In-vitro transcription (IVT) is a reactor-based scheme to manufacture RNA where cell-derived impurities or adventitious contaminants can be avoided thus making the manufacturing process GMP-friendly. The IVT reaction is often carried out at 37°C for 2 hours. During the IVT reaction, T7 or SP6 RNA polymerase binds to the promoter region of a DNA template and catalyzes the formation of complementary RNA using nucleotide triphosphates (NTPs) as building blocks. The IVT reaction should be subject to design-of-experiments (DOE) to optimize the reaction time, temperature, magnesium concentration, DNA template input, etc. to achieve the best results for any given RNA construct.
To make mRNA with co-transcriptional capping, a cap analog is added to the IVT reaction to generate the m7G cap. Since the most widely utilized promoter sequence for T7 RNA polymerase is TAATACGACTCACTATA-GGGAGA starting from -17 position to +6 position, the first-generation dinucleotide cap analogs are m7GpppG derivatives to match the guanosine at the +1 position. Using this unmodified T7 promoter sequence, Ishikawa et al. successfully synthesized mRNA using m7GpppAmG cap analog as reported in his 2009 paper even though the yield and purity were not reported [1]. The paper listed the IVT condition as using 7.5mM ATP, CTP, UTP, 0.9mM GTP, and 6 mM cap analog. Notice the reduced GTP concentration here as it was implemented to minimize the competing reaction using GTP instead of m7GpppAmG as the mRNA starting nucleotide. We tried a similar strategy with the DNA template below using 10mM ATP, CTP, UTP, and 2.5mM GTP where we achieved 2.5ug/ul yield and 98% capping efficiency when synthesizing firefly luciferase mRNA.
Why would anyone use this approach? For two possible reasons –
- You do not want to change your DNA template. Maybe you used ARCA before and want to try m7GpppAmG, CAP4, or CAP5 to get the Cap1 structure. You have the plasmid made already and do not want to start over with a new plasmid. Or you used enzymatic capping before and want to try co-transcriptional capping to reduce manufacturing steps. In this case, your DNA template is likely to have G at the +1 position.
- There is prior art using this promoter sequence so the likelihood of needing a third-party IP license is low.
The downside of this approach is that capping efficiency and yield are not optimal.
An alternative approach is to change the protomer sequence to TAATACGACTCACTATA-AGGAGA by replacing the guanosine at the +1 position with adenosine. This DNA template matches better with the m7GpppAmG cap analog as there are two base pairings at the mRNA start. If you think of m7GpppAmG as an initiating primer, it pairs better with TC than CC in the bottom strand. Using the template below with 10mM ATP, CTP, UTP, and GTP, we could achieve 9ug/ul yield and 98% capping efficiency when synthesizing firefly luciferase mRNA with 10mM m7GpppAmG, Cap4 or Cap5 cap analog.
The advantage of changing promoter sequence is to achieve higher yield and higher capping efficiency. The downside is that you may need to license a third-party IP to use this template.
Once the DNA template and cap analog are chosen, DOE may be performed to select the best IVT condition for higher yield, purity, capping efficiency or lower dsRNA. For detailed IVT protocols, please refer to the Hernderson paper [2] and Ferreira paper [3].
Co-transcriptional capping with m7GpppAmG, Cap4 or Cap5 can generate Cap1 mRNA in a single step with high capping efficiency and yield. Compared to enzymatic capping, this method is more GMP friendly as it requires fewer manufacturing steps and fewer units of operation in the GMP suite.
References
- Masahide Ishikawa, Ryuta Murai, Hiroyuki Hagiwara, Tetsuya Hoshino, Kamui Suyama, Preparation of eukaryotic mRNA having differently methylated adenosine at the 5′-terminus and the effect of the methyl group in translation, Nucleic Acids Symposium Series, Volume 53, Issue 1, September-October 2009, Pages 129–130, https://doi.org/10.1093/nass/nrp065
- Henderson, J. M., Ujita, A., Hill, E., Yousif-Rosales, S., Smith, C., Ko, N., McReynolds, T., Cabral, C. R., Escamilla-Powers, J. R., & Houston, M. E. (2021). Cap 1 messenger RNA synthesis with co-transcriptional cleancap® analog by in vitro transcription. Current Protocols, 1, e39. doi: 10.1002/cpz1.39
- Ferreira, Rafael & Petrides, Demetri. (2021). Messenger RNA (mRNA) Vaccine Large Scale Manufacturing – Process Modeling and Techno-Economic Assessment (TEA) using SuperPro Designer… 10.13140/RG.2.2.34118.80966
- Diana D. Kang, Haoyuan Li, Yizhou Dong, Advancements of in vitro transcribed mRNA (IVT mRNA) to enable translation into the clinics, Advanced Drug Delivery Reviews, Volume 199,2023,114961,ISSN 0169-409X, https://doi.org/10.1016/j.addr.2023.114961