 Hello, everyone, and welcome to the Hereditary Disease Foundation's second research spotlight webinar. I'm Megan Donaldson, CEO of the HDF, and I'm delighted to note that we have a very international group with us today, representing many countries, including New Zealand, Norway, Austria, Brazil, Croatia, Canada, Australia, and many more. Welcome. If this is your first time learning about us, the Hereditary Disease Foundation funds research to identify treatments and cures for Huntington's disease and other brain disorders. Our webinars feature brilliant scientists who are unlocking the secrets of the brain and making game-changing discoveries. We are thrilled to have with us today Dr. Christopher Pearson, Professor in the Department of Molecular Genetics at the University of Toronto and Senior Scientist at the Hospital for Sick Children. Christopher is also a member of the HDF's Scientific Advisory Board. Today Christopher will share his latest scientific findings, including information about a compound that he and his colleagues have identified that could delay the onset and progression of Huntington's disease. For those of you who may have just been learning about Huntington's, it is referred to as a DNA repeat expansion disease because of long repeated sequences in the DNA of the Huntington gene, which caused the disease. In addition to HD, Christopher studies other repeat expansion disorders and says that what he learns from one repeat expansion disease helps him better understand the others. Christopher also works on ALS. A fun fact about Christopher, when he was in his first year of college, the restaurant where he had been working throughout high school offered to send him to a French cooking school in Europe. He loved to eat and he loved to cook, but he also loved science. Unfortunately for the HD community, Christopher realized that he could always cook as a hobby, but he couldn't do science as a hobby, so he decided to stick with the science. It is my pleasure now to introduce Dr. Christopher Pearson discussing towards modifying the Huntington's disease mutation. There will be a question and answer session at the end of Christopher's talk. Please submit any questions in the Q&A box on the bottom right of your screen throughout the talk and thank you for joining us. So I'd like to thank the HDF for inviting me to present my work to you today. It's my great honor to try and convey what I work on. And I'm going to try to do so in a way that everybody would be able to understand it. Hopefully you'll be able to explain what I do and your disease to yourself better after my presentation. So today I'm going to be talking about modulating the Huntington's disease mutation. So I've been working on repeat instability as the basis for human disease for many years, and so I'm going to try and give you an update of what we've been doing. For those of you who want to quick read, there's a pretty lay friendly review article that we recently put out in Lancet Neurology. It's right here. I would be glad to share with you. My email is down below. And for those of you who want to get more in depth, there is a recent issue of the Journal of Huntington's disease, which actually has 12 different articles on targeting repeat instability as a potential therapy. This is a special issue that was put out by myself and co-edited with Leslie Jones and Vanessa Wheeler. So yeah, I'd be glad to share that with you, but it's actually publicly available free as well. So I'm going to be talking to you today about targeting repeat instability by attacking various proteins, DNA repair proteins, and I'll be touching on those. I will also be talking to you about a novel target, which is an unusual DNA structure and how a ligand can bind to that and change the mutation. So before I get into all of that, I'd like to talk about non-mindelian genetics. So Mendel, Gregor Mendel was a monk who many years ago was studying pea plants and he noticed that they made flowers. And when he crossed the two with white flower, this is an oversimplification for sure, white flower with a red flower, he ended up with one shade of pink. And that's what we call Mendelian genetics. However, Huntington's disease and other diseases with repeat expansions tend to be non-mindelian. And this is where genetic anticipation comes in. Essentially, there is a gradient of disease onset and severity in Huntington's families as you go through the pedigree. And that's known as genetic anticipation and that's shown here in this pedigree. So for instance, this individual with 37 repeats in the Huntington's locus had an age of onset at 68 years of age, while their daughter had 42 repeats and they had onset at 50. And then they ended up having a child with 86 repeats and they were born with disease at 30 years of age. So you can see how it progresses through the family getting earlier and earlier and larger repeats. So what is repeat expansion? Well, if we think of the genome, as though are written in three letter words, which in fact it is, genes could be sentences like the cat ate the fat, fat rat. So that would be your gene and mutations that we're familiar with like hemophilia, for example, would be a spelling mistake. The gat ate the fat, fat rat. What is a gat and does it eat fat rats or a loss of a base or an insertion base, which would make the sentence read gobbledygook. These would be stably transmitted and they would not be showing genetic anticipation. However, in