Elimination of HIV-1 Genomes by CRISPR/Cas9 Gene Editing
nature.com
nature.com
For people who are involved in dealing with CRISPR/Cas9, what is the effective rate of gene editing done in vitro vs. in vivo?
Would the molecular machinery of CRISPR/Cas9 encounter any natural defense mechanism of the human immune system when attempting to mutate target sites on T-cells in say, lymph nodes of adult humans (contrasting to changing DNA in the germ-lines of bacteria or even human embryo's)?
Difficult question. CRISPR is ironclad in vitro, but in vivo it's not well understood. For reference, other methods of gene transfer/editing are almost exclusively for in vitro use, with the exception of lentiviral vector-based transduction.
>Would the molecular machinery of CRISPR/Cas9 encounter any natural defense mechanism of the human immune system when attempting to mutate target sites on T-cells in say, lymph nodes of adult humans (contrasting to changing DNA in the germ-lines of bacteria or even human embryo's)?
Yes. The cell membrane, the nuclear envelope within the cell, and the chromatin/chromosomal structure of the genetic material within the nuclear envelope are huge barriers that can't be skipped across easily when in vivo because many of the convenient work-arounds aren't suitable for in vivo use. Bacteria are always much easier.
It will depend in part on the tissue of interest and the delivery vector. In the mouse liver, the Broad group under Zhang has gotten efficiencies as high as 40% using Staphylococcal Cas9 [1].
1 = http://www.nature.com.sci-hub.io/nature/journal/v520/n7546/f...
The answerer I believe is a MD/PhD at Mass General Hospital and before that a research scientist at the Broad working on genomics (and so is the author of the cited paper on mouse liver, small world here on HN). So I can be full of BS, but I don't think the answerer is.
Tbh, I intentionally added a few more jargons more than necessary (I could have just asked "Can gene editing be effective in live humans T-cells vs. cells on a petri dish?") to field my question; but I intentionally did so to filter out the pop-sci, speculative futurists to get answers from people in life sciences research.
>Lentivirus (LV) mediated Cas9/gRNA delivery suppresses HIV-1 infection in human T-cells.
That should be the actual headline of this paper. Yes, it's a big deal that researchers were able to use a lentiviral vector (HIV without the disease causing bits) to genetically alter infected CD4 T-cells. This is a shot at a cure.
Simple infection of a person with the CRISPR-fortified lentiviral vector is a new therapy idea which has the potential to trump the current antiretroviral therapies because it can reduce or remove the HIV "viral reservoir"-- the places in the body where HIV can hang out and survive even when being suppressed by antiretroviral therapy. I can imagine a therapy in which the CRISPR-vector is injected into patient lymph nodes and other similar sites with the intent of curing HIV for good. Won't be cheap for another decade, though.
Such cells in principle would have a survival advantage over native cells and would effectively perform a bone marrow transplant, maybe helped along by some medicinal guidance. Pies in the sky, but using HIV-negative cells may avoid the problem of removing divergent sequences of HIV in different people and ineffective gDNAs.
I've been avidly following the CRISPR/Cas9 developments over the past five years or so, and the possibilities seem astonishing. I can't wait to see where this all leads!
Well, in this case CRISPR wouldn't make much sense for negative eugenics—i.e. trimming the gene pool—vs positive eugenics—purposely introducing or manipulating existing genes.
While the latter is still scary, it's not exactly close to the mass sterilization people think of.
http://www.nature.com/nbt/journal/v34/n1/full/nbt.3439.html
https://medium.com/mit-media-lab/kevin-esvelt-joins-the-medi...
Would my skin colour and gender fall under that definition?
More to the point, talking about sex and gender in the same breath is quite often absurd. Gender theory is a sociological theory, not a scientific one, and while sociology is a useful tool for certain problems, it's hardly reproducible, or falsifiable.
Edit: on second thought, we're not so much in agreement as nothing is fully genetic or environmental. There are, for instance, cases of XY females. I'd agree if you said the genetic link was orders of magnitude stronger between genes and sex, but the "100%" perpetuates a common misunderstanding of genetics.
I suppose you could induce something like that by treating an embryo with finasteride or another SARM but there's a reason such drugs are in pregnancy category X. They tend to produce wider ranging and more severe defects than just changing the sex of the embryo. Hormones are complicated.
