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How to Administer RNA – And How to Do It Again

As the world knows, the mRNA vaccines for the coronavirus are proving to be very effective. That’s welcome news for the obvious pandemic reasons, but it’s also welcome validation for a technique that’s been in the works for many years now. I’ll take a moment to re-emphasize how fortunate we are that so much of this groundwork, all these false starts, re-workings, and dead ends had already been worked through to this degree before a worldwide emergency made the mRNA vaccination platform suddenly so desirable. (And I’ll also re-emphasize another issue, that we’re extremely fortunate as well that the current coronavirus has so many similarities to the 2003 SARS one, because the work that went into studying that and possible vaccines against it has been invaluable this time around).

Let’s talk more about one of the tricky aspects of getting mRNA dosing to work, because it has possible implications for where the field goes from here. As many will realize, RNA-based therapeutics have more applications than just making vaccines against infectious diseases (although that’s certainly a good one). But one of the good things about using it as a vaccine technology is that it’s “self-adjuvanted” – recall that adjuvants are substances that sets off a response of the innate immune system at the site of injection, which can potentiate that later adaptive immune response. Protein subunit vaccines (such as the Novavax candidate in the current pandemic) generally have to have an added adjuvant to get up to the needed immunogenicity. These can be as simple as aluminum salts and as complex as extracts from the rare Chilean soapbark tree.

But mRNA vaccines set off an innate immune response of their own (which is a source of the “sore arm” effect that many have noted). I should note here, by the way, that it’s tempting to decide that a strong site-of-injection reaction must lead to a strong adaptive response, but the two actually don’t seem to be related much). Whether your arm gets sore or not, though, you are getting immediate responses to an mRNA vaccine, and some of that is due to the foreign RNA payload, while some of it is also coming from the lipid nanoparticles themselves.

Balancing all this out is tricky, because while you need some of this innate response, it’s going to affect the eventual adaptive response that’s the whole point of administering the vaccine. The types of antibodies that eventually get generated and the T cell responses that are elicited can be rather different depending on how that innate response fits into the overall immune picture. What’s even more enjoyable is that animal models can only tell you so much about this: there are no animals (engineered or otherwise) that recapitulate the human immune response in all its head-banging detail. To be fair, it works both ways: humans aren’t such great models for immune responses in mice, either, although the eventual market for mouse therapeutics is considerably smaller than the human one, as these things go. But what this means is that you have to run the clinical trials in humans to see what happens.

Another quick digression: this is what gets on my nerves when I see people going on about “Ah, we had the vaccines back last March! It just took soooooo long for the big slow drug companies and the big slow FDA to get them rolled out!” Nope. What we had a year ago were vaccine candidates. Nothing is truly real in drug development until it hits human trials, because we get surprised all the time. Now, the success rate for vaccines against infectious disease is one of the best in the whole industry, true. I mean, historically, only two-thirds of them fail, which is indeed a remarkable rate of success compared to most other things we try. But still. You have to go into humans, which takes time and money (and no small amount of each).

Let’s look into some of those mRNA immune responses. The balancing act is because if you set off too strong a response, you could set off a type I interferon pathway because your immune system thinks that it’s under immediate viral attack. There is a long and complex list of “pattern recognition receptors” that are constantly on alert for signs of this, and unfortunately one of those signs is the sudden presence of odd-looking RNA. I won’t get into all the details – there are separate toll-like receptors (TLRs) that pick up on single-stranded RNAs, double-stranded RNAs, RNA species with unusual cap regions, you name it, and all of them have separate downstream signaling networks. One of the consequences is the induction (via those interferon pathways) of an enzyme called ribonuclease L. That one is a real shredding machine, ripping up every RNA molecule it can find. The idea is that it’s only unleashed when there’s a lot of viral RNA starting to pile up in an infected cell, because RNase L spares not and will tear down your own mRNA molecules almost as cheerfully as it destroys viral ones. It’s an extreme measure, and it’s pretty much the last line of defense before a the apoptotic pathways get triggered and the cell falls on its own biochemical sword in full death-before-dishonor mode.

You clearly don’t want to trigger this process if you’re trying to administer an mRNA, because you will have sent most all of your attempted therapy molecules straight into a cellular buzzsaw instead. To pile on, that interferon response also sets off hundreds of genes that (among other things) damp down translation of mRNA into protein and the rate of protein synthesis in general. Which is more of what you don’t want.

