CRISPR: Genome Editing and Deadly Diseases with Matthew Porteus

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[APPLAUSE] >> Sarah, thank you for the introduction. So just a couple of things. While CRISPR is allowing us to manipulate genes with precision, we're not quite in-patients yet. So what I'm gonna talk to you today about is our progress of bringing this technology as a new therapeutic to patients. And yes, my dad was a professor here, and it was at the old business school which I think is right over there. But he is happily enjoying retired life. I was told by the organizers that that's the social media link if you guys wanna go on. And so, the title of my talk is CRISPR, Genome Editing and Deadly Diseases. And I've kinda co-opted a term here called precision medicine, that you hear widely. Because what I think I'll try to describe to you today is for many diseases we know the exact cause, and yet we don't have precision medicine that is based on that exact cause. What I'm gonna show you I hope, is that with this new tool called CRISPR-Cas9, we're able to change DNA sequences that we know cause disease. And one day we hope what we can do is use that technology to change mutations or change DNA variants that cause disease in patient's own stem cells, give them back to the patient and cure them of the disease. So we're in Silicon Valley, and so I'm gonna start with a quote and end with a quote. So the starting quote is from somebody named K.R. Sridhar. I actually have no idea who he is, but I like the quote. And it says that, supposedly on Wikipedia he's some sort of famous entrepreneur, is a belief that finding innovative ways to make the world better is important. A mind in search of better ideas, even if they sound radical, is more likely to stumble across one. And I think that sort of mantra is not only something that drives Silicon Valley, but certainly drives research in my lab. So I actually, as you heard, I grew up in the area, went away and came back as an associate professor over at the School of Medicine. And after I got here on the MDPHD program, has a seminar series called Medical Mysteries and they said Matt why don't give a talk on sickle cell disease. And I thought [SOUND] and I'll tell you a lot more about sickle cell disease. And it was a little confusing to me because we know a lot about sickle cell disease. It's not a medical mystery. So what I came up with is a title 102 years later: How come we don't have a cure for sickle cell disease yet? And so, why 102? So I gave this talk in 2012. So I would actually have to title it 106 years ago: How come we don't have a cure? And the reason I used 102 years is that it was in 1910 that Dr. James Hera looked at the blood smear of a dental student of his, or a dental student at University of Chicago, named Walter Noel, unfortunately there's no picture of him, and he saw this abnormal blood smear under the microscope. Normally our red blood cells look like this one here. Actually, not even like this one. More like these up here. Round, they have what we call a bioconcave shape and what he was seeing was these abnormally shaped cells and so he wrote a paper and this what he wrote. So there's many sort unique things about this paper. One, is that that's a single author paper. In science nowadays, it's almost impossible to publish a paper in which there's only a single author. Many of my papers have 10, 15, 20 authors on it. And the other thing is this, the language of the times. So he describes, this case is reported because of the unusual blood findings, no duplicate of which I have ever described. Whether the blood picture represents a merely freakish, poikilocytosis, which just means abnormally shaped cells, or dependent on some peculiar physical or chemical condition of the blood, or is characteristic of some particular disease, I cannot present answer. I can tell you if I tried to write that in the second sentence of my paper saying, I don't know what's going on here but there's something weird and I think you guys should all know about it, it wouldn't get past anybody. But he got away with it. And then, of course, the fact that in the history he had to describe the patient as an intelligent negro of 20. Also, I think, points to our times have fortunately changed. Now, of course, this disease, sickle cell disease, was long known before 1910 in Africa. And it goes by many different names, none of which I can say. I mean, I just can't say them. I could say them. But they have this feature in which they have repeating high pitched vowel sounds. And what these words translate as is "beaten up", "body biting", "body chewing". So they're describing a disease that is characterized by frequently painful crisis. Where these people get beaten up. And so, nothing much happened for 39 years. And then, in 1949, Linus Pauling, only person to win two Nobel prizes, took blood from a patient with sickle cell disease and somebody who has sickle cell trait, I'll describe to you what that is, and somebody who doesn't have sickle cell disease. And ran it on a gel and separated the proteins in the red blood cells, the hemoglobins in the red blood cells. And what he found is that the normal hemoglobin in the red blood cell ran with this characteristic shape, and the sickle cell hemoglobin ran at a slightly different rate. And you could see that a 50-50 mixture of A and B, you could see the separation. And so, he deduced from this small shift in migration on a gel, that this molecule had two to four net positive charges than this molecule. It turns out he was right. There's two more positive charges that's it. And so, this has been called the first molecular disease. Eight years later, Vernon Ingram who was then in Cambridge, England who is now a professor of at MIT, actually took the hemoglobin molecule from sickle cell patients and non sickle cell patients and separated the amino acids on a gel. And was able to deduce that the single change was a glutamic acid that was converted to a valine. So on one protein, one amino acid change, turns out the hemoglobin molecule has two copies of this protein, and so that's what gives the two charge change. And [COUGH] then just a short time later, Makio Murayama showed that what happens with sickle hemoglobin under certain conditions the molecule polymerases. So normally hemoglobin is, can be tightly packed into red blood cells. It's normally a crystal in concentration but when sickle hemoglobin looses it's oxygen, and I'll show you schematic of this, it creates a hydrophobic surface, a surface on the surface of the protein. That allows it to polymerize with itself. And it creates these stiff polymers. And it's those stiff polymers that turn the squishy red blood cell into that stiff sickle red blood cell that gives the disease its name. And finally, in 1977, after the discovery of recombinant DNA down here at Stanford, and we won't mention the contributions of Berkeley, >> [LAUGH] >> The actual sequence change that caused this glutamic acid to valine was identified. And it's a single nucleotide change, an