Analysis of the Effects of Different Synthesis and Processing Methods on Circular RNA Integrity,Protein Expression,and Removal of Immunogenic Impurities

Circular RNAs(circRNAs)are emerging as a promising alternative to messenger RNAs(mRNAs)in gene delivery applications due to their enhanced stability and translation.Developing circRNA-based therapeutic platforms requires efficient manufacturing of circRNA with broad scalability.However,the permuted intron-exon(PIE)-based circRNA production commonly used to date involves complex RNA synthesis,circularization,precursor RNA digestion,and impurity removal steps that have limited practical applications.While co-transcriptional circularization could effectively streamline circRNA production,and both cellulose/phosphatase treatment and high-performance liquid chromatography(HPLC)have demonstrated their reliability in mRNA manufacturing,their potential effects on the quality,translation,and reactogenicity of circRNA remained to be fully investigated.Here,using circRNAs systematically manufactured through three independent workflows,we comprehensively examined the utilities of these RNA synthesis and processing methods in circRNA production by comparing the integrity,translation,and immunogenicity of their circRNA products.We began by manufacturing a mNeonGreen(mNG)-encoding circRNA through these workflows and subsequently assessed circRNA integrity via E-gel EX electrophoresis.Protein expression was then monitored in HEK 293T,A549,and DC2.4 cells at 72 hours post-transfection.Finally,we evaluated the immunogenicity of these circRNAs by measuring their interferon beta(IFN-β)induction in A549 cells at 4 hours post-transfection.Using HPLC purification over cellulose and phosphatase treatment resulted in 10-14%higher circRNA enrichment by reducing nicking associated with processing conditions.Protein expression remained consistent across circRNAs from different workflows(P>0.05),demonstrating that co-transcriptional circularization produces circRNA with translation levels comparable to those obtained from the conventional PIE method.Moreover,both cellulose/phosphatase treatment and HPLC purification effectively minimized IFN-βinduction of the purified circRNAs,confirming their reliability in removing immunogenic impurities introduced during in vitro transcription and their compatibility with the co-transcriptional circularization strategy.Collectively,our results provide valuable insights for improving the production efficiency and scalability of circRNA manufacturing that are crucial for addressing key bottlenecks in the development of circRNA-based therapeutic applications.