Mitigating Cellular Dysfunction Through Contaminant Reduction in Synthetic circRNA for High-Efficiency mRNA-Based Cell Reprogramming

Abstract

Synthetic circular RNA (circRNA) holds great promise for biomedical research and therapeutic applications, but impurities introduced during synthesis trigger innate immune responses and significantly compromise its efficacy. In this study, key immunogenic byproducts, including double-stranded RNA, 5′ triphosphates from uncircularized RNA, and hydrolyzed RNA fragments, are identified as impairing circRNA functionality via RNA-sensing pathways. To address this, a multi-step purification process is developed that combines enzymatic treatments and cellulose-based filtration to effectively remove these contaminants. This approach significantly reduces immune activation and increases manufacturing yields of circRNA by over 10-fold. The purified circRNA demonstrates exceptional performance in induced pluripotent stem cells (iPSCs) reprogramming, achieving over 300% reprogramming efficiency with just three OSKMLN circRNA transfection treatments. Additionally, the purified circRNA achieves high levels and persistent expression of chimeric antigen receptor (CAR) in T cells with high cytotoxicity against tumor cells, making it a promising candidate for mRNA-based CAR-T cell therapy. These findings establish the purification strategy as a scalable and reliable platform for producing therapeutic-grade RNA, with broad applications in mRNA-based cell reprogramming for regenerative medicine and cancer immunotherapy.