adenine to a thymidine. And as I mentioned, let me just skip forward one slide, so what happens then is we have this single A to T change that creates the sickle gene, that leads to an RNA sequence that differs by one nucleotide. But that one nucleotide changes the code from a glutamic acid to a valine. And this is a hydrophobic residue, and that allows the plumerzation, which converts this soft red blood cell into this sickle red blood cell. Let's see if I, we don't need to go through that. So why does this cause a disease? Well, it causes a disease through this process of vaso or veno occlusion or vaso-occlusive disease. And what happens is, these stiff sickle red blood cells get trapped in the small blood vessels of our body. They're not able to transverse through these small red blood vessels. There's a characteristic that, actually, the lining of the blood vessels is actually inflamed because of the damage that sickling induces. And so, you create these blockages for blood getting to the distil tissues. And so, the most common manifestation of oxygen not getting to the distil tissues is acute pain in the bones. And I think we, as doctors, are only starting to understand how frequent these bony crises are. And there have been recent studies that have shown that sickle cell patients are taking pain medicines two or three times a week for these bony crises. Sometimes they become so severe that they need to come into the hospital and get our strongest pain medicines. And they sit in the hospital for two to three weeks at a time. And this was a picture that was drawn by a teenage girl, as she's trying to depict the pain she was in. And [COUGH] I first got a sense of how much pain this was, when I was listening to somebody from the Sickle Cell Disease Association, talk about these painful crises. And he had the disease himself. And he said, it's like having your hand slammed in a car door. Except, that instead of the pain lasting for a few seconds, no, it lasts for weeks. And so, yeah, I think, that gives you a sense of what these people are going through. But the problem isn't just that it causes recurrent bony painful crises. The problem is that it causes the inability of oxygen to get to all the tissues. So we don't get enough oxygen to our kidneys and it causes kidney damage. We don't get enough oxygen to the lungs and it causes lung damage. And importantly, you don't get enough oxygen to the brain. And you end up with silent strokes and a decrease in IQ. So not only is there significant morbidity through life, this then leads to early mortality. And 30 years ago, well, actually 20 years ago. But a study that was started 30 years ago, Laura Platt, a pediatrician in Boston. Showed that on average, people with sickle cell disease, both men and women, were only living until their mid 40s. Now, with contemporary care, now, people with sickle cell disease are living to adulthood just like everybody else. So the expectation was, is that if we got people to adulthood better, maybe we would extend their lifespan. But it turns out that, that has not happened. And this recent study that came out of USC has shown that the average life span for people with sickle cell disease, still remains in the mid 40s. I'm not gonna tell you whether that means I should be alive or not. So what that means though, is so that here is the sequence of the protein, in the most common variant of beta globin. And you have no sickle cell disease, and you have one copy of A, and one copy of A. You'll have two copies of S, so this is a recessive disease. You get the sickle cell disease. And if you have one copy of A, and one copy of S, you have no sickle cell disease, in what we call sickle cell trait. And so this is all genetics. But one of the interesting things about this disease, is that change. Is change from the E to the V has occurred four separate times during human evolution. So it occurred by looking at the variance around the change and genome. We can deduce that this change occur four separate times. And has persisted through human evolution. Once here, in what we call the Senegal variant, the Benin variant, the CAR variant, and the Asian variant. So this is interesting, we have a mutation that causes people to die early. That has occurred several different times and persisted in the human population. And the reason for that is it turns out that patients who have sickle cell trait. So one S and one A, have a higher resistance to malaria. And that was first hypothesized when people looked at where people with sickle cell lived. Which is here in Africa and India and parts of Saudi Arabia. And you overlap that with where malaria is. And you can see there's a striking overlap. Now, there are parts of the world with malaria that don't have sickle cell anemia. Turns out, they have other mutations that confer protection against malaria. And [COUGH] so, that was suggestive and the proof has come in many different ways. But the first sort of proof that sickle cell trait is protective, was shown by Tony Allison. And what he showed is, when he looked at survival. Is that patients with SA had a higher survival than patients with AS in a setting. I mean, sorry, this was this person's data, whereas patients with SS had the lowest survival. So this minor survival advantage by being AS sickle cell trait over AA, is why this allele has persisted. Because malaria puts such pressure on the human population. So now, we can go back and say, aha, AA doesn't give you sickle cell disease. But you have a higher risk of severe malaria. AS does not give you sickle cell disease and you have a lower risk of severe malaria. Unfortunately, if you have sickle cell disease and you get malaria, you die like crazy. Now, one other thing that I wanna point out, is those survival curves are based on sickle cell patients in the US. In Africa, right now, where obviously, medical therapy is not as strong. The average life expectancy is on the order of five to seven years. So these patients are dying young and yet, that allele still stays in the population because of this protection against malaria. So what we have now, is a 6 billion base pair DNA sequence that codes for how our cells function. And there is a single typographical error that is causing the disease. The idea is, can we fix that error? Of course, these diseases, sickle cell disease is not the only disease like this. And there's probably close to 10,000 such diseases. And they span all aspects of medicine, from cystic fibrosis, to hemophilia, to bubble boy disease. To different diseases of the heart, different diseases of the skin, the muscles, and the brain. Okay, so the idea is if we can cure one, maybe we would have a mechanism to cure them all. [COUGH] So the disease lies out here in the red blood cell. The problem is, that the red blood cell only lives for 100 days. So if we could fix the red blood cell, that would only give benefit to the patient for about 100 days. And we can fix red blood cells by giving blood transfusions. And so, some of the ways we treat sickle cell disease patients who are