Reading the methods section, it looks the T-cells infected with HIV-1 that are used for targeted for gene editing came from a cell line and incubated in a cultured medium.
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)?
> For people who are involved in dealing with CRISPR/Cas9, what is the effective rate of gene editing done in vitro vs. in vivo?
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].
I found this + the op's comments fascinating purely because I wouldn't have been able to tell if both were real, or just fake technical talk of the ilk my friends and I sometimes do with meaningless software/hardware jargon as a joke, without Googling.
Well, for the record, I am a programmer working at the Broad, a genomics center that is mired in the current CRISPR patent dispute (although I don't work with CRISPR at all).
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.
>For people who are involved in dealing with CRISPR/Cas9, what is the effective rate of gene editing done in vitro vs. in vivo?
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.
The study used Lentivirus as the delivery mechanism, but ex vivo. I imagine an in vivo study would be the next step, and I'm interested in seeing its outcome.
I was in HIV research for a time. I was unimpressed by this paper's title, introduction, methods, etc, until the part where they discussed correcting infected cells derived from human donors. As a general note, a running joke in the field is that "anyone can cure HIV in vitro", so I practically tuned out the sections where they discussed the work in T-cell cultures.
>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.
Seems like it could be effective to use CRISPR/Cas9 to insert the HIV resistance gene mutation (CCR5-delta32, to replace the normal CCR5 gene) into bone marrow stem cells and then inject those into the person after testing the created cells to be HIV negative (or cutting the HIV out by means in this paper). If HIV negative bone marrow cells available were sufficient to do this it could be a 'cure' for the hematopoietic system. Or use HIV-negative induced pluripotent stem (IPS) cell with hematopoietic potential derived from a tissue not normally infected by HIV.
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!
Some cool stuff combined with very scary stuff, like eugenics and large scale food chain modifications with unpredictable effects (already being planned with mosquitos).
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.
It'd be nice to remember original meanings. The "eu" of eugenics means "good" (and how this good is accomplished "positively" or "negatively" doesn't matter), humans have been practicing it for ages though on a much less sophisticated scale (and not as much on our own selves) than things like CRISPR allows for. The thing we should be worried about is dysgenics, "dys" meaning "bad".
Depends on who you ask, right?
Therein lies the slippery slope, and why the word eugenics is uttered in hushed tones among polite people. That and it is typically attributed as a misnomer - human eugenics is more often just oppressive, authoritarian, xenophobic, evil,madness dressed up in pseudo-scientific rationalizations to treat the persecuted underclasses as cattle.
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?
> 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.
Hmm I'm not sure that's accurate. At least, I've seen some claims of gender being linked to genotype in some capacity.
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.
Sure, there may be genetic links, but sex is 100% genetic. Why bring in gender at all when its hard to apply eugenics to it when the link is so poorly understood?
That was my point, so we're in agreement. (I've edited my post to make it clearer).
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.
Generally, individuals with the XY genotype but the female phenotype have an additional genetic anomaly which renders them androgen insensitive. Therefore, I would say that, in most cases, the sex of an individual is fully, or at least a fully as possible, genetic. It's just not as dependent on the presence of an X or Y chromosome as one would think.
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.
Fair enough, but then it should also be possible to environmentally affect gene expression. I'm clearly nitpicking insofar as sex is overwhelmingly determined by genetics, but I feel it's important to hammer in the point that "everything is nature and everything is nurture".
Medicine is dysgenic - I would be dead a few times over without modern treatments for asthma, which runs in my family. My children now have the opportunity to be born with the same bad genes that otherwise would have been selected out of the gene pool. Cumulatively over time, genetic fitness will decline to the minimum level needed for survivability in the environment. Medicine makes the environment much more survivable. Not to mention the more recent habits of agriculture and enough wide-spread cooperation that our tribes aren't constantly murdering eachother. (arguably)
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.
So a gene goes from a huge handicap to a small handicap. That's not ruining the gene pool. Medicine doesn't even remove the selection pressure, let alone apply pressure in the wrong direction.
And you're probably wrong about saying you'd be dead; why did your ancestors survive it?
I wouldn't say that it has an acute immediate negative effect the gene pool by any measure, but it does certainly have an effect on the slow stochastic process shaping the gene pool. It would make intuitive sense that preventable, fecundity decreasing maladies will increase in frequency over time over many generations. Maybe that's not so much a pejorative effect as a beneficial adaptation to a new, more forgiving environment, if you look at it from the right perspective. Still I think that most people would agree that robust health in a low-tech environment is a nice trait to have in a population. Still, we're arguably pretty irreversibly reliant on fire and cooking.
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.
