The structure of mRNA can be divided into five parts, starting from the 5 ‘end, namely, 5’ cap, 5 ‘non-transcriptional region (UTR), open reading frame encoding antigen, 3’UTR and polyadenine tail. Each part is critical to the normal work of the mRNA vaccine.

Sequence Design

  • 5′ cap

One of the functions of the 5′ cap is to prevent mRNA from being detected by sensors that recognize viral RNA in cells, thereby preventing unnecessary immune responses. It also protects mRNA from degradation by exonuclease.

The process of capping mRNA:

  1. The first nucleotide at the 5 ‘end of mRNA removes one phosphate under the action of triphosphatase, and then GTP removes two phosphates to connect to mRNA under the action of guanosine transferase, which is called G cap.
  2. S-AdenosylMethionine (SAM), as a methyl donor, was methylated at position 7 of guanosine to m7GpppNp cap (Cap 0) under the action of N7-methyltransferase.
  3. SAM continues to act as a methyl donor, methylating the second nucleoside of mRNA to m7GpppmNp (Cap 1) under the action of 2’O-ribose methyltransferase.
  4. If you continue to methylate the second position of the third nucleoside, it will become m7GpppmNpmNp (Cap 2).
  • 3′ polyA tail

The length of 3′ polyA tail indirectly regulates the translation and half-life of mRNA. The length of the PolyA tail is generally 100-150nt to effectively form a translation complex and protect the 5′ hat from falling off. However, the long tail will affect the stability of the plasmid. The solution of Pfizer/BioNTech is UGC linker with 10 bp inserted in the middle, that is, A30 (10 bp UGC linker) A70.

  • Codon optimization

The open reading frame that encodes antigens is the most important part. The improvement of this part includes the optimization of mRNA codons, which can improve the level of translation by converting uncommonly used codons into common codons that encode the same amino acid. For example, CureVac’s COVID-19 vaccine CVnCoV uses codon optimization.

When designing the sequence, we also need to remove the binding site of miRNA and the AU enrichment region of 3 ‘UTR to reduce the regions in 5’ UTR which are easy to form secondary and tertiary structures.

Delivery system for mRNA

Because mRNA has a large molecular weight (1-1 million Dalton) and has negative points, it is difficult to penetrate the lipid bilayer on the cell membrane that is also negatively charged. At the same time, in vivo, mRNA is easily digested by immune cells or digested by endonuclease, which leads to degradation. So we need a suitable delivery system.

  • Lipid-based nanoparticles

The advantages of LNP are easy formulation, modularization and large mRNA load. General LNP includes ionizable lipids, solid alcohols, auxiliary phospholipids and PEG lipids.

Cationic lipids can effectively transport negatively charged nucleic acids into cells, but can induce apoptosis and inflammation.

The advantage of ionizable lipid is that they are neutral and uncharged at physiological pH values. Under acidic conditions, it is punctual and combines with RNA. When the endosome (Endosome) is acidic, it is beneficial to fuse with the endosomal membrane and release RNA into the cytoplasm.

  • Polymer complexes and composite nanoparticles

Although the clinical progress is not as rapid as that of LNP, polymers have similar advantages as lipids and can effectively deliver mRNA. Cationic polymers can form complexes of different sizes with mRNA. At present, there are a variety of biodegradable polymer materials for effective delivery of mRNA.

Polyvinyl amide (PVA) is the most studied polymer, which is effective in delivery but highly toxic due to high charge density. The toxicity can be reduced by using low molecular weight structure, combined with PEG, European cyclodextrin and disulfide bond. Similar to ionizable lipids, ionizable conjugates are also in use.

  • Other delivery systems

Peptides can also be used to deliver mRNA because some amino acids carry cations or amphiphilic amino groups that can bind to mRNA. Cationic nanoemulsions based on squalene are also used to deliver mRNA. Some squalene formulations can act as adjuvants.