RNAi Roundtable: HTT02 - ALNYLAM PHARMACEUTICALS INC

Hello, everyone, and welcome to this RNAi Roundtable. We're happy you were able to join us. I'd now like to pass the call over to the company. Good morning, everyone. Thank you for joining us for today's RNAi roundtable, where we'll be discussing Brezosiran, also known as ALNHTTO2, which is in development for the treatment of Huntington's disease. I'm Josh Brodsky, Vice President of Investor Relations at El Nile. With me today are Pushkal Garg, our Chief Research and Development Officer, Kevin Sloan, Vice president of Development Programs and Program Lead for Brazosaran, and Toby Ferguson, Senior Vice President and Head of the Neuroscience Therapeutic Area at El Nile Island. We're also thrilled to be joined by Dr. Jill Bates, Professor of Molecular Neurosciences and co-director of the Huntington's Disease Center at University College London, who will provide perspective on the role of the HTT1A isoform in Huntington disease biology and why targeting exon 1 may be important for HTT lowering strategies. Today's RNAi Roundtable is part of a series of roundtable webinars that we've been hosting to review progress across various clinical stage development programs. Roundtables are archived on both the Capella section and events page of our website if you'd like to access a presentation or view the replay. At the conclusion of today's presentations, Pushkal will moderate a Q&A session. If you'd to submit a question, you can do so at any time during the event by using the Q& A feature directly within the webcast platform. Almilam has engaged Dr. Bates as an independent academic speaking in in her personal scientific capacity. She is not speaking on behalf of University College London or UCL consultants today. Nothing said by Dr. Bates constitutes investment advice, nor should it be relied on in making investment decisions. And as a reminder, we will be making forward-looking statements during the webinar, and we encourage you to read our most recent SEC filings for more complete discussion of our risk factors. And with that, I'll now turn it over to Pushkal to kick things off. Pushkul. Thanks, Josh, and welcome, everyone, to today's RNAI Roundtable on Brazil's Saran, previously known as ALN-HTTO2. This program takes a unique approach to tackling a very devastating inherited neurodegenerative disorder, Huntington's disease. As many of you know, Alnylam spent over two decades pioneering RNA interference as a new class of medicines. RNAI allows us to silence disease-causing genes with a highly specific, potent, durable, and reversible approach enabling infrequent administration. We've translated that science into six approved medicines, and we continue to expand the potential of RNAi into new targets and tissues. Our R&D strategy is focused on maximizing that potential by combining human genetics, advances in tissue delivery, and continued innovation in platform design. We apply those capabilities with discipline, pursuing genetically validated targets in serious diseases where we believe RNAI can have meaningful impact. To date, this strategy has yielded what we believe is one of the most robust pipelines in the biotech industry. With over 25 programs spanning rare, specialty, and prevalent indications across multiple therapeutic areas, we believe this pipeline underscores the depth of opportunity we have at Alnylam to enable growth and deliver more transformative medicines to patients. And we're now entering a period of significant clinical momentum across that pipeline. The Huntington's disease data we're discussing today are part of a growing cadence of important clinical readouts expected over the next several years. These will be accompanied by an increasing number of programs moving into phase three development as we continue to build towards our Alnylam 2030 goals. As I mentioned, a major advance in extending RNAi beyond the liver has been our C16S RNA delivery platform, which opened the door to targeting the central nervous system. We've seen robust targeted engagement and broad biodistribution across the CNS and non-human primates, and we've seen that now reproducibly translate into knockdown of disease-causing proteins in the CNС, first with Mivelsiran, our investigational APP targeting program, and as we've just announced with initial Phase 1b data, now with Verzosiran, which targets Huntington. This successful translation has provided the confidence for us to build a growing pipeline of investigational CNS-targeted RNA and AI therapeutics in collaboration with our colleagues at Regeneron, including programs in Alzheimer's disease, Huntington's disease ALS, and Parkinson's. Grisoceran is part of our efforts to apply RNA to serious neurologic diseases where meaningful new treatment options are desperately needed. With that, I'd like to hand it over to Kevin Sloan, who will discuss the unmet need in Huntington's disease and the approach that we're taking with brazosurin. Kevin? Kevin Sloane, Brazosurine and L-Nylum, L-O-N-I-L-U-M, B-U. Thanks, Pushkal. I'm Kevin Sloanne, the program lead for brazosuran and l-nylum. It's a pleasure to discuss the exciting progress and promise of this program with you all today, as we're working with urgency to improve the lives of people living with Huntington disease. Before I begin, I would like to note that we've been partnering closely with the broader Huntington's Disease community throughout the life of this program and have sought to learn from the many efforts that have come before us. We're very grateful for the support we've received to date and are committed to maintaining that partnership as we move forward. Huntington'S Disease is a progressive, fatal, monogenic neurodegenerative disease. It affects movement, where patients can experience chorea or rapid involuntary muscle movements, incoordination, and falls. It also affects thinking, which can manifest as executive dysfunction and memory loss. It can affect mood, leading to depression, apathy, personality changes, and suicidality. Symptoms of the disease typically appear at midlife during prime working and family years. An average lifespan is shortened to approximately 10 to 30 years from diagnosis. Because HD is inherited in an autosomal dominant pattern, each child of an affected parent has a 50% chance of inheriting the disease-causing mutation. We estimate that there are more than 100,000 symptomatic patients globally, with many more individuals who may be pre-symptomatic or undiagnosed. Huntington is a well-known genetically validated target for Huntington's disease, which is caused by an expanded CAG repeat in the first exon of the Huntington gene. A normal Huntington allele contains 26 or fewer CAG repeats. Individuals who inherit an allele with 40 or more repeats are expected to develop HD during their lifetime. Now, the length of the inherited repeat is only part of the underlying biology. The CAG repeat tract can expand further over an individual's lifetime through a process called somatic instability. Those expanding CAG repeats are thought to drive accumulation of mutant Huntington proteins that are toxic to cells, including full-length mutant Huntingtin, and also a shorter mutant Huntingtin isoform called HTT1a. Both of these protein species are believed to contribute to neuronal toxicity and the downstream pathophysiology of HD. Importantly, the disease process begins decades before symptoms. Changes in CSF or imaging biomarkers can emerge well before cognitive and motor clinical signs, and clinical signs precede the functional decline and increasing loss of independence that ultimately defines later stage disease. This long disease course may provide an opportunity to intervene before significant functional decline. Yet, Huntington's disease is often diagnosed only after clinical signs or symptoms have emerged, underscoring the urgency of identifying and treating earlier in the course of disease. As I've mentioned, the root cause is an expanded CAG repeat in the first exon of the Huntington gene or exon 1. Due to alternative splicing during transcription, the mutant gene can give rise to more than one protein species, a longer, full-length mutant Huntington encoded by 67 exons, and a shorter mutant HTT1A isoform encoded by only that first exon. Both full-language mutant Huntington and HTT2A are implicated as toxicity drivers, and Dr. Bates will go deeper into the biology of HTT3A and preclinical evidence supporting its role as a driver of disease. Importantly, not all past or current Huntington lowering drug development approaches have been able to effectively address HTT1A. That's because many of those target sequences that fall downstream of exon 1 and thereby are only able to lower the longer full-length mutant Huntington protein. When we designed brazosurin, we considered several possible strategies, lowering HTT2A alone, lowering full-ength Huntington alone, or take an inclusive approach to lower both. We ultimately selected the latter, taking an X-on-1 targeting approach designed to lower all relevant Huntington species in the CNS, including both full-length mutant Huntington and HTT1a. Huntington's disease researchers and industry partners have long been trying to tackle this disease, and Huntington lowering has been the primary targeting strategy for therapeutic development since the responsible gene was identified in 1993. However, progress with Huntington lowering has been hampered by technical issues across several different drug development modalities. Unfortunately, the molecule and or platform-related toxicities associated with some early-generation antisense oligonucleotide and small-molecule splice modulators is often conflated with the safety of Huntington-lowering itself. For example, some Huntington–lowering ASOs have encountered exposure-related safety signals, including increases in a biomarker of neuroaxonal injury called neurofilament light, or NFL, even with relatively limited levels of Huntington lowering below 50%. The ASO Tomonersen, which was designed to lower full-length mutant Huntington and wild-type Huntington, was reported to cause dose-dependent NFL increases within six months despite moderate CSF mutant Huntingtin