DNA & RNA Biology, mRNA Vaccines, Vax Contamination & Side Effects, Spike Protein, Ivermectin, Hop Latent Viroid | Kevin McKernan | #149

About the guest: Kevin McKernan is the Chief Science Officer at Medicinal genomics and has been working the the biotechnology sector and conducting genomics research going back to his involvement in the Human Genome Project.

Episode summary: Nick and Kevin discuss: basics of DNA and RNA biology; mRNA vaccines and how they work compared to traditional vaccines; the mRNA vaccine manufacturing process; DNA contamination in the Pfizer and Modern mRNA vaccines for COVID; the SARS-CoV-2 spike protein; vaccine side effects; ivermectin and hydroxychloroquine; and more.*This content is never meant to serve as medical advice.

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* Episode transcript below.

Full AI-generated transcript below. Beware of typos & mistranslations!

Kevin McKernan 4:21

Was that Senator Ron Johnson humble hearing on all those COVID stuff. So you had myself Malone, Jessica Rose, Pierre quarry. long list of people who were just presenting evidence and all of the shenanigans going on in COVID. Yeah,

Nick Jikomes 4:42

we're gonna talk about a lot of that, I guess here. Why don't we just start off with some basic stuff. Want to don't you just tell everyone a little bit about yourself, your background, your expertise, and you know what you do at a high level?

Kevin McKernan 4:57

Okay, sure. Um, so My background started in, in this field in 1995. Actually on the Human Genome Project, I was started there as a member of the research and development team and shortly thereafter to the folks leading it left and left me in the reins, wholly unqualified, and I had to learn on the fly. So I started managing that research and development group to about a probably a 10 or 12 person group inside the Whitehead Institute Center for Genome Research was under Eric Lander and Lauren Lintons guidance. And we built basically the robotic platform and the DNA purification system to purify. We did about 20 million plasmids a year on that thing to do Sanger sequencing for the Human Genome Project. So, my role there was was related to the automation and the DNA purification chemistry and optimizing all the Sanger cycle sequencing stuff. And zash as that project came to a completion a lot a lot of companies were asking how to export that technology and and MIT held some patents on it. So we licensed those and spun them out to a company called Agincourt which became a really large DNA sequencing company. Actually, it was the largest commercial sequencing entity I think in the in the world. By probably 2004. Beckman came to acquire it in 2005. We had beyond just a DNA sequencing facility, it was a we had a bunch of DNA purification technology that was used to purify viruses and a variety of pathogens from blood. So they acquired that but in the process, there was a skunkworks project, we had to build a DNA sequencer that used us to sequence DNA off of single magnetic beads. And that was starting to show some promise, but of course, no one knew how to value it at the time, and so they decided to split it out into its own company called Agincourt personal genomics. And a year later, we presented data sequencing a coli genomes at Eygpt, which caught the attention of ABI and Illumina who proceeded to have a bidding war over the company. And Avi eventually won that bid and purchased Agincourt personal genomics and brought the solid sequencer to market. So I spent from 2006, to about 2011, working at EBI, getting this all sequencer to market. And we also at towards the end of that acquired Ion Torrent, which was another next generation sequencing system that worked on semiconductors. So I worked on that program, helping get that developed and out the door and then decided to split ways and worked on some other passions of mine, one of which was the cannabis genome I had been, I had a very large non compete, the company was now part of life tech, which is a bigger entity, which meant I couldn't really compete. My non compete man, I really couldn't work in that space. So I kind of just went off into the Ag space. And we started sequencing cannabis genomes, because we felt they were the genome had been sequenced at the time, and it had all these therapeutic compounds in there that could be helpful for cancer. And so we figured let's get the thing public and see if that helps kind of mature that field. That field got a little complicated. As you might imagine, growing a business in the cannabis field is difficult from a banking perspective, and the laws keep changing on you. So the company kind of pivoted back into doing clinical sequencing of people who might benefit from cannabinoids, so a lot of epilepsy patients mitochondrial disease patients, autism spectrum disorder. So we were doing exome sequencing on those cohorts as a clinical test. And that went on for about five or six years before we realize that cannabis market started to evolve and mature and we pivoted back into doing cannabis testing. So right now, my role of medicinal genomics is building PCR tests that target all the pathogens in cannabis that can either destroy it from a yield standpoint, or impact patient health, a variety of jurisdictions around the world demand ecoli, salmonella Aspergillus, a host of pathogens get tested for every every pound of cannabis that's sold. And we don't do the testing ourselves. We just make the picks and shovels that other labs used to do this type of testing. So we're mostly involved in assay development and genomic sequencing. Still to this day, we do a lot of genomic sequencing of cannabis genomes, and it's about 2000 of those public now on our website. And we've extended them to other medicinal organisms, philosophy comes to mind because it's has a similar, I'd say therapeutic profile for although it works in very different ways. It's one of these medicinal organisms that is, I'd say under studied in the current FDA regime, if you will, hard to get hard to get patents on these natural products. So a lot of people push them aside. And so we started sequencing. We've been through about 100 of those genomes that are up on our website. and publish some papers on that topic fairly recently. So yeah, I don't belong in sequencing vaccines can fish out of water, I suppose. But it, it came to us somewhat serendipitously. And I think because maybe your podcast and a few others highlighted some of our work in evaluating some of the early COVID tests, we were shocked that these COVID tests came to market without internal controls, because we could never get away with that, and in any other market. And so without those internal controls, it's really hard to gauge your viral load. And it seemed odd that we were racing these tests out the door without that, and perhaps there was a higher positivity rate early on in the pandemic. Many of the companies clean that up over time, but the kind of horses out of the barn at that point. And there's the so I wrote a paper on that, and one with Peter McCullough, which I think caught your attention in the last podcast, just talking about some of the differences between these vaccine mRNAs. And, and what was actually in the virus, which led to some discussions about frameshifting, which I think have recently been shown to be correct. There's a paper from Moroni that came out showing that the shooter yearning can cause some frameshifting, that is probably not the same type of frameshifting that you would see in the virus, because virus doesn't have those types of slippery bases. So

