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].