Mitigating Cellular Dysfunction by Addressing Contaminants in Synthetic circRNA

Synthetic circular RNA (circRNA) shows great potential in biomedical research and therapies, but impurities introduced during its synthesis can undermine circRNA efficacy. This study investigated the immunogenic potential of byproducts generated during its production. We found that trace amounts of double-stranded RNA in high molecular weight impurities, 5’ triphosphates of uncircularized RNA or small intron fragments, and hydrolyzed RNA fragments significantly impact the functionality of circRNA by activating innate immune responses through the sensory molecules involved in RNA sensing. To address this, we developed a novel multi-step purification process that employs enzymatic treatments and cellulose-based filtration to selectively remove these detrimental contaminants. This tailored approach minimizes cellular immune reactions and substantially improves circRNA yields with up to more than 10 times increase. Our findings underscore the critical impact of precise contaminant management in enhancing the expression and potential therapeutic utility of circRNA. This suggests a new direction for optimizing their production for both research and clinical applications.