An engineered T7 RNA polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis

Faraday Discuss. 2024 Sep 11;252(0):431-449. doi: 10.1039/d4fd00023d.

Abstract

Messenger RNA (mRNA) therapies have recently gained tremendous traction with the approval of mRNA vaccines for the prevention of SARS-CoV-2 infection. However, manufacturing challenges have complicated large scale mRNA production, which is necessary for the clinical viability of these therapies. Not only can the incorporation of the required 5' 7-methylguanosine cap analog be inefficient and costly, in vitro transcription (IVT) using wild-type T7 RNA polymerase generates undesirable double-stranded RNA (dsRNA) byproducts that elicit adverse host immune responses and are difficult to remove at large scale. To overcome these challenges, we have engineered a novel RNA polymerase, T7-68, that co-transcriptionally incorporates both di- and tri-nucleotide cap analogs with high efficiency, even at reduced cap analog concentrations. We also demonstrate that IVT products generated with T7-68 have reduced dsRNA content.

MeSH terms

  • DNA-Directed RNA Polymerases* / chemistry
  • DNA-Directed RNA Polymerases* / genetics
  • DNA-Directed RNA Polymerases* / metabolism
  • Protein Engineering
  • RNA Caps / chemistry
  • RNA Caps / genetics
  • RNA Caps / metabolism
  • RNA, Double-Stranded* / chemistry
  • RNA, Double-Stranded* / genetics
  • RNA, Double-Stranded* / metabolism
  • RNA, Messenger* / chemistry
  • RNA, Messenger* / genetics
  • RNA, Messenger* / metabolism
  • SARS-CoV-2 / genetics
  • SARS-CoV-2 / metabolism
  • Transcription, Genetic
  • Viral Proteins* / chemistry
  • Viral Proteins* / genetics
  • Viral Proteins* / metabolism

Substances

  • bacteriophage T7 RNA polymerase
  • RNA, Double-Stranded
  • DNA-Directed RNA Polymerases
  • RNA, Messenger
  • Viral Proteins
  • RNA Caps