David Segal
Description: David Segal is a Professor of Biochemistry and Molecular Medicine at the UC Davis School of Medicine. His research focuses on genome engineering and targeted gene regulation for applications in neurological disorders, particularly Angelman Syndrome. In this episode we talk about the main mechanisms and challenges of molecular medicine – from CRISPR and zinc finger proteins to editing methods and financial barriers. Professor Segal explains the causes of Angelman Syndrome and details how the future of molecular medicine may assist in treating many types of rare diseases.
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Interview with Foundation for Angelman Syndrome Therapeutics (FAST)
Show Notes:
[0:04] Introduction to Professor Siegel
[2:44] The Role of Parents in Research
[6:56] Molecular Medicine Overview
[10:21] Understanding Epigenome Editing
[12:19] Genetic Predispositions and Environmental Factors
[15:06] Evolution of Gene Editing Techniques
[18:37] Applications of CRISPR Technology
[23:00] Addressing GMO Concerns
[25:25] The Impact of Regulation on Innovation
[30:55] Challenges in Gene Therapy Development
[33:43] The Role of Parents in Therapy Development
[51:43] Focus on Angelman's Syndrome
[56:41] Advances in Treating Angelman's Syndrome
[1:06:18] Future Directions in Gene Therapy
Unedited AI Generated Transcript:
Brent:
[0:01] Thank you, Professor Dave Siegel, for coming on today.
David:
[0:04] Really happy to be here. Thanks a lot.
Keller:
[0:06] We'd love to start off by hearing a little bit more about your story and what got you interested in medicine.
David:
[0:11] Yeah, well, that's interesting. So in the beginning, I wasn't that interested in medicine per se. For most of my career, I was kind of a tool builder trying to make molecular tools that could manipulate DNA, try to cut DNA, try to activate gene expression. We could talk more about that later. But at some point, I got a phone call, really kind of out of the blue. This was back before Facebook. So it was actually just a Google group of some concerned parents and researchers that had read some of my papers and wanted to know if the technology that I was working on could be useful for the treatment of Angelman syndrome. Now, like a lot of people at that time, I'd never heard of Angelman syndrome. So I had to look it up on that vast repository of all human knowledge, Wikipedia. And after I figured out, you know, what the disease was about and, you know, how, what they were thinking about, I wrote back to them and said, yeah, you know, I think I probably could design something that would be useful for that disease. But at a late stage in treatments, I'm not sure that it would do any good. And so then they started to connect me up with a bunch of other scientists and, you know, sending me papers, and it just got me kind of hooked into that whole thing. And I just got in deeper and deeper, and it really changed the focus of my career at that point.
Brent:
[1:35] Would you say it's pretty common for parents of people with these diseases to be reading academic papers and trying to contact the academics doing the research themselves?
David:
[1:44] So that is a very important question that you're asking. And the answer is it's not that common, but it's also becoming a new reality that as we are able to make these new tools that can alter DNA sequence in people or change gene expression, something like that, it's now possible to try to design therapies for rare genetic diseases that before just wasn't possible. However and we could talk more about this too it's a, Working in the rare disease field is very difficult. There's not a lot of profit to be made. And so traditionally, it's not been a place where pharma has really delved in. And unfortunately, often parents have to take it upon themselves to reach out to scientists and sometimes to collect funding and try to fund research themselves
David:
[2:42] to get people to work on their disease.
Brent:
[2:45] Yeah, we'll definitely come back to that in a bit.
Keller:
[2:47] And in those earlier applications, before you started working on Angelman's, were you working on more so the genetic engineering systems themselves, or were there other targeted applications that you were looking into?
David:
[2:59] No, I think back when I was a graduate student, I was working in a lab that was studying homologous recombination. And actually, it didn't seem that interesting to me what they were doing. So I went to my primary investigator and I said, do you have any other projects in the lab that I could work on? And he said, well, there is this one thing that I've been interested in, and it has to do with trying to stimulate homologous recombination by making targeted double-strand breaks. And so I said, okay, that sounds kind of interesting. And, you know, that kind of work eventually gave rise to the whole gene editing field. Not just us, right? But we were part of the very early wave of people that were trying to investigate that. And, you know, back in those days, there were no good ways to make targeted double-strand breaks in DNA. So everything was hypothetical or proof of concept. You know, you could set up a system to show that it would work if only you had some kind of targetable nuclease. And then, you know, there were zinc finger nucleases and tall nucleases and now CRISPR and everybody knows CRISPR. So it's a different day.
Keller:
[4:09] What was the big break in that field? What got it to the point where it was no longer just fully in theory?
David:
[4:17] Um... Well, so there were, you know, some, of course, all science, right, is standing on the shoulders of the giants that came before us, right? So there was a group that included Mario Capecchi that had developed a way to target genes. So let's say before the 1980s, basically the main way that we could manipulate genomes was to use some kind of chemical mutagen. You mutagenize at low density, you know, you don't know where it's going, but you can maybe select for something that's useful for you. But there was no way to say, I want to knock out this one gene. And that changed really with Mario Kopecki, who, by the way, won the Nobel Prize for this work. And he came up with a technique that enabled us to make knockout mice and was very effective in making and manipulating the genomes of mice. But unfortunately, that wasn't effective and very efficient in human cells or almost any other organism.
David:
[5:21] And so it led a lot of people like our lab to try to figure out what's the slow step in there, you know, why isn't it working? And I think, you know, our lab, among others, started to come to the conclusion that if you could make a double strand break in the DNA of the cell, that's the missing link. And so that was the aha moment if you will uh that really changed everything and that really said once once we figured out that that's what was needed that kind of set off a, race if you will to try to figure out how you can make a targeted nuclease okay.
Brent:
[5:58] And then uh before we get like more into the science could you give like a brief overview of molecular medicine and then how uh gene editing or gene engineering fits into that.
David:
[6:09] Yeah, so, you know, most of medicine is small molecule drugs. And I'm very happy that we have small molecule drugs. If I was sick, I would take small molecule drugs. But, you know, unless it's some kind of an infectious disease, small molecule drugs usually don't, like, cure people of things.
