Therapeutic Principle and Construction Method of mRNA

Therapeutic principle of mRNA

The use of mRNA as vaccines and drugs to prevent, diagnose and treat certain diseases is gradually becoming the focus of pharmaceutical companies. mRNA-based therapy, in short, is to introduce chemically modified mRNA molecules into the cytoplasm, and use the nucleotides in the cytoplasm for transcription and expression to produce the proteins needed by the body.

The mRNA vaccine uses the target/antigen to encode mRNA, and allows cells to take up and express the encoded antigen through a specific delivery system, thereby causing humoral and cell-mediated immune responses. After vaccination, the mRNA vaccine encoding the spike protein of the new coronavirus encapsulated in lipid nanoparticles enters the cell and synthesizes the protein in the ribosome. The protein is either broken down into smaller fragments (polypeptides) by the cellular proteasome, or transported to the outside of the cell through the Golgi apparatus. The antigen polypeptide fragments enzymatically digested in the cell form a complex with the major histocompatibility complex (MHC) class I protein, which expresses the antigen on the surface of the presenting cell and is recognized by CD8+ T cells to induce cell-mediated immunity (Fig.1 left side). On the other hand, the extracellular spike protein can be swallowed, pinocytosed and broken down into smaller polypeptide fragments by different immune cells, forming complexes with MHC class II proteins, expressing antigens on the surface of presenting cells, and being CD4+ T cells recognize and promote B cells to produce antigen-specific antibodies. (Fig.1 right side)

Figure 1: Mechanism of action of mRNA vaccine (Itziar Gómez-Aguado. 2020).

How mRNA is constructed

Currently, mRNA construction strategies have two directions: nonamplifying mRNA and self-replicating mRNA (SAM) (Fig. 2). RNA is easily degraded in the external environment, which affects translation and expression efficiency. In order to stabilize its structure and improve expression efficiency, non-replicating mRNA has been sequenced with the structure of eukaryotic mRNA as a reference. SAM is to chimeric the coding sequence to a vector with RNA-dependent RNA polymerase (RdRP), and increase the expression efficiency by copying its own mRNA by RdRP. The elements of non-replicating mRNA include 5'cap, 3'Poly(A) tail, untranslated region (UTR), and coding sequence (Fig. 3).

Figure 2. mRNA drug structure

Figure 3. Structural composition of non-amplifying(A) and self-amplifying(B) mRNAs (Kowalski P S. 2019).

How to enhance mRNA stability

Regarding the characteristics of mRNA not being stable enough and having a short half-life, researchers have also found a variety of methods to enhance the stability of mRNA and prolong the half-life. For example:

• Use anti-reverse-cap analog (ARCA) for in vitro transcription, so that all reactions are connected by 5'-5' phosphate bonds, and the product is 100% translatable mRNA;
• The expression of mRNA is positively correlated with the length of poly(A) tail, and the 3'end of exogenous mRNA must contain at least 20 poly(A) tails;
• Use known mRNA non-coding sequence (such as β-globin mRNA) to replace mRNA and the easily degradable AU elements in the 3-UTR region;
• Codon optimization, under the premise of ensuring that the codons encode the same amino acid, use other more stable nucleotides (such as modified nucleotides, 2-thiouridine and 5-methylcytidine) instead of nucleotides that are not stable enough. Unmodified mRNA has innate immunogenicity. Modified mRNA obtained by in vitro transcription technology can further reduce the immunogenicity, making mRNA therapy play a better effect.