Alnylam Pharmaceuticals, Inc. (ALNY) Earnings Call Transcript

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 ALN-HTT02, which is in development for the treatment of Huntington's disease. I'm Josh Brodsky, Vice President of Investor Relations at Alnylam. With me today are Pushkal Garg, our Chief Research and Development Officer; Kevin Sloan, Vice President of Development Programs and Program Lead for brezosiran; and Toby Ferguson, Senior Vice President and Head of the Neuroscience Therapeutic area at Alnylam. We're also thrilled to be joined by Dr. Gill Bates, Professor of Molecular Neuroscience 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's 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 like 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. Alnylam has engaged Dr. Bates as an independent academic speaking 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 a more complete discussion of our risk factors. And with that, I'll now turn it over to Pushkal to kick things off. Pushkal?

Thanks, Josh, and welcome, everyone, to today's RNAi Roundtable on brezosiran, previously known as ALN-HTT02. 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 2 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 6 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 III development as we continue to build to our -- towards our Alnylam 2030 goals. As I mentioned, a major advance in extending RNAi beyond the liver has been our C16 siRNA delivery platform, which opened the door to targeting the central nervous system. We've seen robust target engagement and broad by distribution across the CNS and nonhuman primates. And we've seen that now reproducibly translate into knockdown of disease-causing proteins in the CNS. First, with mivelsiran, our investigational APP targeting program. And as we've just announced with initial Phase Ib data now with brezosiran, which targets Huntington. This successful translation has provided the confidence for us to build a growing pipeline of investigational CNS-targeted RNAi therapeutics in collaboration with our colleagues at Regeneron, including programs in Alzheimer's disease, Huntington's disease, ALS and Parkinson's. Brezosiran is part of our efforts to apply RNAi 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 brezosiran. Kevin?