David:
[6:33] Usually, and I'm grateful that we have the tools that we have, but you can see that some people require drug therapy for their entire life. That can be very expensive. There could be side effects. There could be compliance effects, all kinds of things like that. But that's mostly what we had. And even for genetic diseases, we would still have to rely on small molecule
David:
[6:53] drugs to drug some kind of protein target in the cell. Um and so you know once it became clear that we might be able to do more with these technologies that we could actually manipulate these genes inside living organisms and it doesn't have to be humans it could be plants it could be barnyard animals something like that um you know it just opened up a new world of possibilities so you know i'm more on the medicine side but there are certainly people here at UC Davis and many other places that are using this in agriculture.
David:
[7:27] To try to make crops, say, more resistant to drought, so that as we enter this warming period of our planet, and at the same time that we're having more and more people to feed, we can try to adjust crops faster than natural evolution would. So there's lots of uses for gene editing for these kinds of things. But in medicine, I would say we're just at the very dawn of this becoming a reality for medicine. Gene therapy that came before gene editing is still in its infancy. Only about 18 gene therapies have been approved worldwide. And so it's a new day, and we're all trying to understand what the new day is going to be like.
Brent:
[8:17] Yeah. And I think for gene editing, conceptually, it makes sense to a lot of people. There's a specific area that we want to edit. But how would you describe epigenome editing?
David:
[8:28] Epigenome editing, yes. So there's different ways to control gene expression, and certainly changing the DNA sequence is one of them. And so most of the time when we talk about gene editing, we're talking about going in and changing the DNA sequence. So that could be something like correcting a mutation that somebody has in their DNA. Sometimes that's actually a difficult route to take because they might have that mutation in every one of the trillion cells in their body. And so you might not be able to hit all the mutations. And there are newer versions of even, you know, the kind of gene editing, things like base editing, prime editing. But there's also other ways to manipulate gene expression. And that's to access the so-called epigenetic layer of gene regulation. So this is chemical modifications to the DNA that our cells use when they want to turn on genes or turn off genes. So a good example of that would be.
David:
[9:32] In my brain, in my neurons, I have all the genes to make liver enzymes, but my brain never needs to make liver enzymes. So those genes will be epigenetically silenced. They're not mutated, they're still there, but they're silenced through this chemical method that our bodies have to silence those genes, and they'll be shut off for my entire life. So if we understand how epigenetics works, what are the readers, the writers, the erasers, what needs to go on first, what needs to go on second. If we understand how nature is able to accomplish these tests, then we should be able to design tools that can do the same thing with a short-term treatment, can cause a lifelong effect in gene expression.
David:
[10:17] And that should be, in principle, safer than gene editing because we're not changing DNA sequence. There's less things that could go wrong. So we're very interested in epigenetic editing.
Brent:
[10:26] And then from my broad understanding, that's how a lot of cancer starts. It's just like the genetics gives like the baseline and then how we live our life and the epigenetics is what like ultimately triggers the cancer to start. Would you like have that same approach and use epigenetic editing to like maybe make sure pre-cancerous like DNA never becomes cancerous?
David:
[10:49] Actually, I've had exact conversations about that with people. Um let's just say that you know among the different kinds of diseases uh uh there might be some that have a strong environmental component and cancer is one of them um and there are some that have very little uh environmental component it's mostly just genetics so i mentioned angelman syndrome a little while ago basically if you have the mutation that leads to angelman syndrome you're going to have that disease uh there's nothing you can eat or exercise or run around and do that will help with that.
David:
[11:23] However, fortunately, those are rare diseases. They're bad diseases, but fortunately, they're rare. The more common diseases, things like cancer, cardiovascular disease, some things like that, those are what we call common complex diseases. And they're complex because they have some genetic component, like you said. Often that gives them a predisposition, perhaps, for getting the disease. And then there's a whole of life factors that enter into that. And those life factors could be what you eat, it could be how you exercise, it could be early trauma in your life, or some chemical exposure that you had that you didn't even know you had, or, you know, something of that nature. So there's a lot of research going into what are these things, you know, these other kinds of exposures or experiences, or, you know, dietary things, and how does this affect gene expression.
David:
[12:14] But yeah, you're right, Those kinds of things tend to make epigenetic changes in gene expression. So those diseases are a combination of what happens to you in life, what cards were dealt to you through your genome, and then how this epigenetics plays out.
Keller:
[12:32] And then one more kind of quick terminology point before we keep going. You had mentioned zinc finger proteins earlier. Could you just explain where that comes in into the whole picture of gene editing?
David:
[12:41] Yeah, so with gene editing, especially the early iterations of gene editing, it came from that kind of early work that I was telling you about, that when people found that you had to make a double strand break in the DNA, and that could stimulate this homologous recombination by hundreds of thousands of folds, people were looking for different ways to make a targetable nuclease. And zinc finger was one of the very first ways that people tried to do that. And that's because at the time, that was the best understood DNA binding protein. It's a protein that occurs naturally in our cells. So actually, humans have about 800, I think, zinc fingers in their genome doing various things. And so it was something that we understood.
David:
[13:33] And many efforts, including efforts I've been involved in, aimed at trying to reprogram them to bind to sequences that we wanted to bind to. So that was the whole beginning of my career in that. But, you know, they were always kind of difficult to program. They still are. When you get a good one, it's perfectly good. It's as good as any CRISPR or maybe even better. But, you know, it was a little bit harder out of the box, as we say. And then in 2009, it was discovered that there were these plant bacterial pathogens that had a protein that they called TALs, which stands for something else, but we'll just call them TALs for now. And they seemed a lot like zinc finger proteins, but they were a lot easier to program. And so they basically took over the whole field and everybody was making TAL nucleases. And TALs was the best thing. for about a year and a half. And then the famous paper from Jennifer Doudin, Emmanuelle Charpentier, introduced CRISPR.