That’s why there are so many modifications made to the RNA molecules in any attempted therapy. Bert Hubert takes you through these for the Pfizer/BioNTech sequence here, and it’s a great look at the subject. Modifications get made all up and down the sequence – the cap region, the 5′ untranslated region that comes next, the codons that make up the sequence for the antigen protein itself (the open reading frame) and the nucleosides themselves, the 3′ untranslated region after that, and the “poly-A” tail at that end of the whole thing. This is one of the things I’m talking about when I say I’m glad that we had a chance to explore this stuff over the last ten or twenty years before we needed it for the pandemic: there are a lot of changes that you can imagine making to all these regions, and there’s been a lot of trial-and-error to see which ones are beneficial.

Another thing you have to watch out for is the production of some double-stranded RNA while you’re making your mRNA payload. You will of course have optimized your RNA production platform away from this sort of thing, but the problem is that even very small amounts of dsRNA can set off the toll-like receptor systems and undo everything after your dosing. dsRNA is an unusual chemical species found in very low levels in human cells under normal conditions, and its increased presence is generally interpreted as “funky virus detected”. A further purification step may well be needed, even with an optimized process. Here’s a paper from Moderna talking about just that problem, as well as the use of pseudouridine species to replace native uridine whenever possible. The immune response you get in human patients is very sensitive to both of those factors.

But we haven’t even gotten to the effects of the formulation yet, and here’s where things might get complicated. (I realize that this phrase might cause some people to bury their heads in their hands or go off and clean their basements, but, well, immunology). As is now well-known, the current mRNA vaccines are formulated as lipid nanoparticles, and the lipids themselves include at least one cationic species and at least one that has a polyethylene glycol chain on it. Jonas Neubert lists these for the Pfizer/BioNTech and Moderna vaccines in this great post. Now, there’s a potential problem, in that your adaptive immune system can raise a response to the ingredients of these formulations themselves. That leads to the phenomenon of “accelerated blood clearance” (ABC) after repeated dosing, and this is described in detail in this 2019 Moderna paper, which is open-access.

From that work, it looks like what happens is that many people have antibodies (IgM ones) that already recognize some phosphocholine motifs. That sends some of this antibody-bound form to the spleen, where it’s torn up and the PEG-lipids get presented to B cells. Some of those then get activated to this as an antigen, and that primes the immune system to really rip into another dose with this same formulation (see their Figure 5). Anti-PEG antibodies are indeed a thing (and in fact, there are people who have them even without ever having been exposed to an mRNA formulation).

Here’s yet another one of those balancing acts, though: for a vaccine, some of the adjuvant effect of the mRNA vaccines may well be through the lipids in the formulation. In fact, lipid nanoparticles themselves are being investigated as adjuvants for non-mRNA vaccines. What happens, then, if you’ve been dosed with an LNP-formulated vaccine and then (perhaps a few years later) need to take another LNP-formulated therapy? Does its efficacy decrease because of your prior immune response? On the other side of the question, would an mRNA vaccine whose lipid formulation was somehow completely immunologically silent even work as well? The situation is reminiscent of the potential problems with (for example) adenovirus vaccine vectors. You have populations with pre-existing antibodies to some of those, of course, but after you’ve vaccinated a large population you’ve just created a bunch more of them. There are reports of RNA therapies in animal models that do not elicit immune responses on repeated dosing, but the ABC literature shows that it can be a real problem. And you can also see that people are working on the problem by coming up with formulations featuring “PEG-shedding” to ameliorate the immune response.

I’m not sure yet how this all works out. I’m not sure if anyone is sure. With some of these new therapies and new vehicles, we are leaving immunological footprints, and we’re going to have to keep that in mind. Some of them might end up being like footprints on the beach, and wash away after a while – but others are going to be like the footprints we’ve left on the Moon. . .

42 comments on “How to Administer RNA – And How to Do It Again”

  1. Some idiot says:

    Ok… on the basis of that, is it possible (unlikely or likely) that people who vape could be generating PEG antibodies? If so, that could have consequences…

    1. Anon says:

      You’re thinking propylene glycol, not polyethylene glycol.

      1. Walter Sobchak says:

        What about those of us who use MiraLAX (Polyethylene glycol 3350)?

        1. Glenndc says:

          Shite!!

        2. LeeH says:

          Hopefully you’re drinking it, and not injecting it.

  2. sgcox says:

    “..although the eventual market for mouse therapeutics is considerably smaller than the human one, as these things go.”
    I am not so sure, if you take a longer view according to Douglas Adams.