There's never been a breakthrough like this -- what you edit dominantly gets transferred to offspring. Previous gene therapies didn't necessarily get propagated, which means any mistakes are permanent.... the potential for disaster is huge.
Until we completely understand the risks, perhaps we should make a rule not to breed after genetic manipulation. If you want kids and gene therapy, kids first.
Apparently this doesn't only diminish the infection, but can supposedly confer resistance?
"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 confused me as well. Based on my skimming, the paper doesn't speculate on why this appears to be true, but it's very interesting that the researchers observed this phenomenon at all.
HIV is a virus whose genes are rapidly mutating, even in a single individual (due partially to its genome being based on RNA, not DNA). Due to random chance, it's highly likely that an individual will already have a resistant strain of HIV in their body before they underwent this treatment, so after the treatment, only that strain would be left.
It has been common experience for me to check my internet news feed and see "the cure" for HIV/AIDS/Cancer/Diabetes fairly regularly.
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?
Most HIV treatments center around suppressing the virus. For example, many antivirals used in treatment interfere with viral reproduction. These people are never cured, because part of what HIV does is it inserts itself into the DNA of the infected individual's cells.
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.
if we could create a reasonable number of healthy (uninfected) t-cells, would an individual be left infectious but otherwise asymptomatic (because of the retention of a working immune system)?
This would only make sense if you could make the uninfected t-cells totally resistant to infection. What this paper suggests is that even this would be unnecessary. The combination of antiretrovirals and CRISPR therapy would prevent replication and excise the embedded viral sources. With enough of this combined therapy, eradication may be obtainable, resulting in a properly cured patient.
Figure 1D is said to show "Detection of Cas9 protein by Western blot analysis", so what does "bp" stand for in that figure (above the ladder)? Usually it stands for base pair but that doesn't make sense when looking at protein.
Base pairs. I don't know the exact math and haven't ever done Westerns, but there's almost certainly a way of converting protein size (typically expressed in kDa) to base pairs, since theoretically a large enough quantity of bp would weigh as much as a protein of a given size.
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.
Come on. Having actually done Westerns, there is absolutely zero chance that the "bp"=base pair in that figure is on purpose. Western blot is a protein blot and they are always measured by their molecular weight (kDa). Base pairs would make no sense here. The numbers in the image are obviously kilo daltons.
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.
That's an obvious error. The scale in the figure is kilo-daltons (for proteins). Bp scale is used in figure 1C (RNA). Perhaps they copy pasted that image when fabric... I mean polishing the images.
So it cuts the genomes out, does it keep the cells alive while doing so? I heard that CRISPR/Cas9 doesn't rejoin the dna it cuts. I'm new to this and would love to know from someone more knowledgeable about this.
Cells are always poised to repair DNA breaks. For example: over your lifetime, your cells will have repaired thousands of DNA breaks due to gamma radiation. The breaks due to CRISPR/Cas9 are similar to the breaks caused by gamma radiation and likely to be repaired very rapidly. If the DNA break is not resolved, there are other biochemical pathways to lead to apoptosis (programmed cell suicide).
"...potentially serve as a novel and effective platform toward curing AIDS."
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.
No, that's standard boilerplate these days in papers. You have to "sell" the science a bit to reviewers and connect your work to the broader picture. Those in the field usually ignore statements like this.
When I was a research assistant in college I was encouraged for one of my presentations to emphasize safe hydrogen storage as a vehicle fuel source, when really the research was all about how minuscule amounts of hydrogen change the electrical/optical properties of thin metallic films (and desorption was on the order of days). Selling it as a building block for safer hydrogen storage was necessary to get people engaged.
This paper is in Scientific Reports, which is published by Nature but is not regarded as a top-tier journal. It has an impact factor of 5.5 vs. 41 of Nature.
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.
If you're going to deliver enough lentivirus into an HIV patient to edit all the HIV out of their T-cells, you're going to provoke a massive immune response. This is not the first time people have demonstrated the ability to excise HIV from the genome - I recall a cool system that involved evolving a novel endonuclease that recognized the HIV LTRs. Aha, here: http://www.ncbi.nlm.nih.gov/pubmed/17600219
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.
> If you're going to deliver enough lentivirus into an HIV patient to edit all the HIV out of their T-cells, you're going to provoke a massive immune response
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.
>If you're going to deliver enough lentivirus into an HIV patient to edit all the HIV out of their T-cells, you're going to provoke a massive immune response.
Not if the HIV infection has progressed to AIDS. Even with a regular HIV infection the immune response would be diminished.
And so for that matter are the hep C treatments. They're that rare thing: an effective cure for a serious, chronic disease that was previously only treatable with long term medication with many nasty side effects.
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)?