David:
[14:36] And the real advantage of CRISPR is that you don't need to make a new protein each time. The nuclease basically stays the same, and you just need a small piece of RNA to guide it to the right place in the genome. And it's very easy to make nucleic acids, you know, in huge libraries and huge quantities. And CRISPR works very well, actually. It's a very good nuclease,
David:
[15:02] especially the SV-Cas9 that was first described. It's a very good nuclease. And so then that basically took over and brought us to the world that we're in today. But yeah, that's been the progression of those technologies.
Brent:
[15:16] Yeah. And then with the double strand break, from my understanding of genetics, there's two strands. One typically acts as a template to reproduce. And by cutting it in half, is there a new template being used? Or is that more just to change how both DNA are structured and the recombination aspect to get rid of a certain problem in the genome? Would you describe why the double strand break is necessary to then change the order of things?
David:
[15:51] Yeah, so you mentioned DNA has to replicate and there's often a template strand. And our DNA actually gets a lot of insults every day. The estimates are something like over a thousand strand breaks happening in our DNA in every cell every day. So that's lots of DNA damage that comes in from sunlight, from oxidative metabolism in your cells, maybe other kinds of things, certain chemicals floating around in your blood.
David:
[16:27] So all of those things can cause some damage to the DNA. And so our DNA, our cells have to have very robust repair mechanisms. And that's kind of what we take advantage of when we do gene editing. And so there's different ways to put in a DNA so that it can be repaired in a predictable way. But making these targeted double-strand breaks basically activates those molecular ends of the DNA now to undergo some kind of repair. So the cell hates broken DNA, okay? And it will try very hard to repair it. So if you just break DNA with the nuclease, the first thing that it's going to want to do is just put those strands back together. And it does that in a very quick and dirty way. And sometimes it will actually make mutations at that breakpoint. And this became one of the primary ways that gene editing is made is to actually introduce mutations at a particular site.
David:
[17:30] So like I say, that only requires the nuclease. You can break the DNA. We know that it often reseals perfectly, but then the nuclease is still there. It keeps cutting, breaking, cutting, breaking, cutting, breaking until you get a mutation there. And so people have used this to knock out gene expression, to turn off a gene that's causing a problem, or in some other way, like in the first FDA-approved gene editing therapy that was just approved last December.
David:
[17:59] Treating sickle cell anemia, the goal there is to use the nuclease to target double-strand breaks to mutate a binding site for a protein. So that's one of the primary ways that it's used but there's other ways to make to repair that double strand break for example you can introduce a new template of dna and we'll introduce that new sequence in where the old sequence was and that's the way that we repair mutations in in cells so we can actually go in and you know either repair a mutation or introduce a new a new sequence there.
Keller:
[18:38] Before we kind of go deeper into some of the challenges involved with these technologies, how common are some of the applications of these technologies? Whether it be in agriculture, whether it be with humans or different livestock, how prevalent are these technologies being used?
David:
[18:53] Another very important question that you ask that has a lot of depth to it. So let's just say there's a lot of research going on using CRISPR-Cas. So like many life science labs, when you talk to them, they'll, you know, either have been using CRISPR or use something that has been CRISPR-ed, you know, is becoming like a verb, you know, we'll just CRISPR that.
David:
[19:18] So, you know, that's very widely used. Now, in medicine, I think there's a huge interest in trying to develop these kinds of CRISPR reagents. There's a group now called CRISPR Medicine that keeps track of all the clinical trials that are going on with CRISPR tools. And I don't know what it's up to now, but I mean, it was in the 90s or more clinical trials. For your listeners, when I say clinical trial, that means trials in humans. That's not preclinical trials in animal models. That's trying, you know, some kind of CRISPR therapy in humans. So there's quite a lot of interest in that. Now, the real question is, you know, what is getting approved? And right now there's only been one gene editing therapy approved. Lots of them, as I say, are coming. And we'll be interested to see, like, what's the next thing that gets approved? But let's say, you know, lots and lots of companies and labs and, you know, trying to work in that space. But, you know, we need to make sure that these things are safe. Number one, above all, needs to be safe. And then number two, should be efficacious. So that's the medical side.
David:
[20:29] Then there's the plant side. So again, lots of interest in trying to use these tools to manipulate gene or gene expression in plants for various different reasons. But often, you know, there's a big emphasis in the government to try to think about our food supply in the future. And, you know, one example that I mentioned before was trying to make, say, drought tolerant crops. Another thing which kills more people than the coronavirus, they say, is plant pathogens, because plant pathogens can sometimes try to wipe out entire crop species.
David:
[21:07] And the loss of productivity, the loss of the food supply, the disruptions like that have also ultimately lead to death and malnutrition. And especially looking into the future when we're going to need more food, it's a kind of constant battle with plant pathogens to be able to deliver, keep the food supply safe. And so these gene editing tools there, you know, gives us a chance to get in front of the evolution because whatever you make, there's going to be a pathogen that tries to overcome that, you know, but this gives us a chance to stay one step ahead.
David:
[21:43] And finally, you mentioned livestock. So genetic engineering, for better or for worse, in agriculture, in plant agriculture, has happened before. And by now, there are many crops that are genetically modified, probably not with CRISPR, but with other techniques, that are in the food supply that have been eaten by people for many years. You know, generally, people could say that these are safe. There's a whole bunch of people that are against that. I understand that too. But in the livestock area, they got started on this a little bit later. And for them, it was a very difficult road to get anything ever approved. There weren't even good processes or procedures that they could follow to get a modified livestock approved. I think to this day, it may be true that there's only been one species of fish where they introduced a gene or changed a gene that would make the fish grow a little bigger. And I think that's the only thing that's ever been approved for human consumption like that or been allowed to enter into the food chain.
David:
[22:56] Everything else is all experimental, and it's uncertain if anything will ever get approved ever.
Brent:
[23:01] Yeah. And then would you try to temper people's fears about like GMOs or genetically modified like food? Because if it makes it more like drought resistant, the gene editing is so specific to like what causes like the inability to withstand a drought. So would you overall say it's not a huge deal? Like we're very precise about how we edit the genes and what we're doing. It's not going to really have much of an impact most of the time, like when you consume it.
