Vaccine development based on messenger RNA (mRNA) is a promising new vaccination approach. mRNA based vaccine development is potentially very useful for the production of vaccines against coronaviruses such as SARS-CoV or SARS-Cov-2 (COVID-19). mRNAs based vaccines are a promising alternative to conventional vaccines.
Vaccines prevent many illnesses and save lives every day. With the occurrence of newly emerged viruses such as the coronaviruses or the Hanta virus, there is a need for more rapid development and large-scale deployment of effective vaccines. Presently (as of March 2020), an investigational vaccine for the protection against COVID-19 is in clinical trial phase 1. The mRNA-based vaccine is called mRNA-1273. The hope is that we will have a working vaccine against COVID-19 very soon.
According to Linares-Fernández et al., mRNA-based vaccines enable the design and production of well-controlled on-demand transcript sequences that can be adapted to any pandemic crises. In vitro-transcribed (IVT) mRNA used for vaccine production needs to be highly homogeneous. This vaccine should also be free of DNA, dsRNA, or 5’-triphosphate transcripts in order to avoid any overstimulation of innate immunity.
mRNA is the intermediate molecule between the translation of protein-encoding DNA and the production of proteins by ribosomes in the cytoplasm. The high potency, the capacity for a rapid development, the potential low cost of manufacture, and the safe delivery into cells and tissue makes mRNAs ideal candidates for vaccine development. However, historically, the use of mRNA as vaccines has been prevented due to the inherent instability of RNA as well as inefficient in vivo delivery. Advancements made in recent RNA technologies now help overcoming these hurdles and enable mRNA to become a promising therapeutic tool. Currently, mRNA vaccines are investigated in basic and clinical research.
Earlier reports focused on cancer vaccination however more recent reports also showed that mRNAs have the potential to protect against a wide variety of infectious pathogens, such as coronavirus, influenza virus, Ebola virus, Zika virus, Streptococcus spp. and T. gondii, and potentially the newly emerged coronavirus SARS-Cov-2(COVID-19).
According to Pardi et al., mRNA-based vaccination is non-infectious, does not integrate the coded sequence into the genome, and does not cause an infection or is mutagenic. Normally, mRNA is degraded by cellular processes. Also, the in vivo half-life of mRNA can be regulated by using various modifications including bridged nucleic acids and delivery method. And, inherent immunogenicity can be downmodulated. A careful design is necessary for mRNA vaccines since pathogen-associated molecular patterns (PAMPs) of foreign RNA can be recognized through interactions with pattern recognition receptors (PRRs) in complex ways.
Features of mRNAs for vaccine development
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mRNA vaccines are
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Safety
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Non-infectious.
Non-integrating.
Have no potential risk of infection or insertional mutagenesis.
The in vivo half-life of mRNAs can be regulated through the use of various modifications and delivery methods.
The inherent immunogenicity of mRNA can be down-regulated to further increase the safety profile.
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Efficacy
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Modifications make mRNA more stable and highly translatable.
Efficient in vivo delivery can be achieved by formulating mRNA into carrier molecules;
Carrier molecules allow rapid uptake and expression in the cytoplasm.
mRNA is the minimal genetic vector; therefore, anti-vector immunity is avoided, and mRNA vaccines can be administered repeatedly.
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Production
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mRNA vaccines have the potential for rapid, inexpensive and scalable manufacturing, because of the high yields of in vitro transcription reactions.
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Messenger RNA translation and stability
- The 5’- and 3’-UTR elements flanking the coding sequence strongly influence the stability and translation of mRNA. Both effects are of critical concern for vaccine development.
- Regulatory sequences derived from viral or eukaryotic genes can greatly increase the half-life and expression of therapeutic mRNAs.
- mRNAs require a 5’-cap structure for efficient protein production.
- To synthetic mRNAs, various versions of 5’-caps can be added synthetically or enzymatically.
- Since the poly(A) tail is very important for regulating mRNAs, an optimal length of poly(A) needs to be added. This can be achieved either directly from the encoding DNA template or by using poly(A) polymerase.