having sever manifestation. We give them repeated blood transfusions and replace their red blood cells with red blood cells without sickle. But as I've said, that's only a temporarily solution. So if we really wanna fix this disease for the long term. What we have to do is go back and fix the hematopoietic stem cell. The stem cell that gives rise to all the different types of blood in the body, and this cell lasts a lifetime. And there's a picture, electromicrograph of this round bowling ball that sits in the bone marrow. And the progeny of this cell makes red blood cells, platelets, your immune system and different aspects of your immune system. So if we could give fixed stem cells, we could cure this disease. So one way of getting fixed stem cells is taking the bone marrow. The stem cells from someone who doesn't have the disease. And transplanting them into somebody who does have the disease. And so, the way this is done is we take the patient into hospital. We give them high doses of chemotherapy to get rid of all their own stem cells in their bone marrow. And then, we harvest the blood stem cells from the donor. And we infuse those stem cells through an IV. Those stem cells float through the blood. Find their homes in the bone, their niches and then are able to reconstitute the blood system. And this was first done to cure sickle cell disease in 1984. And actually, what was interesting is that this was a little girl who had leukemia and needed a bone marrow transplant to cure her leukemia. And so she got a transplant from her brother who had sickle cell trait. And what you can see is this the mixture between A and S in the donor was about 60% A and 40% S. And then about seven months after the transplant, the patient actually developed the same hematologic parameters, and was cured of her disease. And so after this, this approach has been broadly utilized. And it turns out that if you have a good donor. By a good donor I mean, a sibling who is an immune match to you, but doesn't have the disease, the cure rate is now around 95% with a bone marrow transplant. And after the bone marrow transplant these patients are cured of the disease. Now unfortunately, this is, most patients only about 10 to 15% of patients actually have one of these donors. And so really we need to find now something better for all the other patients. And so the idea is that instead of using corrected cells from somebody else, is can we take the patients own stem cells and correct them? And to be very simplistic about this, would be the idea of taking a 57 Chevy that had two broken headlights. And somehow being able to fix one of the headlights because we know that would be sufficient to cure the disease. And we call the fixing of headlight, we can fix these genes by a process called homologous recombination. And the idea of using homologous recombination was first described about 30 years ago. Now, unfortunately, while he, Oliver Smithies showed that you could use homologous recombination to fix the mutation. He can only achieve it in about one in a million cells, which was not gonna be sufficient. And so that brings us to genome editing. How do we increase this frequency of homologous recombination? How do we increase this frequency of correcting mutations to a high enough level that we can use it? And the way we do that is through the following process. Is we design a specific protein, that is designed to bind the DNA at a very one site in the genome. And then attached to it is an enzymatic activity, a nuclease that will create a double-stranded break at that site. And if there's a break created at that site, the cell says I have a broken piece of DNA, I need to fix that DNA. One of the ways that can fix the DNA is by just stitching the two ends back together. And we use that sometimes and through that a process of breaking the DNA and stitching it back together over and over again, you can end up with mutations at the side of the break. And so, this is a good way of activating genetic element. So it's not something that we'd wanna do for sickle cell disease but something that's useful as an experimental tool. But the other way cells repair a double-stranded break is by using this Homologous Recombination process. So if we give an extra piece of DNA that shares homology or shares identity to the where the break is but we've introduced small changes in the DNA. The cell will use this piece of DNA through a copy and paste mechanism, and fix the break and introduce this new sequence. And so sort of going back to the car analogy, what we're doing is we're taking a sledgehammer. And we're busting up the broken headlight. And that's inducing the cell, then, to fix the headlight so at least one headlight is fixed. What's interesting about this way of breaking the DNA and then that allows us to rearrange the DNA is actually nature does that itself. And there's multiple different processes in which nature wants to rearrange the DNA in the cell in different ways to create different activities. And nature does this by creating a protein that makes a double-stranded break. And then either repairing it by that non-homologous stitching mechanism or by homologous recombination. And one of the things that we can use is study these to try to better understand how to increase the frequency of the process when we wanna use it. I'm gonna go back just a couple slides. So there's lots of different ways of engineering proteins that can make a double-stranded break. But what has turned out to transform the field is this new class of proteins called the CRISPR/Cas9 protein. And by the way, my lab has worked on all of, well we haven't worked on those but we've worked on all of these. And these we now use exclusively to CRISPR/Cas9 platform. Now where did this platform come from? It's really a fascinating story. So it turns out that we know we have an immune system. So a system that allows us to prevent being infected by bacteria and viruses and other things that wanna invade us. Well, it turns out that bacteria have to do with the same sort of invasive particles. They're viruses for bacteria or DNA for bacteria. And for the viruses for bacteria, we call them phage. And it turns out that when a phage infects a bacteria, the bacteria have this CRISPR/Cas9 system that will take the incoming DNA, chop it into little bits, integrate it into an array. And then express little fragments of this array as an RNA molecule that complexes with Cas9, a protein. And so that then next time the phage comes in that guide RNA molecule complexed to Cas9 will find that piece of DNA, will find the phage DNA and chop it into little bits. And so that's a really interesting discovery. But what has sort of transformed my research is that you can adopt that system to work mammalian cells. So now, what we do is we synthesize a piece of RNA that we've engineered to recognize a specific DNA site in the genome. So we're gonna engineer what we call a guide RNA. That is going to bind to the beta-globin gene, the gene that's involved in sickle cell disease. We're gonna take that guide