David:
[23:29] Yeah, so I fully respect people's opinions and their beliefs in these things. Certainly I have my opinions too, and I'm always very concerned when people's opinions, especially in affluent countries, those opinions tend to set policies in lower income countries where they have less choices. And now they may not be able to use genetically modified foods to feed their people because of, you know, some other restrictions. And it gets very complicated, but I'll just say this. The people that feel strongly that, you know, genetically modified crops and foods, you know, can be advantageous, I think they would take the point of view that we should be regulating the product and not the process. So, like you brought up, um.
David:
[24:27] If we're talking about drugs, we don't talk about, did they use this chemistry or that chemistry? You get a drug and then you test it and you see, is it safe? Is it safe in an animal model? Is it safe in people? You start with a low dose, you escalate the dose. You look over time to see if it was safe. right? That's how we judge things like drugs. And, and, you know, I think the scientists would just say that's the way that we should be judging, you know, the products that come from this other, you know, from plant editing. Let's judge the product. Is this product safe? Is this product going to cause trouble in people? Is it going to overrun the lands with the genetically modified things? Is it going to kill monarch butterflies? Let's ask that question about the product, But let's not demonize the process, because the process could be treating it with radiation. It could be just a selective breeding. It could be genetic modification. And so that's an important point to consider, is let's think about the product and not the process.
Brent:
[25:24] Yeah.
Keller:
[25:25] And kind of touching on that same point, like about policy and about regulation, how does that kind of come into play with the rate in which these technologies are coming out? Because obviously for like FDA or regulatory bodies, they have to do a lot of testing. But are there other policies that might be hindering the rate of innovation with these technologies?
David:
[25:44] Well, clearly in the animal field, livestock animals, they're clearly hindered by regulation. And there's just a lot of politics behind that. They would say every time some new technology comes up, the scientists will give them this small stack of paper, and the opposition will give them a huge stack of paper. And as long as that's the case you know it's they're kind of deadlocked on what they could do, so uh in this field they often say nothing succeeds like success and you know i think if these modified products are perceived to be beneficial then like cigarettes or bitcoin or you know many other things that you know on the face of it you'd say you know maybe these things is actually deleterious to us. But I think if they can be perceived to be useful, then I think there'll be more acceptance.
David:
[26:44] Even with plant genetics, it used to be that any kind of genetic modification done with, say, a CRISPR or something else like that was gonna be, GMO has to be highly regulated, and some portion of the population will not accept it. But a lot of that is changing now. So it now depends on really how you use the gene editing. So I mentioned before, a big way that gene editing is used is to introduce mutations into certain genes. And those mutations could have been introduced through natural means, they could have been induced by radiation means. Um and these are all you know those are all accepted techniques and so if you use the crispr in a way that it makes a mutation in a way that could have been made in nature you know in this case you directed it to a certain spot you know so it's a lot faster streamlined but uh you know if you could do something that could have been happening in nature this is now generally accepted as okay even in the european union which you know before was not not okay with those kinds of things So those kinds of things are acceptable. Certain types of genetic changes, like a manipulation of a gene, might also be okay under circumstances. What still seems questionable is if you introduce a new gene that never would have appeared in that plant, if that's going to be okay. And again, I think that's still out for debate.
Brent:
[28:12] Yeah. And then kind of shifting back towards the human models, what are some of the bigger challenges with getting more genetic engineering for different diseases and making it more widely applicable?
David:
[28:26] Right. Well, so like I say... We have unprecedented capabilities in human history to now be able to go in and treat genetic disease at a very fundamental level, at the level of the DNA. So, you know, that's a tremendous capability that we've really, you know, just gained in the last several years. So while everyone is kind of familiar with CRISPR, you know, moving that into medicine and actually, you know, trying to do that in people, you know, it does, you know, you want to approach that in a very sober way and make sure everything is safe and efficacious, like I say.
David:
[29:08] Um so uh there's um so there's a lot of companies trying to move this forward and uh uh you know working with the fda to try to figure out you know what are the guidelines going to be what are the tests that need to be made you know how much do you need to show about off-target editing and what are the tests that will be accepted for that this kind of thing right um are you just doing your editing in this spot and not many other places in the genome, this kind of thing.
David:
[29:41] We should also make a clear distinction between what we would call germline gene editing and somatic cell gene editing. So the somatic cells are all the cells in your body that don't contribute to the germline or pass down through your shoulder or anything like that. So if people have a disease, you would to treat the people that have disease. We don't usually think about treating future generations through genetic editing. Now, everybody knows that there was already a case of a Chinese scientist that did make genetically modified babies. In doing that, he circumvented all of the institutions that we put into place to keep, society feeling that science is working in their best interest. Those are things like institutional review boards that give oversight on any human experiment that's going to involve people and other kinds of safeguards. So he just bypassed all of that stuff and went ahead and did it. So we can universally condemn those actions. And even in China, he had house arrest and fines and all kinds of things.
David:
[30:55] But I think germline gene editing is something that a lot of people just don't want to talk about or don't want to really have to think about right now because there's so much need for people that desperately want therapies for these diseases that we as a community would have our hands full trying to make these therapies for people that desperately need them. I don't think we need to think today about how we could do germline gene editing and what the ethics are on that. That's my opinion. But in terms of what we can do to treat people with illness now.
David:
[31:33] Yeah, these new tools give us tremendous capabilities. And people like me are very interested in trying to develop that. But as I was alluding to earlier, there are real monetary concerns that are cropping up as significant issues in being able to make these therapies on many levels. So let's just say my journey has taken me on a path ever since that first phone call with the Angelman syndrome people to not just think about what I do in the lab with my students and working with the molecules and making a molecular tool, but also to talk to parents of children that have these disorders and work with foundations that, you know, try to raise money for this research and the plight that they go through. And, you know, we're working on diseases now that most people have never heard of. And I'm happy for that, that they're rare, that most people have never heard of them. But, you know, it also means that they're not getting a lot of attention. And so there are no drug therapies for them. There are no molecular therapies or, you know, many doctors just won't know what to do when someone comes in with a disease like that.
