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

作     者：Shao Wang Tzuen Yih Saw Zebin Hong Kuo Chieh Liao Haiwei Song Yue Wan 

作者机构：Genome Institute of SingaporeAgency for ScienceTechnology and Research138672 SingaporeSingapore Department of Biochemistry and Molecular BiologyUniversity of British ColumbiaVancouverBC V6T 1Z3Canada Institute of Molecular and Cell BiologyAgency for ScienceTechnology and Research138673 SingaporeSingapore 

出 版 物：《Journal of Clinical and Nursing Research》 (临床护理研究（英文）)

年 卷 期：2024年第8卷第9期

页      面：191-200页

学科分类：1002[医学-临床医学] 100214[医学-肿瘤学] 10[医学] 

主　　题：Nucleic acid therapeutics RNA manufacturing Circular RNA Gene delivery 

摘      要：Circular RNAs(circRNAs)are emerging as a promising alternative to messenger RNAs(mRNAs)in gene delivery applications due to their enhanced stability and *** circRNA-based therapeutic platforms requires efficient manufacturing of circRNA with broad ***,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 *** 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 ***,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 *** began by manufacturing a mNeonGreen(mNG)-encoding circRNA through these workflows and subsequently assessed circRNA integrity via E-gel EX *** expression was then monitored in HEK 293T,A549,and DC2.4 cells at 72 hours ***,we evaluated the immunogenicity of these circRNAs by measuring their interferon beta(IFN-β)induction in A549 cells at 4 hours *** HPLC purification over cellulose and phosphatase treatment resulted in 10-14%higher circRNA enrichment by reducing nicking associated with processing *** expression remained consistent across circRNAs from different workflows(P0.05),demonstrating that co-transcriptional circularization produces circRNA with translation levels comparable to those obtained from the conventional PIE ***,both cellulose/phosphatase treatment