the case of Huntington's disease, the mutation would be the cat ate the fat, fat, fat rat. And this would then get transmitted to even larger expansions. And this individual in their affected tissues would have ongoing expansions. So basically what my lab is trying to do is we're trying to put that cat on a diet. So it's not going to eat so much of a fat rat. So this is the brain of Huntington's individual. And I think you'll appreciate it that there's been a lot of cell death in the striatum in the cortex. This is very localized. And these regions of the brain show the largest expansions in an individual after they're born. Now this graph shows the age of onset for Huntington. So shown in the X axis is the size of repeat that's inherited and shown in the Y axis is the age of onset. And you'll notice right away that the larger repeats show very early age of onset, whereas the smaller mutant expansions show a delayed age of onset. You'll also notice that most people have 40 to 50 repeats. So that's 40 to 50 bend. Let's actually space it out 40, 41 each repeat unit. And you'll notice right away that as you increase the repeat size, you change the age of onset. So if you actually were able to take one of those repeats away from an individual, you would actually spare the onset by three years. That's a lot. Moreover, you'll notice that in that 40 to 50 range for any given repeat, let's choose here 47 repeats. These individuals all have 47 repeats, but this person has onset at 57 years. And this individual has onset at 11 years of age. That's a 46 year difference. What is it that makes things different? Well, there's clearly something that's making these individuals experience disease differently. A modifier perhaps. So Genome Wide Association study was done. Now that's a mouthful. What is that? They're looking for modifiers of disease in this study. And basically they found a bunch of DNA repair genes. And those DNA repair genes had polymorphic variants that made them able to delay the pace and disease onset. So that's a big mouthful. What is a polymorphic variant? You ask, whoa. So here's an example of a science paper that was published not too long ago, looking at variations in the color of butterfly wings or the shape of butterfly wing. Small genetic variants change the way these butterflies appear. They all look very similar and you'd say, oh, they might even be the same family or same species, but they're actually different because of the variants. Now variants can be quite dramatic. They don't vary dramatic here, but they can be quite dramatic. Where you actually change the coloring completely between two butterflies, or you could change the shape of the wing or even remove color. For example, this is a very beautiful butterfly. That's my screen saver. So we've noticed that in mice, Huntington's mice, there are naturally occurring variants. And in fact, we mapped a mutation in the mice and the variant in MSH3 of a mouse actually changes the repeat and stability. So what does that mean? That means that the variant allows for high expression of MSH3 versus low high expression, then gives rise to high repeat and stability. More of MSH3, more repeat expansions in Huntington's as well as in myotonic dystrophy. So it seems that naturally occurring variants in mice support the concept that repeat and stability is an important thing to target. And this is actually very satisfying because subsequently it was shown by other groups. So our debris season mostly that showed that this was actually occurring in the human MSH3 for Huntington's individuals. So what's actually happening? So what's happening is the person is inheriting a certain size of repeats. And over time, it's getting bigger and bigger in somatic tissues. And this is hastening the onset. And it's also increasing severity of the progression and of the disease symptoms. So we actually want to target this because it could actually be beneficial. So what you really want to do is you want to stop these somatic expansions or even better yet, you want to reverse them if you could. So how could you do that? Well, after many years of studying from my lab and others, we've identified a series of proteins. These are the same proteins which were identified in the genome wide association study. So they've been validated as good targets. And there's still other proteins yet to be discovered. And these proteins actually interact with these slip DNAs formed at the repeats and drive them into expansions. And so one could think about disrupting the interaction of these proteins with these DNAs so as to actually lead to arresting or reversing these expansions. So that's actually a pretty neat approach. That's pretty much what a lot of drug companies do. They look for ways to disrupt the interaction of the two entities. Well, if we're going to disrupt the lock and key interaction, there's more than just tacking the key. You could attack the lock. So let's think outside of the box. So we could not just target the proteins, but we could actually target the nucleic acid that they're actually going for. We could have a ligand like Na that might bind to this slip DNA. So the good news is we've actually discovered a target. And the target is slip DNAs. Okay. So I'm using big words again. What is slip DNAs? Well, that's a great question. My brother who's not a scientist. He's an animator. He says to me, what is the slip DNA you're always talking about? So I still am