Additional little bit that reminded me of that cell-cell fusion trait... another paper described the SARS-CoV-2 autopsy results, and included this:

Multinucleated syncytial cells with atypical enlarged pneumocytes showed viral cytopathic-like changes, without obvious intranuclear or intracytoplasmic viral inclusions.

Translation: The paper-thin, high-surface-area (for gas exchange) cells wrapping your lung balloons (the pneumocytes in your alveoli) fuse together with each other into an ineffectual, clumpy mess with a way lower surface-area-to-volume ratio. These are fragile cells to begin with; they don't even replicate themselves (other cells have to replace them when they break). They don't seem to be producing virus themselves, but they do seem to be getting badly screwed up by things the virus is doing.

Protein origin shouldn't matter, but mRNAs are not yet proteins. So for mRNA vaccines, they still need a lipid coating for delivery that evades the immune system but will still fuse with cell membranes.

(Normal cell-to-bubble-to-cell delivery involves mostly protein-based tagging and anchoring, and viruses often imitate parts of this process (this is much of what coronavirus spike-proteins are doing, for instance). But if you are using variants of tags that appear unfamiliar to the immune system, you can easily get an immune reaction against them. I'm not precisely sure how these companies have solved this problem, whether it's by using protein-tagging some entirely-lipid-based solution, or what.)

They might not need to be targeting particular cell-types very heavily, it's true. But they still need to be targeting for delivery to cells-in-general.

How can this possibly happen non-pathologically in humans? What really confuses me is... getting DNA outside the nucleus in the first place as a non-freak occurrence requires at least one of:

• Generation of loose DNA partial-transcripts while trying not to generate extra confusion during cell replication
  • If it's handled as ssDNA, that actually would leave it in a form pretty distinct from the usual dsDNA storage. And even as dsDNA, there are epigentic tags you could use to see that it's handled correctly.
  • This does have the benefit of being an "out" channel from the nucleus, not a security-vulnerability "in" channel.
  • I think I've reasoned myself to thinking this is the most likely explanation? I'll have to do more research to confirm/negate this guess.
• DNA cut-outs that permanently leave the nucleus, never to return (resulting in its absence in the next replication cycle)
  • You could come up with a clever working version made from frequently-duplicated genes with extra copies (ex: transposons), but... I've never heard of this. And it's a little evolutionarily-fragile.
  • This does have the benefit of being an "out" channel from the nucleus, not a security-vulnerability "in" channel.
• Reverse-Transcriptase (the most insidious of all viral proteins, that thing that crafts DNA from RNA)
  • Humans do have a special RT for extending telomeres, but it's rarely expressed, and if a random cell is expressing it that's a cancer waiting to happen.
  • There are retrotransposons that copy-paste themselves around the genome. But like... that's basically a nasty virus that got lazy and whose deck is short a few cards.
• Cytoplasmic replication
  • I am under the impression that this is not happening

In eukaryotes, I get the impression that DNA is usually not getting replicated out there in the cytoplasm, at least? DNA viruses usually have to do at least one of two things:

• Get themselves into the nucleus somehow (via small size and/or transport proteins)
• Carry their own replication proteins around with them in the virion, to produce those initial RNA transcripts that produce enough replication proteins for them to get by.

If the treatment involves entering the nucleus of fully-intact undamaged cells, or replicating itself (so really, either of these methods), the alarm bells in my head would be blaring.

But if it's just circles of extracellular DNA... I'm now kind of conflicted and confused? How virus-like do you have to be to make that a viable thing to do?

Some other lingering points of confusion/research:

Plants use ambient restriction-enzymes to make being a random cytoplasmic DNA a hazardous game (on the assumption that ambient cytoplasmic DNA is usually viruses, and the non-viruses will have co-evolved with the particular restriction enzymes to make this system work). I don't actually know that animals do, though. And you could always species-tailor it...

I also have no idea how efficient or inefficient extracellular transcriptase would be for producing mRNA transcripts. If it's inefficient, you might have to use a pretty strong primer, and I find myself a tiny bit concerned about that in a long-lived human therapeutic.

In our previous related thread of related conversation, you mentioned:

Inovio Pharm that also develops a COVID-19 vaccine. It's technology is based on delievering DNA based.

I noticed that I feel more worried about DNA-based vaccines than mRNA based vaccines. I probably should try to articulate some of why.

After doing some preliminary skimming and examining my pre-existing knowledge around this... I'm now kind of conflicted and confused?

(Exploring this turned into a giant sprawl. It would be multiple research projects for me to fully dissolve my confusion here. I'll leave the majority of my thoughts as a comment on this, but it may be so biology-dense that it mostly serves for my own reference.)

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mRNA practically degrades itself. If an mRNA enters the cytoplasm, it might get diced by DICER but it otherwise is probably only good for a limited number of protein-producing ribosome-reads before it gets degraded or digested into unreadability.

DNA is a more robust molecule, befitting the archive-storage of the cell. In eukaryotes, almost all of it is permanently locked up in the nucleus, both for access and regulation reasons and as a protection against UV and other mutagens. DNA outside of the nucleus in a eukaryotic cell usually only comes up only in the context of viruses, cancer, GMOs, or ongoing problems or oddities with that cell.

(But apparently bits of it exist naturally, but have probably been understudied? That's something that I didn't fully realize until today. Pathology and pathogen-imitating GMOs are literally the only context where I have ever heard this come up before.)

mRNA production has fewer dependencies than protein production. To me, it feels reasonably intuitive that this might be a bit faster to assemble, especially at scale and in the face of QC. However, I have a lot of uncertainty around this.

About this next thing, I am more sure. What is more challenging about mRNA, and a good part of why it hadn't been a major method before, is delivery* into cells, and maybe targeting that delivery (if needed). If they've got a great lipid coating already configured for this, at that point it's easier to treat this like a modifiable platform and not just a single product. And that could help turnaround time a lot.