Nick Jikomes 11:22

yeah, I want to get to that, let's spend just a little time giving people who don't have the background, that you have some vocabulary, and just some, some, some basics, some basics that they can keep in mind, let's start super basic, but I don't want to spend too much time here. DNA versus RNA. So there's DNA and all of ourselves, there's RNA and all of ourselves. High level, what's the difference between DNA and RNA in terms of what they do, and what they're made out of?

Kevin McKernan 11:54

I think a good analogy is DNA is like what's on your hard drive on your computer in RNA is like what's in your task manager, like what programs are actively being run from your hard drive. So they're often fragmented and smaller in size, and they're ephemeral, they can go on and go off. So your DNA is this hard drive of all the programs your cell can run. And then the RNA whenever it wants to run a program, it has to turn that program into RNA for the cell to then turn it into proteins. So you have to think of it as all the program is possible that cell can run and not all cells are gonna run all programs at the same time. That's, in fact, what makes the cells very different is they selectively choose certain programs to run in order to be a heart cell versus a liver cell. So you get different programs being run in those two types of cell lines, which you can measure by sequencing the RNA. So the RNA will tell you what, what genes are actually turned on in a given cell? And how, how loud are they being turned on? Do we have one copy of the RNA? Do we have 100,000 copies of the RNA. And now the RNA is supposed to be fairly ephemeral. There's all types of circuitry in the cell to express it and then destroy it so that you don't have something that's constituency turned on all the time. But that process is quite delicate. And is the process that some of these vaccines are trying to hijack to get spike protein made? Yeah,

Nick Jikomes 13:13

okay, so So DNA turns into RNA. The RNA can be likened to the programs, your computer might be running at any given time, for a little while, for longer periods of time they shut down, they shut off. The RNA, the mRNA is made from the DNA, the mRNA can then be used to make proteins from it. In terms of the code here, we talk about the letters of the DNA and the RNA code. What are those letters and how they how do they differ between DNA and RNA? Well, ATC

Kevin McKernan 13:44

and g are the ones that are known to be in DNA. There are some exceptions, you sometimes have methylated versions of these bases, and you sometimes have uracil. But generally, uracil is mostly found in RNA. So when certain bases in the DNA get damaged, they might appear as a uracil. But there's a whole pathway meant to clean that up to get rid of these diamonds that have been turned into yourself. But RNA is always replacing that T with a u. So whenever you see RNA sequence, it would be a use eg there tends to be a U replacing the T. So that's one key difference in the language. You oftentimes find other modifications in RNA as well there's a very rare type of alteration to the use known as pseudo urethane, about point six to 1% of the of the use in any given RNA have a pseudo urethane in them, and the cell can further methylate that into n one methyl Suder urethane with a set of enzymes that methylate the superiority and so there's a pathway for about a very small infrequent percentage of the RNAs have these pseudo eurogenes which are, you know, we don't fully understand their role in biology to be on honest, when you do knockout mice, when you knock out the enzymes that that play in this cascade, really, really weird things happen. But it's I think it's important for folks to know that it's mostly relegated to Sno RNAs and T RNAs. It's very rarely found in messenger RNAs, these pseudo us, but they do exist in nature. And, you know, both of the mRNA platforms leverage the fact that that base was very rare and different, and replaced all of the use in their mRNA with this n one methyl pseudo you reasons of wanting to keep those RNAs around longer. The enzymes that we have that that turn these RNAs on and then destroy them are a little bit slower to act on the pseudo the N one methyl su urien, in the in the Pfizer Madrona vaccines, that was considered a feature not a bug at the rollout of this because their largest concern was injecting these RNAs and then having your cells destroy them before they could express by protein.

