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LNP packaging of dsDNA
Grim Reefer and Vaccines
A recent article downplaying the impact of dsDNA contamination in the vaccines made the assumption that the DNA was not packaged in LNPs and therefor was unlikely to transfect mammalian cells and therefor unlikely to genome integrate. The article was authored by Chitra Pattabiraman who has enough of a background to test some of the cavalier statements presented in the article. This post will contain some experiments to demonstrate the nature of the irresponsible public communication she has embarked on.
You’ll notice this paragraph dwells on any contaminating DNA facing a cell ‘fortress’ as it pertains to transfection.
Little did this author know, that this effect is measurable with well known Live-Dead qPCR tools in the industry. Medicinal Genomics holds a patent on one of these techniques. There are many different Live-Dead qPCR methods published but we prefer the enzymatic method we developed for microbial detection in Cannabis.
In order for qPCR to be calibrated to detect viable organisms you need to separate the dead DNA from the living DNA. The need for Live-Dead qPCR was obvious once Rita Jafaar published her work showing qPCR -PFU discordance in C19 qPCR testing.
To better calibrate for CT to CFU/PFU (colony or plaque forming units) concordance, it is helpful to use enzymes that can digest dead DNA but can’t enter cells to digest live DNA. By eliminating the dead DNA, your CT scores better reflect the viable organism count in the sample. The clearing out of the dead DNA for cannabis pathogen detection is affectionately known as a ‘Grim Reefer’ assay.
Live-Dead qPCR is something the Corona virus fever omitted from its qPCR approach and as result there is now a CDC 90 day moratorium on C19 qPCR once you are C19 qPCR positive. This is because, C19 generates lots of replication incompetent sgRNA that is a template for RT-qPCR. As a result RT-qPCR will continue to pick up this dead RNA for up to 90 days post infection.
Fauci infamously called this ‘dead nucleotides’ as he admitted to Cycling over 35 being irresponsible. This is incorrect as individual nucleotides cannot be amplified. PCR requires fragments 40 bases or larger in order for amplification to occur.
So what does Grim Reefer have to do with DNA in the vaccines? The LNPs are by design, simulating a cell membrane that protects the mRNA and DNA from nuclease attack. How do we measure the DNA in the LNPs versus the DNA outside of the LNPs or unpackaged by the LNPs?
To answer this question, one simply
1)Treats vaccines with DNaseI.
2)Once these are nuclease treated you break open all the LNPs, purify the DNA and run qPCR to measure how much DNA disappears compared to an untreated sample.
Monitoring the DNaseI sensitivity of the sample can help inform on packaged versus unpackaged DNA. In the amount of time it took Chitra to write her poorly thought out article on this topic, she could have picked up a pipette and actually measured this. Maybe her Wellcome Trust funding could pitch in for a few PCR reactions?
To do this properly, you need several controls built into the assay.
If you don’t completely deactivate your DNaseI after such a treatment, the DNase will destroy your DNA during qPCR. To address this, our Grim Reefer assay utilizes a unique control DNA that is spiked in after the chemical kill step used to deactivate the DNaseI. By monitoring the CT of this spiked in DNA, we can ascertain if the DNaseI is completely or partially inactivated compared to an untreated sample spiked with the same control.
This control ‘proof of kill’ DNA is targeted with a 3rd qPCR assay that fluoresces in CY5. So we have Spike sequence tagged with HEX, The Origin/Vector assay tagged with FAM and our Kill step tagged with CY5. This is a 3 color, 3 qPCR multiplexed assay.
Below is a depiction of the DNaseI ‘Grim Reefer’ assay. I have modified an image from Jessica Rose’s recent paper-Halma et al. that helps to depict the LNPs. I have updated the LNPs to contain dsDNA we suspect is present during the packaging process.
LNPs, by design, are nuclease resistant. The lipid bilayer is to protect the nucleic acids from DNaseI activity. DNaseI activity is actually inhibited by actin in the muscle which is one reason to be concerned that any unpackaged DNA is not going to be immediately eliminated upon intra-muscular injection.
A simple 30 minute 37C treatment with DNaseI should eliminate the unpackaged DNA and leave the LNP protected DNA intact (Figure 1).
The DNaseI is then chemically deactivated and a ‘Proof of DNase deactivation’ control DNA is spiked into the sample to track its survival after enzyme deactivation. If the kill step is complete, this DNA should be the same CT as your DNase blank control sample.
If half of the DNA is packaged and half is free circulating DNA in solution, you should see a 1 CT offset from eliminating half of the DNA. We instead see a 0.77CT and a 0.4CT offset which implies less than half of the DNA is in solution and the majority packaged in Nuclease resistant LNPs which will slip right through any ‘Fortress’ door.
What does this look like with a DNaseI that is hard to kill? Below is an example of a DNase (DNaseI-XT from NEB) that fails to deactivate with our kill step. Note the purple CTs below are divergent (Figure 3). This is the spike-in DNA used to monitor the DNaseI kill step. This demonstrates the kill step is failing on DNaseI-XT. As a result, the large offsets we see with Spike and Ori primers can NOT be utilized to quantitate unpackaged DNA in the vaccines as we know the DNase is still active in PCR.
