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With eminent DNA scientist and virologist, Dr. David J. Speicher. Quantification of residual plasmid DNA and SV40 promoter-enhancer sequences in Pfizer/BioNTech and Moderna modRNA COVID-19 vaccines from Ontario, Canada https://pubmed.ncbi.nlm.nih.gov/40913499/ 32 vials representing 16 unique vaccine lots. These data demonstrate the presence of billions to hundreds of billions of DNA molecules per dose in the modRNA COVID-19 products tested. All products tested exceeded the guidelines for residual DNA set by the FDA and WHO of 10ng/dose by 36–627-fold. Total DNA in all vials tested Exceeded the regulatory limit for residual DNA set by the US Food & Drug Administration (FDA) and the World Health Authorization (WHO) by: Pfizer: 36-153-fold Moderna: 112-627-fold 3 Pfizer vials exceeded the regulatory limit for the SV40 promoter-enhancer-ori (p53) The PCR results for the most recent XBB.1.5 Moderna and Pfizer vaccines suggest that DNA residues have not been reduced from previous vaccine versions. Pfizer, Total DNA ranged 371-1,548 ng/dose Moderna, 1,130-6,280 ng/dose Specific DNA of multiple plasmid DNA targets Pfizer ranged 0.22-7.28 ng/dose Moderna 0.01-0.78 ng/dose for Moderna. The SV40 promoter-enhancer-ori(0.25-23.72 ng/dose) was only detected in Pfizer vials. Sequencing of one vial Mean DNA length, 214 bp Maximum length, 3.5 kb Presence of 1.23 × 108 to 1.60 × 1011 plasmid DNA fragments per dose encapsulated in lipid nanoparticles. Our findings extend existing concerns about vaccine safety and call into question the relevance of guidelines conceived before the introduction of efficient transfection using LNPs. This work highlights the need for regulators and industry to adhere to the precautionary principle and provide sufficient and transparent evidence that products are safe and effective, and disclose the details of their composition and method of manufacture. For some of the COVID-19 vaccines, the drug substances released to market were manufactured differently than those used in clinical trials. Rationale for study Manufacturing nucleoside-modified mRNA for commercial COVID-19 vaccines relies on RNA polymerase transcription of a plasmid DNA template. Previous studies identified high levels of plasmid DNA in vials of mRNA vaccines, suggesting that the removal of residual DNA template is problematic. Therefore, we quantified the DNA load in a limited number of Pfizer-BioNTech and Moderna COVID-19 modRNA vaccine vials using two independent methods. This study emphasizes the importance of methodological considerations when quantifying residual plasmid DNA in modRNA products, considering increased LNP transfection efficiency, and cumulative dosing presents significant and unquantified risks to human health.

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