Thread by @Kevin_McKernan

The Dead Man switch has been lifted. Im not dead but the paper has been accepted.
More edits may appear as the Journal typesets this but this stack has the most current versions, Nostr links, Bitcoin links and Zenodo links.

Why this matters-
This paper demonstrates why the regulators cant find the DNA. The nucleases being used to remove the DNA, fail to digest the RNA:DNA hybrids. So they remove the KAN gene, fail to remove the Spike gene and only go looking for the KAN gene. This is why fluorometry sees ~100X more than qPCR.

And once again, you dont have to believe us. @CanningPharm showed you a paper from BioNtech that spells this out.
They knew this but pointed the regulators to an assay that wouldnt find it.

From the BioNtech paper (Lenk et al).

Irony would have it that @BioRXIV censored this.
We had received notice of its acceptance in a Journal the same day.
@DrJBhattacharya @TracyBethHoeg @RWMaloneMD @SenRonJohnson @P_McCulloughMD @RetsefL @weldeiry @KUPERWASSERLAB @JesslovesMJK @CharlesRixey @DJSpeicher

We actually observed this 1st in @DJSpeicher paper. We dug into it more as we could not understand why the regulators would resort to a KAN gene qPCR. Process 1 DOES NOT AMPLIFY that region of the plasmid. So they are quantitating the pre-Amplfied template plasmid. Not the actual Spike region that is amplified. This is absolute clownery.

@DJSpeicher Buckhaults lab also saw this. LOG scale different results with qPCR of KAN vs Spike.

@DJSpeicher Sonia Pekova also saw this.

Log scale different results from vector vs Spike (more spike).

And all of the persistence data is looking at Spike.

You do not want the transcriptionally active ORF DNA floating around a 100X over the limit.

@DJSpeicher open.substack.com/pub/anandamide…

@DJSpeicher For those accusing me of being performative with a Dead man switch.
Publish data on how $100B product contaminated the population with potentially oncogenic sequences and you will earn some haters.

@DJSpeicher The 5 lots in this study are fairly new. A few with expiration in 2026 so the problem has NOT been fixed.

@DJSpeicher Meant to also tag @KMilhoanMDPhD for this thread

@DJSpeicher @KMilhoanMDPhD This is the crux of the problem.
The spike region can’t be destroyed by DNaseI.

The KAN region can be destroy.
Pharma only measures KAN.

Another game of Hide The Ball.

@DJSpeicher @KMilhoanMDPhD Fluorometry using 1000x excess of RNaseA.

@DJSpeicher @KMilhoanMDPhD DNaseI-XT cleans it all up.

@DJSpeicher @KMilhoanMDPhD We ran 27 qPCRs for each vial
3 different targets (spike,SV40, Ori)
3 different dilutions (1X, 1:10x, 1:100x) to rule out PCR inhibition.

All performed in triplicate

@DJSpeicher @KMilhoanMDPhD Qubit vs qPCR
Concordance falls apart with non-spike regions.

@DJSpeicher @KMilhoanMDPhD ~5.3Kb fragments found in the vials.
That highlighted Blue section is one Nanopore read.

@DJSpeicher @KMilhoanMDPhD If you run their plasmid sequence through a cryptic promoter prediction tool, you’ll find a mammalian promoter downstream of the T7 promoter.
Someone needs to RNaseA treat the vaccine, repackage it in Lipofectamine, transfect mammalian cells and look for spike RNA.

@DJSpeicher @KMilhoanMDPhD The above experiment will tell us if the DNA is transcriptionally active.
Recall, it has an NTS so it doesn’t need to integrate to make RNA.

@DJSpeicher @KMilhoanMDPhD anandamide.substack.com/p/cryptic-prom…

@DJSpeicher @KMilhoanMDPhD The Zenodo link is burried deep in the Substack…
I’ll make a link at the top of it so it’s easier to find.

zenodo.org/records/178321…

@DJSpeicher @KMilhoanMDPhD Some people prefer the video of the experiment.

@DJSpeicher @KMilhoanMDPhD Never forget the EMA called them out on the DNaseI problem.
But they warp sped right past that concern.

The Cryptic Promoter isnt so cryptic.

Its well documented in the literature.

I'll search for information about cryptic mammalian promoters in the bacterial ColE1 origin of replication.Excellent question! Yes, the ColE1 origin (and its related pMB1/pUC variants) does contain a cryptic mammalian promoter.

Key Finding:

A 2012 study published in Nucleic Acids Research discovered that plasmids with pMB1/pUC origins (which are ColE1 derivatives) constitutively produce substantial amounts of RNA from a TATA-containing cryptic promoter within the origin of replication that functions in mammalian cells.

Important Details:

The cryptic promoter:
Contains a TATA box within the bacterial origin of replication
Is constitutively active in mammalian cells
Produces readthrough transcripts that can extend into adjacent sequences
Is highly conserved across ColE1 family members (ColE1, pMB1, pUC, and p15A)

The study found:
95% of GenBank plasmid entries use pMB1 or pUC origins (ColE1 derivatives)
The ColE1 and p15a origins are "highly homologous to the pMB1/pUC origin, particularly in the region surrounding and upstream of the transcription start site, and thus may also exhibit promoter function in eukaryotic cells"
This cryptic promoter has confounded countless reporter gene assays
The readthrough transcripts can cause false-positive results when sequences containing splice sites are inserted downstream

Implications for mRNA Vaccine Plasmids:

This is highly relevant to your DNA contamination research because:

The plasmids used for Pfizer and Moderna vaccines likely contain ColE1-type origins
If plasmid DNA contaminates the final vaccine product and enters human cells, the cryptic promoter in the ori could be transcriptionally active
This could lead to expression of adjacent sequences (like kanamycin resistance genes, portions of the SV40 sequences, or other plasmid backbone elements)
The ori region itself becomes a potential source of unintended gene expression, not just the intended mRNA coding sequences

This adds another layer of concern beyond just the presence of residual DNA - the DNA itself may contain elements that are transcriptionally active in mammalian cells.

Would you like me to search for more specific information about which origin of replication is used in the Pfizer/Moderna plasmids?

Here is the full reference for the 2012 paper:

Lemp NA, Hiraoka K, Kasahara N, Logg CR. Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function. Nucleic Acids Res. 2012 Aug;40(15):7280-90. doi: 10.1093/nar/gks451. Epub 2012 May 22. PMID: 22618870; PMCID: PMC3424574.

Additional Access Information:

Journal: Nucleic Acids Research
Volume: 40, Issue 15
Pages: 7280-7290
Published: August 1, 2012 (Epub: May 22, 2012)
DOI: doi.org/10.1093/nar/gk…
PubMed ID: 22618870
PMC ID: PMC3424574 (Free full text available)

Authors:
Nathan A. Lemp (Department of Molecular and Medical Pharmacology, UCLA)
Kei Hiraoka (Department of Medicine, UCLA)
Noriyuki Kasahara (Department of Molecular and Medical Pharmacology, UCLA)
Christopher R. Logg (Department of Medicine, UCLA)

This is an open access article, so you can freely access the full text through:
PubMed Central: ncbi.nlm.nih.gov/pmc/articles/P…
Oxford Academic: academic.oup.com/nar/article/40…

This is a highly important reference for your work on plasmid DNA contamination in mRNA vaccines, as it definitively demonstrates that the bacterial origin of replication sequences commonly used in vaccine production plasmids contain cryptic mammalian promoter activity.