4. GC enrichment and potential G4 (pG4) structures in vaccine
mRNAs
Recently, members of our team investigated possible alterations in
secondary structure of mRNAs in SARS-CoV-2 vaccines due to codon
optimization of synthetic mRNA transcripts [71]. This study has
shown that there is a significant enrichment of GC content in mRNAs in
vaccines (53% in Pfizer BNT 162b2 and 61% in Moderna mRNA-1273) as
compared to the native SARS-CoV-2 mRNA (36%). The enriched GC content
of mRNAs is the result of codon optimization performed during the
development of the mRNAs used in SARS-CoV-2 vaccines, apparently without
determining the effect on secondary structures, particularly the G
quadruplex formation [71].
Codon optimization describes the production of synthetic,
codon-optimized polypeptides and proteins used in biotechnology
therapeutics (such as the synthetic mRNAs used for SARS-CoV-2
vaccination). The altered codon assignments within the mRNA template
dramatically increase the quantity of polypeptides and/or proteins
produced [72]. Synonymous codon replacement also results in a change
in the multifunctional regulatory and structural roles of resulting
proteins [73]. For this reason, codon optimization has been
cautioned against due to its consequent changes causing perturbation in
the secondary conformation of protein products with potentially
devastating effects on their resulting immunogenicity, efficacy and
function [74,75]. Notably, various human diseases are the result of
synonymous nucleotide polymorphisms [76].
In an experiment where GC-rich and GC-poor versions of mRNA transcripts
for heat shock protein 70 were configured in the context of identical
promoters and UTR sequences, it was found that GC-rich genes were
expressed several-fold to over a hundred-fold more efficiently than
their GC-poor counterparts [77]. This is partly because all of the
preferred mammalian codons have G or C nucleotides in the third
position. It is also well documented that AU-rich elements in the 3’
UTRs can destabilize mRNA [78]. What may be of particular concern is
the fact that GC enrichment content in vaccine mRNAs results in an
enhanced ability for potential G quadruplex (pG4) formations in these
structures, and this could cause onset of neurological disease [79].
Remarkably, the human prion protein (PrP) genetic sequence contains
multiple G4 forming motifs, and their presence may form the missing link
in the initial conversion of PrP to the misfolded form, PrPsc [80].
PrP binding to its own mRNA may be the seed that causes the protein to
misfold. This observation is particularly concerning in light of the
fact that the spike protein has prion-like characteristics [81].
On the one hand, the GC content has a key role in the modulation of
translation efficiency and control of mRNA expression in mammals
[82]. Especially during translation initiation, the GC content
operating as a cis-acting mRNA element orchestrates the 43S ribosomal
pre-initiation complex attachment and thereafter the assembly of the
eukaryotic translation initiation factor 4EF (eIF4F) complex. One
representative example of this system in action is the regulation of α
and β globin mRNA expression through their 5’ untranslated regions
(5’UTRs) [82].
On the other hand, the presence of pG4s in RNAs is implicated in cancer
biology as key determinants of the regulation of G4 RNA binding proteins
such as helicase [83]. Generally, the G quadruplexes in RNAs have
essential roles in a) the regulation of gene expression, b) the
localization of ribonuclear proteins, c) the mRNA localization and d)
the regulation of proto-oncogene expression [84].
Regarding SARS-CoV-2, relevant studies reveal overwhelming similarities
between SARS-CoV-2 pG4s, including in RNA coding for spike protein, and
those sequenced in the human transcriptome [85]. Thus, it can be
inferred that synthetic mRNAs in vaccines carrying more pG4 structures
in their coding sequence for spike protein will amplify and compound the
potential post-transcriptional disorganization due to G4-enriched RNA
during natural SARS-CoV-2 infection. Moreover, the cellular nucleic acid
binding protein (CNBP), which is the main cellular protein that binds to
the SARS-CoV-2 RNA genome in human-infected cells [86], binds to and
promotes the unfolding of SARS-CoV-2 G4s formed by both positive and
negative sense template strands of the SARS-CoV-2 RNA genome. A similar
modulation of CNBP on vaccine mRNA G4s and promotion of G4 equilibrium
towards unfolded conformations create favorable conditions for miRNA
binding, and this will have a direct impact on miRNA-dependent
regulation of gene expression [87].