well calling. Slip DNAs are kind of like your genes. My brother's an animator. So he goes, oh yeah, I understand genes. I said, so you have two strands of your zipper. It comes together like Watson and Crick. He said, yeah, yeah, I do that every day. And I put my pants on. I said, that's great. I said, sometimes you have a base base mismatch in your zipper. You have two teeth that don't align and they could cause mutation. That's bad. He says, I said, don't worry, Colin. We have the mismatch repair system. It takes care of these mismatches. It's specifically made to correct those. Oh, that's good. He said, I said, well, if you're wearing unstable genes, you could have problems, particularly DNA. Really? I said, yeah, when you zip the zipper up and on your unstable genes, you could have a slip out of one strand or the other. Oh, not good. He says, I said, don't worry, Colin. We're trying to work at it. And you'll notice, Colin, the zipper has a Watson and Crick and they form different structures. So this is what I want to talk about. So we're targeting the CAG DNA. So we've identified a target. We've also identified a dark that hits that target. And that dark is NA. So essentially CAG DNAs pair with CTG DNAs. And you could have slip outs of CAG or slip outs of CTG. We've actually got some chemistry that binds specifically to this structure. Now, this is beautiful because this structure forms when the repeats being unstable. It's an intermediate. It doesn't form otherwise. So this is actually targeting repeat instability on the CAG slip out. So a lot of what I'm telling you has published in a paper, not too long ago in Nature Genetics. And I'd be glad to share that with you as well. But I'm going to give you the overview. So what did we do? The punchline of this paper is we took a Huntington's mouse with a repeat expansion and we injected into its striatum on the right side of it. Striatum, saline, salt solution. And on the other half of the striatum, we injected saline and NA. And we did this over a course of four weeks with multiple injections. After the course of four weeks, we isolated the brain and we isolated DNA from the saline treated or the NA treated. And we looked at the repeat for size changes. So this mouse inherited 165 repeats and basically over time, each of these little spikes here shows an increasing expansion. And you'll appreciate that this somatic expansion is occurring as the mouse ages inherited this size, but actually changes size. So it's a dynamic mutation. So this peak here is the medium spiny neurons. This is where the most devastating things happen with these expansions. So what happens in the NA treated side? So remember, this is the blue scan coming from the NA treated side. And we've represented the red side here. And you can see that in the same mouse when treated with NA, we have dramatically reduced the size of expansions in medium spiny neurons. Not only we reduced the amounts of them, but we've shifted them over. More importantly, we've shifted the size of expansion by seven repeats down below what that mouse inherited. This is important. This is all mass contractions of the repeat. So what can we say? We can say that NA actually induces contractions with extremely high efficiency in the brain, the central nervous system. It also induces contractions in essentially every cell in the striatum. It's also very effective in the medium spiny neurons, which is the vulnerable tissues. It also induces contractions to lengths that are shorter than the inherited length, and it does so with continued administration. And this is only a four week treatment. So we have the ability to arrest or induce contractions of the expanding DNA repeat. What about the RNA? Because DNA gets transcribed RNA and the RNA is also contracted because it's templated by the DNA and the RNA then gets translated into Huntington protein and the Huntington protein has the polyglutamine tract longer polyglutamine tracks give more aggregates of Huntington and this is a biomarker. So for contracting the DNA and the RNA, we should also be contracting the Huntington proteins repeat. And that should actually have an effect upon aggregation. In fact, this is a slice of a brain of the mouse has been treated either four times with saline or four times with NA. And I think you'll appreciate with the pink dots that they're fairly large and quite intense in number on the saline treated side. However, on the NA treated side in the same mouse over four weeks, I think you'll appreciate that we not only reduce the numbers, but we also reduce the intensity of these aggregates. So we're having an effect on a biomarker. When you actually treat with a drug specificity and hitting the target is really critical. And I can tell you we've done a lot of experiments to assess whether NA is hitting the target. It is. We are not chipping paint off of mom's wall. We are actually hitting the target. We've looked at a lot of different ways. We've done whole genome sequencing. The only thing that we see changes on are the expanded repeat. The non-expanded allele does not change. So how does this happen? Well, we've looked at these proteins that were identified in the G1 and studied by so many for so many years be involved. They're involved in repeatability. Maybe they're involved in the effect of NA. So MSH3 and MSH2, these are mismatch repair proteins. And remember, I told you that they're actually repairing base, base mismatches. So