Synthetic mRNAs; Their Analogue Caps and Contribution to Disease

Kyriakopoulos, Anthony M.; McCullough, Peter A.

doi:10.3390/diseases9030057

Cited by 9 publications

(24 citation statements)

References 92 publications

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“…The spike protein mRNA is further "humanized" with the addition of a guanine-methylated cap, 3' and 5' untranslated regions (UTRs) copied from those of human proteins, and finally a long poly(A) tail to further stabilize the RNA [65]. In particular, researchers have cleverly selected the 3'UTR taken from globins which are produced in large quantities by erythrocytes, because it is very effective at protecting the mRNA from degradation and maintaining sustained protein production [66].…”

Section: Considerations In the Design Of Mrna Vaccinesmentioning

confidence: 99%

“…De Beuckelaer et al (2016) aptly summed up the consequences of such modifications as follows: "Over the past years, technical improvements in the way IVT [in vitro transcribed] mRNAs are prepared (5' Cap modifications, optimized GC content, improved polyA tails, stabilizing UTRs) have increased the stability of IVT mRNAs to such extent protein expression can now be achieved for days after directin vivo administration of the mRNA." [60] However, the optimized analogue cap formation of synthetic mRNAs inevitably forces the recipient cells to undergo a cap-dependent prolonged translation, ignoring homeostatic demands of cellular physiology [65]. The cap 2' O methylation carried out by cap 2' O methyltransferase (CMTR1) serves as a motif that marks the mRNA as "self," to prevent recognition by IFN-induced RNA binding proteins [68].…”

Section: Considerations In the Design Of Mrna Vaccinesmentioning

confidence: 99%

“…The regulatory process controlling mRNA translation is extremely complex, and it is highly disturbed in the context of mRNA vaccines [65,69]. Briefly, the idea is for mRNA vaccines to achieve the intended goal (i.e., production of the modified spike protein) through a stealth strategy that bypasses the natural immunological response to RNA-type viral infection.…”

Section: Considerations In the Design Of Mrna Vaccinesmentioning

confidence: 99%

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Innate Immune Suppression by SARS-CoV-2 mRNA Vaccinations: The role of G-quadruplexes, exosomes and microRNAs

Seneff

Nigh

Kyriakopoulos³

et al. 2022

Preprint

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The mRNA SARS-CoV-2 vaccines were brought to market in response to the widely perceived public health crises of Covid-19. The utilization of mRNA vaccines in the context of infectious disease had no precedent, but desperate times seemed to call for desperate measures. The mRNA vaccines utilize genetically modified mRNA encoding spike proteins. These alterations hide the mRNA from cellular defenses, promote a longer biological half-life for the proteins, and provoke higher overall spike protein production. However, both experimental and observational evidence reveals a very different immune response to the vaccines compared to the response to infection with SARS-CoV-2. As we will show, the genetic modifications introduced by the vaccine are likely the source of these differential responses. In this paper, we present the evidence that vaccination, unlike natural infection, induces a profound impairment in type I interferon signaling, which has diverse adverse consequences to human health. We explain the mechanism by which immune cells release into the circulation large quantities of exosomes containing spike protein along with critical microRNAs that induce a signaling response in recipient cells at distant sites. We also identify potential profound disturbances in regulatory control of protein synthesis and cancer surveillance. These disturbances are shown to have a potentially direct causal link to neurodegenerative disease, myocarditis, immune thrombocytopenia, Bell’s palsy, liver disease, impaired adaptive immunity, increased tumorigenesis, and DNA damage. We show evidence from adverse event reports in the VAERS database supporting our hypothesis. We believe a comprehensive risk/benefit assessment of the mRNA vaccines excludes them as positive contributors to public health, even in the context of the Covid-19 pandemic.

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Section: Considerations In the Design Of Mrna Vaccinesmentioning

confidence: 99%

Section: Considerations In the Design Of Mrna Vaccinesmentioning

confidence: 99%

Section: Considerations In the Design Of Mrna Vaccinesmentioning

confidence: 99%

See 1 more Smart Citation

Innate Immune Suppression by SARS-CoV-2 mRNA Vaccinations: The role of G-quadruplexes, exosomes and microRNAs

Seneff

Nigh

Kyriakopoulos³

et al. 2022

Preprint

Self Cite

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“…How these receptors behave in the presence of both vaccine-derived spike mRNA and viral derived spike mRNAs is a nascent field. However, this field is of interest to many physicians concerned about chronic diseases including cancer 28 . Jiang et al note that the spike protein localizes to the nucleus and significantly alters DNA damage and repair pathways via modified VDJ recombination required for adaptive immunity 29 .…”