reductions. The ASOW-WVE003, which was designed to lower full-length mutant Huntington but spare wild-type Huntington through an allele-selective targeting, was also reported to cause large increases in NSL within six months, despite modest CSS mutant Huntingtin reductions. So a couple of key questions remain unanswered. First, how much mutant Huntingtion lowering may be needed to maximize efficacy? And second, how many mutants are needed to make sure that the mutants that are in the wild-like Huntington lowering can be safely tolerated? Here at LNILAM, we believe there is still tremendous potential in Huntington-lowering. and we're hopeful that any therapeutic program will succeed at bringing forward a disease modifying therapy for patients who are in such urgent need of progress but we believe the technical details of the approach brought to bear on this long-standing challenge are critically important taking into consideration both the underlying platform modality as well as the specific genetic targeting strategy our approach builds upon many learnings in the field with the the potential to avoid some of the challenges that I've just outlined. Rhizocerin is an intrathecally administered investigational C16 RNAI therapeutic for Huntington's disease. It targets a conserved mRNA sequence within the first exon of Huntington, leading to reduced expression of all mutant Huntington species in the CNS, including both full-length mutant Huntingtin and the shorter mutant HT1A. Because the first axon is shared across mutant and wild-type alleles, our approach is also designed to lower wild-types Huntington. RNAi therapeutics are specific and durable, but fully reversible with no permanent changes to the genome. Brezosirin leverages the same C16-sRNA platform that underpins our broader CNS pipeline and has shown a consistently encouraging safety profile to date across five ongoing clinical programs. We're highly encouraged by the preclinical data we've generated over the years. In non-human primates, rosocerin has produced deep dose-dependent Huntington lowering sustained for months after a single intrathecal dose. Importantly, lowering was observed across key CNS regions that are central to Huntington's disease biology, including striatum and cortex. We feel this is important as HD is a whole-brain disease. Early on in this program, we took our time to thoroughly interrogate the safety of Huntington lowering with our C16 sRNA platform. Across a number of single- and multi-dose NHB studies, up to 12 months in duration, and including completed GLP chronic toxicology studies, we have observed an encouraging safety profile for prozocerin, no neurological abnormalities, no elevations in CSF inflammatory markers such as white blood cells or protein levels, no elevation in CSS neurofilament light chain, histological changes, and no adverse findings, even after very deep wild-type Huntington lowering beyond 90%. To summarize, we see three key reasons for hope for the brazosurin program. First, the C16-SRNA delivery platform has been shown to provide a safe, durable approach to target engagement in the CNS. Second, brazosuran gives us the potential to explore deep and widespread Huntington lowering throughout the brain with a durable and reversible approach. Third, its Exon 1 targeting strategy is designed to lower all relevant Huntington isoforms, including both the full-length mutant Huntington and the shorter toxic HTT1A isoforme. With that, I'd like to introduce our guest speaker, Dr. Jill Bates. Dr. Bates is professor of molecular neuroscience and co-director of the Huntington's Disease Center at University College London. She has made numerous contributions to the HDE field over her career, including developing the first mouse model of HDE. She's been an elected fellow of the Royal Society since 2007 and was recently awarded the society's highly prestigious Farrier Medal for her work in understanding the molecular basis of Huntington disease. I'll now hand it over to Professor Bates to tell us more about research on the biology of Huntington's and the potential importance of HG1A. Dr. Bates? Thank you very much, Kevin, for that kind introduction. So I'm going to begin by just outlining some of the basic features of Huntingtin's disease, and then I'm going to tell you what we think of the very first steps in the pathogenesis of Huntingtin disease, steps at the molecular level, and then go on to talk about three experiments that have all been published this year pertaining to lowering Huntington in mouse models of Huntington's disease. So next slide, please. So as we've heard, Huntington disease is an autosomal dominant disorder. It generally starts in midlife to late old age, although there are cases that begin in early childhood or adolescence. So, as you'll see in the video that's about to play, the movement disorder can be quite complex. This lady is presenting with the choreiform dance-like movements that are so clearly associated with Huntington's disease, but you can also see that she has dystonic posturing, she has problems with fine motor coordination, she Has abnormal eye movements but the motor coordination is not always the first sign to begin and Kevin's already told you something about the psychiatric and cognitive impairments and these can often be the first signs many years before the movement disorder kicks in and there are also systemic features like sleep disturbances and weight loss. Next slide, please. So we've heard that the mutation that causes Huntington's disease is a CAG repeat expansion in the first exon of the Huntington disease gene. This is a large gene spanning 67 exons. If you have 35 CAGs or less, you will not develop Huntington'S disease. 40 or more CAGs will result in Huntington's disease within a normal lifespan and people with 36 to 39 CAGs have an increased chance of developing the disorder. So this encodes a large multifunctional scaffold protein that's present in every cell and the CAG repeat encodes a stretch of glutamine residues and the mutation imparts the gain of function to this protein. so whilst the CAG repeat expansion is related to the age of onset of the disorder it's not the only factor and as you can see from the graph on the left hand side that the those early onset cases beginning before the age 20 so do have long repeat have have longer CAGs here going up to 120 and that is clearly associated with the age of onset but for a CAG repeat of 40 or so then onset can begin in any time from 20s up to the 70s or later and so there must be other factors involved and we know that these are some of these genetic modifiers next slide please and if if we look at the neuropathology of Huntington's disease here we're looking at the coronal sections through a brain from somebody who's unaffected with the disease and one of somebody who died from late stage HD. As Kevin said this is a whole brain disorder there's a generalized atrophy across the whole brain and then on top of that you have a selective neuronal cell death so this is very apparent in that region of the striatum or the chordate putamen which here has been reduced to a rim of tissue because 95 percent of the neurons in that région the medium spiny neurons are ones affected by the disorder. But there's cell loss in many other regions of the brain as well. And on top of this, we have an aggregate pathology. This cannot be detected by classical histological stains, as you would see in Alzheimer's or Parkinson's disease, but requires immunolabeling with an antibody to the very end terminus of the protein. And that reveals these inclusion bodies. As you can see in this figure in D, there are inclusions within the neuronal nuclei. Otherwise, they're scattered throughout the neuropil lining up in processes and they're present in pre-symptomatic brains. Next slide, please. So Kevin has mentioned somatic CAG repeat expansion, and we now think this is the first step in the disease pathogenesis. So what is this repeat expansion? Next slide, please. So as we work our way through this slide, the top left hand corner, as I've mentioned, more than 40 CAGs, 40 or more CAGs if we measure in blood is a fully penetrant mutation. But the CAG repeat expands in specific neuronal populations in the brain throughout life. And the first indication that these could be very long repeats came from Peggy Shelbourne's work, this image shown here, where she could show that in the striatum, she could detect rare alleles of repeats of several hundred, up to 700 CAGs. So we've known that for more than 20 years, that some of these very large expansions can be present in the brain. and then genome-wide association studies over the last 10 years or so have produced high quality reproducible data and irrespective of what the phenotype that they're measuring is here shown as total functional capacity it could be age of onset or other symptoms a subset of those at least half of those modifiers turn out to be genes in the mismatch repair pathway So why might that be interesting? It's because we've known since 1999 that if you knock out one of these mismatched repair genes in the mouse, you prevent the somatic expansion from occurring. So putting this together with the modifiers that modify age of onset and progression of disease with the somatic expansion, we now believe that the first step in the pathogenesis of Huntington's disease is this specific repeat expansion that occurs in the vulnerable neurons within the brain. Next slide, please. So if that's the first tip in the pathogens is what might be the second next slide. So here again, we have this the gene with 67 exons. This is transcribed to a pre-mRNA process to produce the mature mRNA as shown on the right hand side of this slide. And then that encodes this large protein. And we know that some proteolysis of that protein occurs. occurs. But we found a few years ago that with the presence of a long repeat, transcription can terminate with intron 1, producing this small exon 1 only transcript called Huntington 1A. And this encodes the Huntington1A protein or exon protein. And we've known for 30 years that this protein is very, very aggregation prone and highly pathogenic. So is it the Huntington 1A protein