they actually protect you from mutations and protect from cancer. But strangely, they're like a Dr. Jekyll, Mr. Hyde. They protect from most mutations, but they actually drive CAG repeat expansions. They are required to actually form that mutation. Kind of an enigma of this protein. This protein complex is required for the mechanism of action of NA. Now, Phan1 is a new player on the field. And this protein has turned out to be a guardian against repeat expansions. And there are multiple studies that have shown that. And there's a lot still yet to be learned about this very interesting protein. And so it's actually thought to protect against hyper expansions. And it's a nuclease. That's why he's carrying a knife. And I'll tell you what a nuclease is in a moment. Basically, we've recently submitted a manuscript to bio archive, which is freely available. And just go to bio archive and pump and Phan1 and it'll come up. And basically it's a nuclease. And we show that it can actually be involved in modulating repeat expansions. So it's actually an interesting nuclease. A nuclease is something that cuts DNA. And this actually has two kinds of activity. It has the endonuclease, which is kind of like a guillotine. It just cuts it off like that. Or it has an exonuclease, which means it nibbles a little bit like a Pac-Man. And we've studied both kinds of nuclease activity in this one protein. Now this is a busy slide, but I can tell you that what it's showing is the endonuclease doesn't really care what the structure is or what the sequence is, whether they're repeats or not, it always cleaves the same way. And when we put NA with our DNA, it cleaves the same way as well. It's not affected. However, when we ask about the exonuclease activity, we actually get cleavage throughout every repeat unit. It's actually nibbling and letting go and pausing at each step. And this is very interesting only to repeat. So it seems that this protein deals with repeats differently. Moreover, when we add NA, it completely arrests that. It protects the DNA. So this is very interesting. So what do we think has happened? We think that there's transcription across the CTG repeat, making a transcript for the Huntington protein. And this is displacing the CAG DNA. And mutase beta is required because it actually forms these structures in the displaced DNA. And fan one, when it sees these, it tries to cut them out. And it does so by nibbling it. And so sometimes it nibbles out some of the expansion, but sometimes there's a little bit that gets away. So expansions are still occurring. But if you take this protein out, you actually have more expansion. So this is required to protect it. So what happens with NA? If we add NA to this system, NA will bind to that. And it will actually bind up all of this. This is unbound and this is bound. And this now blocks fan ones exonuclease activity. It can now no longer nibble at the DNA that's bound with NA. However, the endonucleolytic activity still works. In fact, as it actually cleaves here and it removes this NA. bound region, actually removing more than it would if it was just nibbling one or two nucleotides at a time. It actually removes this and that allows for contractions. So we know that mutase beta is involved in protecting from the base base mutations, but it's also driving these expansions. And we know that the other players that came up in the genome-wide association study, the modifiers of disease, these also participate in mismatch repair and protect the genome from mutations, but they also participate in the same way as does MSH2 and 3 by actually driving CAG repeat expansions. So it seems that these proteins are all Dr. Jekyll's and Mr. Hyde's. They're all involved in the same way. They're automatically giving rise to expansions. However, Fan1 is actually protecting against hyper expansions. So he's the guardian. But does he work alone? That's a good question. There is some data that suggests that these proteins, Fan1, might interact with these other mismatch repair complexes. And there's some recent data that suggests that they do interact. I'm sure that there are several groups that are working on this very interesting aspect of work. And we are looking at this as well. And we're trying to understand this interaction, understanding how these complexes interact and how they actually act together. Where these guys are driving expansions and this one's protecting, even if they're interacting is going to be a very interesting story, I'm sure. And this is what's coming in the future from my lab and others, I'm sure. So I told you about these different proteins that could actually be targets for therapy. And I told you about this unusual DNA structure and how it can actually be a potential avenue to look at inducing contractions. Now it's a long, long wait to get to gene therapy. And so we're patient. We're working very hard at it. So this is a little progress update. So are we there yet? Not quite, but we're on our way. So this is a lot of work by a lot of people and a lot of funding agencies. And so I'd like to thank all the people who contributed to making this happen. And I'd like to thank you. And I'd like to thank Dr. who's a ligand chemistry. He designed N a and Dr. and many other people, including the HDF. And I'd like to take questions now. Hi, Christopher. Thank you for that wonderful talk. I love all of the analogies that you gave the, from the cats eating fat rats and putting the