Thanks, Pushkal. I am Kevin Sloan, the program leader for Brezosiran at Alnylam. 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's disease. Before I begin, I'd 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 [indiscernible] or rapid and voluntary muscle movements, in coordination and falls. It also affects thinking, which can manifest its 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 life span 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 presymptomatic 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 track 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 Huntington and also a shorter mutant Huntington 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 science and clinical signs perceived a functional decline and increasing loss of independence that ultimately defines later-stage disease. This long disease course may provide an opportunity to intervene before a 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 [indiscernible] 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 length mutant Huntington and HTT1a are implicated as toxicity drivers and Dr. Bates will go deeper into the biology of HTT1a 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 brezosiran, we considered several possible strategies: lowering HTT1a alone, lowering full-length Huntington alone or taking an inclusive approach to lower both. We ultimately selected the latter taking an Exon 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 olicnucleotide and small molecules place 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 asotomonersen, which was designed to lower full length mutant Huntington and wild-type Huntington was reported to cause dose-dependent NFL increases within 6 months despite moderate CSF mutant Huntington reductions. The ASO WEV003, 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 NFL within 6 months despite modest CSF mutant Huntington reductions. So a couple of key questions remain unanswered. First, how much mutant Huntington lowering may be needed to maximize efficacy; and second, how much wild-type Huntington lowering can be safely tolerated. Here in Alnylam, 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. Taken 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 potential to avoid some of the challenges that I've just outlined. Brezosiran is an intrathecally administered investigational C16 RNAi therapeutic for Huntington's disease. It targets a conserve 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 Huntington and the shorter mutant HTT1a. Because the first Exon is shared across mutant and wild-type alleles, our approach is also designed to lower wild-type Huntington. RNAi therapeutics are specific and durable, but fully reversible with no permanent changes to the genome. Brezosiran leverages the same C16 siRNA platform that underpins our broader CNS pipeline and has shown a consistently encouraging safety profile to date across 5 ongoing clinical programs. We're highly encouraged by the preclinical data we've generated over the years. In nonhuman primates, brezosiran 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 Strydom 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 siRNA platform across a number of single and multi-dose NHP studies up to 12 months in duration and including completed GLP chronic toxicology studies. We have observed an encouraging safety profile for Brezosiran. No neurological abnormalities, no elevations in CSF inflammatory markers, such as white blood cells or protein levels, no elevations in CSF neurofilament light change, no histological changes and no adverse findings even after a very deep wild-type Huntington lowering beyond 90%. To summarize, we see 3 key reasons for hope for the brezosiran program. First, the C16 siRNA delivery platform has been shown to provide a safe, durable approach to target engagement in the CNS. Second, brezosiran gives us the potential to explore a 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 HTT 1a isoform. With that, I'd like to introduce our guest speaker, Dr. Gill 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 HD field over her career, including developing the first mouse model of HD. She's been an elected fellow of the Royal Society since 2007 and was recently awarded the society's highly prestigious Fairer Medal for her work in understanding the molecular basis of Huntington's 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 HTT1a. 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 Huntington's disease. And then I'm going to tell you what we think are the very first step in the pathogenesis of Huntington's disease steps at the molecular level and then go on to talk about 3 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, hunting disease is an autosomal dominant disorder, it generally starts in midlife 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 showing -- is presenting with the Creaform dance light movements that also 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 Huntington -- the motor coordination is not always the first sign to begin. And Kevin has 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 disease is a CAG repeat expansion in the first Exon of the Huntington's disease gene. This is a large gene spanning 67 Exons. If you have 35 CAGs or -- 35 CAGs or less, you will not develop Huntington's disease, 40 or more CAG will result in Huntington's disease within a normal life span. People with 36 to 39 the 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 in parts to 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 of 20, do have long repeat -- have longer CAGs here they're going up to 120, and that is clearly associated with the age of onset. But for a CAG repeat of 40 or so, then offset can begin at any time from 20s up to the 70s or later. And so there must be other factors involved, and we know that these -- some of these genetic modifiers. Next slide, please. And if we look at the neuropathology of Huntington's disease, here we're looking at the coronal section 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 [indiscernible] and of the core date putamen, which here has been reduced through a [indiscernible] of tissue because 95% of the neurons in that region, the medium spiny neurons are one affected by the disorder. But there's still lost 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 immuno labeling with an antibody to the very end terms of the protein. And that reveals these inclusion bodies, as you can see in this figure indeed, there are inclusions within the -- within neuromal nuclei. Otherwise, they're scattered throughout the [indiscernible] lining up in processes. And that present in presymptomatic brands. 