An important control for such a study is to demonstrate that your Grim Reefer reaction is not anemic and is capable of clearing all of the DNA in a given reaction (Figure 4). Failure to show complete removal in such a control would underestimate the amount of unpackaged DNA in solution.
Even though DNaseI-XT was difficult to deactivate in this experiment, this may be worth further exploration as DNaseI-XT looks like a better nuclease to hydrolyze RNA-DNA hybrids and other purification techniques may eliminate it. Our previous work in this field implies RNA-DNA hybrids are forming and these are more resistant to DNaseI.
Over half of the DNA contamination in the vaccines is DNaseI resistant. This implies the DNA is protected by the LNPs and the DNA is packaged in the LNPs.
This demonstrates the article written on this topic by ORFonline is false. They equated this to ‘Entering a Fortress’. If the DNA is packaged in LNPs then it will be delivered to the cells with the mRNA and much of the DNA may be in RNA:DNA hybrids. I doubt Pfizer or Moderna would claim LNPs are faced with ‘Entering a Fortress’. The whole point of LNP packaging is to provide nuclease protection and easy entrance to the fortress. ORFonline’s argument is thus overt gaslighting as a Person of Ordinary Skill In The Art (POSITA) would assume the DNA is co-packaged with the RNA unless you provided sufficient evidence to counter the Occam’s razor of this manufacturing assumption.
This data also suggests some of the DNA is not packaged. To more precisely quantitate the exact amount of unpackaged or ‘dead’ DNA is in solution, more replicates or digital droplet PCR could be utilized.
In summary, Live-Dead qPCR tools are very versatile and valuable when it comes to measuring vaccines and pathogenic threats. Had these been deployed in C19 testing, we likely would not have quarantined the western world, wrecked the economies and induced iatrogenic death with C19 panic induced modifications to healthcare. Many jurisdictions that did no testing , out performed countries that were swimming in it. This is largely the result of testing asymptomatic people and the test not being able to discern those recovering from C19 with dead RNA from those that had live RNA from an active infection. This blame has been laid on door of qPCR which is a shame given there have been live-dead qPCR tools published for decades.
These same tools can be applied to vaccine quality control to understand the ratio of packaged to unpackaged DNA and RNA. Unpackaged DNA and RNA may induce STING and lead to adverse events and should be monitored closely lot to lot.
Other aspects of Chitra’s article I agree with. Integration has not yet been identified. If it occurs, it is likely rare and occurring in somatic cells that turn over. Nevertheless with billions of DNA molecules being injected across billions of people, we should still be concerned over stem cell or germ cell integration events as these cells can magnify a rare event into a larger one. The bio-distribution studies imply the LNPs are getting to the ovaries. The article made no mention of the nuclear localization signal in the SV40 promoter of the Pfizer vaccine contaminant.
Why is this the Occam’s Razor hypothesis? These are not oil in water emulsions where all of the aqueous compartment is forced into the center of an LNP. These are water in water packaging of RNA and DNA into LNPs and its very pH and temperature dependent. Unless they use nucleases or dialysis, there will always be nucleic acid in the supernatent.
Grim Reefer Assay
20ul of Pfizer monovalent vaccine was added to a DNaseI blank well and DNaseI positive well. Slowly pipette these to avoid LNP shearing.
2.5ul of Grim Reefer 10X buffer (Medicinal Genomics- MGC).
2.5ul of DNaseI (NEB) or ddH20 was added to the positive and blank wells respectively.
Slowly tip mix 5 times with a P200 pipette. You want to mix the DNaseI adequately but not disrupt the LNPs with any more force than the process of injection.
Incubate at 37C for 30 minutes.
Add 2.5ul of MGC lysis buffer.
Add 2.5ul of 10,000X diluted Grim Reefer Internal control (MGC) This is the DNAseI inactivation spike-in control.
Add 54ul of Sensativax magnetic beads. Tip mix 10X. Wait 5 minutes, magnetically separate the beads and 2X wash with 200ul of 70% ethanol. 2 minute RT dry to remove EtOH.
Elute the samples in 30ul ddH20 off magnet.
2ul of eluent + 3ul of ddH20 are used in qPCR with the methods described in our Preprint with one modification.
15ul of the Grim Reefer primer probe (MGC) is added in place of 15ul of ddH20 in the qPCR mastermix.
This requires a CY5 channel for detection in qPCR (Figure 2).
The same method was repeated with 2.5ul of DNaseI-XT from NEB in place of the 2.5ul of DNaseI from NEB (Figure 3).
The DNaseI completion test in Figure 4 was performed using 2ul of Grim Reefer Positive control and 18ul of ddH20. This 20ul was used instead of 20ul of vaccine.
A Japanese group is in the process of replicating some of the work.