Being able to treat this as more of a modifiable-platform than a tailored product is where I suspect the big gains of this method lie.

*Delivery method being a major challenge is generally true of all large-molecule drugs and therapeutics (ex: protein-based), and probably even some of the small-molecule ones. Although I've seen more and more biotech companies specializing into this kind of thing, so humanity is probably getting better at it.

There's something almost comical or ironic about immune response probably foiling most attempts to develop something whose eventual goal is to trigger an immune response, but later. But that's probably a good part of the story of why this was so hard.

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P.S. I was about to recommend you make a high-level post about this. I'm glad to see that you already did! I'm probably going to continue a bit of that conversation here, as this seems like a better place for it.

Ah. Then that is an error on my part because I had no prior knowledge on this topic, and assumed that rebreather oxygen masks were the default form of oxygen masks.

Thanks for the correction!

I've tried to update the relevant bullet-points towards what you described.

Some notes/highlights (but it's not that long and you should read the whole thing)*:

• Generally thought to be more helpful if you can get both oxygen and pressure (pressure helps keep alveoli open)
• At least several oxygen supplementation methods will aerosolize the virus and increase contagiousness nearby, and are not allowed in a healthcare setting as a result. Perhaps particularly methods involving pressure.
  • This is not, or is less of a problem, for low-pressure oxygen (ex: (non-rebreather) oxygen masks). These oxygen masks are probably what is meant by standard oxygen therapy.
  • This is a problem for CPAP machines
  • (Deleted a somewhat-incorrect/confused bullet-point)
  • (Personal thought: Is this part of why hospital outbreaks were so nasty with SARS-1?)

suppose you’re a person with respiratory failure, but you have an oxygen mask, CPAP, BIPAP, or high-flow nasal cannula at home. 40% of the time or so, your noninvasive home equipment is going to fail and you’ll need to be intubated. Usually this happens quite urgently; if you need to take an ambulance to get to the hospital it might be too late.

• Invasive intubation tends to be be urgent, and is a very seek-a-medical-professional do-not-do-this-at-home procedure
• From ~300 pneumonia cases: From the set [oxygen mask (rebreather), high-flow nasal cannula, non-invasive ventillator], high-flow nasal cannulas have the best outcomes associated with them (much better 90-day mortality rates). But for all of them, 30-40% ended up needing invasive intubation within 4 days.
• Given the frequency with which people go on to need invasive intubation, hospitals are probably better than at-home with NIV unless you really can't access the hospital (ex: overun). The rates for needing intubation are still high (~30%-40%).
  • But see also: NIV seems better than standard oxygen therapy (oxygen mask), in the follow-up post

Many of these [open-source ventillators] are automated bag masks, or CPAPs, which aren’t recommended for use in hospitals because they spread COVID-19; and they won’t work on the more severe cases of respiratory distress anyway. Only two projects that I found, the Pandemic Ventilator and the Flometrics project, are explicitly trying to match the specifications of the type of mechanical ventilators found in ICUs.

The follow-up post on non-invasive ventillation (NIV)

• Lot more data surveyed, but across several ventillators and conditions (ex: COPD (definitely helps), pulmonary edema (somewhat less consistently)).
• NIV does seem to improve outcomes by quite a lot, including in pneumonia.
  • Strong results on intubation there (60% changed to 15-20%), but only 136 total pneumonia samples across 2 studies. And one of them noted no difference in mortality.
• NIV makes a pretty big difference on the overall intubation rate, changing 30% to 15%

This means that, in a world where ICUs are overrun, there’s a good chance that a home non-invasive ventilation device could save lives for people with COVID-19.

* Also, I'm not likely to sync this as it updates, I may be out-of-date, errors in bullet-points are mine, etc. etc.

Whoah, lipid-coated mRNA vaccines, not as an intermediate step but as the actual delivery method? That's actually new to me! Sounds like it's mRNAs coding for some subset of the viral proteins, which probably get assembled into proteins in your cells and then get used as something for antibodies to respond against. mRNAs should then just degrade themselves with time.

I have no idea what the most efficient method for producing those is; I am very used to vaccines being protein-based. This probably is in the realm where it's simple enough that modifying PCR-protocols to produce RNA instead might actually work reasonably well, although RNA is generally more fragile and error-prone and that could be a problem.

You'd be using nucleotides, not amino acids, but mRNA from DNA is a short-enough assembly line that you might not need cells to do it.

(Protein production has a lot of dependencies. mRNA transcription should basically just require your DNA of interest, nucleotides (x3), and a transcriptase protein. Maybe add a transcription factor or two.)

HeLa definitely is a human cell line (although that was for Ebola, they may end up using a different cell line). That's good, that probably scales up easily.

...I'm confused about what method you're even trying to gesture at.

They're viruses*, they need a full set of environmentally-provided cell machinery to replicate or produce proteins: ribosomes, transcription machinery (ex: t-RNAs), ATP, the works. They need cells, so you'd need need at least a cell culture. All of biology has heavily optimized protein assembly lines, you're not going to beat it acellularly.

The cells near the outside of an egg are probably used because they're an elegant and self-contained little solution to sterilization (against everything but your virus) and the quality-control problems you'd have to contend with otherwise. It's not really about the protein content, mostly.

(Cell culture is probably more expensive than eggs because 1) bioreactors are kinda expensive, 2) bioreactors are a bit of a pain to maintain, and sterilization is hard, two problems that using an egg pretty neatly solves, and 3) which cell culture will work best is surprisingly hard to predict, you basically have to test it experimentally.)

* Well, technically it's weakened viruses, or single-gene plasmids, or something similar. The need for cells still holds either way.