Nick Jikomes 15:58

So mRNA is Aug and see, most of our RNA most of the time, uses uracil as the EU, but there's a slightly different version that can be incorporated. And that happens naturally a small percentage of the time. And that affects probably, among other things, how long those mRNAs are lasting in the cell.

Kevin McKernan 16:19

Yes, there's some literature suggesting it may play a role in so their localization as well. The ones that are seem to have superiority on them tend to be more nuclear localized. But that's not that's not a hard and fast rule.

Nick Jikomes 16:34

And so in terms of the the mRNA, vaccines, the Pfizer and moderna vaccines that we're pretty much all familiar with at this point that have been used for COVID. At a high level, what was the intention behind these vaccines, how are they meant to work in contrast to traditional vaccines. So traditional

Kevin McKernan 16:50

vaccines would put a protein in your body and be injected into your arm and your body would build immune defenses against that that circulating protein and it made it's not meant to circulate just be in your arms, such that your immune system could see it and build antibodies. The approach here was instead of having to use a protein is was was to inject an RNA and have your cell make the protein for for them. Now that invites a lot more variation, if you will, or variability because not everyone translates RNA at the same speed, we don't know if they're all going to get that when the protein gets made if it's going to fold the same in every person, if it's going to get presented on the cell the same way. So you're several steps downstream in the manufacturing process, that you're sort of outsourcing to the patient and having their cells build these RNAs. Now, I think one key difference here is that when your cells are making these proteins, there's somewhat painting a target on their back for your T cells to come and destroy them. So in the case of a traditional vaccine, you're not decorating your cells with the antigen, to have your immune system attack your own cells, you're just teaching your immune system, how to defend against this antigen, but you're not, you're not really decorating your own cells with it. So I think one of the risks that we're seeing with these mRNA is is that when your cells in your own cells express these foreign proteins, they become targets for destruction. And that seems to be what may be happening. In myocarditis, we have this crossing paper out that shows there's mRNA and Spike protein in heart tissue, and there's a lot of inflammation in that heart tissue. And they can detect it 30 days later. So it may be the immune systems getting turned on against any cell in your body that's expressing these things. And you know that that could be if those are the wrong sets of cells, that can be very damaging. One of the other issues with these is that there's some biodistribution studies that show the SNPs aren't really contained to the arm, they can go all over the body. So you don't really know which which organ you're painting with these things. If you happen to this happens to get into your circulatory system, it could paint the epithelium of your circulatory system and then that epithelium gets destroyed, that could be causing clots. Mark tirado does some interesting work on this as bolus theory, I think has some has some legs to it, that you're stripping the epithelium of your circulatory system. And that's leading to all types of leakage, if you will, that can happen on the blood brain barrier could happen in your, in your aoto. aorta can happen in a lot of places that you don't want it to occur. So I think there's a difference a key differences. The protein is traditionally a proteins injected probably does a better job staying localized, it doesn't express is not made inside your cells, and thus painting targets on your own cells, thus leading to destruction of your own cells. I think those are two very key differences. And the third one is that by asking human cells to make the protein, we're now exposed to all the variability in the human genome, most of the population some people may make those proteins more effectively than others and there may be a much, much bigger, maybe a much wider variety in the expression levels of the actual antigen then just giving someone a really well known concentrated dose of a peptide. So

Nick Jikomes 20:05

So with a traditional vaccine, you're injecting a protein or a set of proteins from a pathogen directly into the body, you know exactly what the dose, how much of each of those proteins is in there. And it's just those literal proteins, you're not, you're not asking your own cells to produce that protein, the proteins are getting into the body directly. And then an immune response comes to that. And that trains our immune system, so that when we encounter the pathogen in the future, if we do, it's pre empting, that immune response, and that's how vaccines work. The mRNA vaccines are saying is we're injecting mRNA, instead of the protein, the mRNA is by design meant to go into our own cells, our own cells machinery, is then producing protein from that, in this case, that would be the SARS cov to spike protein, what you're saying is that now the protein is being made within and as you said, painting our own cells. And one of the other things you said is, there's probably going to be natural variability in how much protein you produce, or which proteins to produce, perhaps you're making a spike protein and some other variants just based on differences in the biology, from person to person in terms of the how quickly the translation is happening, how exactly that protein is folding and that type of thing.

