Effective Synthesis of High-Integrity mRNA Using In Vitro Transcription

• Published in: Molecules (Basel, Switzerland) Vol. 29; no. 11; p. 2461
• Main Authors: He, Wei, Zhang, Xinya, Zou, Yangxiaoyu, Li, Ji, Wang, Chong, He, Yucai, Jin, Qiuheng, Ye, Jianren
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 23.05.2024; MDPI
• Subjects: Amino acids; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; FDA approval; fragmented mRNA; Hydrolysis; Messenger RNA; Molecular weight; mRNA integrity; mRNA Vaccines; mutation; Phosphates; Raw materials; RNA polymerase; RNA, Messenger - genetics; synthesis; T7 RNA polymerase; transcription; Transcription, Genetic; Vaccines; Viral Proteins - genetics; Viral Proteins - metabolism; United States; China; synthesis; T7 RNA polymerase; fragmented mRNA; mutation; mRNA integrity; transcription
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules29112461

mRNA vaccines are entering a period of rapid development. However, their synthesis is still plagued by challenges related to mRNA impurities and fragments (incomplete mRNA). Most impurities of mRNA products transcribed in vitro are mRNA fragments. Only full-length mRNA transcripts containing both a 5'-cap and a 3'-poly(A) structure are viable for in vivo expression. Therefore, RNA fragments are the primary product-related impurities that significantly hinder mRNA efficacy and must be effectively controlled; these species are believed to originate from either mRNA hydrolysis or premature transcriptional termination. In the manufacturing of commercial mRNA vaccines, T7 RNA polymerase-catalyzed in vitro transcription (IVT) synthesis is a well-established method for synthesizing long RNA transcripts. This study identified a pivotal domain on the T7 RNA polymerase that is associated with erroneous mRNA release. By leveraging the advantageous properties of a T7 RNA polymerase mutant and precisely optimized IVT process parameters, we successfully achieved an mRNA integrity exceeding 91%, thereby further unlocking the immense potential of mRNA therapeutics.