The original gene therapies (early 2000s) were essentially RNA therapies (adenovirus). And their unethical rush and subsequent failures caused a bit of a 'gene therapy winter' [1]. We've since made enormous progress on both the ability to safely deliver genes, but also our ability to generate/design new useful genes.
[1] https://www.labiotech.eu/in-depth/gene-therapy-history/
> In 1972, a paper titled ‘Gene therapy for human genetic disease?’ was published in Science by US scientists Theodore Friedmann and Richard Roblin, who outlined the immense potential of incorporating DNA sequences into patients’ cells for treating people with genetic disorders. However, they urged caution in the development of the technology, pointing out several key bottlenecks in scientific understanding that still needed to be addressed.
1) Develop synthesis technique for large scale high purity mRNA without base errors or truncations.
2) Discover pseudouridine modification to decrease innate immune response.
3) Discover and optimize lipid nanoparticles for encapsulation of mRNA to prevent its degradation.
4) Optimize LNPs and miRNA sites in UTRs for localization to desired cell type and to prevent aggregation in undesired or dangerous cells/organs.
Poof, that's it!
https://en.wikipedia.org/wiki/Polymerase_chain_reaction
the emphasis in the wiki article is on DNA, the same basic principles apply with RNA.
for example you can start with RNA and use reverse trancriptase to produce a DNA sequence, amplify that sequence to a large copy number by repeatedly replicating it then transcribe the DNA to produce large quantities of RNA.
or you can start with the DNA [in large quantities of purity] then create many copies of the RNA by repeatedly transcribing the DNA.
The practice of artificial [in vitro] gene synthesis can create arbitrary sequences for input to the process[es].
https://en.wikipedia.org/wiki/Artificial_gene_synthesis
liposomes are manufactured via biochemical-mechanical process.
This is not my understanding. PCR works to produce DNA because the DNA polymerase enzyme creates DNA copies from DNA templates, and those copies become templates themselves, feeding back into the chain reaction.
I'm not aware of similar enzymes capable of making RNA copies from RNA templates (RNA polymerases use DNA templates AFAIK).
I was imagining the RNA vaccines would have used a fully synthetic oligo production method, to get the extreme purity required. But it might be that some sort of PCR-like amplification process is used. Would love to read more if someone has details.
to wit : https://chempedia.info/info/viral_rna_polymerase/
I was making reference to the process of cyclic amplification, in a manner palatable to non biology literate individuals
The same _basic_ principles
as can be read in the chempedia example the process of RNApol replication of RNA from RNA template is error prone, thus it is superior to create a large copy number of DNA template corresponding to the mRNA desired as DNA amplification is self correcting for the most part.
dependent upon experimental or procedural requirements it may be desirable to produce error prone replication variants of the RNA template, however it is quite possible to go from RNA template to RNA product with RNApol.
due to the fragile nature of RNA there must be some shielding or , a low cycle rate is used in conjunction with immediate harvest and stabilization of the product.
the sars-2 covid19 virus replicates by way of an RNA dependent RNApol [rdRNApol]
Producing RNA from DNA amplicons (PCR products) makes sense, I'm curious if that is the source of material from in the vaccines, or if it's artificially synthesized?
Would that be a trade secret?
when you have determined a sequence of RNA corresponding to a desired protien you may construct a DNA template with modification to suit your purpose.
the DNA polymerase used for DNA PCR is originally from a high tempurature tolerant organism, [thermophillic bcterium] and it is possible via searching high and low to find an organism that bears high temperature tolerant rdRNApol
https://www.sciencedirect.com/topics/biochemistry-genetics-a...
and BTW the original inventor of PCR [kary mullis]
was awake AF on LSD when he conceived of this technology while sitting on a park bench.
https://en.wikipedia.org/wiki/Kary_Mullis
i believe the original PCR was performed MANUALLY! with glassware and perfectionist tedium.
https://en.wikipedia.org/wiki/Kary_Mullis#Use_of_hallucinoge...
you may find this to be interesting:
Read the history section of the rna vaccine wiki: https://en.m.wikipedia.org/wiki/RNA_vaccine
You’re right, we’ve understood that this could be done for a long time. Not quite the 70s, but.. at least the 90s. But believing something is possible and knowing how to do it are different:
1989 - injected rna maybe goes into cells
1990 - proof injected rna creates proteins
1994 - proof injected rna creates immune responses
Then:
“2005 they published a joint paper that solved one of the key technical barriers by using modified nucleosides to get mRNA inside human cells without setting off the body's defense system”
That kicked off a ton of research, but:
“Up until 2020, these mRNA biotech companies had poor results testing mRNA drugs for cardiovascular, metabolic and renal diseases; selected targets for cancer; and rare diseases like Crigler–Najjar syndrome”
But why did they spend from 2005 to 2020 working on mRNA drugs and not vaccines? Capitalism. Vaccines are not generally profitable (take once, you’re done) - so vaccines are not an appealing target for a startup with investors wanting big returns. (Source: https://www.statnews.com/2017/01/10/moderna-trouble-mrna/)
Medical research needs to be driven by what can help people, not by what can make the most money.
If we had a different administration in the USA we may have been able to conquer that quickly with sheer cash and coordination since the knowledge how to build melt blown N95 machines is there. For whatever reason we just didn't.
The immune system is incredibly dangerous to its own host if mishandled. By stimulating response, you are trying to light a cigarette using a white phosphorus flamethrower, so to say.
It took a lot of time to find the optimal way of mRNA delivery that a) really does something but b) does not provoke a massive, counterproductive response. This is a very narrow rocky ledge with precipices on both sides to walk.
Figuring out what sequence of mRNA will be the right one to get a cell to produce the right antibodies for the job, getting that sequence sliced out of the viral RNA, getting that all into a form where it can be absorbed by cells and not just instantly degrade are all nontrivial tasks.
The devil’s in the details: compare to the field of software or cpu engineering... seems straightforward enough to just have more instruction decoders, but due to complexities only ditching x86 has actually made it possible for Apple to do this.
There was quite a lot of development on basic techniques of working with RNA necessary before that could even in isolated circumstances be easier than, or even competitive with, “isolate the antigen, inject it, done”.
> The question is, how did this start just now?
It didn't.
Getting a treatment to market isn't the start of application of a new technique in medicine; its usually something that happens many years, often decades, into work using the technique.
I found this video (from 2013) in the other thread today about the vaccine Moderna Vaccine taking two days to make: https://news.ycombinator.com/item?id=25468959
Reading how Pfizer and Moderna worked on it together, they needed detailed gene sequencing to understand how to design a potential vaccine. Even then they were left with a lot of potential options they still had to whittle down. Finally even with all that work they’re left with a vaccine with complex storage requirements.
So it’s entirely possible that we just didn’t have the surrounding technical ability even if theoretically it was possible. The gene sequencing to sequence it quickly and share that across the entire world, the compute needed to do try different experiments at scale, the manufacturing capabilities, Moderna having invested in the space for the preceding 10 years, existing experience with developing a SARS vaccine, etc.
Moderna's vaccine is a great deal easier to store than the BioNTech/Pfizer vaccine (requires 'normal' freezers for storage, can be at refrigerator temperatures for a longer period).