Kevin McKernan 21:26

Yeah, and a few years ago, these were hypothetical concerns, because we're asking the cells to do the manufacturing for us. But the pharmaceutical companies didn't do a very good job proving that the cells made those proteins faithfully, they just showed we have antibody response, which there's we now know there's, there's there is high variability in this due to this base that they put in. So they put in this in one metal suit or urethane so that it would evade the immune system and then last longer. But that came with some compromise, which is that the ribosomes that read RNAs that have that many modifications, get confused, and they sometimes slip and get out of frame. And make, I think, I think the Moroni paper said 8% of the proteins were frame shifted. So we're already taking an 8%, loss and fidelity, making unknown proteins to get the spike manufactured by ourselves, that wouldn't be the case, if you injected a purified protein, you'd be able to make it outside of the body, purify it, quantify it, to put in only what you're looking for. But when you start asking for your cells to perform his manufacturing for you, and you have to, you know, put some camouflage on the RNA to sneak it through the immune system, you're inviting some some fidelity issues with the translation process that has now been exposed through the Morrone paper. And

Nick Jikomes 22:45

so just to tie some of this stuff together for people mRNAs naturally, are, as you said, they're ephemeral molecules, they're not supposed to last very long in the body, they're supposed to be produced for set periods of time, you don't want them sticking around too long. So our bodies have lots of enzymes to quickly break them down. It sounds like what you're saying in terms of the mRNA vaccines that were manufactured for COVID. They use this pseudo Euro Dean in place of the Euro cell, so they use that slightly different version of the EU in the RNA code. And that's because my understanding is if you use the normal you that the mRNA is not going to last really long enough, it's gonna get broken down right away, and you won't actually generate an immune response. So it has to do with increasing the stability of the mRNA. So that actually sticks around a little bit longer. Yeah,

Kevin McKernan 23:29

so there's a whole class of rnases that localize in different cell compartments and tissues, but the one that Carrico was was really concerned about was something known as RNase L. And they demonstrated that RNase l was less active on mRNA that had this modification to it. So they thought this is a great way to get the RNA to last longer, and they were right about that it does last longer. But I think the maybe the concern that the models didn't predict is that clearly in some patients, it's lasting a lot longer than even a forecast, they were suggesting 48 hours, we've now had papers out showing it 28 days in plasma 30 days in the heart five days in breast milk, 10 days and placenta. So they're picking up this mRNA you know, anywhere between five to 30 days later and various tissues that have been surveyed. The spike protein itself is sticking around longer. And there's one paper out showing 187 days where they're picking up spike protein. So I don't know if that's the RNA is still around and we're not detecting it and it's still expressing or if the Swype proteins really hard to degrade. I think there's still a lot of questions to be answered as to what's the mechanism of action of that of that persistence. But it's possible that these LPs are getting to stem cells which are immune immune privilege, so immune system won't attack your stem cells. And if you happen to get an LMP into a stem cell, well then it's you kind of have it camouflage inside your body and it could be expressing spike protein for much longer than

Nick Jikomes 25:02

what is an L L MP?

Kevin McKernan 25:05

Oh sorry, lipid nanoparticles are the, it's kind of the fat bubble they put these RNAs into so they can get into your cells. That means it's protected from a lot of the lot of the nucleases that might degraded outside of the cell. And it kind of Trojan horses its way right into a cell. Okay, so

Nick Jikomes 25:21

a lot of a lot of the design of these vaccines was aimed at making sure that the mRNA actually got into the body and lasted long enough to do what we wanted it to do. Yes,

Kevin McKernan 25:33

yeah, that's, that's key. And I don't know how much attention was put into understanding the clearance of it. You know, how having something lasts for for a long time may not be a desired outcome, you may, most immune responses are finding a small antigen, and preparing the body to amplify its response to that the second time it sees it. So you don't necessarily need a lot of antigen to deliver, to get to build a response, I think they were in such a new space here. Their concern was, let's make sure we at least get a response. So let's make the mRNAs last long, without as much concern over what happens if they last too long. And this persistence, create disease, it maybe wouldn't create disease, if it wasn't expressing a protein as notorious as spike protein, maybe it's maybe the platform is fine if it had some other type of, you know, benign protein in there. But the combination that we that there's a protein that now has a lot of publications on its toxicity, and persistence is one concern. Now there's, there's there's other concerns out there that well, what if you just had naked LLPs? With nothing in them? What damage? Would that do? We don't have an answer to that. It could be that just these NNPS, bombarding the cells with any foreign peptide turns the immune system against them, and you're really just inviting the immune system to erase a certain percentage of the cells. I mean, there's, there's some, you know, numbers on how many LPs are in this, I've seen literature that it's anywhere between like 50 billion to a trillion, I tend to think the 50 billion numbers more accurate based on just surface area volume calculations I've done but that's still, you know, 50 billion, you probably have, you know, 40 trillion cells. So you're talking about one and 1000 cells getting painted for destruction, which wrong cells, that can be a problem.

Nick Jikomes 27:22

So when we talk about the the mRNA, vaccines for COVID, they contain the mRNA, which encodes the spike protein, but it's not the it's not identical to the native mRNA that's in the virus, it's using this modified you in the code. And they're also encapsulated in these lipid nanoparticles. So it's not just like, we took the straight mRNA chunk of mRNA. From the virus that encodes the spike protein, we took that we modified and tweaked it, we wrapped it in these protective lipid nanoparticles. And that's what goes into the body.