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 the 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 penetrated 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 of this image shown here where she could show that in the striatum, she could detect rare allies 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 [indiscernible] 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 mismatch repair genes in the mouse you prevent the sematic expansion from occurring. So putting this together with the modifiers that modify age of onset and progression of disease with the [indiscernible] expansion, we now believe that the first step in the pathogenesis Huntington disease is this specific repeat expansion that occurs in the vulnerable neurons within the brain. Next slide, please. So if that's the first step in the pathogenesis, what might be the second? Next slide. So here, again, we have this -- the gene with 67 Exons. This is transcribed to pre-mRNA processed produce the mature mRNA, as shown on the right-hand side of this slide, and then that encodes this large protein. And we know there's some proteolysis of that protein occurs. But we found a few years ago that with the presence of a long repeat, transcription can terminate within TRON 1 producing this small Exon 1 only transcript called Huntington 1a, and this encodes the Huntington protein or Exon protein. And we've noted for 30 years that this protein is very, very aggregation prone and highly pathogenic. So is it the Huntington 1a protein that's the effect 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 polyacites within first intron become activated in the transcription terminates at those sites. So in human Huntington's disease first [indiscernible] in [indiscernible]. And in both cases, there are 2 sites activated in humans, 2.7 to 7.3 into the intron and in [indiscernible] base pairs. And in both 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 1 with different [indiscernible] CAG repeats. These were made in the labs of Marcy McDonald and Scott Siglin, 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, [indiscernible], 111, 140, and the one called 175 actually has stabilized at 190. So we've used this -- select this collection 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 homozygous mice. And all of these mice were the same age at an early -- the young mice, they're all the same age. And so the blue line shows you the amount, the blue bar show you the amount of Huntington 1a wild type mice, the orange ones in heterozygous line, you can see it's increasing with increasing CAG repeat length and the silver ones in [indiscernible], 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 Huntington 1a protein. And again, we're seeing that increase with increasing CAG repeat length. Next slide, please. So Huntington 1a transcript and protein is made at higher levels as a CAG repeat expense, can we detect this in human sales or tissues. Next slide. So we've used digital droplet PCR. We developed digital [indiscernible] PCR to specifically to the human Huntington 1a messenger RNA, and this is showing the levels that have been detected in postmortem brains in the sensorimotor cortex and in the HIPAA campus. And the CAG repeat length is on the bottom of the -- along the x-axis, it's moving, actually, I think -- we -- so we -- you can see there are increasing levels -- at the level -- the amount of Huntington 1a 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 adolescents. Next slide. [indiscernible] next slide, 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 Makara Lab who've taken postmortem brains. And in this case, in this sorted the nuclei from the striation 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 up with around 40 or 42 CAGs from in the blood as it would be measured in the blood, the CAG repeat has expanded in most cells. But in most cases, it's still in this range of less than 100 here with the median of around 70. And it's only 2% to 5% of the sales that have these very longer repeats more than 100. So given that the CAG, 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 some of the contingencies. And on the right-hand side, it's the control brain. The top panels are immuno probed 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. On this slide, on the left-hand side, we have the nucleus in gray and the cytoplasm on the right-hand side of that, there is the Huntington DNA. And then within the nucleus, we have the Huntington 1a pre-mRNA and full-length Huntington pre-mRNA that still contains the interims. So the full RNA is sliced and exported as a mature mRNA in cytoplasm on the Huntington 1a is transported to cytoplasm and there, of course, they translated to generate proteins. So if we wanted to target the full length Huntington alone, as Kevin said, we can target the transcripts from Exon to downwards. So you might be able to -- want to do that with antisense oligonucleotides or small molecules and they would work in the nucleus or with RNAi, siRNAs, microRNAs and they might work and they would work in the cytoplasm. So if you target from Exon to 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 this intronic region that's left on that Huntington 1a 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, Huntington 1a alone or full-length Huntington and Huntington 1a? Next slide. So I'm going to describe 3 pieces of work that were all published this 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 use the Q1 50 knocking mice and removed the cryptic polyasites from intron 1 so that transcription could not terminate in intron 1. And this was to prevent the generation of Huntington 1a and the mice with the dilution in the internal [indiscernible] delta intron mice. Next slide, please. So these western blocks show how much we've managed to reduce the Huntington 1a protein. This is Huntington that has been immunoprecipitated with an antibody to the muted proteins, 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 antibody that [indiscernible] detect all fragments. We have full length Huntington at top, Huntington 1a down below. Then there's 2 antibodies to Huntington 1a that is specific to Huntington 1a. You can see that in the delta intra-mice, there's very little left and then the final panel shows you an antibody to Exon 2, which doesn't detect Huntington 1a. And again, now we don't see it. Next slide, please. So what is the effect of removing Huntington 1a or decreasing the decreasing the level so much on aggregation. So if you look at the top 2 rows, there's striatum, the bottom 2 rows, deep layers of the cortex. The top one in each case is the knock-in mice, the one below is the delta intran-mice, and we're looking at aggregation from 2 to 17 months of age. If you look at the top 6 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 intramysis 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 to Huntington's disease. So one of these in neurofilament light chain and the other is one called [indiscernible] in humans. And here, it's BRP-39 and mice. So Kevin talked about neurofilament as a safety biomarker, but it's also a biomarker 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 [indiscernible], knock-in nice and the knock-in mice [indiscernible] in the intron working at 12 and 17 months of age. And if you look at the blue dots, you can see that the [indiscernible] both increase with disease progression. And when you look at the red dots, which is for the delta intra mice, 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 the -- 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 Carls lab at the University of Washington, and he used the [indiscernible], 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, Exon 2 and [indiscernible] to Exon 42. And the bottom schematic is the knock-in allele. And I told you that Exon -- mouse Exon 1 is replaced with human Exon 1, part of the intron is also humanized. So he had an antisense oligonucleotide to the humanized part of the intron. So that targeted Huntington 1a because it was before the cryptic polyacites and also the full-length mutant transcript. So here is red ASO muted was targeting Huntington 1a and full light mutant. And then you compare this to a pan ASO, which is the purple one in Exon 42 that just regulated the full-length transcripts, and not Huntington 1a. Next slide, please. So he gave these ICV injection at 3 months, again at 6 months and then mice was sacrificed at 9 months. If we look at aggregation in the [indiscernible], 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 neuron. So it's shown 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 9 months of [indiscernible]. The pan ASO, which down regulates full length mutant Huntington really had a negligible effect on aggregation. But when he looked with the mutant ASO that [indiscernible] Huntington 1a and full length mutant, there was a negligible aggregation