Cryptic Promoters

Can the Pfizer plasmid make spike in mammalian cells?

This has always been a question. Now that we have shown there is failed linearization in the Pfizer plasmid the next excuse we should expect delivered from the defenders of this manufacturing slop, is that T7 Promoters do no transcribe in Mammalian cells so even if plasmids stick around, they will be ‘inert’.

That is not a safe assumption and even biased AI tools will call out this fable as there is an entire bioinformatics field dedicated to identifying Cryptic Transcriptional start sites (TSS).

So I asked ChatGPT5.o

“Is there any evidence of SV40 plasmids with T7 promoters cryptically expressing a gene proximal to the T7 Promoter in a mammalian cells”

“I’m interested in pcDNA3.1 but where the CMV promoter is replaced with a T7 promoter and the plasmid is Dam+/Dcm+ methylated.

Notice how even ChatGPT advises to remove SV40 to prevent Cryptic transcription.

Also notice that if you put the OR134577.1 Pfizer reference sequence through Promoter2.0 it highlights a highly likely cryptic transcriptional start site at Base 3000, just in front of the spike sequence.

The Top track has the m6A methylation data in Yellow and Dark Purple with the GATC sites annotated. The Middle black track shows the Dcm (CCWGG) methylation sites. The bottom most track is the Promoter 2.0 output (Eukaryotic or mammalian predictions). Note, this isn’t all of the data. I downsampled it 10X and filtered for Q20 reads which leads to less uniform coverage.

You can run the sequence through their web tool here. You should get the output below. OR134577.1 can be downloaded from NCBI.

So this risk cannot be ruled out. Ultimately, one needs RNA-Seq data from these plasmids transfected in mammalian cells without the modRNA to see if these plasmids in fact make spike RNA. If they do, then that might explain persistent spike expression in patients. Once RNA is made, it already has the proper Kozak sequences to initiate translation in mammalian cells.

This is less clear that such an RNA would be translated in bacterial cells as it would require Shine-Dalgarno sequences in the RNA to attract bacterial ribosomes.