleoside modification and rolling circle translation (RCT). However, it remains challenging to produce protein-encoding circRNA with modified nucleosides. Here we identified a cap-independent translation enhancer (CITE) element from black beetle virus, termed BBV, which could drive the translation of nucleoside-modified RNA and RCT of engineered circRNA. In addition, we developed a system to produce ‘scarless’ circRNA via in vitro transcription (IVT). The resulting circRNA vaccine outperformed m1ψ-mRNA vaccine in inhibiting tumour growth in mice. With nucleoside modification, circRNA exhibited reduced expression of pro-inflammatory cytokines. Further, nucleoside-modified circRNA encoding glucagon-like peptide-1 (GLP-1) peptides reduced blood glucose levels with efficacy comparable to that of commercial semaglutide, and ameliorated liver damage in obese mice. Moreover, nucleoside-modified circRNA encoding myelin oligodendrocyte glycoprotein (MOG35–55) peptides induced immune tolerance and alleviated disease progression in an experimental autoimmune encephalomyelitis (EAE) murine model. Collectively, we established a nucleoside-modified circRNA platform that facilitated efficient translation with minimal immunogenicity, expanding the application scenarios of circRNA beyond vaccines. A suite of tools to improve translation and immunogenicity of circular RNAs, including nucleoside modification and rolling circle translation, for applications in cancer vaccines, protein replacement and autoimmune therapies.