left. So it was delayed by lowering Huntington 1a, but not by full length Huntington. Next slide. And another market [indiscernible] transcriptional disregulation. So a large proportion of the transcriptome becomes it's regulated in Huntington's disease, and we know that this is caused by that aggregated Huntington in the nucleus. So first fully compared in the top left here, the saline treated mice with the ones where he had just knocked out full length. So what we're looking to see is whether or not the ASO treatment had improved the disregulation in any way, and there was really no effect. But when you compare the saline treated mice with the ones that have not done Huntington 1a and mutant Huntington, then there were a lot of gene changes. And [indiscernible] focused on 188 consensus genes within the straight and that are always changed in Huntington's disease, and showed that when he used knockdown just for 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 like mutant Huntington have 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 in here, we used the 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 for Lantuntington alone with Huntington alone. Next slide, please. So this work was done with Anastasia [indiscernible], who is the Director of the RNA therapeutics Institute at UMass. And she had a very potent siRNA that targets for length Huntington, and with her lab, we identified some that targeted the interim before the first [indiscernible] because these are acting in the cytoplasm, then they are specific to either Huntington 1a or full length Huntington. And again, so the full length one is very potent and will knock down up to around -- up to 80% after one month in vivo. The one targeting Huntington 1a was much less potent and really only if we never saw more than 50%. Next slide, please. So we designed a fairly complex study with these. The -- it was ICV, bilateral ICV injection, there was an untreated group, a group treated with a non-targeting siRNA, the group -- cohort with the one targeting full-length Huntington and one with the targeting just Huntington 1a. And we had a baseline set started 2 months take 3, just to make sure that the siRNAs are working as they should do. And then an early cohort, so we take -- we injected 2 months and take it 6 months. The double cohort 2 months, 6 months and again 6 months and 10 months and then the late cohort at 6 months and 10 months. And these siRNAs were most potent and durable in the [indiscernible] campus. And so that was -- that is 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 CA-1 region of the HIPAA campus. And if we look at the untreated images on the left. The early cohort was stocked at 6 months and the [indiscernible] later 10 months. So that shows you what aggregation looks like in the parameter cells of the CA-1 at 6 and 10 months. We have this diffuse staining again and the inclusion bodies are more pronounced at 10 months than they were at 6. The non-targeting siRNA had no effect. The one targeting full-length Huntington have no effect. 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 6 months, then going in later has less of an effect. Next slide, please. But it's a little more complicated than that. So I'd just highlight what we saw in the CA-3 region. Again, if you look at the untreated region, we're looking the untreated samples on the left hand side, early is at 6 months, double and late 10 months. We have a diffused stain in the nucleus and then we see inclusion bodies at 10 months [indiscernible] as well. But we also have a layer of extra nuclear 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 nuclei inclusions and it did a lot to remove or slow down the generation of the extra nuclei aggregates. So once again, Huntington 1a [indiscernible] 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 with the top 2 of the double cohort treated at 6 months and taken at 10 months. And then the -- sorry, 2 months and 6 months and 10 months, the latest 6 months internments. And you can again see that although less potent, the Huntington 1a [indiscernible] 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 less potent siRNA than the one time full length Huntington was generally more effective. But the onetime full length Huntington, was very potent and it had some results in some improvements. Next slide, please. So in summary, we -- 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 as 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 a molecular basis of Huntington's disease with us. My name is Toby Ferguson. I'm a neurologist, a neuroscientist and [indiscernible] neuroscience therapeutic area at Alnylam. I'll now take you through our initial Phase I clinical data, which we just shared recently at [indiscernible] as well as our next steps for our clinical development. Here you can see the general design of our Phase Ib study. It's a randomized, double-blind, placebo-controlled study designed to [indiscernible] the safety and pharmacology of one dose of [indiscernible] brezosiran. The trial enrolled people with HD between ages of 25 and 70 with over 39 CAGs. We also had to be diagnosed in Huntington stage 2 or early stage 3, [indiscernible] had critical signs and symptoms or evidence of early functional [indiscernible]. The study is ongoing in Canada, Germany and the U.K. 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 2 important questions in parallel. How much Huntington lowering can we achieve with a single dose of brezosiran and what is the safety profile associated with that lowering? Patients were randomized to one dose of single intrathecal brezosiran 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 6 months, followed by a variable duration follow-up that allowed us to characterize the durability of the molecule and then patients are able 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 5 dose cohorts through 3 months. The study's primary endpoint is safety and tolerability. Secondary endpoints include CSF and plasma PK profiles as well as importantly, change from baseline and CSF mutant Huntington levels, a key biomarker of target engagement. Additionally, we are measuring exploratory endpoints, such as clinical scales like the PSC or UHDRS, imaging biomarkers and biomarkers of disease progression and safety such as neurofilament. Turning to the next slide. The population included in 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 a repeat number and measures of disease severity, including total function capacity and CAP 100. CAP 100 is an estimate of the cumulative exposure in [indiscernible] 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 II or Stage III 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 a traditional SMC assay. In this context, you can see a clear dose-dependent reduction of CSF Huntington as we increase the dose. And we observed a mean level reduction of the top cohort of 64% at month 3 with individual reductions up to 80%. However, when we dug into assay performance, it became clear that a significant percentage of individuals in this trial, in this case, about 1/3 fell below the lower limit of quantification, especially after deep Huntington lowering. Given the sensitivity limitations, that it was important to confirm more robustly our measurement of CSF [indiscernible]. To that end, we also measure CSF Huntington with a new, more sensitive mutant Huntington revised assay. This asset was developed in collaboration with CHDI, Alamar, Evotec, and we help to develop further the assay for clinical trial use. Here on the right, you can see with this assay, we had no participants below the lower limit of quantification. It enable us to measure mutant Huntington across all study participants even those with very low levels. We saw similar dose-dependent reductions in mutant Huntington