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

Circular RNAs (circRNAs) exhibit extended stability and enhanced,sustained protein expression. Nucleoside modification and rolling circle translation are supposed to reduce the immunogenicity and enhance the translational efficiency of circRNAs. However, it remains challenging to produce encodable circRNAs with modified nucleosides rather than the original nucleosides. Here, we identified a 39-nucleotide Cap-independent Translation Enhancer (CITE) element, termed BBV, which effectively drove the translation of nucleoside-modified RNAs with minimal immunogenicity. Subsequently, we demonstrated that the in vivo-produced circRNA harboring BBV could achieve efficient and sustained rolling circle translation lasting two weeks. Furthermore, we developed an extracellular vesicle-based delivery platform, termed EPM-EABR ASsisted Vesicle Yield (EASY), to deliver circRNAs without introducing viral Gag-Pol proteins. Additionally, we developed a Permuted Intron for Clean CircRNAs (PICC) system to produce scarless circRNAs via in vitro transcription and achieved efficient rolling circle translation driven by BBV. In a murine model, we demonstrated that the PICC-produced circRNA vaccine outperformed the N1m?-mRNA vaccine in inducing anti-tumor immunity. Importantly, we succeeded to achieve complete substitution with modified nucleosides in circRNAs via Twister ribozyme self-cleaving, dramatically reducing the immunogenicity of circRNAs, but still retaining the ability of rolling circle translation. Collectively, we established a nucleoside-modified circRNA platform that facilitates efficient translation with minimal immunogenicity, offering a safer and more effective platform for applications in vaccine development and disease treatment. Overall design: RNA-seq profiling of A549 cells treated with unmodified or nucleoside-modified circRNA and single cell suspension obtained by percoll of tumor from OVA+ CT26 tumor-bearing mice treated with PBS, modified mRNA encoding OVAFL or circRNA encoding OVApeptide generated by PICC system.