- Also, codon usages impact protein translation. Replacing rare codons with frequently used synonymous codons having abundant cognate tRNA in the cytosol can increase protein production.
- Enrichment of G:C content apparently also increases steady-state mRNA levels in vitro and protein expression in vivo.
mRNA Design
Biological Function
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Cap
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5-UTR
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ORF
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3’-UTR
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Poly-A
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Modified in eukaryotes.
Important for translation initiation, mRNA stability, and nuclear transport.
If suboptimal, recognized as PAMPs by the innate immune system.
RNA Closed-Loop.
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Recognized by translation machinery.
Recognized and scanned by ribosomes.
Important for mRNA translation and stability.
RNA Closed-Loop.
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Sequence encoding the gene of interest.
In mRNA-based vaccines, the sequence encodes the antigen.
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Important for translation initiation and mRNA stability.
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Important for mRNA stability.
Recognized by Poly-A binding proteins (PARB).
Recruitment of translation factors.
Important for translation initiation (RNA Closed-Loop).
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Region optimization of mRNAs
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5’-Cap
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5-UTR
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ORF
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3’-UTR
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Poly-A
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Natural Cap-1 needed to avoid recognition by pattern recognition receptors (PPRs).
Use enzymatic capping for higher capping efficiency.
7-Methylguanosine (m7G, m7GpppN structure)
Anti-reverse cap analogs (ARCAs) can be added for superior translation.
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Inclusion of Kozak sequence.
No strong secondary structures.
No other start codon.
Polysome profiling to count the ribosome loading in sequences in silico.
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Codon optimization increases translation.
Low optimal codons may be important for adequate folding.
Remove sources for antigenic peptides.
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Optimal sequences derived from highly stable mRNA (e.g. α- and β-globin).
2x copies in tandem
RNA Closed-Loop.
Two β-globin in head-to-tail orientation.
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Poly-A sequences of 120 to 150 units.
Adding a poly-U sequences.
Providing a dsRNA in the poly-A region increases adjuvant effect.
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Optimization of the whole mRNA molecule
Avoid binding sites of miRNAs present in target cells.
Uridine depletion to avoid recognition by the innate immune system.
Production at high temperature (50 °C) using a thermostable polymerase and/or low magnesium concentration to decrease levels of dsRNAs.
Purify mRNA using HPLC to decrease amounts of dsRNA.
Avoid highly stable and long secondary structures at could activate PRRs.
{Adapted from Linares-Fernandez et al., Pardi et al., Schlake et al., and Sahin et al.}
Reference
Amarante-Mendes Gustavo P., Adjemian Sandy, Branco Laura Migliari, Zanetti Larissa C., Weinlich Ricardo, Bortoluci Karina R.; Pattern Recognition Receptors and the Host Cell Death Molecular Machinery. Frontiers in Immunology 9, 2018, 2379. [Link]
Sergio Linares-Fernández, Céline Lacroix, Jean-Yves Exposito, Bernard Verrier; Tailoring mRNA Vaccine to Balance Innate/Adaptive Immune Response. Trend in Molecular Medicine Vol. 26, 311-323.
NIH-Clincal-Trial-COVID-19-Vaccine Begins
Pardi N, Hogan MJ, Porter FW, Weissman D. mRNA vaccines - a new era in vaccinology. Nat Rev Drug Discov. 2018 Apr;17(4):261-279. doi: 10.1038/nrd.2017.243. [PMC]
Pathogen Associated Molecular Pattern
Sahin U, Karikó K, Türeci Ö. mRNA-based therapeutics--developing a new class of drugs. Nat Rev Drug Discov. 2014 Oct;13(10):759-80. [PubMed]
Schlake T, Thess A, Fotin-Mleczek M, Kallen KJ. Developing mRNA-vaccine technologies. RNA Biol. 2012 Nov;9(11):1319-30. doi: 10.4161/rna.22269. [PMC]
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