with a mean reduction of 60% at month 3 and individual reductions up to 83%. Thus, these results provide an [indiscernible] confirmation of robust and durable target engagement following a single dose of brezosiran. These initial Phase Ib data are clearly encouraging and suggest that Brezosiran can successfully knock down [indiscernible], supporting the key objective of our study. We also highlight the other key goal was to determine, is this Huntington knockdown tolerated? So let's turn to the safety experience thus far. As a reminder, the study is still ongoing. So the safety analysis is pooled across both placebo and treated participants. In this analysis, adverse events recorded and most participants across all doses as is common in neurogenerative disease studies. However, across the 5 cohorts, including analysis, we observed only a limited number of serious and severe adverse events. And importantly, it did not increase with frequency with increasing doses. There were 2 serious adverse events -- excuse me, 2 severe adverse events, in dose group 4, a radius fracture and likely [indiscernible]. The 2 severe events were also reported at [indiscernible], and there was one participant in group 2 had a headache that has since resolved that was reported severe but nonserious. In addition to the severe and serious events I just noted, there were 2 additional serious adverse events in dose group 4 and acute myocardial infarction that is resolved with [indiscernible] 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 type 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 have seen us inflammation are altered, given that in some other Huntington lowering programs, evidence of CNS inflammation was observed, and it could be potentially ready to either Huntington's lowering or the platform or study drug tested. We've seen this 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 showing today, you can see that both CSF white cells and [indiscernible] measurements were similar between treated and placebo participants. Additionally, most observed values are within the normal range as denoted by the dash 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 whether or not we see increases in neurofilament with Huntington [indiscernible]. As both Kevin and Gill have mentioned, [indiscernible] is important biomarker for both safety and in some cases, efficacy. To remind, [indiscernible] is a cytoskeletal structural compound that in many CS neurons, it's released into 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 Huntington [indiscernible] programs observed increases in neurofilament within the first 6 months after dose initiation. Here we show both the absolute and full change in CSF performance over the course of the ongoing study. We've also demarketed in gray, the range of values observed in the placebo treated participants. Clearly and importantly, we've not observed any full change increases in CSF [indiscernible] in individual [indiscernible] brezosiran that exceeded the range [indiscernible] placebo. [indiscernible] absolute changes differ meaningfully from placebo. Furthermore, even with increasing doses, we observed no change in [indiscernible] levels. Given the emerging importance of neurofilament as a tool to measure neuronal injury, these data clearly suggests over these time points and with this analysis, but neither brezosiran nor Huntington lowering increases neural injury or degeneration. We feel this is an important observation given the product concern that Huntington lowering [indiscernible] injury or the potential treatments may increase [indiscernible] levels independent of target reduction. Finally, we measured brain ventricle volumes using MRI. The important implication of change in ventricle 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 Huntington-lowering agents. So there is a concern in the field. This can potentially reflect either increased both atrophy or inflammation that disrupts the CSF flow which result in increasing ventricle size. In contrast to those other Huntington lowering programs, we did not observe any dose-dependent increases in the size of ventricles after brezosiran treatment. As [indiscernible] size, increases observed appeared consistent with the placebo group. Overall, these initial results provide an encouraging safety profile of brezosiran 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, brezosiran, unlike most other Huntington lowering agents being investigated, aims to reduce both full-length and mutant -- both full-length mutant Huntington and the shorter 1a [indiscernible]. The emerging preclinical data and the human data suggests that 1a could be an important contributor to pathogenesis as you heard from Dr. Bates. Finally, initial data suggests that brezosiran has the potential to deeply and durably reduce Huntington and people with Huntington's disease and so far as well tolerated. These results give us confidence to advance brezosiran in 2 complementary studies in parallel. The ongoing Phase b open-label extension will be continued and will allow us to -- for all participants to receive Brezosiran every 6 months. This will enable us to continue to characterize the longer-term safety of sustained [indiscernible] in production 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 brezosiran can slow Huntington's disease progression. And to the best of our knowledge, will be the first placebo-controlled test the [indiscernible] hypothesis. Advantage of these studies in parallel reflects both our confidence of the profiles we observed 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 brezosiran offers a differentiated approach to under lowering, supported by 3 key findings. First, our inclusive Exon 1 targeting strategy is designed to lower all forms Huntington, including the HTT1a isoform, which may be quite important in disease pathogenesis. Second, our initial Ib results, we've seen patients -- we see patients on brezosiran experience deep and durable Huntington lowering after a single intrathecal dose. Across the first 5 cohorts, dose-dependent reductions of CSS Huntington were observed with a mean reduction of 60% to 64% in the highest dose group evaluated and maximum individual reductions up to 83%. Third, and importantly, we're encouraged by the safety profile observed to date with no clinical evidence of resource rate-related safety events, no evidence of dose-dependent increases in either markers of CNS inflammation or markers of CNS injury. And finally, we saw no evidence of dose-dependent change in ventricular volume. Our findings to date do not establish clinical efficacy. [indiscernible] longer, larger studies are required for that, put together that 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, like a moment to comment on the unmet need and the opportunity in Huntington. The unmet need in Huntington's disease is substantial and clear. We estimate there are about 100,000 diagnosed symptomatic individuals worldwide, about 32,000 in the U.S. alone. There are no approved disease modifying therapies currently available. Not included in those estimates are of the more than 100,000 carriers in the U.S. and even more globally who may be symptomatic or undiagnosed. The patient population is [indiscernible] identifiable [indiscernible] is often concentrated to 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 effective disease-modifying therapy, we believe this can create an efficient path, patient identification through treatment delivery as well as the potentially meaningful and commercial opportunity. However, most importantly, we think brezosiran represents a major opportunity to address a devastating neurologic disease for patients and families who 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 have may missed it. [Presentation]

