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CRISPR eliminates HIV-1 infection in live animals

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Re: CRISPR eliminates HIV-1 infection in live animals

#221

Earlier quoted context omitted.

This is fascinating. How in the world does the body "raise antibodies" that are effective against arbitrary proteins it hasn't seen before? How does this get "remembered" and how does the memory get communicated through the body? If there's an ELI5 (or, ELI-college-101) I'd be interested to read it.

Very over-simplified: You have a random library of many billions of cells each making a single unique antibody that was created via random combinatorial genetic shuffling early on. The ones that accidentally bind to your own natural proteins are filtered out by killing them before they leave the bone marrow, so the circulating cells remaining form a library that could only bind -foreign- proteins. When one of these f…

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Re: CRISPR eliminates HIV-1 infection in live animals

#222
post #124

Earlier quoted context omitted.

I dedicated a significant fraction of my life to the idea that gene therapy would become a viable medical treatment. It seems unlikely to really become a high-impact treatment. Being able to change DNA in cells is one thing; actually being able to show your treatment does what it's supposed to and didn't have negative effects is hard. There are a limited number of diseases where gene therapy should work great, but th…

Would you elaborate on what makes a disease a good fit and these grand-challenge class problems?

A disease is a good fit if you can just inject the therapy into a localized region (say, the eye, or an organ) and the treatment works for a reasonably long period of time (months+). Typically another requirement is that the target is a defective gene where the phenotype can be repaired through addition of a "corrected" form of the gene, without the defective gene needing to be removed. This is the case in X-linked retinitis pigmentosa, where the genetic cause of the disease has been understood for some time, it's relatively simple underlying mechanism (so we think), and you can deliver the medicine to the retina with periodic injections.

Treating a disease where you have to remove an inserted retrovirus from a large number of freely circulating or "hidden" cells (which is the case in HIV) is far more challenging- you need a way to recognize the cells of interest, access all of them, and get 100% transversion. All without causing negative side effects.

Re: CRISPR eliminates HIV-1 infection in live animals

#223
post #75

Earlier quoted context omitted.

My scientist friends tell me China is paying well, making funding available, and has a lower go-to-market burden. Innovation appears to be literally moving to China as talent is being drained away from the US & Europe.

They have a lower go-to-market burden because the regulators don't much mind if you literally fabricate long-term clinical trial results out of whole cloth. The only innovation occurring is in the field of separating fools from their money.

Russia, too. http://www.cardiobrief.org/2017/04/26/serious-questions-rais...

Re: CRISPR eliminates HIV-1 infection in live animals

#225

Earlier quoted context omitted.

>There's a reason some ideas are left unexplored by industry. But your argument essentially boils down to "We haven't yet discovered an effective delivery method, therefore this technique will never work". Isn't that one of the basic problems facing all clinical genetic modification research? Is it unreasonable to assume that this problem could be solved by some future breakthrough, or does it somehow violate the law…

I fully support basic science pursuing crazy ideas. I think this is a very interesting piece of basic science, it's just at an incredibly speculative stage that's unsuitable for clinical investment. Efficient delivery of novel proteins into a cell by genetic methods, nanoparticles, or direct transduction is -the- challenge for a lot of novel ideas. Massive effort is ongoing to find breakthroughs here. The proteins in…

Seems XKCD was on point this week: https://www.xkcd.com/1831/

Re: CRISPR eliminates HIV-1 infection in live animals

#226
post #132

Earlier quoted context omitted.

And yet, if you're not really planning on selling the techniques you find on the open market could you save yourself a lot of time by ignoring the FDA?

You can spend hundreds of millions of dollars before you even get to the point where you can ask the FDA if you can start human trials. Even having spent all that time and money, you still won't have any guarantee that your drug will prove to be safe, effective, or better than any existing drugs.

> You can spend hundreds of millions of dollars before you even get to the point where you can ask the FDA if you can start human trials.

