Disingenuous Integrity

RNA lost its integrity with N1' methylpseudouridine

I was delighted to see more folks combing over EMA documents this week. For those in the weeds on the mRNA vaccines, you know the EMA raised many valid concerns over the odd bands on Western Blots and the worsening RNA integrity number as they scaled up production. A common hymn at this point is to talk about how unstable RNA is.

This is a magic trick. Squid ink. A decoy. It reminds you RNA is so fragile that it will never survive in the jungle of your blood stream. Yet we detect this RNA weeks to months later in some patients. These two ideas are at odds with one another. Yes, RNA is not as stable as DNA but poor cold chains is not degrading 100ug of RNA at a meaningful rate.

The mRNA is truncated not because it is decayed, but because the RNA polymerase seized up on its replication when it was fed a non-native nucleotides. It is truncated because it never finishes faithful transcription of the DNA plasmid.

The real reason for the -80C cold chain isn’t because RNA is this fragile. It is to avoid the LNP emulsion from forming syncytia. Much like a Paul Newmans Salad dressing, Emulsions ,given enough time at 1G, will eventually break and form an aqueous and hydrophobic phase. If LNPs agglutinate and merge they turn BBs into cannonballs. Your cells can tolerate a few BBs but a cannonball will make the Surface Area/Volume of the cell unsustainable and cytotoxic. Even empty payload Syncytia can be cytotoxic. No Spike required for damage to be done.

For perspective, LNPs experts aim to maintain at 200-800nm liposome in size. Capillaries have a diameter of 5-10um. If these LNPs merge and get too big they will melt the ends of capillaries.

A good review of liposome stability can be found at NIST.

Increased temperature leads to instability and more oxidation.

This doesn’t mean loss of RNA integrity doesn’t matter. Just pay attention to the actual cause. I doubt it is the RNA molecular stability at these temperatures. It is far more likely that the RNA polymerase is stalling and failing to synthesize full length mRNA when it is choking on pseudouridine, than the RNA is degrading like a radioactive substance.

This is an important point. You will notice a recent Nerf paper on Pseudouridine that I critiqued for using Reverse Transcriptase assays. If you are trying to hide Pseudouridine issues, Reverse transcriptase are a good way to do it. But these turn RNA into DNA and are not used in the manufacturing of RNA templates from DNA plasmids. In other words, the model isn’t relevant for the methods used to make mRNA vaccines.

We want high RIN numbers for these mRNAs and vaccine lots properly sequence QC’d to understand the transcriptional error present in the synthesis of these mRNAs. Ideally, translational error would be measured as well in relevant cell lines. But the LNP polydispersion and stability is the blind spot. Few are talking about this and it likely plays its own roll in the presentation of adverse events seen in VAERs.