that's the effector through which the expansion causes these pathogenic consequences? Next slide, please. So drilling down into this just a little bit more detail. What happens is that when you have a long CAG expansion, cryptic polyacytes within the first intron become activated and the transcription terminates at those sites. So in human Huntington's disease first intron is 12 kb, in mouse it's 20 kb and in both cases there are two sites activated in humans 2.7 and 7.3 kb into the intron and in mouse 680 and 1145 base pairs and in those cases it's called Huntington 1a. Next slide please. So I'm just going to introduce the mouse models that we work with as all of the work I'm going to be talking about has gone on in mouse. So these are knock-in mouse lines. And the mouse exon 1 has been replaced with a human exon one with different length CAG repeats. These were made in the labs of Marcy MacDonald and Scott Zeitlin. And there's an allelic series of these mouse models with CAG repeat expansion shown in this table. So there's a line with 20, 50, line 80, 111, 140. And one called 175 actually has stabilized at 190. So we've used this selection of mouse lines to say what would happen as that CAG expands through a somatic expansion in a single neuron to its effect on the Huntington 1A transcript and protein levels. Next slide please. so here we have as you can see on the x-axis of these graphs these various lines with the expanding CAG repeat and in each case we're looking at wild type heterozygous and homozygious mice and all of these mice were the same age at an early that's their young mice they're all the same age and so the blue line shows you the amount the blue bars show you the amount of Huntington 1A in wild type mice, the orange ones in heterozygous mice, you can see it's increasing with increasing CAG repeat length, and the silver ones in homozygus, which is twice as much as the heads. And in the panel on the right hand side, we have an assay that is specific to the Huntington1A protein. And again, we're seeing that increase with increasing CAG repeat length. Next slide, please. So the Huntington 1A transcript and protein is made at higher levels as the CAG-REPEAT expands. Can we detect this in human cells or tissues? Next slide. So we've used digital droplet PCR. We've developed digital droelet PCR specifically to the human Huntington 1a messenger RNA and this is showing the levels that have been detected in post-mortem brains in the sensory motor cortex and in the hippocampus and the CAG repeat length is on the bottom of the along the x-axis it's moving actually I think we so we can you can see there are increasing levels at the level the amount of Huntington1a is increasing with increasing CAG repeat length. And it's most apparent in those brains from people who have had longer repeats and have developed the disease in childhood and in adolescence. Next slide. But it's challenging. Next side, please. It's challenging to identify this inpatient brains because we have to think about what the CAG repeat length is in most of those cells. So some of the outstanding work in this area has been done by the McCarroll Lab, who have taken post-mortem brains and in this case, in this sorted the nuclei from the striatum of those brains and then measured the CAG repeat length in those cell nuclei. and so if you're looking at a brain if we look at that plot along the top if you looking at brains that start off with around 40 or 42 CAGs from in the blood as would be measured in the blood the CAG repeat is expanded in most cells but in most cases it's still in this range of less than 100 here with a median of around 70 and it's only two or four two to five percent of the cells that have these very longer repeats, more than 100. So given that the Huntington 1A is associated with the long CAG repeats, and that's where we're seeing the most generated, when we're looking at bulk RNA, this is going to be diluted out by the fact that most of the cells have shorter repeats. And of course, there's all of the cells that are not the vulnerable neurons in which no expansions have occurred. And this is why we see more of it in the brains from people who are starting with longer CAG repeats. Next slide, please. Can we see the protein? So we can see the protein in the fact that it has aggregated. So if we're looking at on the left-hand side, we have brain sections from someone with Huntington's disease. And on the right-hand side, it's a control brain. The top panels are immunoprobed with an antibody to the beginning of the Huntington protein, and we can see these inclusion bodies. And if we use an antibody that is specific to Huntington 1A, it only detects that small protein. Again, we can now see those inclusion bodies, so that means that within these cells, there was sufficient protein to aggregate and form these very large structures. Next slide, please. So, if we want to lower Huntington, what strategies are available to us? Next slide. So, on this slide, on the left-hand side, we have the nucleus in gray and the cytoplasm on the right-hand of that. There is the Huntington DNA. And then within the nucleus, we have the Huntington 1A pre-mRNA and the full length Huntington pre-MRNA that still contains the introns. So the full-length RNA is spliced and exported as a mature mRNA to the cytoplasm, and the Huntington-1A is transported to cytoplasm. And there, of course, they're translated to generate proteins. So if we wanted to target the full link Huntington alone, as Kevin said, we can target the the the transcripts from exon two downward so you might be able to want to do that with antisense oligonucleotides or small molecules in this in and they would work in the nucleus or with rnai si rnas micro rnas and they might work and they Would work in a cytoplasm so if you target from exxon two downwards you'll just reduce the level of full-length huntington alone If, as Kevin mentioned, you target exon 1, either with ASOs or small molecules in the nucleus or RNAi in the cytoplasm, you'll reduce the levels of both Huntington 1A and full length Huntington. If you want to target Huntington 1A alone, you're really restricted to using RNAi in the cytoplasm against the intronic region that's left on that Huntington1A messenger RNA because those intronic regions are still left in the nucleus. So you would, if you target that in the nucleus, you'll again knock down both species. So next slide, please. So the question then is, what is the relative pathogenesis of Huntington 1A and the full-length mutant Huntington, and should we target full- length Huntington alone, Huntington1A alone, or full-lenght Huntington and Huntington-1A? Next slide. So I'm going to describe three pieces of work that were all published this year. year. I'm not going to have time to show very much of the data so I've just selected a couple of results from each study but the reference for the papers is on the bottom of the slide so if you want to follow up and look at it in more detail then the papers are available. So the first approach we used was to try and prevent the Huntington 1a protein being generated in a mouse model and for this we used the Q150 knock-in mice and removed the cryptic polyacytes from intron 1 so that transcription could not terminate in intron one and this so this was to prevent the generation of Huntington 1a and the mice with the deletion in the intron are called the delta intron mice. Next slide please. So these western blots show how much we've managed to reduce the Huntington 1A protein. This is Huntington that has been immunoprecipitated with an antibody to the mutant protein. So the wild type lanes are all negative because there's no mutant protein and then in the first panel we have probed with an antibodies that will detect all fragments. We have full length Huntington at the top, Huntington 1A down below. Then there's two antibodies to Huntington 1a that are specific to Huntington one aims you can see that in the delta intron mice there's very little left and then the final panel shows you an antibody to exon two which doesn't detect Huntington y and again now we don't see it next slide please so what is the effect of removing Huntington1a or decreasing the decreasing the level so much on aggregation so if you look at the top two rows, those are striatum. The bottom two rows are deep layers of the cortex. The top one in each case is the knock-in mice. The one below is the delta intron mice. And we're looking at aggregation from two to 17 months of age. If you look at the top six-month panel, you can see that diffuse staining. That is an aggregated form of the protein. So within the nuclei, you get this diffuse aggregation accumulating, which resolves as a nuclear inclusion as the disease progresses. And you can see in the delta intron mice, this has been slowed down considerably. Next slide, please. And in these genetic models, one of the things we were able to do was look at biomarkers that are measured in the CSF for Huntington's disease. So one of these in neurofilament light chain and the other is one called YKL40 in humans. And here it's BRP39 in mice. So Kevin talked about neurofiliment as a safety biomarker, but it's also a biomarking that accumulates with disease progression in HD. So here we're looking at we have a wild type, the wild type mice with the deletion in the intron, the knock-in mice and the knockin mice with a deletions in the entron. we're looking at 12 and 17 months of age and if you look at the blue dots you can see that the nfl and the white mouse ykl40 both increase uh with disease progression and when you look at the red dots which is for the uh delta intramyse there is absolutely no nfl or ykl 40 there so it's completely rescued the levels of these translatable biomarkers in the csf if you're not making the Huntington 1A protein. These mice, of course, still have all of the full-length mutant Huntington. Next slide, please. So the next project I'm going to tell you about was done in Jeff Carroll's lab at the University of Washington. And he used the Q111 knock-in mice. And He used antisense oligonucleotides. And he was able to target the full-length Huntington gene, both the mutant and wild type, and compared that