What's a rough breakdown of costs? Salaries certainly don't seem to be the dominant factor. Is it lab equipment & facilities?

Re: CRISPR eliminates HIV-1 infection in live animals

#227

Earlier quoted context omitted.

The expensive part of getting a drug to market isn't proving that it can kill cells in a petri dish. Lots of stuff can do that, bleach, hydrogen peroxide... The expensive part is the clinical trial. You try out the compound in the chemical woodchipper that is the human body, and see what happens. Almost all drugs fail at this point: http://blogs.sciencemag.org/pipeline/archives/2017/01/23/i-d... >The timing of this r…

Huge amounts of the cost of clinical trials could in theory be cut by automating many of the tasks. A lot would be done if all computer systems across hospitals (and lab equipment) could seamlessly talk to each other, medical records were completely standardized and contained all necessary information in machine readable formats etc, not that I see this happening in the near future though.

An aside -- I think machine-readable formats will get less and less relevant. Machines can almost read what humans can read. Just screen-shot it.

Re: CRISPR eliminates HIV-1 infection in live animals

#229
post #57

Earlier quoted context omitted.

Well, as CRISPR was originally found as a kind of immune system, there do exist a number of anti-anti-Cas9 systems against that evolved alongside it. There are a number of small inhibitors of Cas9 [1] (which themselves could be used to tune Cas9 in therapeutics). However up-taking such a defense is admittedly an unlike route for a virus like HIV to take to evolve resistance to a CRISPR-based therapy. More practically…

"More practically, HIV has such a high mutation rate, that it's likely very difficult to target every HIV sequence with a sequence-specific Cas9 therapy. If the Cas9 guide sequence is too generic it'll take out stuff besides HIV (stuff you need). " Unless I'm grossly misunderstanding how CRISPR works, there's no conceptual reason why you couldn't target multiple sequences at the same time , with a cocktail method. Th…

I think ∼3E−5 per base per replication. Might be a more useful number [1].

If you use 10mers, that only gives you 1048576. I'd be almost certain that >90% of those sequences also exist in the human genome. So your target isn't specific enough (take out stuff you need as the parent suggested).

So you need to use a longer sequence, perhaps 25bp. Maybe there's a stable region or set of regions you can target (in which case the high overall mutation rate doesn't matter). Or a cocktail of sequences, specific to the global HIV population (I doubt this, HIV mutates more in a single individual than Flu does in the global population).

But if not, then you first need to figure out what the viral population in this individual looks like. So you sequence a subset of population, and come up with a 25mer or set of 25mers that target this population.

That might be a lot of sequences (significant problem). Which you then need to get synthesized (will take weeks).

Now. It's taken days to run your sequencing experiment, and weeks to get your CRISPR stuff synthesized. In this time the viral population has been generating 10E11 new virions per day. You're population has moved on, and almost certainly contains members which don't have your previous cocktail of 25mers in them and will survive the treatment.

Because HIV mutates so much, there was some interesting work I saw a while back on guiding the evolution of the population. You'd use drugs which don't wipe out the infection, but push the population toward specific genotypes. Specifically those which you have good treatments for, in the hope that you can wipe out most of the population at once.

[1] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3530041/

Re: CRISPR eliminates HIV-1 infection in live animals

#230
post #222

Earlier quoted context omitted.

Would you elaborate on what makes a disease a good fit and these grand-challenge class problems?

A disease is a good fit if you can just inject the therapy into a localized region (say, the eye, or an organ) and the treatment works for a reasonably long period of time (months+). Typically another requirement is that the target is a defective gene where the phenotype can be repaired through addition of a "corrected" form of the gene, without the defective gene needing to be removed. This is the case in X-linked r…

We learned early on that one of the difficulties in eliminating HIV is that it hides in the nervous system and can reemerge at any time. This article show promise in that it can reduce HIV viral load during active shedding. I think it is less likely that it could ever eliminate HIV entirely (i.e. cure)
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