The real question is whether or not we, as a species, can be trusted with the unprecedented power and responsibility of direct control over our own evolution. Modern medicine is fundamentally dysgenic. My line alone has tendencies towards bipolar disorder, poor vision, asthma, and crippling congenital foot defects. I was fortunate to have escaped most of them. However, the mere fact that I was born carrying these traits can be credited to the fact that my grandparents were treated for them (club feet in the case of my grandmother) and survived to pass them on. Great for the individual, but in the cold calculus of survival, it can only be said that the more serious genetic maladies that we can correct for with medicine and surgery, the more they will build up in the gene pool.
The promise of these new gene modification techniques is that we can put the hands on the wheel of our own genetic destiny rather than leaving it to the Ouija-board control of whatever god one would choose to cede them to. One might well expect this thought to make a great many people very uncomfortable. This is entirely justified given the human track record.
However, another ethical standpoint to take might be this: If I am told that my child, in utero, has a 100% chance of being born with cystic fibrosis, but that a reasonably priced (hey, it could happen) treatment could apply a patch to that nasty little mutation, would that somehow be less ethically sound than abortion, abstaining from childbearing, or allowing the child to come to term to a life of certain, now trivially preventable, suffering?
Contrarily, if the burden depressive moodiness is 'cured' from our genetic makeup, what cost do we pay in losing the creative pearls built up around the pain of the melancholic soul? A world without Poe, van Gogh, or even Morrissey?
Tough questions.
No. Check back in 50-100 years.
The interesting case, is that our long-term survival is now more a question of information, culture, and knowledge than it is of our biological capacities. Our most interesting evolution is happening in the rapid and ephemeral'software' of shared knowledge and technology, and not the fleshy hardware ruled by genes.
And you're probably wrong about saying you'd be dead; why did your ancestors survive it?
Cave dwelling fish don't lose their eyes because of any immediate selection counter pressure, but from the slow reversion to the mean from no pressure either way. Or maybe the small pressure of nutrients spent on maintaining a useless organ.
Perhaps my example of my own case was misguided - I was turning blue and rushed to the emergency room as an infant, though. It was a single case provided to demonstrate a point where no single case matters that much.
I hope I'm wrong, but how can anyone see that working out?
"When evaluating a therapeutic strategy based on CRISPR/Cas9, it is critical to understand that not only will HIV-1 be eliminated from latently infected cells, but the majority of uninfected cells will become resistant to HIV infection. Thus, there is a high likelihood that rebounding viral infections will be contained by the resistant cells."
This article does not appear to claim to be "a cure", but does seem promising. Can anybody comment on the significance of this and why the layman should care about it?
The technique proposed in this article would be revolutionary because it suggests that we may eventually be able to use CRISPR to selectively cut that HIV DNA out of infected cells, leading to a permanent cure, if 100% eradication of HIV DNA can be achieved. Unfortunately, doing it in a petri dish is much different than doing it in vivo, particularly with 100% eradication.
The trick here is to understand that Western blots use gel electrophoresis to detect protein size, meaning that heavier proteins will not traverse as far as lighter proteins.
How do we know the numbers are kilo daltons? Well, they say they're using β-tubulin as a loading control. β-tubulin's molecular weight is about 50kDa and that's what you can see in the image.
Also, look how the headers are not aligned properly in the image. As if the image is a draft.
Researchers are usually quite conservative and the media are usually the ones to sensationalize titles. Given the above quote, this is likely a major step forward.
Sure my work could be extended in that direction, but I had done zero work to pursue it.
But I would say that "selling the science" occurs more often for high-profile journals because the authors need to convince the reviewers that the paper has a significant impact. Papers in more field-specific journals tend to write conservatively.
This is 2007 - doing this again with CRISPR is, frankly, just using a flashy new toolkit. The fundamental problems with gene therapy remain and are, as usual, the major hurdle to any of this shit being effective. It's easy to edit genomes in a dish; doing it in the human body is orders of magnitude harder.
From a layman's point of view: isn't that exactly what HIV does in the first place, only faster? As I understand it, on initial infection there can be a major immune response, causing skin rashes or worse.
I'm actually really interested in that kind of work.
"The most recent published estimate of lifetime HIV treatment costs was $367,134 (in 2009 dollars; $379,668 in 2010 dollars)."
http://www.cdc.gov/hiv/programresources/guidance/costeffecti...