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The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
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The gene expression in cells is regulated at different stages: (i) transcription, (ii) RNA processing, (iii) RNA localization, and (iv) translation. Transcriptional regulation is mediated by regulatory proteins such as transcription factors, activators, or repressors—these control gene expression by initiating or inhibiting the transcription of genes. Once a precursor or pre-mRNA is produced, it undergoes post-transcriptional modification, including 5' capping, splicing, and the...
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MicroRNAs01:22

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Exploring the Impact of In Vitro-Transcribed mRNA Impurities on Cellular Responses.

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Manufacturing messenger RNA (mRNA) therapies requires understanding impurities. This study links specific RNA impurities to cellular responses and reactogenicity, emphasizing advanced analytical methods for quality control.

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Optimization of In vitro Transcription Reaction for mRNA Production Using Chromatographic At-Line Monitoring

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Immunology

Background:

  • Messenger RNA (mRNA) therapies represent a significant advancement in medicine, utilizing a standardized in vitro transcription (IVT) manufacturing process.
  • Downstream purification is crucial for removing impurities that can impact the safety and efficacy of mRNA therapeutics.
  • A clear understanding of critical mRNA impurities and their correlation with therapeutic outcomes is still needed.

Purpose of the Study:

  • To establish structure-function relationships between specific in vitro-transcribed mRNA impurities and their impact on cellular responses.
  • To investigate the role of different bacteriophage T7 RNA polymerases in generating various mRNA impurities.
  • To correlate mRNA impurity levels with therapeutic efficacy indicators such as protein expression and reactogenicity.

Main Methods:

  • Production of in vitro-transcribed mRNAs using wild-type and engineered T7 RNA polymerases.
  • Assessment of mRNA integrity, purity, and functional activity via advanced physicochemical and cellular assays.
  • Quantification of impurities like abortive transcripts, partial poly(A) tails, and double-stranded (ds)RNA using mass photometry and native mass spectrometry.

Main Results:

  • Structure-function relationships were established for abortive transcripts, partial poly(A) tails, and dsRNA byproducts by monitoring cellular responses.
  • Differences in T7 RNA polymerase variants directly influenced the levels of sense-antisense dsRNA byproducts, correlating with immunological reactogenicity in dendritic cells.
  • Native mass spectrometry precisely resolved short 3'-loopback dsRNA byproducts, highlighting T7 RNA polymerase influence.

Conclusions:

  • The study underscores the critical need for sensitive analytical methods to characterize IVT mRNA impurities.
  • Understanding the interaction between mRNA impurities and cellular machinery is essential for ensuring the quality and safety of RNA-based therapies.
  • Optimizing the IVT process by selecting appropriate T7 RNA polymerases can help minimize critical impurities and enhance therapeutic performance.