to the full length mutant as well as Huntington 1A. Next slide. So just looking at where these are targeted in a little bit more detail. so the top schematic is the wild type allele with exon 1 intron 1 exon 2 and down to exon 42 and the bottom schematic is knock-in allele and i told you that exon mouse exon one's replaced with human exon part of the intron is also humanized so he had an antisense oligonucleotide to the humanized part of intron so that targeted huntington 1a because it was before the cryptic polyacytes, and also the full-length mutant transcript. So his red ASO mutant was targeting Huntington 1A, a full-lang mutant, and then he compared this to a pan-ASO, which is the purple one in exon 42, that just regulated the full length transcripts and not Huntington1A. Next slide, please. So he gave these by ICV injection at three months, again at six months, and then mice were sacrificed at nine months if we look at aggregation in the cytoplasm in the striatum this is immunofluorescence in this case so the green is indicating this is huntington you've got again the diffuse aggregated huntington and inclusion bodies and the purple in this case are neurons so it's showing that the aggregation is in the neurons and the first panel is with saline, so that's the level of aggregation reached in these mice by nine months of age in the striatum. The pan-ASO, which Dan regulates full-length mutant Huntington, really had negligible effect on aggregation, but when he looked with the mutant ASO that Dan regulizes Huntington 1A and full- length mutant, there was negligible aggregation left, so it was delayed by lowering Huntington 1A, but not by full length Huntington. Next slide. And another phenotype that he looked at was transcriptional dysregulation. So a large proportion of the transcriptome becomes dysregulated in Huntington's disease. And we know that this is caused by that aggregated Huntington in the nucleus. So first of all, he compared in the top left here, the saline-treated mice with the ones where he had just knocked down full length. So what we're looking to see is whether or not the ASO treatment had improved the dysregulation in any way, and there was really no effect. But when he compared the salline-treated mice with the ones that had knocked down Huntington 1a and mutant Huntington, then there were a lot of gene changes. And Bobby focused on 188 consensus genes within the striatum that are always changed in Huntington's disease and showed that when he used knockdown just full length, there was no real effect on these, but knocking down Huntington 1A and mutant Huntington rescued or partially rescued about 50% of the transcription levels. So again, now the targeting Huntington 1A as well as full length mutant Huntingdon had profound effects as compared to just the full length Huntington alone. Next slide, please. And then the final study I want to tell you about is one that was performed in my lab. And here we use these AQ175 knock-in mouse models. And here, we were using siRNAs. And we were able to compare the effect of targeting mutant and wild-type full-length Huntington alone with Huntington-alone. Next slide, please. So this work was done with Anastasia Kvorova, who is the director of the RNA Therapeutics Institute at UMass. And she had a very potent siRNA that target full length Huntington. And with her lab, we identified some that targeted the intron before the first poly A. And because these are acting in the cytoplasm, then they are specific to either Huntington 1A or full length huntington. and again so the the full length one is very potent and will knock down up to around eight up to 80 percent after one month in vivo the one targeting huntington 1a was much less potent and really only we never saw more than 50 percent next slide please so we designed a fairly complex study with these um the it was icv bilateral icv injection there was an untreated group a treat a group treated with the non-targeting si rna a group target a cohort with uh the one targeting full-length huntington and one with the um targeting just huntington 1a and we had a baseline set start at two months take at three just to make sure that the si rnas were working as they should do and then an early cohort so we take it to we inject at two months and take it six months the double cohort two months six months and again at six months in that 10 months and then the late cohort at six and ten months and these siRNAs were most potent and durable in the hippocampus and so that was the those are the data I'm going to show you next Next slide, please. So what does this do to aggregation? This is a bit complex. I'll work you through it. So this is the CA1 region of the hippocampus. And if we look at the untreated images on the left, the early cohort was stopped at six months and the double and the late at 10 months. So that shows you what aggregation looks like in the pyramidal cells of the CA1 at six and ten months. We have this diffuse staining again, and the inclusion bodies are more pronounced at ten months than they were at six. The non-targeting siRNA had no effect. The one targeting full-length Huntington had no affect. But when we looked at the one targeting Huntington 1A, even though it was a much less potent siRNA, the effects were quite profound. but if you look at the ones the late group targeting at six months then going in later has less of an effect next slide please but it's a bit more complicated than that so i i just highlight what we saw in the ca3 region again if you Look at the untreated region we're looking at the Untreated samples on the left hand side early is at six Months double and late 10 Months We have the diffuse stain in the nucleus. And then we see inclusion bodies at 10 months of age as well. But we also have a layer of extra nuclear aggregate aggregates alongside the nuclear layer in this region of the brain. Again, non-targeting had no effect whatsoever. But now targeting full length Huntington, it did. it slowed down the production of the nuclear inclusions, and it did a lot to remove or slow down the generation of the extra nuclear aggregates. So once again, Huntington 1A, although less potent, was more effective, but full-length Huntington certainly did have consequences. Next slide, please. And then if we look at the transcriptional dysregulation here, this is a chart showing the number of genes that were altered at this is only looking at 10 months of age we're at the top two of the double cohort treated at six months and taken at 10 months and then the sorry at two months then six months then 10 months the late at six months and 10 months and you can again see that although less potent the Huntington 1a lane in the double cohort was more protective than the full-length lane, and treating late, actually we had a lot of transcriptional changes were exacerbated in those mice. So essentially, lowering Huntington 1a was, although a less potent siRNA than the one targeting full-ength Huntington, was generally more effective, but the one targeting full-length Huntington was very potent and did have some result in some improvements. Next slide please. So in summary from the work that we've done in mice we think that there's targeting strategies for lowering Huntington should include Huntington 1a but right at the moment we don't know whether or not targeting full length mutant Huntington as well gives any added benefit to targeting Huntington 1a. So I'll just end by the next slide, please. Just acknowledging all the people who work in my group and all of the people who have collaborated with us on the projects I've described in this presentation. Thank you. Thank you, Dr. Bates, for sharing your insights from your research on the molecular basis of disease with us. My name is Toby Ferguson. I'm a neurologist, a neuroscientist, and I lead our neuroscience therapeutic area at Alnylam. I'll now take you through our initial phase one clinical data, which we just shared recently at EHDN, as well as our next steps for our clinical development. Here you can see the general design of our phase 1B study. It's a randomized, double-blind, placebo-controlled study designed to diet for safety and pharmacology of one dose of intrathecal rosacea. The trial enrolled people with HD between ages of 25 and 70 with over 39 CAG repeats. They also had to be diagnosed with hunting in stage 2 or early stage 3, meaning they had clinical signs and symptoms or evidence of early functional decline. The study is ongoing in Canada, Germany, and the UK. Here we show a more detailed overview of our study. As Kevin had noted, understanding the safety of Huntington knockdown has been confounded by technical issues, and therefore this study was designed to evaluate two important questions in parallel. How much Huntington lowering can we achieve with a single dose of prozoceran, and what is the safety profile associated with that lowering? Patients were randomized to one dose of single intrathecal prozaceran or placebo, with doses increasing by no more than 3x when escalating from each cohort to the next. The double-blind portion of the study was six months, followed by a variable duration follow-up that allowed us to characterize the durability of the molecule. And then patients are eligible to enroll in an open-label extension. Of note, the study is ongoing, and the initial data we are sharing is all the available data so far, which includes up to five dose cohorts through three months. The study's primary endpoint is safety and tolerability. Secondary endpoints include CSF and plasmid PK profiles, as well as, importantly, change from baseline and CSF mutant hunting levels, the key biomarker of target engagement. Additionally, we're measuring exploratory endpoints, such as clinical scales like the TSC or CUHDRS, imaging biomarkers, and biomarkERS of disease progression and safety, such as neurofilament. Turning to the next slide, the population, including this initial data cut, had baseline patient characteristics in the study that were generally similar across the cohorts and reported dose ranges. The cohorts were well-matched on repeat number and measures of disease severity, including total function capacity and CAP100. CAP100 