RNA and complex it to the Cas9 protein. And it's this Cas9 protein that then makes the break in the DNA. And amazingly, this simple bacterial system of Cas9 protein with a guide RNA works in a mammalian cell. It can find that target site and it can break that target site incredibly efficiently. So now we have some tools. So some of, now we need to understand how to use those tools. A couple things that we've learned is that, in order to get high frequencies of editing in a cell, you need to get a lot of nuclease in the cell. So if you only give a little bit of nuclease to the cell, in the green bar here, you only get a little bit of editing. And if you give a lot of nuclease to the cell, you can get a lot of editing. So when we find that we're not getting a lot of editing in cells, what we have to focus on is how do we get more nucleus into the cell to create that break and to create the edits? If we're creating the break, then how can we get the break to get repaired by correcting the break rather than repairing the break by enjoining? And the simplest way we found to bias the way the break gets repaired is by delivering lots of that donor DNA. So if we swamp the cell with donor DNA, the cell for reasons we don't fully understand, will more often use that donor DNA to fix the break than just putting it back together. And if we measure the ratio of how many times it fixes it using our donor DNA versus just breaks using the other pathway. When we give lots of donor DNA we can find that we get over one of one HR of M per every NHEJ event. And this worked great in cell lines. So cancer cells lines are easy to work with but they of course are not what we wanna modify in patients. And so when we took the CRISPR system and we moved it into the cells we wanted to modify, we found that it didn't work. And what we thought is that we weren't getting this crisper system expressed at high enough levels in the hematopoietic stem cells, in those stem cells, to get the activity we wanted. So we began a collaboration with Agilent Technologies, a biotech company right down the road, and they have the ability to synthesize this RNA molecule in a test tube and make large quantities of it. And so we made some different flavors of this large RNA molecule. And we put modifications on the end to protect that RNA molecule from being degraded. And we made different sorts of modifications where there was just one modification, or two or three, with the idea of creating more and more resistance so that the molecule, once we introduced it into the cell, would not be chewed up. And we said, okay, will this finally allow us to modify the stem cells? And it did. And so, at two different genes, that gene involved with sickle cell disease and the gene involved with bubble boy disease, what we found is, is that if we delivered the Cas 9 as a MRNA, and we delivered the guide RNA as the stabilized forms of that small RNA. So, Cas 9 MRNA will get translated into protein, the protein will then complex with the guide RNA, and what we find is that we're able to get insertions and deletions at our gene. So our first step, we can make breaks at our target gene. So then the next question is, is how do we get our donor DNA into the cell? It turns out that if we take naked DNA and put it into our cells, those cells see that naked DNA as an invading pathogen and they'll actually react to it and secrete all the things that make us feel sick as stink when we get the flu. So they make tons of Interferon and they make themselves sick because they don't want the invading pathogen to take over the cell. So when we introduced DNA into these cells, we were making them sick, and that wasn't working. So what we had to do was figure out how to get this donor DNA into our stem cells without making them sick. And the way we've done that is we now packaged our donor DNA into a virus, a recombinant adeno associated virus. This is what my colleague, Mark at Standard calls nature's nano particle, cuz it is evolved as a way of delivering DNA into the nucleus of cells without activating that interferon response. So what we do is we take millions of these cells, we put them on a little. We mix it with our Cas9-guide RNA complex, and we zap that mixture, we create holes in the cell's membrane. This will float into the cell and get to the nucleus. And then we pull it out the cuvette and we add our virus, and then the virus delivers the donor DNA. And so, the first thing we did is just to see how well this would work, is we made a virus in which we were gonna insert what we call a cassette that would turn the cell green. So we were gonna insert a promoter and the GFP gene so it'd integrate into the beta globin gene. And if it integrated into the beta globin gene, it would turn our not-green cell into a green cell, which we can measure by microscopy or flow cytometry, or other techniques. And what we found is, is that using this strategy, we could get 30% of our stem cells to turn green. So now we have an ability of making DNA changes at the betaglobin gene. Now, of course, turning stem cells green is cool, but it's not actually gonna cure sickle cell disease. So how are we gonna actually cure sickle cell disease? Well, first we have to show that this same process of turning cells green in wild type stem cells will work in stem cells that come from a sickle cell patient. It could be that the sickle cell patient's stem cells are different and everything we worked out in normal stem cells won't apply. But fortunately, we found that that was not true. And what we found is, is that using that same system I just described to you, using sickle cell, what we call CD34 stem cells, we get 40% of the cells to turn green. So not even 30%, but 40%. Now, again, that's not gonna do, I mean, again, we can make green cats and green animals, but what we really wanna do is correct that sickle cell mutation. And so now, instead of making a donor DNA that made cells green, is now, we had a donor DNA that only had a few DNA changes, the most important of which is that was gonna change that T back to an A. So I'm gonna just switch that one nucleotide back. Now, for technical reasons we introduce these other changes, and they allowed us to monitor it, and they allowed us to increase the efficiency of the process. And then we took sickle cell stem cells and we introduced our nuclease, and we introduced our donor DNA. And then we measured what happened to all the beta globin genes? 