David:
[32:50] And so it's unfortunate that parents often need to take the initiative to try to get a scientist interested in their disease to do any kind of research, let alone try to put together some kind of a therapeutic program to treat their population.
David:
[33:14] So... And it's difficult on many different levels. And then, you know, also like once a therapy is made, you know, there's the question of is there going to be enough profit to make it profitable to produce the therapies?
Brent:
[33:29] Yeah, I can see that being a huge issue because especially if it cures the disease, it's a one-time treatment versus a lifelong of taking a drug. And we know pharmaceutical companies do enjoy people being on their drugs for a long time.
David:
[33:44] So for example you know and i hate to say this but uh a lot of you know we're we're interested in trying to do our part in this so i i work very closely here at uc davis with three other labs we consider ourselves an interventional genetics team and we are mostly interested in trying to develop molecular therapies for a small subset of neurodevelopmental diseases i mean we could use it for a lot of things but you can't be a jack of all trades so we have to focus in on like one area and really make an impact in that area so angelman syndrome is a good example of that some of the diseases we work with they don't have syndromic names they just have a gene name like cdkl5 uh adnp syngap1 um and uh you know just just to give you an idea of like the plight Of the people, you know, for cancer, like if somebody had cancer, right, they would go to the doctor, they would expect that number one, the doctor has heard of stomach cancer, you know, and that there's a treatment and then they would, you know, maybe talk to their doctor about what's the best treat, maybe there are 15 treatments, what's the best treatment for you, right? Like for someone with a rare disease like that, they would often go to their doctor. The doctor would have no idea like what to do about it. There are no approved therapies. And the advice that they would get is.
David:
[35:07] Well, it's a terrible disease. Maybe you could start a Facebook group, try to gather other families that are afflicted with this disease, and then maybe you could start a foundation. And then with your foundation, maybe you could raise some money, mostly through walkathons and bake sales, to try to raise some money for research and try to find any scientist anywhere in the world who might do some research in your disease. That's that's what they're told you know um now consider those scenarios and then just consider if i tell you that rare disease although individually rare so rare disease means it's less affects less than 200 000 people in the united states so each one is individually rare but collectively they affect more people than cancer and aids combined so you just have to let that sink in with your listeners and say, this is a huge unmet medical need.
Brent:
[36:05] Yeah.
Keller:
[36:07] Then kind of back to what we were talking about earlier, I had a question about, for the clinical trials, if you were to get to that stage, you mentioned the delivery methods. How does that, if I were to be a patient that had one of these rare diseases, I get what we're talking about with the editing, but the actual like me being a part of the treatment, there's still a kind of a gap. What would that look like from the patient's perspective?
David:
[36:35] That's another good question. So, I'll tackle it on a technical level first.
David:
[36:46] So, the people in this field, in the therapy field, often like to say that the three main challenges facing this genome editing field is delivery delivery and delivery so you know we're able to make the tools you know relatively easily but getting them into the cells where they need to go inside the body is a real challenge and so there's a number of techniques but all of these techniques have strengths and weaknesses and so i don't want to nerd out too much on your podcast here but let's say uh, One of the most powerful methods for delivery is putting it into some kind of viral vector. And we all went through the pandemic, and we know that viruses are very good at delivering their nucleic acid contents into cells. So often these viral vectors, and the most common one that's used in gene therapy now is something called adeno-associated virus, or AAV. It's very good at bringing some DNA into cells. Especially neurons, because we're interested in brain disease. So today, it's the most efficient vector that we know of to do that. And so if you're treating brain disease, it's like the go-to virus. However, AAV has some drawbacks.
David:
[38:07] It has a limited packaging capacity. So things like CRISPR just barely fit inside it. If you want to do epigenetic editing, that makes the protein even bigger. Sometimes you have to use two AAVs to deliver everything. so that gets more complicated. And the other thing is it's immunogenic, meaning that once you inject someone with an AV, they develop an immune response against it, so you can't re-inject after that. So if you can't re-administer, you basically have to deliver everything you've got in one shot. That causes the injection titers to be very high, so they inject a very high concentration of virus, basically as high as they can that the patient will tolerate. Because they only get one shot at it. And sometimes that dose might not be enough or sometimes that dose may be high and lead to some toxicity.
David:
[39:02] So what would be much better, the golden egg that we're all looking for is something that could be re-administered, that would be less toxic, less immunogenic and have a larger packaging capacity. So there is another tool that's widely used called lipid nanoparticles. And lipid nanoparticles are all synthetic. They're created in laboratories. They don't come from any living thing. You don't have to grow them in cells. They are generally a lot less toxic. They can be readministered. And they have a larger packaging capacity. And they sound great. Like, that's exactly what we want, right? But today, they're mostly only useful in the liver. So, if you can treat liver disease, like many clinical trials have used this in liver disease, that's a great choice. But if you want to treat brain disorders, we can't really use LMPs today. Now, I have unqualified hope that clever chemists will be able to re-engineer those so that we can get good uptake into the brain or distribution or whatever the problems are that we have to solve. But today, we don't have that.
David:
[40:19] So this brings up, when we think about treating patients, this creates a situation where you have to think about how could I put the best tool in the best delivery system to be able to make the biggest impact in disease. So it's not surprising that the first gene editing therapy, for example, is what we call an ex vivo gene therapy. So to treat sickle cell, they take cells out of the body, they treat them in a dish where they can control a lot of things, and then they reintroduce those back into the body. That's the so-called ex vivo gene editing approach. Because that does not involve putting a virus into the body and getting it all biodistributed and everything like that. Because that's a trickier thing. But lots of those are coming. And most of them are using AAV to do their delivery. And yeah, they face a higher bar to demonstrate that it's going to be safe and effective.
Brent:
[41:22] Yeah. I think that gives a pretty good overview of the different methods uh, Do you have a high-level explanation of why the lipid-packaged cells? Lipid nanoparticles. Yeah, those. Why are those not able to be treated for other diseases outside the liver? What's the issue with them getting into different places?
