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Chemical Secret to Stunning Success of Pfizer & Moderna mRNA Vaccines; Decades of Painstaking, Slowly Progressing Research Underlie “Warp-Speed” Development of Miracle Vaccines for COVID-19

Dr. Gerald Zon’s latest “Zone in with Zon” blog post, dated May 25, 2021, and published by TriLink BioTechnologies of San Diego (https://www.trilinkbiotech.com), discusses the key component for the Pfizer-BioNTech and Moderna mRNA vaccines, namely the chemically modified mRNA (modRNA) synthesized by in vitro transcription (IVT) reactions, wherein one of the natural (aka wild-type) A, G, C, and U nucleotide 5’-triphosphates is replaced by a base-modified analog. Dr. Zon notes that interest in chemically modified mRNA (modRNA) (https://onlinelibrary.wiley.com/doi/10.1002/jmv.26924) can be traced back to a 2005 report by Karikó et al. (https://pubmed.ncbi.nlm.nih.gov/16111635/) demonstrating that modRNAs comprised of 5-methycytidine (5mC), N6-methyladenosine (m6A), pseudouridine (Ψ), 5-methyluridine (m5U), and 2-thiouridine (s2U), either separately or in combination, can reduce immunogenicity mediated by toll-like receptors (TLRs). Dr. Zon notes that “subsequently, it was shown that Ψ modification, in particular, could increase the translational capacity and biological stability of mRNA (Karikó et al. 2008) (https://pubmed.ncbi.nlm.nih.gov/18797453/). Along the lines of the familiar saying ‘good, better, best…,’ later studies by others (Andries et al. 2015) (https://pubmed.ncbi.nlm.nih.gov/26342664/) found that simply adding a methyl group to the N1 position in Ψ (N1-methylpseudouridine, N1mΨ) in modRNA via IVT with the corresponding 5’-triphosphate produced N1mΨ-mRNA that outperformed Ψ-mRNA.

Dr. Zon goes on to say “Perhaps the most compelling evidence of the performance enhancement provided by N1mΨ-mRNA is the COVID-19 mRNA vaccines developed independently by Pfizer-BioNTech and Moderna, both of which are comprised of mRNA with complete replacement of uridine by N1mΨ. In view of this unarguably proven utility of N1mΨ-mRNA, the Zone (Dr. Zon’s blog) is featuring N1mΨ-mRNA in two blogs. The current blog post (May 25, 2021) is Part 1 and provides some backstory on the initial discovery of the benefits of N1mΨ-mRNA, as well as a subsequent investigation into optimizing the performance of IVT mRNA using N1mΨ. Part 2 will follow soon and feature the use of N1mΨ to optimize mRNA for heart gene therapy.”

In providing the back story, Dr. Zon said that “following the development of the modRNA platform, various pre-clinical studies demonstrated its potential for therapeutic applications. These notably included studies by Warren et al. (2010) (https://pubmed.ncbi.nlm.nih.gov/20888316/), which used 5mC/Ψ-mRNA to reprogram and differentiate human cells, and the work of Zangi et al. (2013) (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4058317/), which used 5mC/Ψ-mRNA to treat a mouse model of myocardial infarction.”

Dr. Zon continued by saying that “based on these promising in vitro and in vivo findings, and as part of her 2015 PhD thesis at Ghent University in Belgium, Oliwia Andries (photo) states that she ‘sought to identify RNA base modifications that could further reduce the immunogenicity and increase the translational capacity of mRNA by using mRNA containing Ψ as a benchmark.’ Dr. Andries’ study, with colleagues, was published on November 10, 2015 in the Journal of Controlled Release. The article is titled “N1-Methylpseudouridine-Incorporated mRNA Outperforms Pseudouridine-Incorporated mRNA by Providing Enhanced Protein Expression and Reduced Immunogenicity In Mammalian Cell Lines And Mice” (https://www.sciencedirect.com/science/article/abs/pii/S0168365915300948).

Dr. Zon said that, briefly, this study “demonstrated that N1mΨ, long known in the biosynthesis of hypermodified 18S rRNA of yeast (Brand et al. 1978) (https://portlandpress.com/biochemj/article-abstract/169/1/71/11448/Biosy...), markedly improved the translational capacity of mRNA compared with Ψ-mRNA and 5mC/Ψ-mRNA in A549 cells (human lung epithelial cells), BJ cells (human foreskin fibroblasts), C2C12 cells (murine myoblasts), HeLa cells (human cervix epithelial cells), human primary keratinocytes from neonatal foreskin, and in mice after intradermal or intramuscular injection of modRNAs. It did so when incorporated alone or in combination with 5mC (5mC/ N1mΨ). 5mC/ N1mΨ-mRNA was also shown to have reduced toxicity and reduced activation of the intracellular innate immune response in the various cell lines compared with 5mC/Ψ-mRNA. Finally, the superiority of 5mC/ N1mΨ-mRNA over 5mC/Ψ-mRNA was shown to be at least partially due to its enhanced ability to avoid activation of toll-like receptor 3.”

Dr. Zon said that “although Andries et al. concluded that 5mC/ N1mΨ-mRNA might be a new benchmark for modRNA-based therapeutic applications, subsequent studies (discussed later in his blog) found more complex relationships between 5mC/ N1mΨ-mRNA composition and its biological effects.

Please go to Dr. Zon’s full blog (https://zon.trilinkbiotech.com/2021/05/25/optimizing-the-performance-of-...) to see additional details of the incredible advance of science that set the stage for the very-hard-won success of the Pfizer and Moderna vaccines.

In the conclusion of his first of two blogs on IVT modRNA and N1mΨ-mRNA, Dr. Zon suggested that “although investigations and applications of IVT synthesized modRNAs have advanced rapidly in recent years, the Zone’s view is that it is still “early days” in terms of gaining a comprehensive understanding of all the factors that influence the biological functionality of IVT modRNAs.”

“By analogy to how the functional ‘rules’ in the early days of antisense oligonucleotide and small-interfering RNA therapeutics changed with new data from further experimentation, the field of IVT modRNAs will also likely go through periods of changing rules based on experimental results in other biological systems and the possibility of system-by-system dependencies.”

BioQuick News is proud to count Dr. Zon among the publication’s distinguished board of Science & Medicine Advisors (http://www.bioquicknews.com/node/34).

[Zone in with Zon blog]