Viral vaccines immunise us against infectious viral diseases by triggering a specific protective
response, and historically have done so by containing a weakened or inactive virus, or
a part of the virus called the antigen.
However, the COVID-19 vaccines by BioNTech/Pfizer and Moderna contain RNA instead.
Although they were the first of their kind, their development was the result of nearly
three decades of research.
I will be leading you through how mRNA came to be in our vaccines, and what their future
may be.
In 1961, mRNA was isolated, its theoretical biological role published, and its function
proven by Nirenberg and Matthaei in their experiment where they matched codons to amino
acids through translation.
In 1990, it was demonstrated that injecting in vitro transcribed mRNA expressed the encoded
protein in mice, but their therapeutic use was not explored due to mRNA having a short
half-life and unfavourable immunogenicity, in addition to interest in plasmid DNA and
viral DNA.
In 2004, it was found that only certain forms of RNA stimulated human dendritic cells, which
play a key role in immune response.
Dendritic cells were also the subject of the 2011 Nobel Prize in Medicine alongside the
discovery of toll-like receptors, proteins in dendritic cells which play a key role in
innate immunity.
In 2005, mammalian tRNA was identified to not induce a detectable immune response whereas
mitochondrial RNA and bacterial RNA did, suggesting an inverse correlation between immunostimulatory
potential and the extent of nucleoside modification.
Nucleoside modification involves replacing the standard adenosine, cytosine, guanosine
and uridine nucleosides with non-standard nucleosides such as pseudouridine.
The finding that nucleoside modifications to mRNA reduced their immunogenicity was pivotal
in advancing the use of mRNA therapeutically.
In 2008, incorporating pseudouridine into in vitro transcribed mRNA was demonstrated
to increase translation and biological stability in addition to reducing immunogenicity, addressing
the problems of mRNA use therapeutically.
The translation inhibitory effect was suggested to be due to RNA-dependent protein kinases,
or PKR, as it was only observed in cells containing PKR.
PKR is a mammalian enzyme with a variety of cell functions, including regulation of translation
during conditions of cell stress.
It activates when bound to double stranded RNA, and subsequently phosphorylates eukaryotic
translation initiation factor 2 subunit alpha, which inhibits local translation.
In 2010, Anderson and colleagues identified the role of PKR in the translation of in vitro
transcribed mRNA. in vitro transcribed mRNAs were produced by T7 RNA polymerase using linearised
plasmids for the reporter genes firefly luciferase and Renilla luciferase.
Luciferases are light emitting enzymes that can be quantitatively measured using a luminometer.
The pseudouridine-modified RNAs were assembled by replacing uridine triphosphate with pseudouridine
triphosphate.
Both firefly and Renilla luciferase-encoding mRNAs were transfected into HEK293T cells
to show that translational inhibition extended to modified transcripts because translation
levels decreased if one transcript was modified.
HEK293T cells are immortalised human embryonic kidney cells commonly used for their efficient
transfection and protein production.
Total cellular protein synthesis was suppressed more by unmodified mRNAs, as measured by quantifying
the incorporation of radioactive methionine and cysteine.
Using purified PKR and measuring activation using phosphorimagers, they found that modified
transcripts had less PKR activation compared to unmodified transcripts, which was supported
in HEK293T with western blotting using an antibody that detected activated PKR.
Viral proteins known to reverse PKR-mediated inhibition of translation were used to confirm
PKR’s role when translation of unmodified transcripts increased but not modified transcripts.
This was further confirmed using PKR-deficient mouse embryonic fibroblasts where both modified
and unmodified RNA had similar translational activity.
Pseudouridine-modified mRNA was demonstrated to not competitively inhibit PKR when it failed
to inhibit PKR activation when mixed in high concentrations with a known activator.
This suggested a lack of binding between PKR and modified mRNA, which was confirmed using
biotinylated RNA pull-down and detected using western blotting.
PKR activation was suggested to be due to the formation of intra- and intermolecular
secondary structures of RNA, as this was influenced by nucleoside modifications.
In 2015, N1 methylpseudouridine modification was shown to outperform pseudouridine modification
by further reducing innate immunogenicity and improving cellular stability.
In 2020, just 64 days after the sequence information for the coronavirus RNA was available, clinical
trials for Moderna had started.
The speed of mRNA-based COVID-19 vaccines was much faster due to not needing cell cultures,
biohazard materials, or complex purification processes.
These vaccines contain N1 methylpseudouridine-modified mRNA encoding for a prefusion spike glycoprotein
which stimulates the immune response, alongside lipid nanoparticles to protect the mRNA from
degradation and enable their delivery into cells without toxicity.
The precedent set by the COVID-19 vaccines opens avenues for other mRNA vaccines against
infectious diseases such as influenza, HIV and malaria.
mRNA-based technology also has potential in applications such as cell reprogramming to
pluripotency, immune checkpoint therapy for cancer treatment, genome editing using engineered
nucleases.
These three applications specifically mentioned have each led to a Nobel Prize in Medicine.