cats on a diet and Dr. Jekyll and Mr. Hyde and the knight in shining armor, armor, locks and keys. You, the darts and the dart board, you really covered everything to illustrate all of your concepts so well. So thank you for that. So we have a number of questions that I will go through. And I want to start sort of down, down towards the, the bottom and everyone who's watching, please do feel free to continue asking questions and love it if you want to put your name in so we can know who you are. So the first question I'd like to start with Christopher is from an anonymous attendee asking, are there a significant number of scientists working on HD modifiers at this time? And are there diseases other than ALS that are similarly impacted by repeats with research also underway? Yeah, those are great questions. So the, the modifiers has turned out to be a very exciting area because of the realization that the modifiers all seem to be categorized in the same class. Most of them are actually DNA repair proteins, proteins that actually modify the DNA when the DNA is damaged. And in this case it seems that they are actually driving or protecting against the mutations. So there's a lot of interest in this because modifying the disease age of onset or severity or its rate of progression would be a good way to actually help individuals who are affected with this disease. And as to other diseases, it seems that these modifiers are also playing a role in other repeat expansion diseases. So that increases the interest of affected families in different diseases as well as of pharma in trying to drive this forward. And there are multiple other diseases. There's at least 50 different repeat expansion diseases. So yeah, the interest is pretty high. And ALS is one of the diseases that is also caused by repeat expansion, a different kind of repeat. And it's not all kinds of ALS. It's a certain particular kind in one particular gene known as C9ORF72. So these are good questions. Thanks for that, Christopher. I'm sure you could probably talk about the connection with ALS in much more depth, but I guess that's a topic for another time. Another question from another anonymous attendee who says, I have two questions. The first is the interesting number of three years. I'm curious as to how you were able to calculate this. I assume it was on a rather large sample size. Do you expect there would be a variation so that it might be perhaps five years for other people? The second question is where do you think this research might be ideally and realistically in 10 years? Thank you for your work and taking my question. Those are good questions. So let me deal with the number. So the number is based upon a very large sample size. In fact, the sample size that I showed you is a smaller subset of what's known. And the reality is it appears that if you lose the repeat, it would actually be that benefit. But it's really what it is is the ages of individual people have those on sets. And so the thought is that one, if one could remove that repeat, one would have such a benefit. And the point about whether it might differ from one person to the other, that actually is also a possibility because the thought is that individuals who have the lower, for example, 47 repeats at 11 years of age onset versus the individual with the same repeat at 50 plus years of onset. This individual have different DNA repair variants. And so if you remove a repeat from one, you might have a different effect in the number of years. What that exactly is we don't know right now, but this is what the hope is, is that we would have a benefit at that level. Did I cover all of the aspects of that person's question? You did. You did. 10 years. Where will the research be in 10 years? This is a great question. As a scientist, we always imagine where things are going to go scientifically. And we hope that they go along a certain path and a certain timeline. I would hope that if it's not NA, some other compound like NA would hopefully be identified that would be safely delivered to be able to arrest or reverse the expansions in affected individuals. And I'm also hopeful that other approaches towards treating Huntington's or other repeat expansion diseases will also be identified. For example, Huntington lowering, either lowering at the protein level or at the RNA level could actually be successful. And this would be a combinatorial approach. We could use something like NA to treat the DNA and you could also treat the RNA and the protein. So a multi pronged approach is probably a good thing. And I'm hopeful that in 10 years or less, we will be somewhere that will be able to actually treat individuals affected with repeat expansion diseases, including Huntington's. Terrific. Thank you for that, Christopher. I've got two questions that kind of cover the same topic. First one from Fernando Squitieri in Rome. And the second one from Ali Koshnan, who is one of our grantees at Caltech. So Fernando says, hi, Chris, very nice presentation. Do you expect such kind of methodology may affect differently large expansion compared with mild expansions? Mice all the time mimic pediatric HD. What do you think? And Ali asks a similar question. What are the minimum CAG repeat required for NA? Does it work in the range found in HD patients? Right. Okay. Both good questions. So hi, Fernando. Hi, Ali. So the length effect of the repeat instability, we know that the repeat is more unstable as it