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Harnessing the potential of mRNAs to express proteins of interest has emerged as a powerful tool in biotechnology and medicine, particularly in the development of vaccines. Such mRNAs can be in-vitro transcribed (IVT) in a controlled laboratory environment using a DNA template that encodes the protein of interest. Key components required include the DNA template, T7 RNA polymerase, ribonucleoside triphosphates, and necessary cofactors. The resulting mRNA is then purified and can be loaded directly into target cells ex vivo or formulated into lipid nanoparticles for in vivo delivery as, e.g. a vaccine. IVT-produced mRNA vaccines offer rapid development, high efficacy, and a versatile platform, as demonstrated by the success of COVID-19 vaccines. In this chapter, we describe the production and purification of unmodified IVT mRNA for ex vivo generation of dendritic cell (DC) mRNA vaccines or T cells engineered with chimeric antigen receptors.

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Large-Scale Production of Unmodified mRNA for Reprogramming Human Dendritic Cells and T Cells

  • 实验方案

  • 发表日期：

    2025年

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  • Stein Sæbøe-Larssen,
  • Mouldy Sioud

摘要

Harnessing the potential of mRNAs to express proteins of interest has emerged as a powerful tool in biotechnology and medicine, particularly in the development of vaccines. Such mRNAs can be in-vitro transcribed (IVT) in a controlled laboratory environment using a DNA template that encodes the protein of interest. Key components required include the DNA template, T7 RNA polymerase, ribonucleoside triphosphates, and necessary cofactors. The resulting mRNA is then purified and can be loaded directly into target cells ex vivo or formulated into lipid nanoparticles for in vivo delivery as, e.g. a vaccine. IVT-produced mRNA vaccines offer rapid development, high efficacy, and a versatile platform, as demonstrated by the success of COVID-19 vaccines. In this chapter, we describe the production and purification of unmodified IVT mRNA for ex vivo generation of dendritic cell (DC) mRNA vaccines or T cells engineered with chimeric antigen receptors.