Kevin McKernan 27:57

Yeah. And that actually is an important point, because as I'm sure we'll get into, if you start having contaminants that are in these LPs, they're you some of your defense mechanisms to get rid of them can't do their job. So when you wrap this in a fat bubble like that, it protects it from a lot of the nucleases in the blood. That's true for the RNA and any potential contaminating DNA that's in the shots. Typically, if you inject DNA into somebody that has like a 10 minute Half Life in the blood, it's not a big deal. They have a lot of previous vaccines that have had DNA contamination in them when they're injecting those peptides. But that stuff is gets destroyed pretty quickly. The moment you package it into the LNP. You're bypassing that whole defense mechanism when you're delivering that DNA and then RNA straight to a cell. So now it's there's an unknown as to whether you know what the tolerability is how much DNA can we tolerate under those circumstances that hasn't really been addressed by the FDA?

Nick Jikomes 28:54

In basic terms, can you walk us through just the basic process by which Pfizer and moderna manufacture the mRNA vaccines? How do they go from raw materials to the final product? Well, it

Kevin McKernan 29:07

starts with your initial question. So they start with DNA. And they take an RNA polymerase to express RNA off of that DNA. And so they use that DNA almost like a, let's only call it a template, but it's almost like a printing press, you have a system that you can just print RNA off of. Now, there's two different ways you can generate that DNA. And you know, Maderna, from the beginning, had a plasmid that was making your RNA. So trial went on with the plasmid there, they took a different day they took their their their clinical trial reflected their mass production. Pfizer made it made a bit of a switch here, they started not having enough DNA, so they PCR amplified the region of DNA that they wanted the RNA to make out of a plasmid. So usually what a plasmid is is a circular piece of DNA that allows that DNA to replicate in stores very well, because it's circular, you can put them in bacteria and bacteria can harbor these plasmids. And you just grow the bacteria out. And it creates about 100 of these plasmids per cell. And the coli cells double every 30 minutes if you give them the right temperature and nutrients. So it's a great system has been used for ages in the biotech system to replicate DNA inside of another organism. So Pfizer ran their clinical trial amplifying PCR amplifying off that plasmid DNA. And then they made RNA from the PCR product. Now, the reason that's materially different is that when you amplify a plasmid like that, you can then your your amplified material was about a million times higher in concentration than your background, you could put a very, very small amount of plasmid in amplify it and get a million fold amplification, and about 20 cycles, PCR. And that means that your contamination is a million fold diluted. So then you can then take that very clean DNA and make RNA from it. And then when you're done making that RNA, you now have a pot that has DNA, some DNA template and lots of RNA that you just made. And conventionally, they would like to erase that DNA and they use some enzymes like nucleases to get rid of that DNA. And that is something that seems to be failing in their process, there seems to be a failure to get universal DNA since the enzyme they use so to completely eradicate this DNA. So in the process of scaling this up, they they went, they did the trial on this PCR generated material, which is very clean. And then when they had to scale up, they switch the process to process two, which didn't which skip that amplification step and they tried to get the plasmid DNA directly into the into the RNA generation process. And since they skip that step, what that means is the complexity of the background DNA is now the entire plasmid, not just the region, you amplify. So this means another like 4000 bases of DNA come through, that have an antibiotic resistance gene that have an SB 40 promoter that have to they have a variety of other components in the backbone of this plasmid. So there's more background genetic material that comes through when you do this, you skip that PCR step. Now they were supposed to do as a study comparing process one to process two across children 52 people, and they threw the towel in saying it's not gonna matter. It's not big enough of a study to really find anything. And the EMA looks like they let them off the hook on that. So we don't really know if there is a different adverse response rate that would be witnessed and Process Two versus process one. That's been something that Richard Levy And Josh blitzscale have brought up in the BMJ showing that this is, this is unusual in the biotech space when you it was compact, complex biologicals, like this, the actual process is the product, because there are so many different components in living systems like this, that when you're when you're using a coli to amplify your DNA, you can have a host of different contaminants that you can't necessarily measure that can come through the process. So whenever there's a process change, they consider that to be a new product, because you can't fully characterize everything that might be in that background. Now, one, one background in particular, a lot of people highlight is when you're working with plasmid DNA, and you don't amplify it, you have to crack open those E. coli cells and get your DNA out of those cells before you make RNA from it. And that process can be prone to leaving a lot of coli guts in the equation. By guts. Most people are concerned about endotoxin that comes through on the coat of the E. coli cells crack open those cells. And now endotoxin, which is known to be really aggressive immune stimulator comes through with the plasmid DNA. That can be very tricky to measure just nature that compound, but that's something that is if it is there. We don't know how much of it's there, because most of the documents we're finding have the endotoxin levels redacted. But if it's there, that is known to create anaphylaxis, anaphylactic shock. So that is something that could be responsible for some of the acute reactions that people see. I don't think the DNA contamination is creating any of these people fainting or anything that cute, it's something that might be more long term of a concern.