is an estimate of the cumulative exposure in Newton Huntington, where a score of 100 is usually consistent with motor symptoms or functional impairment. Consistent with our intent to enroll relatively early stage HD participants, the cohorts had a mixture of participants with stage 2 or stage 3 disease. Now to the pharmacologic data. Accurate measurement of mutant Huntington levels in the CSF has been a long-standing challenge for the Huntington's field. Here we show data using the traditional SMC assay. In this context, you can see a clear dose-dependent reduction of CSF-Huntington as we increased the dose. And we observed a mean-level reduction at the top cohort of 64% at month three, with individual reductions up to 80%. However, when we dug into the assay performance, it became clear that a significant percentage of individuals in this trial, in this case about a third, fell below the lower limit of quantification, especially after deep Huntington lowering. Given the sensitivity limitations, it was important to confirm more robustly our measurement of CSF Huntington. To that end, we also measured CSF huntington with a new, more sensitive mutant Huntington reliza assay. This assay was developed in collaboration with CHDI, Alamar, and Evotech, and we helped develop further the assay for clinical trial use. Here on the right, can see with this assay we had no participants below the lower limit of quantification enabling us to measure mutant hunting across all study participants even those with very low levels we saw similar dose dependent reductions in mutant hunting with a mean reduction of 60 percent at month three and individual reductions up to 83 plus these results provided orthogonal confirmation of robust and durable target engagement following a single dose of prozosaran. These initial phase 1b data are clearly encouraging and suggest that prozosarane can successfully knock down mute country, supporting the key objective of our study. We also highlight the other key goal was to determine is this hunting knockdown tolerated. So let's turn to the safety experience thus far. As a reminder, this study is still ongoing, so the safety analysis is pooled across both placebo, and treated participants. In this analysis, adverse events were recorded in most participants across all doses, as is common in neurodegenerative disease studies. However, across the five cohorts, including the analysis, we observed only a limited number of serious and severe adverse events, and importantly, did not increase with frequency with increasing doses. There were two serious adverse event, excuse me, two severe adverse adverse events in dose group 4, a radius fracture, and a likely bacterial meningitis. The two severe events were also reported as serious, and there was one participant in group 2 who had a headache that has since resolved. That was reported as severe but non-serious. In addition to the severe and serious events I just noted, there were two additional serious adverse events, in dose Group 4, an acute myocardial infarction that was resolved with sequelae, and it resolved post-lumbar puncture syndrome. We did not observe any deaths, and there have been no study discontinuations due to adverse events. It's also important to remind you that all adverse events have been reviewed by our DMC, which includes external experts, and the study is ongoing. Overall, these data demonstrate at this time, these time points, and in this analysis, that robust reduction of both mutant Huntington and wild-top Huntington has been well-tolerated so far. We've seen from the initial clinical and safety observations that reduction of Huntington appears to be well-tolerated. However, it's important to understand whether biomarkers of CNS inflammation are altered given that in some other Huntington lowering programs, evidence of CNF inflammation was observed and it could be potentially related to either Huntington's lowering or the platformer study drug tested. With CNS information, you can observe moderate, in some cases marked, increases in both CSF protein and white blood cells. However, with these initial data we are sharing today, you can see that both CSF white cells and protein measurements were similar between treated and placebo participants. Additionally, most observed values are within the normal range as denoted by the dashed line. Furthermore, even with dose increases, we saw no evidence of elevations of either white cells or protein to date. These observations in total suggest we do not, as of this analysis, have any evidence of CNS inflammatory process that is ongoing. So let's turn to the question of whether or not we see increases in neurofilament with Huntington lowering. As both Kevin and Jill have mentioned, neurofilment is an important biomarker of both safety and in some cases efficacy. To remind, neurofilm is a cytoskeletal structural compound found in many CS neurons. It's released into the CSF and blood after injury or during neurodegeneration. Notably, we've recently come to understand that increases in neurofilament are an important safety biomarker. And as Kevin discussed, several previous hunting and lowing programs have observed increases in neuro filament within the first six months after dose initiation. Here we show both the absolute and full change in CSF neurofilment over the course of the ongoing study. We've also demarcated in gray the range of values observed in the placebo-contributed participants, clearly and importantly, we did not observe any full change increases in CSF neurofilament in individuals receiving brazosaran that exceeded the range observed in placebo, nor did the absolute changes differ meaningfully from placebo. Furthermore, even with increasing doses, we observed no change in neurofilment levels. Given the emerging importance of neurofiliment as a tool to measure neuronal injury, these data clearly suggest over these time points and with this analysis, but neither brazosuran nor hunting and lowering increases neuronal injury or degeneration. We feel this is an important observation given the prior concern that hunting and lowering may exacerbate neuronal injuries or that potential treatments may increase levels independent of target reduction. Finally, we measured brain ventricle volumes using MRI. Though the importance and implication of change in ventricles size is not fully understood, there is a slow increase in ventricle size typically observed in people with Huntington's disease over time. In addition, a few other programs investigating Huntington lowering as a therapeutic approach have observed more pronounced dose-dependent ventricular enlargement over time in participants with Huntingdon lowering agents. So there is concern in the field that this could potentially reflect either increased atrophy or inflammation that disrupts the CSF flow, with the result of an increase in ventricle size. In contrast to those other hunting and lowering programs, we did not observe any dose-dependent increases in the size of the ventricles after the Zosaran treatment, as the ventricular size increases observed appeared consistent with the placebo group. Overall, these initial results provided an encouraging safety profile of the Zoseran in terms of clinical observations, as well as biochemical markers and imaging measures. in the context of robust Huntington lowering. I'd like to quickly recap a few key points. First, Rosoceran, unlike most other Huntington-lowering agents being investigated, aims to reduce both full-length mutant Huntington and the shorter 1A isoform. The emerging preclinical data and the human data suggest that 1A could be an important contributor to H2 pathogenesis, as you heard from Dr. Bates. Finally, initial data suggests that brazosurin has the potential to deeply and durably reduce Huntington in people with Huntington's disease, and so far is well tolerated. These results give us confidence to advance brazosuran in two complementary studies in parallel. The ongoing Phase B open-label extension will be continued and will allow for all participants to receive brazosurun every six months. This will enable us to continue to characterize the longer-term safety of sustained Huntington introduction, as well as explore potential efficacy trends over longer periods of time. At the same time, I'm excited to confirm that pending regulatory alignment, we're investing in a placebo-controlled pivotal study, which we plan to initiate in 2027. This study is intended to provide a definitive test of whether or not rosocerin can slow Huntington's disease progression, and to the best of our knowledge, will be the first placebo- controlled test for the HTT1A low-A hypothesis. Advancing these studies in parallel reflects both our confidence in the profiles we observe to date and our commitment to move with urgency for people with Huntington's disease and the broader community. We look forward to sharing more about the pivotal study design following our discussions with regulators. In summary, we believe Brazosurant offers a differentiated approach to Huntington lowering, supported by three key findings. First, our inclusive exon 1 targeting strategy is designed to lower all forms of Huntington, including the HT1A isoform, which may be quite important in disease pathogenesis. Second, our initial 1B results, we've seen patients on risotriene experience deep and durable Huntington lowering after a single intrathecal dose. Across the first five cohorts, dose-dependent reductions of CSS Huntington were observed with a mean reduction of 60 to 64 percent at month three, and the highest dose group evaluated, and maximum individual reductions up to 83 percent. Third, and importantly, we're encouraged by the safety profile observed to date, with no clinical evidence of rosoceran-related safety events, no evidence of dose-dependent increases in either markers of CNS inflammation or markers of CNС injury. And finally, we saw no evidenceof dose-dependent change in ventricular volume. Our findings to date do not establish clinical efficacy. We believe longer, larger studies are required for that. But together, they give us confidence in the molecule, the targeting strategy, and the path ahead. Now that we