David:
[41:47] Yeah, so when foreign things are injected into our bloodstream, they go quickly to the liver, and the liver basically functions as a filtering organ. Spleen also kind of does some of that and spleen is another place where a lot of things go so even injected if we were to inject this adeno-associated virus into into blood so it would say that's a systemic injection it would mostly go to the liver and in fact you can get dose limiting toxicity in the liver before it even gets to your organ of interest so you know because av's been around for a while people have worked on clever ways to try to detarget it to the liver or reduce its expression and lots of things that people try to play there but still has the same problems that i mentioned earlier yeah but yeah unfortunately everything tends to go to the liver first even.
Brent:
[42:38] With like site-specific injections.
David:
[42:40] Well, site-specific injections can help quite a lot, actually, yeah. But, you know, it really depends on what the disease is. So you might be able to, you know, inject into the kidneys. You might be able to inject into the heart directly.
David:
[42:56] But then, you know, you have to wonder if your injection will, like, get all over the heart. So, you know, is it going to spread from your injection site?
David:
[43:05] Or, you know, is it going to be just very focal in a little spot? So, you know, that's another choice, for example, for brain delivery is, you know, you can inject right directly into the brain. But typically what you would see is that you would just get, you know, a big bolus of stuff injected at the injection site and not that far away. So for brain delivery, yeah, we face additional challenges because the brain has this thing called the blood-brain barrier, which is a barrier that basically tries to keep many things in our blood from crossing into the brain. And so when we want to apply drugs to the brain, for example, they have to be able to permeate the blood-brain barrier. And AAV does not cross the blood-brain barrier. So there was a big leap forward a few years ago now where a group in Caltech was able to show that they could redesign an AAV that was able to efficiently cross the blood-brain barrier. And that kind of sent a shockwave through the entire community that this is going to be the way that we're going to treat brain disorders now. We're going to use these new viral capsids for AAV, and they're going to be able to distribute this directly to the brain. It's.
David:
[44:31] Systemic injection and uh the first the first setback was that the the initial work was done in mice and they found out that actually um it really didn't work in any other animal uh, the same way that it did in mice and in fact it really only worked in the species of the strain of mouse uh not even a different strain of mouse yeah so uh so that became limited but it told everyone that it's possible to make these new viruses that could go, to the brain, but also to maybe many other places. So now this is a very hot area of research now, trying to make new capsids, and getting ones that cross the blood brain barrier more efficiently, you know, to go to other other organs. And the hope is then you could just inject into someone's arm. And this thing will find its way to the brain and go and treat the cells in the brain and edit the DNA and like everything's going to happen. And that's one possible future. But I would just point out that there's other people that would say, okay, you fix that problem with AAV, but it's still immunogenic, you still only get one injection, it still has a small packaging capacity, so we haven't solved those problems.
Brent:
[45:43] And by immunogenic, you just say there's an immune response that then targets and tries to kill the viruses you've injected?
David:
[45:50] That's right, yeah, there's an immune response so that either it will try to intercept the virus on the way to where it has to go with antibodies. Or when it gets into the cell, there's another kind of immune system that takes over that will surveil the cells and look to see if they're producing any viral proteins. And if they see that it looks like it's infected, they'll try to kill it.
Brent:
[46:14] Yeah. And then I think people are generally scared of viruses because of our typical encounter with them getting us sick. But for these types of treatments, should people just think about it as a way to deliver a type of like a little bit of dna that the cell can then use and then be beneficial.
David:
[46:30] Absolutely yes that's absolutely true and i would say the least thing that someone would need to worry about is are they going to get sick from like an av uh the same way you would get it from from the uh uh the av in nature absolutely not all that stuff has been taken out of the virus, Another widely used virus, not as widely as it was used previously is like a lentivirus that's based on the HIV virus. Everybody knows about HIV, nobody wants to get that. But like, that's just out of the picture, that's not going to happen.
Keller:
[47:05] And with the rare diseases, you've mentioned that here in Davis, you guys are working on like a few different ones other than Angelman's and that obviously they're very, at scale, very prevalent and impact a lot of people. Is there a way or are people like pairing certain rare diseases together in their studies so that let's say you found out a gene that impacted like Angelman's? Is there diseases that we know to be similar enough to where those research like findings can carry over or is it still very one-off by each disease?
David:
[47:35] Yeah, that's also another big question because in the field, there is a concern about how much of this kind of gene therapy or gene editing stuff is going to be necessary to treat these diseases and what's going to be the cost of that. So we already talked a little bit about cost, but certainly if we had to treat every mutation as an individual thing, you know, it's impossible to even think of a future like that. So for example, if we think about BRCA1 and BRCA2, so these are breast cancer genes that are very well known. There's, you know, lots of studies for that. Women that have a family history of breast cancer, you know, might be at risk of this, and they would get that checked.
David:
[48:26] Each one of those genes has over 600 mutations associated with it and what i mean by that is you know when we talk about a gene that's mutated right the gene you know is a very long thing and the mutations can happen almost anywhere within that gene so a mutation maybe at the end of the gene is not as severe as a mutation that happens early upstream in the gene something like that, And so, you know, depending on which mutation you get, you might have a higher risk or a lower risk or no risk, right? So we have to think about what each mutation means. But, you know, if you were going to try to make a therapy where you repair each one of those mutations when it comes up, have a therapy that's going to change the mutation in this person, you know, you would need just over a thousand different kind of therapeutics just for those two genes, right? And if there's 20,000 genes in the body, when you do the math, it just doesn't work.
David:
[49:21] So we have to come up with strategies where one treatment will fix all the problems that that disease has somehow. That's got to be the goal, because otherwise you can't just take a mutation by mutation. Sickle cell anemia is actually a rare case where basically all the mutations, there's only like one mutation. Almost all the disease is based on one mutation. So another possible reason why that sickle cell anemia gene editing therapy was a.
