Potential Adverse Cellular Influences From N1-methyl-pseudouridine (m1Ψ)
'Spikeopathy' and other mRNA injection components and disease manifestations
Image 1: The above image shows the structural difference between uridine, pseudouridine, and N1-methyl-pseudouridine. Notice that pseudouridine has two amines (-NH) within the heterocyclic ring, but the N1-methyl-pseudouridine is methylated (-CH3, Me) at nitrogen 1. A Comprehensive Review of the Global Efforts on COVID-19 Vaccine Development - https://www.researchgate.net/figure/Chemical-structures-of-uridine-pseudouridine-and-N1-methyl-pseudouridine_fig2_350480428
The mRNA injections are meant to deliver an mRNA instruction template for a host cell to use in the creation of a spike protein copy of SARS-CoV-2. The spike protein of SARS-CoV-2 is used in its attachment to host cell ACE2 receptors for viral entry. The spike protein whether from the virus or induced into production from within the host cell is an immunogenic protein. As discussed by A. Rubio-Casillas, et al. in a 2024 review article titled “N1-methyl-pseudouridine: Friend or foe of cancer?” there have been many technical challenges over the years that have impeded the ability to produce the immunogen spike protein, including:
Degradation of an RNA molecule by ribonucleases before arriving at the ribosome (the ribosome is where proteins are produced).
Endosomal capture
Difficulty for RNA to cross biological membranes because of its negatively charged backbone.
These problems where overcome when scientists successfully encased the RNA in a lipid nanoparticle (LNP) which allowed for easier distribution to the ribosome. The LNP also increases the cargo within the LNP (mRNA) to pass through the cell membrane.
There were two other major obstacles facing mRNA technology for successful use within cells:
Nuclease breakdown
Immunogenicity induction comparable to infection by pathogen
These additional technical problems where resolved with the incorporation of pseudouridine (Ψ) into the RNA amino acid chain increasing its stability and reducing the anti-RNA immune response.
Pseudouridine has been widely recognized to alter RNA improving its stability against degrading enzymes and reducing its immunogenic potential. These influences where discovered beneficial through in vitro studies, but a more advanced form of pseudouridine was desired to potentially improve overall functionality within the cell. The authors of this paper (i.e., A. Rubio-Casillas, et al.) describe how investigation into other derivatives of pseudouridine occurred with the final discovery that N1-methyl-pseudouridine (m1Ψ) worked even better than pseudouridine when applied in animal studies. Essentially, the scientists found that m1Ψ enhanced overall translational efficiency and reduced cytotoxicity of the modified mRNA.
The problem, as you can imagine, is that what occurs in vitro (in an artificial environment) or an animal model (e.g., mice) does not always translates well when applied to humans with regards to long-term safety. Such is the case with N1-methyl-pseudouridine, and although more research will be done in the coming years there are a number of concerning mechanisms at the cellular level which have been discovered so far that could pave the way for various diseases, including cancer.
This short article is meant to provide an overview of some of these mechanisms as they relate to cellular alteration of important systems involved in immune regulation.
RIG-1 signaling impairment - RIG-1 (retinoic acid-inducible gene 1) is a type of intracellular receptor (aka cytoplasmic receptor) capable of identifying internal viral double-stranded RNAs of viruses. These receptors when activated initiate a signaling process which induces production of Type-I interferons (IFN) and pro-inflammatory cytokines. Activation of RIG-1 (and other similar receptors in the receptor family called RIG-like receptor, RLR) has been shown to have anti-tumor properties. With regards to m1Ψ:
m1Ψ attaches to RIG-1 and can change its conformation which alters its ability induce a strong innate immune response.
This conformation change may appear to have benefits because of a dampened innate immune response and reduced secondary inflammation. However prolonged alteration could increase immune suppression favoring reactivation of bacterial, viral, and fungal infections.
Unrestrained multiplication of cancer cells could occur with prolonged conformation changes of RIG-1 by m1Ψ.
Programmed Cell Death protein 1 (PD-1) - this protein expressed on T-cells interferes with receptor mediated activities, resulting in T-cell dysfunction or exhaustion:
Animals injected with m1Ψ experienced an increase in PD-1+ CD8+ T cells, but they displayed a “non-functional phenotype with reduced ability to produce interferon-gamma and a poor clinical outcome for patients with head and neck cancer, colorectal cancer, melanoma, prostate, breast, and gastric cancer.”
Exhausted CD8+ T cells can allow for cancer growth. With a decrease in PD-1+ CD8+ T cells the likelihood of survival is increased.
Toll-like receptor (TLR) interference - these receptors detect foreign antigens (i.e., from bacterial, fungus, virus). There are downstream cellular effects linked to inflammation as part of a normal and efficient immune reaction for pathogen clearance. However, high level activation of these receptors is associated with cancer development. Some tissues or tumor cell lines express certain toll-like receptors at high levels:
It is possible that TLR4 expression on tumor cells may directly or indirectly enhance tumor progression via inflammatory influences. This is suspected with certain tissues such as colorectal tissue with regards to neoplasia development.
The situation is complex because other toll-like receptor disruption can compromise innate immune activation necessary for anti-cancer surveillance. For example, TLR7 and TL8 influence M1 macrophages (pro-inflammatory) needed for phagocytosis, and the attraction of cytotoxic T-lymphocytes. m1Ψ blocks TLR7 and TLR8 activation (see image below).
There are other concerning mechanisms involved with m1Ψ such as:
+1 frame-shifting which alters normal protein synthesis. This has potential broad-sweeping effects such as activating T-cells against host tissue expressing an abnormally altered protein. Another concern is the development of abnormal protein aggregations that cause cellular function dysfunction, and prion protein formation.
Increased IgG4 production which in regulated amounts assists with anti-IgE effects linked to allergy. However, excessive production of IgG4 is linked to various cancers.
G4 quadruplex and R-loop formation within the genome can lead to DNA instability, fragmentation, and overall DNA damage. The image below is an example of a G-quadruplex showing one layer and a full stacked structure.
Conclusion
In summary, there are multiple mechanisms involved with the introduction of a 100% m1Ψ modified mRNA. On one hand the mRNA modified with pseudouridine improves its stability against cellular degradation enzymes, but its other effects are significantly problematic.
The final image is Table 1 from the A. Rubio-Casillas, et al. article. For a full reading of this article go to - https://www.sciencedirect.com/science/article/abs/pii/S0141813024022323?via%3Dihub.