10% of the beta globin genes were unchanged, they didn't do anything to it to them, okay? 40% of the beta globin genes, the nuclease came, it cut the gene, and the gene mutated. So we broke the gene even more. But 50% of the beta globin genes were fixed. So we've now turned 100% of the beta globin genes into 50% fixed, 40% broken even more, and 10% unchanged. And then, what if we took this population of cells and analyzed individual cells and said what happened to the two genes in each cell? We got this distribution. We got a distribution in which about 2% of the cells had neither gene change. But importantly is we go about 50% of the cells in which at least on of the sickle genes was converted back to the hemoglobin A gene. It was fixed. Okay, now sometimes both of them were fixed. Sometimes it was A over S. So we created that sickle trait, and sometimes it was A over the further broken. And by the way, when we break it further, that creates a disease called beta thalassemia, but we know that A over beta thalassemia is good. So we know that 50% of the stem cells have at least one gene corrected. And when we turn those stem cells into red blood cells, what we find is that 84% of the hemoglobin coming out of those stem cells making the hemoglobin A, and we're only getting 16% hemoglobin S. Whereas in the unmodified sickle stem cells, 100% of the hemoglobin is S. And the important thing to know is that when we keep the level of Hemoglobin S below 30%, we're here at 16%, we keep it below 30%, patients are cured of their disease. So we've hit a number that we think would cure patients of the disease. This was all done in a test tube, so a really important aspect of all this is did we create stem cells that can actually make blood if you put it back into an animal? So we can't put it back into patients yet, we're still in the laboratory. So what we did is we take the human stem cells and we put them into a mouse that has been engineered to accept human stem cells, all right? The mouse immune system has been ablated, and it allows our human cells to graft, and we can see if our human stem cells behave like human blood stem cells. And what we find is, is this that they do. So our input correction frequency is about 44%. And when we look at three mice that had gotten these cells, on average, the correction frequency was 40%. So, now we have an ability to correct the mutation of stem cells, we can show those stem cells make blood, and we can show those stem cells act like stem cells when we put them into an animal model. So that puts us in the position to actually think about taking this process that we're doing at about a million cells per time and actually scaling it up Into a patient. And so we can define sort of some criteria. So we will harvest the stem cells from a patient, and we're not gonna do babies first. The FDA, the regulatory agencies, will say they're too special. You're gonna have to do adults. But babies are cuter. So we'll harvest the stem cells when we know we have to harvest a lot of them on the order. We're gonna have to, for an adult, we're probably gonna have to harvest on the order of five hundred million stem cells. And then we're gonna have to bring them into a specialized facility that is designed to allow you to manufacture cells. And we know we're gonna have to make casein protein and the guide RNA and the virus and we'll have to have a delivery device that all have been qualified as appropriate ways to modify a cell that you would give back to a patient. And then we would analyze this modified cell population and make sure that we achieve the right correction frequency. Make sure that we didn't do anything bad to the cells like create a translocation or something that might harm the patient. And then we transplant these cells back into the patient after giving the patient chemotherapy to make room for our corrected cells. So now instead of using corrected cells from someone else, we would use the patient's own corrected cells. And so that really puts us right here at what we call The Valley of Death. Is how do we go from here, and that is what my lab looks like. It's not my lab, but I can tell you that's what it looks like, a bloody mess. But it means stuff is going on. And how do we bring across so we can get these cells to a patient? And we're doing something at Stanford that has not been done at Stanford, which is let's try to do it all at Stanford. So normally what we would do is what I would do is I'd say we've proved that we can cure, we can do this in a model. I will then license it to a company, or I'll start my own company. And we'll do it through a company, and then I give up everything. And instead what we're gonna try to do, or we're doing, is creating a new infrastructure here at Stanford called the Center for Definitive and Curative Medicine. And the idea is to create an infrastructure that will allow people like myself and others to bring their discoveries and not have to license them out to a company. But bring these discoveries and develop them and bring them to the patients right at Stanford, so in a wholly internal system. And so the idea is that we have discovery research and then there's pre-clinical development and clinical development. And we wanna be able to do everything up to these first phase one to the first in human clinical trials. And if those work then idea is to say, okay, big pharmaceutical company, we've shown that it works. Now you figure out how to bring it to the hundreds of thousands of patients. Sorry, I didn't tell you this. So there's about 100,000 patients in the US with sickle cell disease. There's about 100,000 patients in Western Europe with sickle cell disease, but there are tens of millions of patients in places like Ghana, Nigeria, India, and so on and so forth. [COUGH] So we need to have that facility to manufacture those cells, and Stanford has built a facility in the last year to do that. Let's see, California Avenue is that direction. It was an old research building, and they remodeled i. And it's now got special air filters, and there's ISO 9 and ISO 8, which just means that there's only one dust particle per ten to the ninth particles of air. It's run by David DiGiusto. And so now it gives us the infrastructure to actually manufacture these cells. And so what we're hoping over the next year and a half is to take that process that we developed in the lab and move it into this facility. Such that we can create a large number of cells that have been done in what we call GMP grade fashion, good manufacturing practice fashion, so we can give them to patients in the new children's hospital that is being built as well. So to end I just wanna end on a few slides discussing the ethics of genome editing because it's been in a lot in the press. So to me, one of the big ethics is it's all well and. So by the way, this is a picture of Packard Children's Hospital, which is where I work when I'm not in the lab or here talking to you. And I think that we will be able to do this very sophisticated manufacturing process. By the way, the GNP facility costs $10 million. Actually it cost $9,999,999 because if it went over $10,000,000 it was gonna need the trustees approval, and no one wanted to go through that process. And so they kept the budget $1 under 10 million. So you guys all know how Stanford works. But it's really, I showed you that the patients, most of the patients with sickle cell disease are here and here and here. And places in the world where they're not gonna build a $10 million facility. So how are we gonna take a complex process done in some place like Palo Alto and Stanford, California, and bring it to this patients. And of course I have