Section: Fidelity Of Spike Protein Expressionmentioning

confidence: 99%

“…The vaccine mRNAs prolong their translation due to robust capping resisting natural mRNA decay pathways. This can trigger cancer initiation and progression 28 .…”

Section: Toxicity Of Spike Proteinmentioning

confidence: 99%

Differences in Vaccine and SARS-CoV-2 Replication Derived mRNA: Implications for Cell Biology and Future Disease

McKernan¹

2021

Preprint

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Codon optimization describes the process used to increase protein production by use of alternative but synonymous codon changes. In SARS-CoV-2 mRNA vaccines codon optimizations can result in differential secondary conformations that inevitably affect a protein’s function with significant consequences to the cell. Importantly, when codon optimization increases the GC content of synthetic mRNAs, there can be an inevitable enrichment of G-quartets which potentially form G-quadruplex structures. The emerging G-quadruplexes are favorable binding sites of RNA binding proteins like helicases that inevitably affect epigenetic reprogramming of the cell by altering transcription, translation and replication. In this study, we performed a RNAfold analysis to investigate alterations in secondary structures of mRNAs in SARS-CoV-2 vaccines due to codon optimization. We show a significant increase in the GC content of mRNAs in vaccines as compared to native SARS-CoV-2 RNA sequences encoding the spike protein. As the GC enrichment leads to more G-quadruplex structure formations, these may contribute to potential pathological processes initiated by SARS-CoV-2 molecular vaccination.

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On‐column capping of poly dT media‐tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and improved stability against RNase

Che,

Feng,

et al. 2023

Biotech & Bioengineering

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The messenger RNA (mRNA) 5′‐cap structure is indispensable for mRNA translation initiation and stability. Despite its importance, large‐scale production of capped mRNA through in vitro transcription (IVT) synthesis using vaccinia capping enzyme (VCE) is challenging, due to the requirement of tedious and multiple pre‐and‐post separation steps causing mRNA loss and degradation. Here in the present study, we found that the VCE together with 2′‐O‐methyltransferase can efficiently catalyze the capping of poly dT media‐tethered mRNA to produce mRNA with cap‐1 structure under an optimized condition. We have therefore designed an integrated purification and solid‐based capping protocol, which involved capturing the mRNA from the IVT system by using poly dT media through its affinity binding for 3′‐end poly‐A in mRNA, in situ capping of mRNA 5′‐end by supplying the enzymes, and subsequent eluting of the capped mRNA from the poly dT media. Using mRNA encoding the enhanced green fluorescent protein as a model system, we have demonstrated that the new strategy greatly simplified the mRNA manufacturing process and improved its overall recovery without sacrificing the capping efficiency, as compared with the conventional process, which involved at least mRNA preseparation from IVT, solution‐based capping, and post‐separation and recovering steps. Specifically, the new process accomplished a 1.76‐fold (84.21% over 47.79%) increase in mRNA overall recovery, a twofold decrease in operation time (70 vs. 140 min), and similar high capping efficiency (both close to 100%). Furthermore, the solid‐based capping process greatly improved mRNA stability, such that the integrity of the mRNA could be well kept during the capping process even in the presence of exogenously added RNase; in contrast, mRNA in the solution‐based capping process degraded almost completely. Meanwhile, we showed that such a strategy can be operated both in a batch mode and in an on‐column continuous mode. The results presented in this work demonstrated that the new on‐column capping process developed here can accomplish high capping efficiency, enhanced mRNA recovery, and improved stability against RNase; therefore, can act as a simple, efficient, and cost‐effective platform technology suitable for large‐scale production of capped mRNA.

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Synthetic mRNAs; Their Analogue Caps and Contribution to Disease

Cited by 9 publications

References 92 publications

Innate Immune Suppression by SARS-CoV-2 mRNA Vaccinations: The role of G-quadruplexes, exosomes and microRNAs

Innate Immune Suppression by SARS-CoV-2 mRNA Vaccinations: The role of G-quadruplexes, exosomes and microRNAs

Differences in Vaccine and SARS-CoV-2 Replication Derived mRNA: Implications for Cell Biology and Future Disease

On‐column capping of poly dT media‐tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and improved stability against RNase

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