have highlighted a molecule, emerging clinical data, and next steps, it's like a moment to comment on the unmet need and the opportunity to hunting this. The unmet needs in hunting this disease is substantial and clear. We estimate there are about 100,000 diagnosed symptomatic individuals worldwide, with about 33,000 in the U.S. alone. There are no approved disease-modifying therapies currently available. Not included in those estimates are the more than 100, 000 gene carriers in the U. S., and even more globally, who may be symptomatic or undiagnosed. The patient population is genetically identifiable. Care is often concentrated through specialized Huntington's disease centers, and there's growing familiarity among providers with intrathecal injections. Importantly, there's also a strong and well-organized patient advocacy community supporting both awareness and engagement. For an effective disease-modifying therapy, we believe this can create an efficient path to patient identification through treatment delivery, as well as the potential for a meaningful commercial opportunity. However, most importantly, we think Prozostrin represents a major opportunity to address a devastating neurologic disease where patients and families are eagerly waiting for options that can change the course of their disease. Before we close, I'd like to return to the people at the center of this work. Some of you may remember Alyssa's story from our 2025 R&D day about 18 months ago. We're going to share a brief version of her video now for those who may have missed it. i'm 34 years old and my husband and i live in baltimore maryland i am a physician assistant i work in orthopedic surgery my father was diagnosed with huntington's probably about 10 years ago i had a really close relationship with my dad growing up he was my softball coach He held a really high-level job at a hospital. I was proud of him. And watching him decline over the past decade with Huntington's disease has been really hard. I decided to get tested for Huntington disease in 2020. I had seen multiple family members deteriorate. I found out in November of 2020 that I was gene positive for Huntingtons disease. I have 42 abnormal repeats, which is equivalent to my dad's. I will probably become symptomatic in my fifth or sixth decade of life. My husband has been immensely supportive. He has been my rock through all of this. My husband and I chose to do IVF. They were able to test the embryos specifically for the Huntington's disease gene, and then we ultimately decided to proceed with implantation of a Huntington's disease-free embryo. I'm currently 18 weeks pregnant with a baby boy due in June. We're super excited that our baby is in fact Huntington disease- free. It's hard not to feel that time clock over your head, but you need to just live life to the fullest while you can. Alyssa's story reminds us of the incredible need of this community for transformative therapies and it further motivates us to advance BrazosRain with urgency, rigor, and a close partnership with the broader HD community to determine if Brazos Rain can fill the need for a meaningful therapeutic for people with HD. I'll now turn it back to Pushkal to moderate the Q&A. Pushkal? Thanks, Toby. We are indeed indebted to Alyssa for sharing her story. Hearing her experience and from others like her is incredibly motivating as we advance programs like Prezoceran to help patients with Huntington's disease. So we're going to now move into our Q&A session. As a reminder, please submit your questions through the dialogue box located within the webcast platform. We'll answer as many questions as we can within the time remaining. Joining me today for Q&A are Kevin Sloan, the Brazil Saran program lead, Toby Ferguson, who's the neuroscience therapeutic area head, and Christy Kelsey, who is the Senior Vice President of Value and Pipeline Commercialization at Alnylam. And as well, we're very privileged to have Dr. Jill Bates, Professor of Molecular Neuroscience and Co-Director of the Huntington's Disease Center at University College of London. She'll specifically speak to the preclinical biology of Huntington disease and research on the ACT1A isoform. So we've gotten a number of questions, a lot of interest, clearly, in this program. So I think probably the number one question that I've now seen a few times is what level of HTT knockdown is necessary or optimal to be clinically meaningful in this disease? So maybe, Dr. Bates, I can ask you to start from your preclinical evidence. What do we understand about how much HTT lowering is likely to be helpful. And then maybe, Kevin, you can speak to what we've understood internally from some of our preclinical data and supplement what Dr. Bates has to say. Yes, thank you. So I think you would have seen from our mouse studies that knocking down Huntington 1A by about 50% was having quite profound effects. So, I think one of the things you have to also remember is that mice we are using have CAG repeats that are very long in all cells. So this is an effect we're seeing in mice in which the disease's onset is going to be early and very progressive. So I think our preclinical data is very encouraging and i think perhaps one other thing to say is though it is also that in our experience when we've looked at agents that are targeting x on one the level of knockdown of full length huntington and of huntington 1a um is relatively comparable although we have we have i have to say we have not looked at your agent but um i think that that that's our pre-clinical experience. Great. And Kevin? Yeah, I mean, look, I think the short answer is that no one really knows for sure. At this stage, a clinically meaningful threshold for hunting lowering has not been established. This has been a hot topic of debate in the field for many years. And the question is, how much is enough for efficacy, but how much might be too much from a safety perspective? And with a monogenic disease like Huntington's, we assume greater lowering of mutant Huntington would lead to better outcomes. We know from animal models, like the work that Dr. Bates has talked about, that 50 percent, you know, seems to give a good effect. On the safety side, we also know from human genetics that heterozygous loss of wild-tip Huntington appears to be tolerated from birth, which may be similar to 50 percent lowering. So you kind of coalesce on that sweet spot at 50 percent seems like a reasonable place to start you know based on the totality of the data that we have um you know we think approximately three percent lowering would be a good place to start we don't think this is a florid kind of target 90 95 is necessary um and then maybe i'll just also highlight the preclinical work that i spoke to earlier you know really really deeply interrogated the safety of wildlife huntington lowering in preclinical studies and non-human primates, to really ask, is there any phenotype associated with very deep wild-type honeycomb lowering in primates? And we have not seen one. So that gives us confidence that we can maybe start trying to target fitters that aren't lowering, and maybe there's room to push beyond that. Great. Thank you, Kevin. And then maybe I think the other sort of really common question, and Dr. Bates, I think a lot of favorable feedback coming back from your just deep discussion around ACT1A. But question saying, you know, look, if ACT1a is central to your therapeutic hypothesis, what do you know about how much you're actually lowering 1a? Are we inferring that? Are there preclinical data? And so, and why can't we measure it? So maybe Dr. Bates, I think you did touch on this, but you could spend a little bit time in terms of, it seems like we're able to measure this in certain mouse models, but there's maybe some limitations you highlighted in terms of being able to measure this in actual clinical studies. So maybe you could speak to that. And then, Kevin, you could also then expand on what we understand about whether bezosan is in fact lowering the 1A species. All right. So yes. So when we're working with these mouse models again, as I say, we're working with very long repeats in every cell. And as you saw from the presentation, as the CAG repeat increases, the amount of Huntington 1A produced is increasing. So in the mouse models, we have Huntington1A levels being generated in every cell. So we're able to measure Huntington2A easily in these mice, and we can tell very easily and accurately what the level of knockdown is. Now this is much more difficult when we come to people. So most of the work that informed us about Huntington 1a in people at the moment is coming from post-mortem brains and when you look at the level of the CAG repeats, if you remember the work I showed you from Steve McCarroll's lab when then taking a post-mortem brain and sorting cells and looking to see what how long the repeat is there's only a small proportion of cells that have these very long repeats where those vulnerable cells are starting to get into a into a range whereby they are going to become sick and and as steve's work predicts will die so the amount of huntington 1a produced in the whole brain is going to be it's not like the mouse where you have a lot generated from all cells it's going to be very small because it's only being produced in quantity from these small number of cells and those are only the vulnerable cells with the long repeats so we can see it because we we know the proteins there because we can See the aggregates but actually being able to detect the protein in a clinical sample would probably be quite difficult also you know in the mice as the mice age and get more aggregates or the amount of soluble protein you can detect decreases because it's all getting sucked into the aggregates so i think in terms of um so measuring soluble huntington 1a in the csf i think is very unlikely to be possible i would um i'd be very very happy if if it was seen but i think it's unlikely tobe possible so but you can measure the not you can measure full length huntington decrease in the csf as i mentioned earlier in our experience when we've worked with an agent that targets exon one the level of knockdown of huntington 1a and full length