    1. Roland says:

      In the short term the lab mice need to be allowed to unionise and demand hazard pay, then all that extra disposable income will be available to stimulate the mouse therapeutic economy and create more human jobs. Win-win #musequality

      1. Barry says:

        Oh, in the U.S., not only are rats and mice not have unions. They’re not even “animals”; they’re “laboratory equipment”

        https://www.apa.org/monitor/julaug02/rats

    2. Eugene says:

      How many countless times have a cure for a disease been reported in the press that turns out to work in Mice but not humans.

  3. idiotraptor says:

    Vaping liquids commonly contain ethylene glycol, the monomeric diol. Is PEG present as well?-perhaps. There are many other constituents in vaping liquids that pose an immediate risk of toxicity.

  4. Polynices says:

    I can’t be the only person who reads articles like this and is reminded to again be grateful that we’re able to make any drugs or vaccines work, ever. Based on the ludicrous complexity of all of this I’d quite believe an alternate reality where it’s simply impossible to create vaccines or immunotherapies of any kind.

    1. metaphysician says:

      You remind me of “A Mote In God’s Eye”, where that is essentially the case for the alien Moties. Just with their reproductive system rather than their immune system. Evolved in a way that is really, really direly unpleasant and counterproductive to the happiness of a sentient race with an advanced civilization.

      1. Charles H says:

        Read the sequel, “The Gripping Hand”. It wasn’t that their reproductive system couldn’t be hacked, it was that they didn’t have any researchers, only highly skilled technicians.

        Don’t want to say much more, but the resolution seems more plausible than that such skill in technicians could be genetic.

  5. Doug H MD says:

    DereK; I searched and was unable to find any good discussion of why the mRNA therapy for Crigler-Najjar failed. All that is mentioned is that repeated doses caused serious problems. Is that related to this?

  6. PV=nRT says:

    In 25 years the fountain of youth will turn out to be IM injections of some PEGylated growth factor, and folks who went for the JNJ and Oxford vaccines will be the only viable recipients.

    1. Aleksei Besogonov says:

      It’s possible to remove antibodies to a substance, at least temporarily. It’s not something that is done lightly, but it’s possible.

  7. idiotraptor says:

    This is a correction to my errant chemistry above; I meant to say propylene glycol.

  8. Michael Koehler says:

    My reading of the paper Derek linked to in the statement, “a strong site-of-injection reaction must lead to a strong adaptive response, but the two actually don’t seem to be related much)” is exactly to opposite of what he seems to have gotten from it. In figure 2, the authors show that Cervarix, which uses alum and MPL as adjuvants, gives a much stronger neutralizing Ab response than Gardasil, which uses only alum. Figure 3 then shows that Cervarix produces exaggerated local side effects, while the systemic side effects are similar for the two vaccines. Ergo, the stronger site of injection response might well be linked with a stronger immune response.

  9. Jonathan says:

    Given all this, why aren’t they moving to PEG alternatives? BioNtech has known about this already: https://pubs.acs.org/doi/10.1021/acsanm.0c01834

    1. Stephen says:

      well you then have a whole new vaccine – lets wait another year to test and validate and manufacture. And many alternative stealth materials have also shown to elicit antibody response. To my knowledge polysarcosine has not been tested in humans, and animal trial are a poor predictor for this sort of thing.

  10. DT says:

    Pegylation is utilized to evade immune response for certain therapeutics. Are these different weights or am I missing something?

  11. debinski says:

    Can anyone explain the physiologic mechanism behind the sore arm effect vs systemic side effects and how they are different? For both Pfizer and Moderna’s trial, systemic AEs increased substantially after the 2nd shot as compared with the first (~15% increase for Moderna and 11% for Pfizer). However, injection site reactions didn’t change as much. They were slightly higher after the 2nd Moderna dose (4% increase) and slightly lower after the 2nd Pfizer dose (5% decrease). I also have heard from dozens of people who said they got much worse systemic effects after the second dose of either vaccine. Apparently the cause of local and systemic side effects are different? Is it just a matter of innate vs adaptive immunity (but could adaptive immunity be kicking in quickly enough to cause systemic AEs with 12 hrs of the first dose)?