Nick Jikomes 34:20

So so when we think about so someone goes in and get gets their vaccine, the mRNA vaccine, you know, they want to talk about what's in that syringe. So there's the stuff that's supposed to be in there by design. And then there's the potential that other things that we don't want to be in there that aren't supposed to be in there are also in there. Starting with the first group, the stuff that's in there by design, you've got obviously the mRNA, which has this modified you in the code that we discussed. You've got the lipid nanoparticles, the little fat bubbles that are like little Protective Shells around the mRNA give us a sense in a single dose of say the Pfizer vaccine, how many mRNA molecules are in there? How fool is it with these lipid nanoparticles and what else is in there by design?

Kevin McKernan 35:04

So there shouldn't be about 13,000,000,000,013 to 14 trillion mRNAs and Pfizer dos Maderna has got three times that amount, the closer to 42 trillion mRNAs. And those are we estimate are probably in, you know, 40 to 50 billion lipid nanoparticles. So do the math on that it's couple 100,000 of these mRNAs per for LNP. There's some cholesterol and peg and other ingredients that help stabilize these LPs. But I think those are the two key things is that that mRNA is there and the lipid nanoparticles there, what we discovered is that there's also DNA inside those LPs. And that's broken up, it's fragmented, but it's at there's billions of copies of those as well, not trillions, but billions.

Nick Jikomes 35:51

So so you've got billions with a B of lipid nanoparticles in say, a Pfizer vaccine dose, each one of those is going to contain on the order of hundreds of mRNA molecules. So you got billions of little fat bubbles, trillions. It sounds like of mRNA molecules. How did you guys go about looking for DNA contaminants that were in there? What was that process? How did it

Kevin McKernan 36:15

Yeah, so when we were we were actually studying hop latent viral infections in cannabis, this is something that's devastating the cannabis field. And we're doing just boatloads of RNA sequencing of plants that were infected at different points in the infection cycle. And when you do RNA sequencing, as we mentioned before, you should get sequencing that lines up only over the genes. But if you get sequences that aren't in the genes, there's probably something wrong with your RNA sequencing system. And one, one week we came in, and that's what happened, we saw sequencing that was all over the genome, and we're like, okay, something's broken, we're not capturing mRNA, we must be capturing genomic DNA or, or we're somewhere there's a problem, we shouldn't be getting sequencing all over the genome like this. So to solve that problem you typically do is you spike in a known mRNA as a control. If you can't capture that, then you can pinpoint, okay, the magnetic beads that pull down the RNA are broken, or maybe the DNA step is broken. So I needed an mRNA that had that was pharmaceutical grade had a poly a tail and said of ordering one, I was like, Well, I've got one of these on the shelf, someone shipped me, that's a Pfizer vaccine, that should be that should be pharmaceutically pure, let's pop that thing in there. And if that doesn't come through our RNA sequencing pipeline, then we can figure out what's broken about it. It did come through the sequencing process, we did identify, we had a bad DNA base enzyme that wasn't chewing up the background DNA, which is why we're getting sequencing everywhere. But in the process, it also revealed that there was the Pfizer's vaccine plasmid was still in the vials. So we ended up with the

Nick Jikomes 37:48

piece of circular DNA that they use to amplify to get the mRNA pieces

Kevin McKernan 37:54

that was still in there, that was still in there billions of copies of it per vial, or per dose, I should say. So that was a bit shocking, because we were expecting to find any of that we got you know, we got these assemblies back that had spike protein in there. And we're like, well, Spike should be 4200 bases. Why the heck is this thing 7800 bases long, and we threw it into snap gene. And that's when we saw Oh, there's an SP 40 promoter. There's a kanamycin gene that this is the expression vector you

Nick Jikomes 38:19

saw, you saw the you saw the carcass of the DNA plasmid and the things that we know are in it. Yeah,

Kevin McKernan 38:25

there's obviously a blueprint of how to make it basically.

Speaker 1 38:29

Oops, not so how, how many

Nick Jikomes 38:33

of these experiments did you do? How fresh was that vaccine batch that you had is how confident are you basically that? Oh, yeah,

Kevin McKernan 38:39

so that's a good, that's a great point. That's something that people always bring up. So they weren't very fresh, actually, people shipped these to us. And I ignored the request to sequence them for probably six months, chucked them in the freezer and forgot about them. And then when I had an emergency, I was like, Ooh, that that thing will work. And by the time I pulled this stuff out and used it, it was in fact, an expired vial. Now people have since gone back and replicated this with non expired vials. Philip buckholts Did some of this work. David speaker did this work in Canada as well.