David:
[49:56] Um, let's say that, that, that's one of the reasons why there's a lot of interest in sickle cell also is, uh, is that feature, but most other genetic disease will have lots of mutations. So one of the ways that we're thinking to approach this kind of thing is, um, to work with a family of, of diseases that have a similar kind of genetic problem. And a very typical kind of problem that we see in genetics is, um, they actually have one good copy of the gene and one mutant copy of the gene. I'm so used to talking about this with people that can see I'm holding my hand. So let's say you have a good copy of the gene and a mutated copy of the gene. If that mutated copy leads to complete loss of function of that gene, we have a fancy word for that. It's called haploinsufficiency. And what it really means is that the system, these two genes now are only making half as much protein as they used to make because one is not making its protein anymore. So it's like you only have half of the level, and that's not sufficient. So we call it haploinsufficient.
David:
[51:03] So we think a way to address this kind of on a systemic level is if we can just turn up the level of gene expression from that one good copy, then we just need to raise it from a 1x level back to a 2x level as if there were two genes. And then if it's really haploinsufficient that should fix the problem. No matter where the mutation was that was causing the problem, as long as it's that case that we could raise the level of the good gene, we should be able to address all of the mutations in that disease. And in fact, any haploid insufficiency should be able to be treated in the same way.
Brent:
[51:44] Yeah. And I think we have a pretty good background now, like genetic engineering as a whole. Could you talk a bit more about what Angelman's is and what you're doing for that disease?
David:
[51:56] Yeah, so Angelman's syndrome is a rare disease, affects about 1 in 15,000 births. So there's still 25,000 or more people that are living with Angelman's syndrome. And there may be more than that because a lot of pediatricians even today might not recognize it and it might be misdiagnosed. I'd like to say that in this day of genome sequencing, getting cheaper and cheaper, that, you know, that's a very quick tool that people would turn to. But still, often the time it takes to actually get a genetic diagnosis for these rare diseases, they call this the genetic odyssey. Because often, you know, they'll think it's one thing, they'll test for that, it wasn't that. So then they'll think it's this other thing, they'll test for that, it wasn't that. And this genetic odyssey still typically goes on for three to five years before someone gets a diagnosis. So.
David:
[52:57] But then when they get a diagnosis, we can think about what we can do. So for Angelman syndrome, it's a disease that causes severe mental impairments. There's a lack of communication because the kids struggle with expressive language, so they can't really speak. A lot of Angelman children will never speak.
David:
[53:30] Nowadays, they can try to communicate through like touchscreen things and stuff like that, but still very difficult. I think the frustrations of not being able to communicate leads to a lot of behavioral issues as well. They also have some characteristic movement problems. Some of them will learn to walk when they're 13. Some of them will be wheelchair bound for their lives. Um then there's also seizures some kids don't have a lot of seizures some will have multiple seizures every day uh that can be some of them will be treated with epileptic drugs and some some not so much um and so um but another characteristic of that disease is that the children tend to be very happy and so when you meet enjulman kids they're always you know very you know, outgoing and like to explore a lot and laugh a lot. And some people would say that that's something that really makes this community different is that, you know, the kids seem happy.
David:
[54:34] In many, many situations. So that's kind of an outline of the disease part. On the genetic level, there's some very complex disease, complex genetic situations behind this disease. So again, without nerding out too much, let's just say that the problem is caused by loss of expression of a gene called UBE3A in the brain. And in most of the parts of the body, of this gene is expressed from both copies of our genes. So I would get half of my genes from my father and half of my genes from my mother, right? And for most of our genes, they're either expressed from both germlines or they're not. In this particular region, UV3A is only expressed from my mother's chromosome.
David:
[55:36] It's present in my father's chromosome, the one that he gave me, but it's shut off. And it's only shut off in the brain. So that's the neurotypical case. Where Angelman syndrome comes from is if that one remaining copy on my mother's chromosome is either mutated or lost in some other way. So now that that maternal copy is not there, all that's left is the paternal copy, and that one has been epigenetically silenced. And so now they don't make any ube3a in their brain and that's what results in angelman syndrome so our therapeutic approach is to try to reactivate the father's copy of ube3a which is you know perfectly intact and um will allow this gene to be expressed at its natural level under its natural physiological you know control whatever that is uh uh so the hope is that if we could just you know turn that gene back on that now ub3a will will be made in the brains of of these people,
David:
[56:39] and um and hopefully give them a better quality of life.
Brent:
[56:42] Yeah and then can you talk about where you guys are at that point like are you have you been able to do it or you have you done it in models humans like.
David:
[56:49] Yeah so um again uh a lot of science as i said earlier is is standing on the shoulders of the people that came before. And so earlier work has shown, you know, earlier pioneering work, I would say, you know, showed, you know, like what the genetic, the very complicated genetics there, and, you know, looked at all of that. And earlier work showed that if you could somehow artificially.
David:
[57:24] Set up a genetic situation that would allow that paternal copy of that gene to be turned back on, that that could restore a lot of problems for Angelman, or at least it would turn UB3A back on. And so then the question got to be like, what kind of tool could you use to do that in a person's body instead of just in a cell or in a mouse? Because you can make transgenic mice, but you can't make transgenic people. So we have to be able to take the genetics that they have and then somehow turn this gene back on. So one of the first ways that that's been done is with something called an antisense oligonucleotide. So strictly speaking, not gene therapy, but it falls within the realm of what we might call molecular therapy. And antisense oligonucleotide is a whole thing in itself that I could talk a lot about. But let's say the way that this antisense oligonucleotide was used, is it basically was used to turn off the brakes that keeps this paternal copy of UB3A off. And so, by releasing the brake, UB3A was able to come on by itself, and that works.
David:
[58:38] Because antisense oligonucleotides, or ASOs, as they're sometimes called, they're just like a chemically synthesized stretch of something that's kind of like DNA. So they can be easily manufactured, easily purified. It's not like AAV, not immunogenic like that. And amazingly, you can inject these things and they get into cells by themselves. So they have many features that make them a very good first choice for therapy. And so the fact that it has these features that make it good for first-line therapy, and it worked, worked in the animal models and things like that, that one was the first going to therapy. I'll just tell you as a quick aside.