Alyssa's story reminds us of the incredible need of this community for transformative therapies and [indiscernible] with urgency, rigor and in close partnership with the broader HD community to determine if brezosiran can fill the need from 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 and hearing her experience and for others like her is incredibly motivating as we advance programs like brezosiran 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 Brezosiran program lead; Toby Ferguson, who's the Neuroscience Therapeutic Area Head, and Christi Kelsey, who is the Senior Vice President of Value and Pipeline Commercialization at Alnylam. And as well, we're very privileged to have Dr. Gill 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's disease and research on the ACT 1a isoform. So we've gotten a number of questions, a lot of interest clearly in this program. So I think probably the #1 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.

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 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, so it is also that in our experience, when we've looked at agents that are targeting Exon 1 the level of knockdown of full-length Huntington and the Huntington 1a is relatively comparable, although we have -- I have to say we have not looked at [indiscernible]. But I think that's our preclinical experience.

And Kevin?

Look, I think the short answer is that no one really knows for sure. At this stage, a clinically meaningful threshold for Huntington lowering has not been established. This has been a hot topic at the date 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 the monogenic diseases like Huntington's, we assume greater lowering of [indiscernible] would lead to better outcomes. We know from animal models like the work that Dr. Bates as talked about, the 50% seems to give a good effect. On the safety side, we also know from human genetics that heterozygous loss of wild type of Huntington appears to be tolerated from birth, which maybe similar [indiscernible] is not lowering. So you kind of coalesce on that sweet spot at 50% seems like a reasonable place to start. Based on the totality of the data that we have, we think approximately 30% lowering would be a good place to start. We don't think this is a floor it kind of target 90%, 95% is necessary. And then maybe I'll just also highlight the preclinical work that I spoke to earlier, we really, really deeply interrogated the safety of wild-type Huntington lowering in preclinical studies and nonhuman primates. To really ask, is there any phenotypes associates with very deep wild type Huntington in primates, and we have not seen one. So that gives us confidence that we can maybe start trying to target that it was not lowering and maybe there's room to push beyond that.

