Creating Synthetic mRNA

Synthetic mRNA has so many uses now in biotechnology. It is one of the great genetic engineering tools available now that can transform a static microbial cell into a dynamic mini factory.

The applications are steadily rising all the time. We have:-

• cancer immunotherapy,
• protein replacement therapy,
• genome editing,
• pluripotent stem cell generation (Breda et al., 2023)
• vaccines against infectious diseases (Baden et al., 2021).

Creating synthetic mRNA involves several steps, including in vitro transcription, capping, and purification. We shall see that the current manufacturing methods for mRNA are produced in standardized in vitro transcription (IVT) systems. These systems are made up of an RNA Polymerase biocatalyst, DNA template, modified nucleosides, magnesium-containing buffer, and a capping enzyme/analog (Ourandis et al., 2022).

 The whole manufacturing process of synthetic mRNA is generally seen as simple and modular in structure. It does not need cells and so is described as a cell-free production platform. These manufacturing systems are also fairly flexible and now have great predictability (Whitley et al., 2022).

The main issue why mRNA synthesis is not conducted in microbial cell systems is because of cellular pathways that encode and encourage rapid RNA turnover. The average mRNA half-life in E.coli is a short 5 minutes. One way to retain double-stranded RNA (dsRNA) to create g/L quantities is achieved by removing RNase III which is a nonessential dsRNA-targeting endonuclease (Pertzev, 2006).

Here’s a step-by-step guide on how to produce synthetic mRNA:

Materials Needed

1. DNA Template: Linearized plasmid DNA or a PCR product containing a promoter for RNA polymerase (e.g., T7, SP6).
2. RNA Polymerase: Corresponding to the promoter on your DNA template (e.g., T7 RNA polymerase).
3. Nucleotides: ATP, GTP, CTP, and UTP.
4. Capping Enzymes: For the addition of a 5′ cap (Cap 0, Cap 1).
5. Poly(A) Polymerase: For adding a poly(A) tail.
6. Transcription Buffer: Appropriate buffer for in vitro transcription.
7. RNase Inhibitors: To prevent RNA degradation.
8. Purification Kits/Reagents: To purify the synthesized mRNA.
9. DNase I: To remove the DNA template after transcription.

Step-by-Step Procedure

1. Preparation of DNA Template
   • Ensure your DNA template includes a promoter recognized by your chosen RNA polymerase.
   • Linearize the plasmid containing your gene of interest downstream of the promoter using a restriction enzyme, or use a PCR product with the promoter and gene.
2. In Vitro Transcription
   • Set up the reaction: Mix the following components in an RNase-free tube:
     • Linearized DNA template (1-2 µg)
     • RNA polymerase (T7, SP6, or T3)
     • Transcription buffer
     • NTP mix (ATP, CTP, GTP, UTP, typically 1 mM each)
     • RNase inhibitor
   • Incubation: Incubate the mixture at the recommended temperature (typically 37°C) for 1-2 hours.
3. Capping the mRNA
   • After transcription, add the capping enzyme and reagents to the transcription reaction to cap the 5′ end of the mRNA. This is crucial for stability and translation efficiency in eukaryotic cells.
   • Incubation: Follow the enzyme manufacturer’s instructions for incubation time and temperature.
4. Poly(A) Tail Addition
   • Add poly(A) polymerase and ATP to add a poly(A) tail to the 3′ end of the mRNA. The poly(A) tail enhances stability and translation.
   • Incubation: Again, follow the enzyme manufacturer’s instructions for incubation.
5. DNA Template Removal
   • Add DNase I to degrade the DNA template. This step is important to ensure that no DNA contaminates the mRNA preparation.
   • Incubation: Incubate at 37°C for about 15-30 minutes.
6. Purification of Synthetic mRNA
   • Purify the mRNA using a suitable method, such as:
     • Spin column-based RNA purification kits.
     • Phenol-chloroform extraction followed by ethanol precipitation.
   • Ensure all buffers and reagents are RNase-free to prevent degradation of your mRNA.
7. Quality Control
   • Verify the size and integrity of the mRNA by agarose gel electrophoresis.
   • Assess the concentration and purity using a spectrophotometer or a bioanalyzer.

Example Protocol

In Vitro Transcription Reaction Mix:

• 1-2 µg of linearized DNA template
• 2 µL of 10X transcription buffer
• 2 µL of NTP mix (1 mM each)
• 1 µL of RNA polymerase
• 1 µL of RNase inhibitor
• RNase-free water up to 20 µL

Capping Reaction Mix:

• Add 5 µL of the capped mRNA mix to the transcription reaction.
• Follow specific instructions from the capping kit.

Poly(A) Tailing Reaction Mix:

• Add 5 µL of the poly(A) tailing mix to the capped mRNA.
• Follow specific instructions from the poly(A) tailing kit.

Purification:

• Follow the steps provided in the RNA purification kit.

This protocol provides a general framework, but specific details and conditions may vary depending on the kits and reagents used. Always refer to the manufacturer’s instructions for the most accurate and successful results.

References

Baden, L. R., El Sahly, H. M., Essink, B., Kotloff, K., Frey, S., Novak, R., Diemert, D., Spector, S. A., Rouphael, N., Creech, C. B., McGettigan, J., Khetan, S., Segall, N., Solis, J., Brosz, A., Fierro, C., Schwartz, H., Neuzil, K., Corey, L., … Zaks, T. (2021). Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. New England Journal of Medicine, 384, pp. 403–416

Breda, L., Papp, T. E., Triebwasser, M. P., Yadegari, A., Fedorky, M. T., Tanaka, N., Abdulmalik, O., Pavani, G., Wang, Y., Grupp, S. A., Chou, S. T., Ni, H., Mui, B. L., Tam, Y. K., Weissman, D., Rivella, S., & Parhiz, H. (2023). In vivo hematopoitic stem cell modification by mRNA delivery. Science, 381, pp. 436–443 (Article)

Ouranidis, A., Vavilis, T., Mandala, E., Davidopoulou, C., Stamoula, E., Markopoulou, C. K., Karagianni, A., & Kachrimanis, K. (2022). mRNA therapeutic modalities design, formulation and Manufacturing under pharma 4.0 principles. Biomedicines, 10, pp. 50

Pertzev, A. V. (2006). Characterization of RNA sequence determinants and antideterminants of processing reactivity for a minimal substrate of Escherichia coli ribonuclease III. Nucleic Acids Research, 34, pp. 3708–3721.

Whitley, J., Zwolinski, C., Denis, C., Maughan, M., Hayles, L., Clarke, D., Snare, M., Liao, H., Chiou, S., Marmura, T., Zoeller, H., Hudson, B., Peart, J., Johnson, M., Karlsson, A., Wang, Y., Nagle, C., Harris, C., Tonkin, D., … Johnson, M. R. (2022). Development of mRNA manufacturing for vaccines and therapeutics: mRNA platform requirements and development of a scalable production process to support early phase clinical trials. Translational Research, 242, pp. 38–55.