David:
[59:28] And this is just the history of Angelman syndrome, um, it was realized that this kind of approach could work, and it was shown in mice. At that point, there was a famous company that makes antisense oligonucleotides got involved in that. And when that happened, we thought, okay, well, this is it. We have a really good company that knows how to make these antisense oligonucleotides. They're involved in this. You know, they're trying to make a therapeutic. It's done. We're just going to sit back. We'll watch. And, you know, years, years went by. And, and, you know, it wasn't clear that that project was advancing forward. And I don't want to single out, you know, this company in any way. But let's just say this is an issue in the industry. And to bring it back around to what we were talking about earlier, you know, companies have an agenda, they need to make, you know, they need to be able to make progress on projects and have an income and make a return to their investors. And that's what keeps this enterprise going. So, they need to make a return on their people that invested in them. So, they have to set their priorities on what they're going to work on. And if this project doesn't seem like it's going to be a blockbuster for them, they might prioritize a different project. And that's just a hard cold reality of, you know, any kind of production.
David:
[1:00:57] So, you know, that project was not moving along that fast. And the next company that came along was, was a big pharmaceutical company, I won't mention their name, but everybody knows this company. And when they got involved, we're like, okay, wait, they're one of the biggest pharmaceutical companies in the world. They're making an antisense oligonucleotide also. This is done, you know, forget it. These guys, when they come in, it's all, it's finished, you know, just sit back and watch. And, you know, years went by and, you know, we didn't really know what was happening. So this is, you know, I'm talking as an academic. Now, if you were.
David:
[1:01:37] Parents with kids, and these kids are getting older, right? Years go by, your kids get older, and you're watching this happen, and you're saying, is there anything I could do to make this go faster, right? Because you're just sitting and watching, and it must be really painful. So a group of Angelman parents got together, they formed a for-profit company, and with that for-profit company that had investor income, they were able to develop their own ASO. They got a scientist, and he developed an antisense oligonucleotide for them. It went through all the same kinds of clinical trials, preclinical trials, animal models, safety, have to go to FDA, the first meeting, second meeting, the final meeting, the IND enabling study, you know, all of this stuff. They went through all of that in their own company that they had established. They became the first group to take their ASO into clinical trial.
David:
[1:02:34] And so, the first clinical trials were carried out by a group of parents of affected kids. So, this is a heartwarming story of people that went from being a victim of this disease to being kind of victorious over it and having, you know, children actually being treated in clinical trial with this drug that they, you know, basically, you know, through their hard work and effort and fundraising efforts, were able to make this thing happen. So on the one hand, it's a fantastic story in Angelman. On the other hand, it's also a horrific story.
Brent:
[1:03:10] Yeah.
David:
[1:03:11] Because I think no parents should ever have to lift a finger to get a therapy for their kid.
Keller:
[1:03:18] Yeah. And that's a beautiful story. What do you think is the future for these gene therapies? because obviously you're like, it shouldn't fall on the parents, but there is a very difficult relationship between business and academia. What does that look like in your idea? What's like the perfect future to get these technologies to where they need to go?
David:
[1:03:39] I don't know. I'll be honest. There's a lot of people working on this now. And with the understanding that, you know, now making the tools, it's still not easy, but like we we know that this can be done but the business part of it it's not clear that this can be done um and so what it will probably require is a rethinking and reimagining of how these therapies can be funded really and so um uh you know one of one of as just one of the many efforts um uh jennifer dowdner at berkeley has set up this institute for innovative genetics or the IGI. And this is one of the issues that they've been tackling. They have literature on this. They convened lots of discussions by stakeholders of all sorts. Um, and, uh, you know, trying to think about ways to reduce the cost of goods to, um, you know, incentivize companies to get into the space instead of getting out of the space, cause it doesn't look profitable. Uh, and to, you know, think about what can be done with in the nonprofit sector, uh, where maybe, you know, they don't, they don't need to make a return on investment to wall street, but maybe they're beholden to pay to make a return on investment to, to governments.
David:
[1:04:59] To NGOs, to something like this, you know, funded in a different way with a different idea. You know, that could be part of the future. I'm also a member of the American Society for Gene and Cell Therapy, and this is a major focus that their policy arm has been working on. And so, you know, they're working both in front of the scenes and behind the scenes with drug companies, with legislature, you know, talking to people at the nation's capital, at state capitals, trying to understand, you know, how can we get these things funded? You know, how can we set policy that will, again, attract the right people to be there? We need, you know, UC Davis can do many things. We can make drug therapies here. We can maybe even do a phase one clinical trial here. I don't know if we can make a phase three clinical trial here because that's very expensive. And, but we're never going to manufacture drugs, right? We're not going to be manufacturing and labeling drugs and selling it, you know, on the market and getting cash for that. Like, that's just not a university's mission. So it's going to be beyond what a university could do. But I think academic institutions have an important role to play in the early parts. Because like we were saying, there's so many of these diseases, it's such an unmet medical need.
David:
[1:06:14] And people can make these things. People in my lab can make these therapies. So we have a role to play here, but we have to get this other part sorted out, and that's very much in play today.
Brent:
[1:06:27] Well, as we wrap up here, do you have any closing thoughts, any messages beyond what you've already said, or maybe even specific to students who are interested in this type of field?
David:
[1:06:37] Yeah, I would say, I really feel like in medicine, we're just at a real turning point now, where it's really possible to be able to address genetic disease on a very genetic level, like really for the first time in human history. So in that sense, the future is very hopeful, that we might be able to address diseases that in the past we just had nothing to offer. But now we can do it, and we could do it relatively rapidly. But we need to work out the end part. And that part is really important because it's not enough just to make it a therapy if the people that need that therapy don't get access. So we have to make sure that we can make the therapy, that it's safe, that it's effective, and above all, there has to be a path to access.
Keller:
[1:07:33] Sweet. Well, thank you so much for your time. This is great.
David:
[1:07:36] Thanks for having me. It was a pleasure talking with you today.