The team took white blood cells out of a patient's body, used CRISPR to knock out a gene which suppresses immune response, then injected those same cells back into the patient, hoping that they would attack the cancer without that inhibiting gene.
If this were a live CRISPR treatment in a human, it would probably make more sense to just knock out the activated oncogene(s) in the patient's cancer cells and/or repair the deactivated tumor suppressant gene(s).
I've been wondering about that for the past couple of years, since I heard of CRISPR basically. Who is working on this aspect most seriously right now? Is there any interesting published progress? I understand it will take a while to make real progress, but mostly I'm just curious as an outsider to the industry.
This isn't ready for in vivo use yet due to off-target effects (CAS9 is a very permissive protein that often binds to the wrong part of the DNA).
In the US, Carl June's group at U Penn is working on editing adoptive, engineered CAR T cells for cancer therapy, going into clinical trials. They're probably bummed to have been scooped (first to do an adoptive CRISPR engineered T cell transfer), though their work is certainly more clinically relevant than Lu's.
The problem as I understand it is that right now, CRISPR requires some manual steps in the lab to make it work well. You can design the protein and guide RNA and spacers until you're blue in the face, but if you can't find a specific enough cutting site on the genome, or one that is close enough to your target, or something like that, you'll wind up with a lack of specificity in that cut, which can lead to unwanted mutations. And since the CRISPR system was originally a sort of bacterial immune system, it's really geared towards 'knocking out' specific genes by (I think, but I'm really shaky on this part) introducing a bunch of extra mutations when the cut it makes in the DNA gets repaired. But there are two kinds of repair mechanisms, and apparently there's a way to encourage the more accurate one, although this is really an area I need to read more about.
Anyways, the technique has also been used to introduce entirely new genes, but I think that involves using modified CRISPR proteins called 'nickase's to make single-stranded cuts in the DNA, rather than double-stranded ones. You introduce plasmid DNA with your genes to match up with the cleavage sites, and ideally it gets taken up.
Whatever the approach, you still aren't going to get 100% expression or transfection, and stable transfection either requires invasive techniques like biolistics (shooting DNA through membranes on accelerated nanoparticles) or delivery by viruses, which has a lot of potential, comparatively minimal side effects, and can even be targeted somewhat to certain types of cells. But I think the FDA is nervous about, especially in humans.
Again though, I'm a layman here too, so someone please correct me where I'm wrong :)
I am not a biologist, but nevertheless I found out something interesting today regarding the problem of designing good guide RNA via machine learning.
The work that I learnt about today addresses 2 key questions via machine learning approaches: off-target effects of CRISPR (http://biorxiv.org/content/early/2016/10/05/078253) and optimization of the guide RNA sequence to maximize on-target activity and minimize off target effects (http://www.nature.com/nbt/journal/v34/n2/full/nbt.3437.html).
I learnt of this stuff from one of the authors who works at MSR New England (http://nicolofusi.com/).
Also of possible interest is their project page https://www.microsoft.com/en-us/research/project/crispr/, where they are planning to release a cloud-based service for end-to-end guide design by incorporating the approaches developed in the above work.
You are completely correct, in fact one thing that was notable about it was how much work intensive it is, she was forced to miss lots of family events because she had to go in the lab work on the cells, it requires lots and lots of manual labor.
That said, the stuff IS awesome, after you figure how to get it right, it can get right a lot. (but she did this with animals, where she CAN attempt trial and error until she figures the right reference gene sequence to use to guide the proteins, I dunno how that would work when you are racing against time to save a live patient for example, specially if you want to edit his heart or some other area that is hard to access).
By the way, one of the motivations of MIT new variations of CRISPR (that don't use Cas9) is that Cas9 technique require big needles if you want to work in a living organism, the new ones would allow you to use much smaller needles, that are more practical to stab into a patient... and it is very forward thinking, because people didn't even tried stabbing CRISPR needles into someone yet, it is just that MIT guys realized it would be impractical with Cas9 and decided to invent another technique before actually trying it.
The poison is a bicyclic polypeptide that shuts down ribosomes in your liver. Destroying or removing the poison is as difficult as destroying or removing all the protein. (like dealing with mad cow prions) It's just not going to happen unless you like your mushrooms charred pure black all the way through. Well, that or dusty white ash.
Just half a mushroom will kill an adult human. This isn't something to take chances with.
This study was in vitro (and then in vivo!)
I would agree, though, that most people would have thought "in vivo" from the title alone, since the prospect of and actual practicality of doing it in vivo is what really differentiates CRISPR from the tech that came before it.