ideas, but I don't know the complete solution. And so whenever I talked to undergraduates and high school students and medical students, that's your job. Figure it out. I think we can do it, but it's gonna be hard. Now the second thing about genome editing that I want to bring up is it's been incredibly powerful. The CRISPR system has been incredibly powerful at creating genetically modified organisms. So we can take yeast or cells in a tissue culture dish or worms or flies or mice and rats and pigs and dogs and monkeys, non human primates. And we can use the CRISPR system to make mutations in their DNA that mimic human disease. And so don't have much problem creating mutations in these things or these things. But I think as you go up the mammalian landscape, and perhaps I'm primate centric here. I think we all have to ask ourselves is that while a disease like Huntington's disease is devastating in humans and we need to find a cure for it, is it ethically right to create a monkey with Huntington's disease to allow us to find cures for human disease? And I'll leave it at that for you guys to come up with your own answers. Finally, the other thing about genome editing is because we have found that there is an efficiency to be able to create genetically modified organisms, it raises the question of how far should we go with this technology? So I just talked to you about using somatic cell editing to cure disease. And so we would take a patient's own somatic cells out, we'd modify them, give them back to cure a disease. Those somatic cells won't get passed on to future generations, and they will cure disease. So I put that in green. I don't think there's much ethical problems with that. Where people are starting to get into really have discussions about this is what about using somatic cell editing for enhancement. So let's say, we've identified a gene, let's say erythropoietin. We know erythropoietin. If you have more erythropoietin, you make more red blood cells. You make more red blood cells, your endurance will be better, and you have a better chance of winning a gold medal, or winning the Tour de France, or any other things that require endurance. And so if we get really good at this, what do you think about engineering the EPO gene, or the growth hormone gene, to allow people to be Enhanced? And as you can see, I sort of think that's maybe not so good. But other people say, why not? Let's make the human race better. We're not perfect people. Let's figure out how to make our physiology better. And then there's this big divide right here. So this is still somatic cells so it wouldn't get passed on to future generations. What about editing the zygote? Or the stem cell that gives rise to sperm and egg? And so therefore, the change would be passed along to future generations. Should we use that to cure disease? So for example there are diseases that are so disseminated in the body it is unlikely that we'd be able to use somatic cell editing. To get at all those tissues and cure the disease. And perhaps the only way to cure that disease is by doing editing much earlier, that would result in passing on of that change into future generations. Now, one would argue, we don't need to do that, there's a process called pre-implantation genetic diagnosis where you create a bunch of embryos. And you screen for the ones that don't have the disease, and plant those that don't, and that people have done that. It's not paid for by insurance in the US, so I'm talking to patients with Huntington's disease, which is an autosomal dominant neurologic disease. They said they have a 50% chance of passing on their disease to their children, and they don't have the resources or the funding to pay for PGD out of their own pocket, and so they just take a chance. And you can imagine the angst that puts people through. And then finally, we get into this lower quadrant here about we do heritable editing for enhancement to create a super human race. And I put that in bright red. But there are other people out there who said, no, we should actually do that, we should make the human race better. So anyway, I wanna thank you for your attention and the opportunity to discuss some of our work. I get to come up here and talk in front of you all. But to have a great group of people in the lab who'll actually do the work, great collaborators both within my clinical division at our new GMP facility, collaborators around the country. We can't do this without money and multiple different funding sources, including the federal government, the California Institute for General Medicine, and some great philanthropy that has supported the lab. We all know that Stanford is a great place. Silicon Valley is a great place to work as is Stanford as is Packard Children's Hospital. Here's the Medical School. Here's the low-key building for stem cell research where my lab is. Here's some of the people, a part of the team. I told you I'd end with a quote, so this is the quote I wanna end with. So this is a picture taken. That's my dad, that's me, my brother, and my sister, when my dad was on sabbatical in Australia. It turns out my brother and his family were here yesterday but didn't have this picture to recreate it. So anyway, but I like this picture because it highlights a quote from Kurt Vonnegut that says, I want to stay as close to the edge as I can without going over. Out on the edge, you can see all kinds of things you can't see from the center. And I think that's what drives us to think about trying to cure diseases in ways that have never been cured. And with that, I'd be happy to take any questions upon us. Thank you very much. >> [APPLAUSE] >> So there's microphones up here for those of you who have loud voices. >> When I first read about the science news a while back, they were talking about the possibility of taking the mosquito that carries Malaria and modifying it so that all of its prodigy would also have that thing to not carry Malaria. >> Yeah. >> And they're concerned about all the repercussions. Can you address that? >> Yeah, so I can a little bit. So that would be the ethics of genome editing for. It has more to do with ecology, I think, although it is focused on disease. I think if you could, yeah, it's complicated, right? So I think, technically, we could engineer. So there's been two thoughts about this. One is there's a specific type of mosquito that carries malaria, and people have proposed, why don't we use the CRISPR system to create a bunch of infertile males that will breed with females and will just wipe out the species? And so we'll no longer have mosquitoes that can carry malaria around. And you can imagine, the way I'm saying it is I don't know what the ecologic ramifications of wiping out an entire species that's actually low on the food chain. So therefore, who knows what their ramifications would be? But people said, look, malaria is an awful disease, this should be worth killing. What you're proposing is another, and so I think that's technically possible. Once that species is out in the environment, there's no bringing it