becomes longer. And it becomes more likely that it will form unusual structures as it becomes longer. And so the instability rate increases with longer repeats and NA, the mechanism of action, is that it targets those unusual structures that are formed in the repeat. So one would imagine that a certain point, the repeat might get short enough that it is no longer forming these unusual structures and hence no longer unstable. And so that's when I would suspect that NA or a compound like NA would no longer be effective. And I would imagine that would be coincident with the instability and the disease length because it's the disease lengths that are actually showing the ability to form these. So those are both good questions. And it's true in mice. They do have longer repeats because when one makes a mouse model, they're doing it because they're trying to model it. A certain aspect of the disease, not every model models all aspects. And the mouse models for instability are relatively longer repeats. So we've used mouse models with 120 to 150 repeats, 160 repeats or so. And so yes, the instability is relatively high in those mice. So working with human cell lines, we've used NA on the mice. So working with human cell lines, we've used NA on that where the repeat length in the cell lines that we've used has 43 repeats and NA is able to contract the repeat at the Huntington's locus. This is an important experiment because it shows the length effect, but it also shows the action of the compound on the disease locus. So that's in a human cell at the human chromosome where the mutation is there. So those are good questions. Thank you. Okay. I've got a couple of comments. I'll give you a breath opportunity to take a breath. So I've got some compliments here from, and pardon me, Ava Svirakova. Not sure where Ava is from, but she says good evening, fellow scientists here. I just want to compliment the awesome way you present complicated concepts with amazing simplicity. This is so refreshing and so nice to watch and listen. Thank you. And one more really nice compliment from someone named Elizabeth Aime saying, this is not a question, but rather an appreciation of your incredible ability to explain these things to a retired MD for whom the terminology is an unknown foreign language almost your cartoons are the best I have ever seen. Thank you. So some great compliments. Let's see what else we have here. We have some very technical questions and we have some that are not quite so technical. And so I'm going to go to this anonymous attendee who asks what is a biomarker? Okay, that's a good question. So I'm sorry, I do use a lot of big words in my science presentations. So a biomarker is a known signal. It could be it could be something the way the cells look or it could be a protein or it could be an RNA or some sign that's actually known to correlate with disease. And the case that I was presenting is the Huntington aggregates of the Huntington protein itself forms these aggregates of the polyglutamine protein. And you can see these aggregates in the brain and that's a biomarker of disease. If you get rid of that, you get rid of disease, presumably if you don't have that you don't have disease. And so this is a marker and indicator of the disease. Okay, I hope that that was a great answer. Got another question from Rita Chiang asking, does the phenotype of the HD mice get better when they get NA? That's a good question. So the biomarker definitely goes away. The DNA gets smaller, the RNA gets smaller and the aggregates get less. We have unpublished data that suggests that the mice actually behaviorally get better and their motor aspects get better, but this needs to be reproduced. And so I think that, you know, people are thinking, okay, so a lot of the experiments that we do are in mice. And this is actually an important aspect of science. You can't go to humans right away. So you want to do your experiments in mouse models. And so then people say, well, that's in a mouse. Is that going to be what happens in humans? And that's an important question because I mean, in a lot of cases an experiment in a mouse could be great, but it may not be reproduced in humans, which is why we have to take those small steps from the bench to the bench. And to learn if it actually makes a difference. The fact that it actually is having an effect in a mouse model is actually very promising, but it's not actually the end of the track towards the bedside. Okay. So I've got a question, another question from an anonymous attendee who says on the slide where you explain the time to get the disease and the impact on generations. Could you please tell me how you obtain the data for people with a great deal of longevity? If I read the graph correctly, it appeared to go well past the age of 100. If someone gets it at a really advanced age, do they experience difference in how the disease presents itself? Yes, so the rate of progression does change between individuals and the graph. I've drawn the graph, the graph was drawn so that you can see what the length is, but there were no individuals who were over 100 in that presentation. So I don't think that the repeat size extends an individual's life. If that's what you're inferring, I think that what it is is it shows that there is a rate of disease progression difference between different individuals who inherit different sizes. And for a given size, it isn't always the same rate between