Optimization Of In Vitro Transcription For mRNA Production

By Millicent Lin, R.K. DeLong, Mathew Brier, Manish Tandon, Li-wen Wang, Yun Chen, Radouane Zouaoui, Aviva Joseph, Eli Kraus, and Gregg Nyberg

In vitro transcription (IVT) is a biochemical process used to synthesize therapeutic RNA macromolecules, typically over 200 ribonucleotides long, which cannot be efficiently produced through automated solid support synthesis. The reaction, catalyzed by the T7 RNA polymerase at 37°C, is buffered near neutral pH, with cations to stabilize the mRNA-enzyme interaction. IVT performance is measured by RNA yield relative to DNA template input, or turnover. After IVT, key steps include 5'-end capping, DNase treatment, and purification, followed by tangential flow filtration (TFF) and mRNA lipid nanoparticle formulation.

To optimize IVT, we explored alternative salts and surfactants, achieving up to a 208-fold turnover and reducing the enzyme ratio while maintaining a 183-fold turnover, enhancing scalability. Standard agarose gel electrophoresis was compared to a newer HPLC assay to monitor mRNA purity and integrity across stages like capping, DNase treatment, and TFF buffer exchange, showing no significant impact on mRNA quality. The expression activity of our IVT-produced mRNA encoding green fluorescent protein (GFP) was confirmed through cellular delivery and fluorescence analysis. Our progress aims to develop a GMP-compatible process for clinically relevant mRNA production, with optimized IVT reactions and comparable expression activity to purified mRNA standards. Ongoing efforts focus on improving downstream purification, analytics, lipid nanoparticle formulation, and expanding IVT-based RNA production.