Huntington is equivalent so you would expect that to be able to infer in in a in a person the amount you were knocking it down by measuring the knockdown a full length Huntington. In the mice, we can measure it precisely, but it's going to be a challenge in people, but I think you should be able to infer. Thank you, Dr. Bates. Kevin, anything you want to add from what we understand about that? Yeah, maybe just a reminder, we designed brazosurin to target a conserved sequence in that exon one that is present in all forms of Huntington, including HT1A. We have confirmed target engagement both in vitro and in vivo in mouse models but hd1a is not present in normal primates we can't do that work in the monkey work we've not yet tried to measure hd-1a and csf from our ongoing phase one trial and so far as we just heard from dr bates 1a has only been detected in post-mortem brain tissue and aggregates tied up in cells and co-localizing with cells that contain the elongated cg repeats it's not yet been detected in the csf of living patients and in our trials we're limited to sampling the csv we can access the brain tissue itself also maybe a reminder that full-length mutant huntington is present at very low levels in csf making it very difficult to measure as we talked about earlier with the different assays we think 1a may be less abundant and even harder to detect as it's thought to be produced in cells a subset of cells with those elongated cut repeat expansions that are very short-lived or may also remain trapped in aggregates within cells leaving very little or no protein uh available for detection the field is working to try to develop even more sensitive assays for 1a that could potentially be used to look for 1 a in the csf in the future for now we're just banking csf from our trial participants and as that sort of technology evolves we may give it a shot and look in the but you know as the appointment dr bates made it's unlikely we will ever be able to find it in the css thanks kevin um tell me maybe there's a question for you i think uh people commenting on the reproducible pharmacology and the dose dependent pharmacology across the two assays but there is a question about are we reaching a ceiling effect as you look across these two and assays and they're not seeing incremental additive knockdown so why are we continuing to escalate the dose um as we as we kind of look at this maybe you could comment on how you're thinking about the pharmacology that we're seeing thanks push call i think i think it highlights sort of overall our ghost our goal really is to identify, as we've highlighted, a regimen that can really achieve deep and durable knockdown to target what we've heard at least as a target 50% lowering of Huntington's. In the data we shared today, we saw with both assays reduction in the mean range of about 60%, particularly those two top doses. But I think it's important to note that we really haven't yet begun to explore the overall durability of these data, and at those higher doses, we think we'll both explore not only depth of knockdown, but durability. If you put together the data we've seen in the clinical studies, the data that we've seen pre-clinically, I'd note we highlighted in multiple doses the non-human primates that we get about 80 percent knockdown in CNS tissue after multiple doses. We put this together, we model it, we really think that we need to explore these doses to potentially move forward with a biannual dosing, and we think the higher doses really will support this goal. Thanks, Toby. And I think maybe a related question that's kind of coming up around the dosing is, you know, you're measuring this in CSF, but, you know, we think we need to actually get the sRNA into the striatum, into the putamen and caudate. So what do we understand, and is that part of the reason for escalating the dose to get into those deeper brain structures, and what do we understand about the biodistribution? So maybe you can touch on this, and then, Kevin, you can add on top of that. Sure. I'm happy to have that. Where this molecule is going. So when this molecule's administered in the intrathecal lumbar space, and what we know, we've added the C6 in Moiety that allows that robust biodistribution, but what we do know is there's a gradient from the site of injection in the spinal cord up into the cortex into the deeper tissue. So conceptually, we do know that we need to really achieve this knockdown in the key regions of interest, which are the striatum. So we're not driving for a knockdown per se in CSF, but we want to drive for that knockdown in those deeper tissues. And that may be another reason to exploit these higher doses. And maybe I'll just add, you know, we've got the benefit of multiple programs now in the CNS where we've been able to do, you Know, preclinical work in non-human primates where we can measure tissue lowering and measure CSF signals, and then we can build modeling across all of those. And what we've seen consistently is dose-dependent lowering in brain tissue, and as we push the dose up, we are able to get target engagement in various deep brain tissues, including the cate and butamine and the striatum, which are relevant for this indication, and other deep regions may be relevant for other indications. And then, you know, we really use that modeling data across all the programs to think about how to extrapolate from CSF lowering to tissue lowering. But sure, in clinic, you know, one of the challenges is that CSF is a very imperfect measure of what's happening in the brain. And we really have to rely on that totality of data to choose our doses. And we'll talk more about that in the future. Very helpful. And then maybe one other question around pharmacology is, you've highlighted, Kevin, Toby, that we're knocking down both the full-length and the shorter HTT1A, but also the wild type. And so what level of wild-type lowering do we know to be safe? So maybe, Dr. Bates, you could speak what we're understanding preclinically, and Kevin, you can speak a little bit about what we've observed in our non-clinical studies around wild-otype lowering. Yeah, well, the preclINICAL data that we have is just in mice, and I can only really speak to the siRNA study that I presented. And there we have a very potent siRNA knocking down both full length and full length mutant and wild type. And so treatment from two to 10 months of age, we didn't see any adverse effects, but the knockdowns predominantly in the forebrain. I think you probably have more data in primates that is useful than the mouse data. but we haven't seen any adverse effects. Thank you. Kevin, do you want to add? Yeah, I think it's a fascinating question. And, you know, when we first got into this field around 2018 and we're attending conferences, a lot of people raised this issue. You know, what is the safety? We're not sure. Does the strategy make sense? I think there's probably some nuance here about the safety of wild-type Huntington lowering during development of the brain versus in an adult setting, you Know, after the brain has largely become developed. So there may be multiple answers to this question. When we look at human genetics, you know, there really isn't any evidence of humans walking around with complete knockout, like homozygous Huntington's knockout or complete loss of function, loss of expression. There is, though, evidence in adults, the sort of heterozygous population, small, that has been documented that don't appear to have a phenotype. And so we think that's probably equivalent to, let's roughly say, 50% loss of function. So that gives you some confidence, at least in the adult setting, you can probably get to that level. The question will be between 50% and 100% ultimately, and again, where, as we've just been talking about. Now, in our primate studies internally, you know, we've done quite a lot of work. I think we've conducted now at least five individual primate studies over the last several years. Those have been for various purposes, various depths of knockdown for various periods of time. But, you know, for as deep as 90 percent or more tissue level Huntington lowering for as long as 12 months in duration, you Know, in very rigorous toxicology studies where we're working up the data and really looking at the tissue at a histological level, we are not seeing any adverse effects. So that's really given us confidence that there may be a path forward, specifically with this S-RNA-mediated honeyism lowering approach. Super. So really, you know, as best we can see from the extensive non-human primate work we've done, we can get to pretty deep knockdown of wild type, we believe, and we're not seeing any adverse consequences even with that over an extended period of time. So let's flip to a bit of the path forward um uh toby you highlighted uh sort of the ongoing phase one and the open label extension i think a number of questions about when might we see some updated data from that that that you know might give us an inkling around um efficacy when will we see either clinical data, imaging data, more NFL data, et cetera, you know, and will we be comparing that to natural history at all to infer what might be going on in terms of on the efficacy side of the equation? Thanks, Pushkal. So I think the overall we've highlighted the design of the OLE is such that participants of the study can roll over to get dosed every six months. We've also highlighted, as you noted, some key measures will include clinical scales such as the TFC and the CUHDRS, will include important biomarkers like neurofilament. You've heard that can both be a safety biomarker, also possibly an efficacy biomark. I think as we've seen the – so these are going to be useful over time, I think particularly the longer time. What we've see in the field as a whole has evolved over the last few years with a number of therapeutic trials ongoing, is that we really think that we need longer duration studies because of the nature and the progression rates of honeydews to really understand if there's potential efficacy. So we're certainly interested in this over time, but we do think we'll need a relatively long period of time, 24 months to 36 months, to really