Development and comprehensive evaluation of scarless circularization systems for circular RNA therapeutics.

Circular RNAs (circRNAs) are promising candidates for RNA-based therapeutics due to their enhanced stability and sustained protein production compared to linear mRNAs. Traditional circRNA production methods, such as the Anabaena-based permuted intron-exon (Ana-PIE) system, often introduce extraneous sequences, referred to as "scars." However, a comprehensive evaluation of the functional consequences of incorporating or omitting extraneous "scar" sequences during circRNA production is lacking. In this study, we developed two scarless circRNA circularization systems, SCAP and mSCAP, based on Ana-PIE. We systematically compared these systems, along with the scarless Clean-PIE approach, across key performance metrics: circularization efficiency, protein production, stability, and immunogenicity. By quantifying these parameters using multiple reporter genes, we provide a comprehensive evaluation demonstrating that removing scar sequences, particularly with the SCAP system, can enhance protein production while preserving stability and maintaining minimal immunogenicity. Our comprehensive evaluation establishes a framework for the rational design of circRNA therapeutics.