back. There's no way we can suck all those genetically modified mosquitoes back. Same thing with the idea of, well, we're not gonna make the mosquitoes infertile, we're not gonna the mosquitoes. But we'll make them resistant to malaria, so they can't pass along the disease. That seems like a little more reasonable approach. Falciparum malaria, no one really knows what it's good for, but again, what would happen if we wiped out falciparum? Hard to know what that would happen. I think that would be a more cautious approach. Maybe a more reasonable approach because this malaria still is an awful disease. I think, again though, the issue is that, how could you do it in a way that if there were adverse consequences, you could contain the process? And unfortunately, mosquitoes are not gonna respect national borders. All of you guys fly on planes. So containment, as you understood whether you've created something devastating, is something we really have to think about. I don't know if I fully answered your question, I probably would never answer your question so really that quick. >> [INAUDIBLE] >> Exactly. I think technically, it is possible, exactly. But there's a question of I think we've gone beyond the can we to the should we. >> Is it possible to harvest a small number of stem cells and then let them grow and divide until you have the half a billion that you need? >> So the question is this. >> [INAUDIBLE] >> Yeah, so I've talked about taking a large number of stem cells and modifying the bulk population and then infusing that large population in the cells. But another strategy would be to take a small number of stem cells, modify them, and then expand those up. What's the pros and cons? So for hematopoietic stem cells, it turns out that we don't yet know how to expand them. So for that type diseases in which we need to transplant hematopoietic stem cells, that's not a possibility. But there are other stem cells that we can grow and expand. Mesenchymal stem cells, airway stem cells, pluripotent stem cells, even neural stem cells. We can grow in a dish and expand them. So there's a possibility. Adipose stem cells, which are a variant of mesenchymal stem cells. Yep, yep, all those we can do. Now, what's the potential downside of doing it? Well, it turns out that every time a cell divides in culture, it requires mutations. And the question would be if you started from a small number of cells and you ended up growing into several billion cells, would you then have created a population that has acquired so many mutations that one of them may go bad? No one knows that. But that's the potential downside of starting from a small number of cells and expanding up to a large number of cells. So I think it's important to look at what is the distribution of mutations at that end product, and how would that compare to the distribution of mutations that might occur by just modifying a large number of cells all at once? Yup? Yeah, yeah. So I didn't show the slide, cuz one of the issues in the CRISPR technology is while it's designed to make a break in a very specific site, it's about chemistry. And it has a probability of making a break at a site you don't want and creating mutations there. And so I spend time at meetings, and I will be at a meeting next week where we have active discussions about, how much should we worry about that? I don't worry about as much as others because when we measure those off target mutations they're very low. But also I don't worry about as much because I contrast that to a number of mutations that are occurring in us all the time. So on average, every time a cell divides in our body It acquires one new mutation. So if we have 40 trillion cells, and maybe 10% of them are dividing every day, we're acquiring 10 trillion mutations per day. If we give somebody, I am sure that there's no one in this audience who smokes, so I don't even wanna talk about that, By for instance. If you go out on sunlight, you are acquiring 10,000 DNA, 10,000 DNA legions in your skin per second from sunlight. So now our cells have evolved, we've evolved in suns. We know how to fix most of them. The point is, is that our cells are very good at fixing DNA damage. So I think putting this expansion or the CRISPR off target effects into the context of our ongoing mutation frequency is really important. And then I'll step back and I know there's another question, and then is we're all born with an incredible number of differences. So, people at Stanford here, but others have done the same thing, where they've sequenced mom, dad, and a kid and it turns out each of those people have 3,500,000 variants from each other. So, we're all born with an incredible number of variants to begin with So again is putting the initial variation, the ongoing variation, into the context of what we're willing to tolerate as we're trying to engineer therapies. Yeah, great question. So adeno-associated virus, there's actually a lot of different flavors of AAD. There's one through nine, and then I think we're creating more flavors. And the different flavors, different serotypes, have different tropisms for different cells. And so what I didn't go into is, we're using a serotype that has the particular ability to get into hematopoietic stem cells. It actually has the ability to get into a lot of different cells. It seems to not have a lot of specificity so yes, you need to find the right flavor to get into your cell type. And different cell types will need different flavors. So, one of the reasons that we like the AEV, is that when we've looked at the cells after they've been exposed to the AEV and said, how did you react? Did you change the way expressed different genes? There's some changes in gene expression but not very much and when we look into the cells divide and replicate and signal like their sick they have none of the signatures of being sick. After the recombination process, there is no viral DNA left in the cell. It's just the changes we left in and the other pieces of DNA have disappeared. And that's why we like this genome editing approach to modifying cells rather then using other viruses which do leave some of the viral DNA into the genome of the cell that will stay there permanently. Well I'll look into it. Thank you for letting me know and no were always looking for ways of tweaking the system, and I showed you the summation of a lot of small. There were some bigger improvements but really it was a lot of little improvements as we went from 1% to 4%, to 8%, to 12% and eventually getting the 50%. So little improvements all the way along. We're always looking, so we're always on the lookout. So I'll look into that, thanks. >> Using this technology to cure disease is really exciting, but is there anything we can do to try to slow the possibility of somebody using this for bad purposes? >> Yeah [LAUGH]. Yeah, Well, I mean, you guys probably have just as many good ideas and maybe better than I. So I think one thing, I've been fortunate enough to be on a committee, the National Academy of Science is how a diverse community that's