Nick Jikomes 39:05

So other people independently made this observation using different fresh batches,

Kevin McKernan 39:09

which has been very helpful because people had good reason to throw tomatoes at us, if you will, when we published this. They were mad that we had an expired vial that we that we sequenced, but you know, these, there's no reason to believe it. And the expiration date would actually destroy the DNA or make more DNA in there.

Nick Jikomes 39:27

Right, right. Yes. 20. You wouldn't expect an old batch to have a DNA contaminant that wasn't in a fresh patch. Right,

Kevin McKernan 39:33

right. Unless someone some Gremlin got on there and put it in there. But the fact that it was Pfizer's expression vector was a pretty good fingerprint that Pfizer put it in there and had their spike sequence in it, and it had what looked like a about expression vector. And the other thing to know is that the expiration dates weren't some hard science, they often would just change them announced that oh, this expired vial can now be used. So expired viruses were injected into people that didn't stop them from using them. In the field, it just was something that was a critique of the, you know, the way we went about sequencing this. And that's just because we didn't, we didn't set out to sequence this as any type of grand experimental plan, it was kind of an accident. And so as we did to try and help there as we built PCR assays to make it really easy for other people to replicate this in other places, and so they wouldn't have to go through this expensive sequencing process, and that, that indeed, helped the replication of the work in other places. So

Nick Jikomes 40:30

the implication here is that in the mRNA, vaccines that were actually used, you had not only the mRNA, and the lipid nanoparticles, all the stuff there by design, but you also had remnants of the DNA from the plasmids used in the manufacturing process of these vaccines. I guess the next question is, how big of a concern is that? Is it? Is it plausible that those are going to cause an issue in a human being? Or are these DNA sequences likely to be pretty inert and not really doing much of concern?

Kevin McKernan 41:05

So I think that's where a lot of the debate lies is what's the clinical implications of this? I don't think anyone's doubting their existence anymore. Now that there's been so much replication. We've had the EMA the FDA, and Health Canada come out and admit that okay, yeah, this, this could be in there. They trust the manufacturer who's measuring this to say it's below some certain limit. I mean, Philip buckholts, brought up a very good point on this, which is your limits were set based on the decay rate of natural DNA being injected in traditional vaccines, this is a different beast, we have them in LMP. So they're not going to decay. And the transfection efficiencies of this DNA is going to be very high. So the prior regulations on this around 10 nanograms of DNA per dose, now those those guidelines have changed 1000 fold over the last couple of decades since the end, after in the Reagan era, they put in the nCVA Act, which is the National Cancer National Vaccine Injury act. And that gave pharmaceutical companies a bit of liability shield on vaccines. So since then, the regulations have moved from 10 pico grams up to 10 nanograms with traditional vaccines that don't have LPs. So we're in a different world now, where we have LPS that are facilitating the CNAs entering so And arguably, that limit should be revisited. There's another thing that I think we've learned in this process, which is that maybe those regulations shouldn't be just about any DNA, what if the DNA is a plasmid that can replicate? You know, now, now, you can slip something in that through that loophole, and get the DNA to make more of itself once it gets in the cell. So if we have the capacity today to sequence every piece of DNA, and it's in there, we didn't have that back in 1984, when they were conceiving of these liability waivers. But today, the cost of sequencing has gone down 100,000 fold in the last decade. So there's no reason why we can't know precisely what type of DNA is in every single contamination event. So a lot of things have changed since those roll those those rules were written, and they probably need some some revision. So alright, let's get to the clinical implications. What could happen this DNA, if it gets in, I'm a little bit less concerned with Maderna is only because the plasmid the nature of the plasmid they have contaminating has, it doesn't have a few of the features that are in Pfizer, and they have they seem to have a better job. They're lower in DNA contamination levels than Pfizer. So if you get if you get through the patent literature, you'll you might understand why that that Maderna is actually has a patent out there, that speaks to the residual DNA risks, and they invented technologies to get rid of it. And it looks as if those technologies work because they have less of it. But inside that patent from Maderna, it'll point out that this DNA is a risk of insertional mutagenesis, which means it can insert into your genome and cause can cause cancer. It's a hypothetical risk if they didn't present data showing it's causing cancer and people that just knowing molecular biology, if you put DNA into a cell, and it can get to the nucleus, it can integrate into your into your, your genome through a process known as either Hamilton non homologous end joining or micro homology mediated and joining us, it sounds