    1. debinski says:

      And then there is a third category of reaction, the delayed injection site reaction known as “covid arm” What’s going on there?
      https://www.nejm.org/doi/full/10.1056/NEJMc2102131?query=TOC

      1. Marko says:

        Most likely a result of the developing adaptive response, as described in your linked paper:

        “…Our suspicion of delayed-type or T-cell–mediated hypersensitivity was supported by skin-biopsy specimens obtained from a patient with a delayed large local reaction who was not among the 12 patients described here. Those specimens showed superficial perivascular and perifollicular lymphocytic infiltrates with rare eosinophils and scattered mast cells…”

        1. debinski says:

          Yes, at least there is a “suspicion” on that. But weird since apparently this is not reported with other vaccines (?). But what is going on with the immediate injection site reactions vs systemic reactions?

  12. Barry says:

    RNase-L is a last-defense-but-one except in brain neurons, where apoptosis is not an option.

  13. /df says:

    How should we differentiate between a sore arm from the AZ SARS-Cov-2 vaccine (personal experience), from mRNA vaccines, or just from having a needle stuck in your arm?

    1. Marko says:

      You shouldn’t. Plenty of people in the placebo cohorts of vaccine trials report sore arms.

      Anti-vaxxers will try to make a mountain of any such molehill, however. It’s how they stay in business.

  14. sgcox says:

    I think for all practical reasons you can not really discriminate between mRNA, adenovirus or any other type of COVID vaccine. As it has been discussed in this forum many times before, it is an innate immune response to the sudden large load of foreign antigen. I had similar minor reactions to Oxford/AZ, flu and pneumo shots in last few months. All of them are of course totally different types of vaccines.

  15. asd says:

    Maybe it’s just me, but this reads like a vaccine advertorial…

    1. Lappan says:

      Nope, it’s a description of some of the complexities of C-19 vaccine mechanisms, and of vaccines overall and how they interact with the human immune system, written by a practitioner in the overall field. He’s not selling you vaccines, he’s taking it for granted that his audience wants to know more about how they work and what their limitations are.

  16. lizzy says:

    Depoprovera injections for birth control have been used for the last 30 years in millions of women worldwide. Young women get this shot IM into their deltoid or gluteus maximus muscle every three months for years, many for more than 5 years at a stretch. Each depoprovera injection contains 28.9 mg polyethylene glycol 3350, No peg antibody problems have ever been detected to my knowledge.

    So why the worry? Same route, same peg, dozens of dosages in the same individual.
    What don’t I get here?

    1. Marko says:

      You’re never gonna make it in the fear porn business.

    2. Doug H MD says:

      Does depo contain lipid nanoparticles?

  17. lizzy says:

    On adjuvants: Yes Derek, I looked up the Chilean Soapbark Tree. It tickles me….all the things made from inner tree bark or its sap, from Euwell Gibbons, to Pycnogenol, to Taxotere.
    Here’s another interesting type of adjuvant. “Hitches a ride on albumin directly to the draining lymph nodes.” What a concept!
    https://www.medscape.com/viewarticle/946906?src=wnl_edit_tpal&uac=130465ER&impID=3232703&faf=1

  18. J Curwen says:

    You mentioned the dsRNA problem. However, it seems to be a well kept secret that all of these mRNA vaccines can contain up to 300ng DNA/mg RNA as an impurity from the in-vitro-transcription process. I wonder how much of this residual template DNA you can find in the individual batches (after DNAse digestion and purification). To my knowledge no paper exists which describe the genomic integration probability of such a multitude of DNA fragments in a lipid nanoparticle environment. Will there be the same concerns as with DNA vaccines?

    1. Doug H MD says:

      and what concerns would that be?

      1. J Curwen says:

        The activation of oncogenes or the inactivation of tumour suppressor genes via genomic integration. I would guess that the lipid nanoparticles promote such a process. But we will soon see, the field experiment already started.

        1. Doug H MD says:

          do you know of any evidence that such a thing COULD happen let alone has happened?

          1. J Curwen says:

            Well, it resembles a transfection process (or lipo-NP-fection in this case) and therefore is a basic mechanism used routinely in most labs. I dont think that human muscular tissue or blood cells do have an unknown transfection protection. I am further sure that the modRNA producers did not waste time on such a research (they also did not look for effects of the nucleoside analoga on protein homoeostasis after degradation of the modRNA).

  19. SmallMolChemist says:

    Why are the mRNA vaccines swirled but not shaken? Is it to avoid forming bubbles, or does shaking measurably increase liposomes leakage/fusion?

    1. J Curwen says:

      The modRNA is quite fragile, the protocol allows up to 50% fragmented species, you surely dont want to have more than that.

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