mRNA Vaccine Platform: mRNA Production and Delivery

Abstract

Vaccination is the most efficient way to prevent infectious diseases. mRNA-based vaccines is a new approach to vaccine development, which have several very useful advantages over other types of vaccines. Since mRNA encodes only the target antigen there is no potential risk of infection as in the case with attenuated or inactivated pathogens. The mode of action of mRNA-vaccines implies that their genetic information is expressed only in the cytosol, leaving very little possibility of mRNA integration into the host's genome. mRNA-vaccines can induce specific cellular and humoral immune responses, but do not induce the antivector immune response. The mRNA-vaccine platform allows for easy target gene replacement without the need to change the production technology, which is important to address the time lag between the epidemic onset and vaccine release. The present review discusses the history of mRNA vaccines, mRNA vaccine production technology, ways to increase mRNA stability, modifications of the cap, poly(A)-tail, coding and noncoding parts of mRNA, target mRNA vaccine purification from byproducts, and delivery methods.

Keywords: RNA; chemically modified nucleotides; mRNA delivery methods; mRNA vaccines; untranslated 5'- and 3'-regions.

Fig. 1.

Schematic representation of mRNA degradation. mRNA degradation occurs in the cytoplasm within the ribonucleic complexes called P-bodies, which contain 5'-3'-exonucleases, decapping and deadenylating enzymes. Once the poly(A)-tail is shortened to 12 residues or less, mRNA degradation occurs through cap cleavage and 5'→3' or 3'→5' cleavage [21]. Endonucleases (not shown in the figure) may also be involved in mRNA degradation.

Fig. 2.

Schematic outline of in vitro transcribed (IVT) modified mRNA production: 5'-UTR, 5'-untranslated region, 3'-UTR, 3'‑untranslated region, ORF, open reading frame, ARCA, Anti-Reverse Cap Analog, IRES, Internal Ribosome Entry Site, AUG, start-codon and STOP, stop codon [5].

Fig. 3.

Cap structure (cap0). Cap is a N7-methylguanosine ribonucleotide linked by a 5'-5'-triphosphate bridge to the first nucleotide residue in the transcript.

Fig. 4.

Closed mRNA loop model. (a) In vitro transcribed mRNA (IVT-mRNA) can recruit translation initiation factors which bind to the 5'-cap and 5'-untranslated region (5'-UTR), promoting mRNA entry into the ribosome and mRNA translation and also recruit the poly(A)-binding protein (PABP) to the poly(A)-tail; (b) in the closed loop model, strong interaction between the translation factors on both sides of the mRNA induces the formation of a stable loop, which protects the transcripts from RNA degrading enzymes and facilitates mRNA reentry into the ribosome thereby enhancing translation [22].

Fig. 5.

The Structure of Anti-Reverse Cap Analog (ARCA).

Fig. 6.

Principle of Anti-Reverse Cap Analog (ARCA) action.

Fig. 7.

The structure of the second-generation CleanCap analog^®.

Fig. 8.

The structures of modified nucleosides.

Fig. 9.

Lipid nanoparticle structure.

Fig. 10.

Intracellular barriers for in vitro transcribed (IVT) mRNA delivery: (1) interaction between the delivery system and the cell membrane, (2) endocytosis, (3) exit from the endosome and mRNA release to initiate translation. Endocytosis is a mechanism by which extracellular components and fragments of the plasma membrane internalize with the formation of the endocytic vesicle. This process involves vesicles with the internal pH of ~5 known as endosomes which develop from the early endosomes to the late endosomes before fusing with the intracellular organelles called lysosomes. In such a way, particles entering the cell via endocytosis are captured by the endosomes and eventually appear in the lysosomes, where active enzymatic degradation takes place [5, 46].

Fig. 11.

The structures of the LNP components: (a) SM-102—(heptadecane-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate)); (b) ALC-0315—((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyl decanoate); (c) DMG-PEG2000—1-monomethoxypolyethylene glycol-2,3-dimyristylglycerol with polyethylene glycol with the average molecular weight of 2000; (d) ALC-0159—2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.