Nick Jikomes 44:13

like anyone engaged in this type of molecular manufacturing process. It's, it's a known thing that you are probably gonna get some amount of DNA contamination in here. It's known to the extent that Maderna actually invented methods to reduce the levels of contamination from that residual DNA. So they had less of it, it appears in the Pfizer vaccine. But the other the other complicating thing here is, you know, even with other injectables that are known or could have DNA contaminants, some of those thresholds you mentioned around what we allow, are, are based on the idea that if it's naked nucleic acid in there, it's going to degrade pretty quickly at some known rate or some rate that we can estimate but because we're using these lipid nanoparticles to shield the nucleic acids with these new vaccines. If you have DNA contaminants in there, they themselves might be protected, protected by the lipid nanoparticles which might, which could hypothetically enable them to stick around long enough to do something where they would simply be degraded if they weren't shielded. Yes,

Kevin McKernan 45:20

yeah, and we've done some work. To move this from hypothesis to a little bit more sound theory. One thing you can do with these vaccines to estimate how much DNA is in the lipid nanoparticles or outside of lipid nanoparticles is you can take them and treat them with the enzymes that Pfizer is using to try and get rid of this stuff known as DNase. One. And it's an enzyme that destroys DNA, if you treat their vaccines with DNA is one you won't see a CT shift and PCR for the vaccine DNA, which tells you that most of this DNA is actually protected from the nucleus, probably inside the inside the LPS. The other thing that we've done very recently with Uli Commonwealth in Germany is she's taken these vaccines and treated ovarian cancer cell lines with them, and then grown them in flasks, and then passage them into several rounds of growth, to show that the DNA persists inside the cells through several passages, so that tells you as well, that the DNA is in the cells. Now, for those not familiar with cell passaging, you treat these cells with the vaccines, a small amount of them a third of a dose, I think was what she used in her case. And then you see those cells into a dish and let them replicate a couple times to go to Confluence, then you rinse all the stuff off the cells to clean off any the residual vaccine, take a portion of those, PCR what's in the supernait and PCR the cells and then put the new cells into another flask, let them grow out again, rinse them off, and then PCR the supernatant in the cells. When we do that we can track how much RNA is outside the cells and how much is inside the cells. And we can see this DNA inside the cells through several passages. That tells us that the LPs are in fact delivering this DNA into the cells going through cancer cell lines. They're not they're not patients, because it's more complicated to this work on patients. So

Nick Jikomes 47:09

yeah, so let's let's really break this down for people. So you've got human cancer cell lines growing in a petri dish, you put the mRNA vaccine, you dose them with mRNA vaccine, you just spray it on to the cells, you let them divide some number of times, and then you're saying you can find the residual DNA contaminant from within the vaccine inside of the cells. Yes. And is that are they inside the cells in a lipid nanoparticle? Are they integrating into the genome inside the cells? That's a good question. I

Kevin McKernan 47:39

don't think our experiments really address that. So we were doing PCR of the supernatant and of the cells, and we could see the PCR signals in the supernatant in the cells in passage one and passage two, and that told us that there's a good portion of this DNA that's actually in the cells. The other thing she did is she stain the cells with immunohistochemistry for spike, to see like, okay, it's in the cell are they expressing, and she and she got the cells to be about 50% SPIKE IHC positive, which is what she's aiming for, it's not to have every cell transfected, but maybe half of them transfected. So those are the those are the two bits of information we have, we then went and did whole genome sequencing on those cells. And that revealed some other interesting information. The whole genome sequencing gave us about 3,000x coverage over the vaccine. So in sequencing coverage is the number of times you sequence the molecules. So we at any given base in the vaccine, we had at least 3000 reads covering the vaccine. And in the actual ovarian cancer genome, which is a much bigger genome, we only have about 3030 fold redundancy and sequencing. So there's about 100 to one ratio of plasmid to to the actual ovarian cancer cell line. That's perhaps not too surprising when you think about how big the human genome is, and how small this plasmid is. And when you deploy this much sequencing, you should expect to see more of the of the actual vaccine there than the human genome, at least from it. From a coverage standpoint, these are only 7000 letters long the human genome is 3 billion bases long. But I think what was most shocking to us is that we could see that there were variants in the vaccine plasmid backbone that didn't exist in the vaccine that we sequence that was outside of the cell. So as a control, we sequenced the vaccine directly. And then we sequenced the cells that were treated with the vaccine. And when you look at the assemblies of the vaccine, in the cells versus outside of the cells, they're different. There's a fair number of variants that are only in the origins of replication in the plasmid that tells us this, those are doing something with that DNA, perhaps replicating it.

Nick Jikomes 49:47

Because the plasmid DNA remnants that were in the vaccine, get into the cells and persist for some number of cell cycle divisions and replications And because this you found variants. So the the sequence inside of the cells sometimes did not match the original sequence that you found in the vaccine itself, that implies that perhaps the DNA, this contaminated DNA is being replicated in the cells. And as a natural consequence of being replicated some number of times there's going to be some amount of mutation.

Kevin McKernan 50:20

Yes, and and all those mutations were concentrated in three regions in the plasmid that are known as origins of replication. There's an f1 origin of replication, there's an SP 40,