The negative-sense RNAs are intermediate molecules produced by the
replicase transcriptase complex (RTC) formed by the nonstructural
proteins of coronaviruses (including SARS-COV-2) to provide efficiency
in replication and transcription [88,89]. This, however, introduces
another potentially serious complication associated with vaccination.
Co-infection with other negative sense RNA viruses such as hepatitis C
[90] or infection by other coronaviruses contemporaneous with
vaccination periods would provide the necessary machinery of RTC to
reproduce negative sense intermediates from synthetic mRNAs and
therefore amplify the presence of pG4s by negative sense templates. This
would result in further epitranscriptomic dysregulation [91].
Summarizing the topic to this point, the enrichment of GC content in
vaccine mRNA will inevitably lead to an increase in the pG4 content of
the vaccines. This, in turn, will lead to dysregulation of the
G4-RNA-protein binding system and a wide range of potential
disease-associated cellular pathologies including suppression of innate
immunity, neurodegeneration, and malignant transformation [83].
Concerning the post translational deregulation due to emergence of new
G4 structures introduced by vaccination, one other important issue
related to miRNA regulation and pG4s arises. In miRNA structures,
hundreds of pG4 sequences are identified [92]. In their unfolded
conformation, as during binding to their respective targets in 3’ to 5’
sequences of mRNAs, miRNAs switch off the translation of their
respective target mRNA. Alternatively, when in the presence of a G4
ligand, the translation of their target mRNAs is promoted [93].
Moreover, a vast number of putative miRNA binding sites overlap with G4s
in 3’ UTRs of mRNAs as there are at least 521 specific miRNAs that are
predicted to bind to at least one of these G4s. Overall, 44,294 G4-miRNA
potential binding sites have been traced to possess putative overlapping
G4s in humans [87].
As described elsewhere, during the cellular translation of vaccine
mRNAs, an increased assembly of a number of RNA binding protein
helicases, such as eIF4A bound to eIF4G, will occur [65]. The
presence of increased pG4s in synthetic mRNAs can potentially amplify
binding of RNA binding proteins and miRNAs. This form of molecular
crowding of protein components (helicases) with great affinity for G4
binding [87] will decrease the number of RNA binding proteins
binding G4s normally available for miRNA regulation. This loss of RNA
binding proteins as well as miRNA availability for regulation by binding
to G4s can dramatically alter the translational regulation of miRNAs
present in cells and thereby disrupt essential regulation of oncogene
expression. An example is the p16-dependent regulation of the p53 tumour
suppressor protein [87,94].
This process is exceedingly complicated yet tantamount to cellular
homeostasis. So, again, it merits summarizing. If pG4s accumulate, as
would be expected with an increased amount of GC content in the vaccine
mRNA, this would have an effect of increasing potential G4 structures
available during translation events and this can affect miRNA
post-transcriptional regulation. This, in turn, would either favor
greater expression of the oncogenes related to a range of cancers or
drive cells to apoptosis and cell death [95]. The case study
described earlier in this paper strongly supports the hypothesis that
these injections induce accelerated lymphoma progression in follicular B
cells [56].
miRNA binding recognition patterns are imperfectly complementary to
their target regions, and for this reason they are referred to as
“master regulators,” since one miRNA affects a plethora of different
targets [92]. The multitude of pG4s in the mRNA of the vaccine would
predictably act as decoys, distracting miRNAs from their normal function
in regulating human protein expression. The increase in G4 targets due
to the vaccine would decrease the availability of miRNAs to target
human-expressed G4s for regulation of gene expression. This can result
in downregulation of miRNA expression which is implicated in
cardiovascular pathology [96], onset of neurodegeneration [97],
and/or cancer progression [98].
In most respects within epitranscriptomic machinery, miRNAs are involved
in translation repression. One example, vital for cellular normal
housekeeping is that of Mouse double minute 2 homolog (MDM2), a physical
negative regulatory protein of p53. P53 itself is considered the master
regulator of the cellular tumor suppression network of genes. P16
controls the expression of many miRNAs, and, via miR-141 and mIR-146b-5p
binding to MDM2 mRNA, it induces the negative regulation of MDM2, thus
enabling p53 ubiquitination and promotion of cell survival upon DNA
damage events [94]. Deregulation of miRNAs that control MDM2
suppression of p53 would predictably lead to an increased risk to cancer
[99].