start to tease this apart. There are natural history data sets that are available that can inform these comparisons, and we certainly will use them. But really, I think the key point is that time is critical here. Our OLE is just starting, so there will be some time until we can really share any potential look at efficacy based on the OLE. And, Toby, I guess on a related point, some questions, I mean, people noted that Unicure might be pursuing a potentially accelerated approval using a natural history comparison. Do we think, do you think that the data that you're generating out of this OLE might offer that same opportunity? It's really an excellent question, but I think simply at this point, it's really way too early as to whether or not the data from the OLE could support an accelerated approval. The appropriate path, regulatory, will really depend on the totality of the data, the discussions we have with health authorities, the evolving regulatory landscape. That said, we've highlighted we're pursuing two parallel tracks. The OLE, as I've noted, is intended to provide long-term safety and exploratory information. That's important. We've highlighted tools like the clinical scales and neurofilament. That being said, we've also highlighted we're investing in a large placebo-controlled pivotal study that we plan to start in 2027, pending regulatory discussions. It's really that study that review as the definitive test of whether or not prozosterone can slow disease progression. And ultimately, our real goal here is to advance prozostorone in the most efficient way possible while definitively testing the efficacy and safety. Our current belief is that the adequate and well-controlled study remains an important part of that for demonstrating the efficacy and safety profile. We believe that generating that robust placebo-controlled data is critical both for regulators and those discussions, but also for physicians, patients, and payers. Super. So this is actually a perfect segue to the next sort of set of questions that we're getting, which is really around that pivotal trial design. You know, you spoke at high-level terms. Are you able to provide any more color in terms of the population? You know, I think there's questions around whether we're looking at stage 2 or stage 3 patients. What will be the nature of the endpoints? Do we know anything about the duration of that study? What color can you provide today around any of that? These are all critical and really important questions, and we've certainly engaged with regulators early and often around our phase 1 data sets, and that's ongoing. But fundamentally, as we said, we plan to initiate the placebo-controlled study in 27, but that really is pending a regulatory discussion. So really, once we have those discussions, we can share more detail in the study design and the key features such as the inclusion criteria, the populations, and the key endpoints. I think it's fair, based on what you said around the OLE, that this is likely to be a several-year pivotal trial with one of the commonly accepted sort of clinical endpoints as the primary endpoint and then a series of secondary endpoints is that is that all fair that's quite fair i think in the context of the field it's coalesced around a number of tn points such as the chrs and tfc and and already highlighted the longer durations are likely needed good good um so the other question we've started to get is a little bit about i think people have seen that there are advances being made in terms of using a for example tfr shuttle to administer agents systemically, you know, rather than going through the intrathecal route. So I guess a question about, you know, A, is that sort of necessary to move forward in this kind of an approach? And where we are with our own shuttle approaches. So maybe, Christy, I'd like to invite you to come into the conversation and just speak a little bit about how critical it is to do that or, conversely, the acceptability of the current administration approach of Brazil's Saran for patients with Huntington's. And then, maybe, Toby, you can elaborate on what's happening at Alnylam in terms of bringing forward a brain shuttle and the timing of that. No, it's a great question. And maybe the question behind the question there is, do we see the intrathecal administration as a key commercial challenge. And I would say, in short, no. You've heard about this disease, how severe it is. And we know that for diseases like Huntington's, it's efficacy that's ultimately the driver for treatment decisions. It's early days, but we're encouraged by the data presented today. It brings us one step closer to bringing forward innovation at pace. And we've heard so far that an infrequent, potentially twice-severe intrathecal delivery is acceptable. We'll, of course, work with specialists to make sure they have the product information to have the best patient experience possible. But IT is only our first offering, and I'll pass it off to Toby to talk about our efforts related to shuttle. So, Toby, over to you. Thank you, Christy. We've been excited to see the evolving landscape on the various CNS shuttles. I think we'd highlight that, of course, we're actively working on a number of brain shuttle platforms across our CNS portfolio, And we'll talk more about those in the future when we have data to share. I think what I'd comment at a high level that for each target and program, you'll want to carefully look at the trade-offs between a shuttle approach and an IT approach. What is the tissue you need to deliver the SRNA2? What's the depth of knockdown you need? Is there a safety or a broader knockdown in peripheral tissues versus safety of a local knockdown In the CNS? So I think there are a number of factors, but I think you've heard from Christy that there's not an immediate barrier to proceeding with our IT program. I think our real focus at this moment is to establish proof of concept that Prozos ran in a strategy that targets both hunting and lowering of 1A and the full length and slow hunting disease. Once we've established that, it's a great platform and we'll be well positioned to optimize from there for people with HD. Thanks, Toby. um dr bates one for you i think um you know you showed us some really interesting uh data on pre-clinical models uh there's a question around what they tell us and is there a time that it may be too late to intervene in this disease based on the pre-clinical evidence that you're that you've generated or and you and your colleagues and what might be the ideal time to intervene um if we want to have effective therapies any insights that you can share from what the research is telling us there i think that's a very difficult question um so again if i come back to the fact that the mouse models you know have a very early onset and rapid progression and that but most importantly the fact that the very long cag repeat is present in all of the cells so the data we have suggests that you know the if you if you you if you administer the therapeutic very early then you have a more pronounced effect than if you go in a little bit later um and i think that's a kind of um that's the message that people are very receptive to generally with these treatments for huntington's disease but i think we'll only find out really um with with time with clinical studies because We really don't understand the trajectory of these molecular changes in the patient brain when the somatic expansion is occurring in a stochastic manner throughout the brain and throughout life. So, you know, within the mice, it says earlier is better than later, but I think, you know, we don't really, it's hard to predict at the moment whether there's a time that would be too late and when that would be. Okay. Thank you, Dr. Bates. And maybe just the last question for this morning. Christy, this one's for you. I think Toby briefly covered just the epidemiology, but I don't think there's any question about the unmet medical need here, but maybe just people are trying to understand what is the sort of overall market opportunity here. Are all the patients identified? And so is this sort of a fixed size population or is this like other sometimes rare diseases where we identify a lot more patients once there's effective therapies? And obviously there are on now? That's absolutely the right question. You heard the epi data from Toby talked about there's in the U.S. 33,000 patients that are diagnosed and symptomatic. What we haven't talked about a lot is if you think about the U。S. there's a hundred thousand gene carriers. So if you just do the math that shows a very very low diagnosis rate. Unsurprisingly patients are hesitant to get diagnosed if there's no therapies available. You heard Alyssa talk about, you know, her situation and seeing what her dad went through. She was one of the few patients who did get diagnosed early, but as agents are expected, or agents have brought forward to the market, we do expect this to change. Patients will get it diagnosed earlier, as we saw with Alyssa, and also more patients will seek diagnosis. So there's a lot of potential upside here, as, obviously, new treatments come available. There's a high in-med need, high efficacy aspirations of our profile, and what's very encouraging is the data that we saw today, and this just gets us to one step closer to bringing a sizable opportunity forward to patients, and it's fully aligned with our 2030 strategy of additional transformative medicines beyond PTR. So very happy to have the opportunity to see this data brought forward today is it gets us one step closer to a very sizable opportunity. Well, I think that's a fantastic way to end this roundtable. So thank you, Christy. Thanks, Kevin, Toby. Special thanks, Dr. Bates, for your insights and contributions to today's discussion. This concludes today's RNAi Roundtable and the 10th season of our RNAi roundtable series. You can access the replay of the webinar and download the slides in the Capella section of Alma-Alam's website. Thank you all for joining and have a great day. Thank you for joining this RNAi Roundtable event. This concludes the meeting. 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