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Published on: November 9, 2019
Effective Synthesis of High-Integrity mRNA Using In Vitro Transcription
Wei He1,2, Xinya Zhang2, Yangxiaoyu Zou2
1College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China.
Abstract:
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.
Insights
Researchers developed a new method to improve messenger RNA (mRNA) vaccine production. By modifying T7 RNA polymerase and optimizing processes, they significantly reduced mRNA fragments, enhancing vaccine efficacy.
Area of Science:
- Biotechnology
- Molecular Biology
- Vaccine Development
Background:
- Messenger RNA (mRNA) vaccines are rapidly advancing but face production challenges, primarily incomplete mRNA fragments.
- These fragments, arising from hydrolysis or premature termination, hinder in vivo expression and therapeutic efficacy.
- Full-length mRNA with 5'-cap and 3'-poly(A) structures is essential for successful mRNA therapeutics.
Purpose of the Study:
- To identify and address the causes of mRNA fragmentation during in vitro transcription (IVT).
- To enhance the integrity and efficacy of mRNA for therapeutic applications.
- To improve the manufacturing process of mRNA vaccines.
Main Methods:
- Investigated T7 RNA polymerase during in vitro transcription (IVT) to pinpoint sources of mRNA impurities.
- Identified a critical domain on T7 RNA polymerase linked to premature mRNA release.
- Utilized a T7 RNA polymerase mutant and optimized IVT process parameters.
Main Results:
- Achieved mRNA integrity exceeding 91% through process optimization and polymerase engineering.
- Successfully reduced the prevalence of mRNA fragments, a key impurity in IVT products.
- Demonstrated a viable strategy for controlling product-related impurities in mRNA synthesis.
Conclusions:
- The study successfully identified a key factor in T7 RNA polymerase activity leading to mRNA fragmentation.
- Optimized IVT processes and T7 RNA polymerase mutants significantly enhance mRNA integrity.
- These advancements hold promise for unlocking the full potential of mRNA therapeutics and vaccines.
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01:10Transcription
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