different individuals who have that same size. They might actually have different modifiers that actually advance the disease more or faster or slower. Okay, I have another question here that interestingly enough is actually from Christopher Pearson. So it says, hey there Christopher, I understand that this research is still a long way from people, but in theory, what would the success rate be when applied to a person? In theory. Right. If we are lucky enough to get to the point where we're able to administer something like NA to humans, I would hope that it would have the same rate of contraction as it does in the mouse and I would hope that it would continue. But actually when that might be, I mean, I work with hope in my heart that what we do in the lab will actually lead towards treatment. But whether I can say that it's going to happen, I can't be sure. I can tell you that I promise I'm going to work the hardest I can to make it work, but I can't guarantee when it will be in humans. Okay, I'm going to go to one of the technical questions. So, from Jonathan Monkmeyer saying awesome research in C9-ORF 72, the CAG repeats are in the introns and introns get excluded in the final assembly of the messenger RNA protein. Since introns are not making protein, does that mean that the actual toxicity is only in the RNA of the intron? CAG repeats and RNA produced DS RNA hairpins that look like a virus. So everyone who is not a scientist, please bear with us as Christopher answers this one. That was a big question. So I actually stand back and I look at the results and I know that there are a lot of toxic entities present for the C9-ORF 72 repeat expansion diseases. The RNA is definitely being expressed from both strands. So it could be either of those. And there's a barren splicing of the transcript mutant RNA. And there's also ran translation proteins that are being produced from both strands and could be the ran peptide, but it could also be the downstream peptides. But the data so far do not so exactly what it is. That is the toxic disease causing aspect. I mean, all of those entities are there. There's even reduced expression. So it's not really clear what it is. I think that, you know, we are in a sense as scientists, scientists lucky that the C9 was not the first disease discovered because it's probably one of the most complicated of all the repeat expansion diseases. So yeah, I'd say we're still waiting for more results to be sure as to what the toxic entity is for the C9-ORF 72 expansion. Okay, and we've got two questions from a person named Nikhil Ratna saying, asking, could these mechanisms, and this is touching on what you talked about earlier, could these mechanisms be shared among all repeat disorders? And if so, why different types of repeat mutations? HD, SCA, FRDA, et cetera, do not co-occur? Or is it specific to the type of repeat? Right. Okay, so let me address the latter part first. So a person with Huntington's disease is born with an expansion in the Huntington's repeat in the Huntington gene. They are not born with a CAG expansion in the SCA1 gene, for example. And so co-occurrence would be having those both. And there are rare individuals who have had, for example, myotonic dystrophy and Huntington's. And that's just because they actually have expansions in both genes, but that is not a likely situation. It's very unlikely and would be rare. Whereas the question about the modifiers at the DNA repair level, it is very likely that the same proteins are participating in somatic expansions as well as in transmission expansions for one repeat to the other. And that doesn't just limit it to CAG CTG repeats. So it's also known that a lot of these DNA repair proteins hacked both on CGG, CCG, CAG, GAA. And it encompasses a lot of the CAG CTG diseases, including the CGG, Fragilex and the Friedrich's Ataxia. So a lot of those will be modified by the same DNA repair protein. So targeting those proteins is likely a good approach for those diseases. And about targeting the nucleic acid, every one of them are likely to involve a different DNA structure. Whether it's going to be NA or some other compound like NA is something worth looking at as a scientist, I would say. Great. Thanks for that. I'm going to go to an anonymous attendee who asked, are there any pharmaceutical companies who are involved with NA research? Yes. That's a great question. So we are working on partnerships with this right now. And we're open to partnering in looking at other opportunities to find other compounds for other repeat expansion diseases, including ALS. Terrific. We got a couple of new questions coming in, which are kind of similar from a Diego Castillo saying, Hi, Dr. Pearson, great presentation. My question is, if there is treatment, what would be the delivery system to administer it? And then a similar question from Carl Leventhal saying what are the pharmaceutical properties of NA, such as solubility, crossing blood, brain barrier, et cetera, doesn't have known toxicity? All right. So the more technical questions, I'd be glad to answer those in an email. And we do have some information, and they are pertinent to the first part of the other question by Diego, the delivery mechanism. What we know experimentally, and a lot of this is included in the NA, the Nature Genetics manuscript that's been published, and I'd be glad to share that with you, the mechanism of action suggests that you don't need to completely saturate