coming together will put out some recommendations about how to address some of these issues that I've brought up. So I think first thing you can do, is you can start to define how this process should be regulated. So that anybody who wants to work in the bounds of regulation and society will follow that. How do you prevent somebody going rogue? That's a tough one. I will say that as much as I made this sound simple, maybe it doesn't sound simple, it's not really that simple. I know that you can buy crisper kits that you're supposed to be able to use in your garage to really engineer stem cells, and do this in a way, that's not so easy. So, there is some technical hurdles to this. And then the question is, and so then the other thing is that the Department of Defense is very concerned about this issue. And has put out what they call requests for applications, which means send us a grant proposal about just this. How do we regulate it, how would we make it safer, how would we detect if somebody was genetically engineered? So, we'll see. And I think it's complicated, there's no one answer, so it's a great question. Somebody up here, you had a question, yeah, or comment yeah? Also we didn't expect, so GFP is a piece of DNA from jellyfish and we put it into human cells. Now we don't intend to use that therapeutically, we use that as a tool to allow us to measure it's easy to see green cells or not. But yeah, I think people are thinking about taking pieces of DNA from one species and put in into human cells and I'm gonna give you an example. And maybe you're gonna throw food at me. And so turns out that HIV which affects 30 million people around the world. There's no cure, no vaccine we have some good drugs. Doesn't effect, doesn't infect monkeys and people have identified genes in monkeys that are similar to the genes in humans but they confirm resistance to HIV. And so what we and others have thought about doing is taking those monkey variants, or the small changes that were in the monkey gene and putting them into the human cell. So now we create an immune system that is resistant to HIV. And so that would fall into the rubric of what people call synthetic biology in which we're engineering cells to adopt new features and phenotypes and where you get that DNA from then is complicated we are actually using. Viruses, so not genome medicine, but viruses that contain pieces of DNA from other species and introducing them into human cells. And since they don't seem to have any problems, they stay there. So, yes? >> I have a very basic question about genome editing. So how does when you modify a cell or a subset set of cells, how does it actually spread throughout the body, because the goal is to get it everywhere right? >> Right, so what we do is we take cells outside the body and modify them. And the only way it'll spread is if that cell divides and its progeny spread throughout the body. But other people are working on can you deliver the crisper editing machinery to cells within the body and modify the cells directly in the body and we'll spend some progress on that too. You won't be able to actually edit like all the cells in the body, right? You still depend on it being spread. >> I think, as I said there's 40 trillion cells in us. I don't think we can get at 40 trillion cells. So as we think about using a secure disease, we have to understand which cells and at what frequency and what way can we use editing on to cure that disease. Different diseases will have different frequencies that will move together. Turns out, one of the diseases, bubble SCID, bubble boy disease, we might only need to correct a small number of cells, whereas other diseases we might need to correct a large number of cells. So every disease, I think is gonna have different criteria. >> Hi, assuming that your CRISPR technology is successfully applied to human beings, how soon can we expect a cure for sickle cell? >> So what I will say is taking, we're shooting to take what the data I just showed and scaling it up and doing safety studies that would satisfy the FDA, and start a clinical trial in early 2018. And then, they won't allow us to treat 100 patients. We'll have to treat one patient and make sure that person does okay and then treat another. But I would expect that granted me that everything would work, all right? I'm gonna assume everything works, that I would expect, that say, in five to ten years, we'll be at a point where we're starting to treat hundreds of patients. And maybe in ten years, thousands of patients and so on and so forth. So I think this is, I'm biased, right? Go to Midas, get a muffler. Go to somebody who does cell and gene therapy, I'm gonna tell you cell and gene therapy is great. But I do think that this is one of the next new horizons in medicine, the ability to get modified cells to cure disease rather than a drug or a biologic, really opens up a whole host of diseases to be treated in ways we never thought of before. >> Unfortunately, we only have time for one more question. >> Yeah, and I'm happy stay after and answer questions as well. >> My question is about the target audience for the sickle cell therapy. If sickle trait provides some protection against malaria, and you administer this, and it's successful in Africa or India, do you then put the people at risk? >> Yeah, yeah. I mean, I think that's a great question. So let me just go back here. Remember, we're gonna end up creating a population of cells in which the patient will have some sickle trait cells, some not sickle cells, some sickle fat, I mean, some beta thal cells. These cells are also resistant to malaria. So I don't know, now, the best way of not getting malaria, is to sleep with a bed net at night. So perhaps, if we're gonna give someone a therapy that costs $100,000, we might be able to find $2.50 to have them sleep in a bed net and make sure they don't get bitten by a mosquito. Sorry, I mean, there could be simple solution. But I think that the disease is severe enough. Look, as I said kids in Africa are dying at age five with sickle cell disease. Let's get them to 25 with bed nets and good antimalarial drugs, and we'll deal with that slight change in mortality. Remember, we're not changing it because we're doing this semantically. We're not changing the frequency that will be passed onto future generations as well. So it will still be in the human population. All right, well I'm happy to take any more more questions, and thank you for your great questions and attention. >> [APPLAUSE]
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Channel: Stanford Alumni
Views: 2,892
Rating: 4.891892 out of 5
Keywords: stanford, stanford alumni, stanford university, matthew porteus, crispr, genome editing, disease, molecular disease, structural biology, sickle cell disease, gene mutation, hematology, gene therapy, stem cells, DNA, corrected cells, deadly diseases
Id: D6gRcdGcu-o
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Length: 60min 8sec (3608 seconds)
Published: Wed Nov 02 2016
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