Great. And then maybe I think the other sort of really common question in Dr. Bates, I think a lot of favorable feedback coming back from your deep discussion around HTT1a. But a question saying, look, if HTT1a 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 brezosiran is, in fact, lowering the 1a species?

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 Huntington 1a levels being generated in every cell. This is where we're able to measure Huntington 1a 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 postmortem brains. And when you look at the level of the CAG repeats, if you remember the work I showed you from Steve [indiscernible] 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 range whereby they are going to become sick 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 a 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 the small number of cells, and those are only the vulnerable cells with the long repeats. So we can see it because 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 in the mice as the mice age and yet 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 -- so measuring soluble Huntington 1a in the CSF, I think, is very unlikely to be possible. I would I'd be very, very happy if it was seen, but I think it's unlikely to be possible. So -- but you can measure the -- you can measure full-length Huntington decrease in the CSF. As I mentioned earlier, in our experience, when we worked with an agent that targets Exon 1, the level of knockdown of Huntington 1a and full length Huntington is equivalent. So you would expect to be able to infer in a person, the amount you were knocking it down by measuring the knockdown of full-length Huntington. In the mice, we can measure it precisely, but it's going to be a challenge in people, and I think you should be able to infer.

Thank you, Dr. Bates. Kevin, anything you want to add from what we understand about that?

Yes. Maybe just a reminder, we designed brezosiran to target a conserved sequence in that Exon 1 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 HT1a is not present in normal primate, so we can't do that work in the monkey work. We've not yet tried to measure HT1a and CSF from our ongoing Phase I trial. And so far, as you just heard from Dr. Bates, 1a has only been detected in post-mortem brain tissue and aggregates tied up in cells and colocalizing with cells that contain the elongated CAG repeats. It's not yet been detected in the CSF of [indiscernible] patients. And in our trials, we're limited to sampling the CSF. We can't 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 not to be produced cells -- subset of cells with those elongated CAG repeat expansions that are very short-lived, and they also remain trapped in aggregate with themselves, leaving very little or no protein available for detection. The field is working to try to develop even more sense of assays for 1a that could potentially be used to look for 1a 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're -- we may give it a shot and look in the future. But as a point that Dr. Bates made, it's unlikely we will ever be able to find it in the CSF.

Thanks, Kevin. Toby, there's a question for you, I think, people commenting on the reproducible pharmacology and the dose-dependent pharmacology across the 2 assays. But there is a question about, are we reaching a ceiling effect? As you look across these 2 assays, they're not seeing incremental additive knockdown. So why are we continuing to escalate the dose as we kind of look at this, maybe you could comment on how you're thinking about the pharmacology that we're seeing?

Thanks, Pushkal. I think I'd highlight overall, our goal really is to identify, as we've highlighted, a regimen that can really deep and durable knockdown to target, 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 at those 2 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 we've seen preclinically, I'd note -- we highlighted in multiple doses in the nonhuman primates that we get about 80% 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're measuring this in CSF, but we think we need to actually get the sRNA into the striatum into the [indiscernible]. 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. [indiscernible] where this molecule is going.

So when this molecule is administered an intrathecal lumbar space and what we know, we've added the C6 [indiscernible] that allows that robust bio distribution. 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 were this triatum. So we're not driving for [indiscernible] per se in CSF, but we want to drive that knockdown in those deeper tissues, and that may be another reason to exploit these higher doses.

And maybe I'll just add. We've got the benefit of multiple programs now in the CNS, where we've been able to do preclinical work in nonhuman 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 branch issues, including the [indiscernible], which are relevant for this indication and other deep regions may be relevant for other indications. And then 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 the clinic, one of the challenges is the 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 HTT 1a, but also 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 nonclinical studies around wild-type lowering.