We've already seen previous methods of attempting to "knock out" or "Repair" genes with gene therapy; it's an entirely non-trivial problem for a large number of different reasons.
I can't wait to get a rakunk, myself.
Lu’s team then cultured the edited cells, increasing their number, and injected them back into the patient, who has metastatic non-small-cell lung cancer. The hope is that, without PD-1, the edited cells will attack and defeat the cancer."
Or maybe it was this:
>"The researchers removed immune cells from the recipient’s blood and then selectively killed most cells containing a certain sequence using CRISPR–Cas9, which combines a DNA-cutting enzyme with a molecular guide that can be programmed to tell the enzyme precisely where to cut. The targeted gene codes for the protein PD-1, which normally puts the brakes on a cell’s immune response: cancers take advantage of that function to proliferate.
Lu’s team then cultured the surviving cells, increasing their number, and injected them back into the patient, who has metastatic non-small-cell lung cancer. The hope is that, without PD-1, the selected-for cell population will attack and defeat the cancer."
Since there is no paper (only press release) we can't say much more about which explanation is most plausible in this case.
Endpoint Classification: Safety Study
Intervention Model: Parallel Assignment
Masking: Open Label
Primary Purpose: Treatment
Progression free survival - PFS [ Time Frame: From date of randomization until
the date of first documented progression or date of death from any cause, whichever
came first, assessed up to average 10 months ] [ Designated as safety issue: No ]
Overall Survival - OS [ Time Frame: The time from randomization to death from any
cause, assessed up to 2 years ] [ Designated as safety issue: No ]
I suspect the randomization relates to the allocation to the experimental arms as described on the reporting page:
This is a dose-escalation study of ex-vivo knocked-out,
expanded, and selected PD-1 knockout-T cells from
autologous origin. Patients are assigned to 1 of 3
treatment groups to determine the maximal tolerant dose
Look it up, in every paper where they report on that aspect, the vast majority of the cells die. Also, they detect small numbers of mutants in the control (non-"modified") cells.
"Plasmids encoding Cas9 and sgRNA targeting phosphatase and tensin homolog (Pten) (fig. S6A) were delivered into MCF7 cells, followed by culture for 48 hours and puromycin selection. More than 80% of the cells survived the selection process, indicating the high delivery efficiency of our method.
Similar to Pten knockout, more than 80% of 53BP1 knockout cells survived the
You have to look at figure 5B, where the caption reads:
"Cells (5 × 10^4) from (A) were seeded in 60-mm dishes in complete medium and cultured for 7 days. Cells were trypsinized and collected for cell count in a Countess II FL Automated Cell Counter (Life Technologies) daily for 7 days."
So they started with 5 x 10^4 cells but if you look at the first day after being in culture, it is much less than that. I can't tell how low from the chart which shows it as basically indistinguishable from zero. They do not seem to report those values anywhere.
Also, you can look at figure S5 and see that they saw GFP get knocked out in >2% of control cells (Cas9 only). They don't do a similar quantification for the Pten, but if they had, I am pretty sure some low percentage of cells would be shown as already knocked out for Pten.
That should be 1%.
It seems that we already have good drugs which inhibit interaction between PD-1 and PD-L1 is there really enough benefit from this?
The field is extremely promising and it's worth exploring fully, especially considering that even good responders can suffer relapses due to subclonal evolution.
It will be interesting to see if there are differences in side effect profiles in the PD-1 knockout T-cells vs our current biologicals, however.
Checkpoint blockade alone is a great development, but having treated patients with these drugs, I can tell you it is far from a cure for 80% of people.
Also, there is toxicity associated with gene modification. Take a look at the patient deaths with CAR-T therapy.
I always thought the editing was implied even if it wasn't explicitly called out.
Jerome was different in that he was not edited.
Roman road system allowed for flow of goods and people within empire on scale never before seen in acient world, but also for their legions to strike empire's enemies.
I'm talking about germline gene editing, beyond gene therapy.
It will have consequences that span multiple generations, and are self-replicating. It will completely change the fabric of society. We are going to be changing what we are as life forms, without really understanding life in the first place.
That's laughable after this election.
https://en.wikipedia.org/wiki/CRISPR -> https://en.wikipedia.org/wiki/Gattaca
https://en.wikipedia.org/wiki/CRISPR -> https://en.wikipedia.org/wiki/Gremlins_2:_The_New_Batch
I hope we cure a lot of diseases before we get into the bad side of genmods.