the DNA with NA to actually have an effect. So that might suggest that you don't need a lot. Delivery, it is a small molecule by definition of the molecular weight. So delivery should be facilitated. However, when we do experiments to get it into a mouse brain, it gets in, but the experiments that we did, we injected it stereotypically. So delivery into the periphery into the blood system would not get enough into the brain of a mouse, but in non-human primates, we do have some evidence that it can get in. But how much is necessary is a good question, and we don't know that yet. So we're thinking about other modes of delivery or modifying the chemistry of NA or NA-like compounds so that it would have better delivery. These are good things to think about. So we still have a lot of great questions, and we've only got seven minutes left, and I just want to remind everybody that any unanswered questions will be sent to Christopher, and he will answer them directly to you if you've provided your email. I want to just acknowledge here Patricia Hillman, and thank you so much for writing Patty. So Patty writes, hi, Patty Hillman here, and this is a really difficult question, and I'm sorry to put this to you, but I did want to acknowledge Patty saying, Thanks for presenting this at a level everyone can understand. I'm 59 and have been dealing with cognitive impairment for over 10 years. I'm now experiencing physical symptoms in my gait balance, dropage, etc. My expectations are what you've described will probably be available, if at all, around the time I'm wheelchair bound. Do you believe that what might be available to the public will still be available to help HD symptoms that are advanced? Thank you. And thanks, Patricia, for writing. Appreciate that. Okay, that's a tough question, Christopher. It is a tough question. So, I'm going to be really honest, most of the community knows that in the scientific world, when you treat a mouse model, for example, the best results come from animal models that are pre-symptomatic. And if one were to do such an approach where you would want to contract the repeat, you'd want to contract it when it's not too large. But that doesn't mean that there is no hope. There is there is hope in learning about whether it can be contracted and if we can do it. And clinical trials would move slowly. You wouldn't actually go to the most severe cases. Initially, you'd go for cases where you might have some effect. And then you'd go further beyond that. So, I would hope that we would be able to do that. But I, again, I'm kind of cautious in my expectations. So thank you for that question. And I'm glad that you were able to understand better what's happening scientifically. It is an exciting time, but we're all working great hard at it. So, I'm going to do one more question. There are so many, we could go on for another hour. There's so many great questions about cancerous side effects and the more about your drug. And, but I'm going to do a final question from an anonymous attendee who asks, I'm not a medical scientist, so please forgive me if this is a naive question. What role has the mapping of the human genome played in your research? So I think this is a great question. I think that's a great question as well. And there are no bad questions. So I'm going to try and answer the question. The mapping of the human genome has had a huge, huge effect. The ability to do these studies where people identify modifiers is unbelievably empowered because of that. In fact, what was done in the genome-wide study was known variants that already exist. People were looking for those known variants. Looking at the sequence level, we can identify new things that aren't known. And in earlier this year, a paper was published where autism spectrum disorder was looked at by a colleague of mine, Dr. Ryan Ewan, in my institute. And basically, he sequenced the genomes of individuals with autism and looked for disease-causing mutations and found repeat expansions as associated with autism. And this actually is powered because of the ability to do sequencing of the human genome. So I think that it's a very powerful thing that we've been able to learn and harness from that. So thank you for that question. And that wraps it up for all of the questions. Thank you, everybody, for asking all of these great questions. And again, Christopher will receive copies of the questions and he will answer them directly. So I'd like to say thank you for the Redditorial Disease Foundation. We'd like to thank Triplet Therapeutics for underwriting our research spotlight webinar series. And we would also like to thank Neurocrin, Biosciences, Novartis, and Unicure for sponsoring today's webinar. Thank you, Christopher, for your extraordinary research and the time you've spent with us today. With your research, we're better able to serve the patients and families with Huntington's disease and so many other brain disorders. And thank you to all of you for attending today, this meeting, to learn and share with us. To continue our research spotlight webinar series, please join us in September when Dr. Brent Fitzwalter from the Broad Institute of MIT and Harvard and Dr. Rachel Harding from the Structural Genomics Consortium at the University of Toronto will discuss their work. Registration will open on our website soon. Thank you again, Christopher, and each of you for joining us today. We really appreciate it. Have a great rest of the day.