Introduction

According to COVID data from World Health Organization (2022/02/28), total COVID cases have reached 400 million, and the COVID-induced death is close to 6 million people. On the bright sight, the COVID vaccines have been administered over 10 billion doses, and around 4 billion people are fully vaccinated now. Among the FDA-approved COVID vaccine, the primary modality is mRNA-based vaccines from Moderna or Pfizer/BNT. In February 2022, Moderna announced establishing subsidiaries in four Asian countries, including Taiwan, and planning to manufacture COVID vaccine and other mRNA-based drugs in Taiwan. What is the procedure to make an mRNA vaccine and mRNA-based drugs? Here I will explain how the lipid nanoparticles with mRNA are manufactured.

Mechanism of gene therapy

First, we need to know what mRNA is. In our cells, chromosomes composed of DNA and proteins in the nucleus control the gene expression by different DNA fragments. Additionally, the mechanism is that first, the DNA in the nucleus transcripts into messenger RNA (mRNA), and the mRNA would translate into protein to be expressed in the cells (Figure 1 ). Also, that’s why mRNA plays an essential role in molecular biology. To produce the mRNA, we would need a lot of DNA templates for in vitro transcription (IVT), and the easier way to collect a large number of DNA templates is to clone the desired DNA template into a plasmid and transforms it into E. coli, to use E. coli to grow and produce a lot of plasmids for IVT. After collecting the plasmids from E. coli, we would use enzymes to isolate the DNA template to make the RNA through IVT reactions. (Figure 2) The mRNA structure comprises the RNA coding region, untranslated region, capping, and poly-A tail. (Figure 3) Therefore, we need to add 5′ capping and 3′ poly-A tail to the IVT RNA before purification. The mRNA production is a simple reaction, but several essential elements affect the mRNA efficacy.

Key elements in IVT reaction

First, we need to add a T7 promoter in the DNA template because T7 RNA polymerase is required in the IVT. Second, the RNA coding region should be designed by codon-optimization to prevent the single-strand RNA from forming secondary structure to decrease the efficacy. Third, there is an untranslated region (UTR) on each side of the coding region. Although the UTR would not be translated into protein, these regions could affect the efficacy of mRNA in the cells. Additionally, a good UTR sequence could improve protein production in vitro and in vivo and alleviate the immune response triggered by the mRNA. In the biotech company, the UTR sequences are confidential because they could highly affect mRNA therapeutics’ efficacy. Last, the 5′ cap is also a crucial element in mRNA synthesis. In the COVID vaccine from Pfizer and Moderna, the capping system, CleanCapⓇ is from another mRNA biotech company, Trilink, to minimize the immune response in the body and increase the vaccine efficacy. Therefore, the whole production of mRNA is pretty complicated. I will prepare another blog to introduce the IVT reaction and the following procedure to produce mRNA.

Lipid nanoparticles production

Next, after purification of capped mRNA with polyA tail, we could mix the mRNA and lipid-like materials to manufacture the lipid nanoparticles (LNP). In the previous blog (introduction to gene therapy), I have introduced how cationic lipid-like materials form LNP under acidic conditions by electrostatic interaction. The critical factor in LNP production is mixing because well-mixing could allow the cationic materials and anionic nucleic acids to form a more compact structure and smaller nanoparticles. To make those mRNA-based LNP with less than 100 nanometers in diameter, we used a microfluidic device with a micromixer to better mix the organic phase (lipid-like materials) and aqueous phase (nucleic acids) by chaotic advection (Figure 4). The smaller size of LNP could prevent liver filtration after administration. Furthermore, another essential step is how we purify the LNP and remove the residuals during the process to minimize the side effects of the mRNA-based vaccine.

Conclusion

Although the whole process looks pretty simple, every step in manufacturing is critical to maintaining a safe and efficient vaccine. There is still a lot of work we could do in research to improve the mRNA therapeutics: How we design the coding sequences UTR sequences. How we develop a new capping system and nucleotides to alleviate immune response. Also, currently, all the FDA-approved mRNA-based vaccines are used non-degradable materials to deliver mRNA. Most scientists and biotech companies are working on new degradable materials in RNA therapeutics. Therefore, if you are interested in gene therapy, maybe you could consider contributing to the field of mRNA therapeutics.

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