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 Centre at University College London, who will provide perspective on the role of the huntingtin 1a 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.
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. 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 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. 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 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 targeting engagement and broad biodistribution across the CNS in non-human primates. 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 huntingtin. 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'll discuss the unmet need in Huntington's disease and the approach that we're taking with brezosiran. Kevin?
Thanks, Pushkal. I'm Kevin Sloan, the program lead 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 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 in midlife during prime working and family years. Average lifespan is shortened to approximately 10-30 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. Huntingtin 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 huntingtin gene. A normal huntingtin 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 huntingtin proteins that are toxic to cells, including full-length mutant huntingtin and also a shorter mutant huntingtin isoform called huntingtin 1a. 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. 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 huntingtin 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 huntingtin encoded by 67 exons, and a shorter mutant huntingtin 1a isoform encoded by only that first exon. Both full-length mutant huntingtin and huntingtin 1a are implicated as toxicity drivers, and Dr. Bates will go deeper into the biology of huntingtin 1a and preclinical evidence supporting its role as a driver of disease. Importantly, not all past or current huntingtin lowering drug development approaches have been able to effectively address huntingtin 1a.
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 huntingtin protein. When we designed brezosiran, we considered several possible strategies. Lowering huntingtin 1a alone, lowering full-length huntingtin alone, or take an inclusive approach to lower both. We ultimately selected the latter, taking an Exon 1 targeting approach designed to lower all relevant huntingtin species in the CNS, including both full-length mutant huntingtin and huntingtin 1a. Huntington's disease researchers and industry partners have long been trying to tackle this disease, and huntingtin lowering has been the primary targeting strategy for therapeutic development since the responsible gene was identified in 1993. However, progress with huntingtin 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 huntingtin lowering itself. For example, some huntingtin 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 huntingtin lowering below 50%. The ASO tominersen, which was designed to lower full-length mutant huntingtin and wild-type huntingtin, was reported to cause dose-dependent NfL increases within six months, despite moderate CSF mutant huntingtin reductions. The ASO WVE-003, which was designed to lower full-length mutant huntingtin but spare wild-type huntingtin through an allele selective targeting, was also reported to cause large increases in NfL within six months, despite modest CSF mutant huntingtin reductions. A couple of key questions remain unanswered. First, how much mutant huntingtin lowering may be needed to maximize efficacy?
Second, how much wild-type huntingtin lowering can be safely tolerated? Here at Alnylam, we believe there is still tremendous potential in huntingtin lowering, and we are 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 longstanding 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 potential to avoid some of the challenges that I have just outlined. Brezosiran is an intrathecally administered investigational C16 RNAi therapeutic for Huntington's disease.
It targets a conserved mRNA sequence within the first exon of huntingtin, leading to reduced expression of all mutant huntingtin species in the CNS, including both full-length mutant huntingtin and the shorter mutant huntingtin 1a. Because the first exon is shared across mutant and wild-type alleles, our approach is also designed to lower wild-type huntingtin. 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 five ongoing clinical programs. We are highly encouraged by the preclinical data we have generated over the years. In non-human primates, brezosiran has produced deep dose-dependent huntingtin 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 huntingtin 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 chain, no histological changes, and no adverse findings even after very deep wild-type huntingtin lowering beyond 90%. To summarize, we see three 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 deep and widespread huntingtin lowering throughout the brain with a durable and reversible approach. Third, its Exon 1 targeting strategy is designed to lower all relevant huntingtin isoforms, including both the full-length mutant huntingtin and the shorter toxic huntingtin 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 Centre 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 Ferrier 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 huntingtin and the potential importance of huntingtin 1a. Dr. Bates?
Thank you very much, Kevin, for that kind introduction. 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 steps in the pathogenesis of Huntington's 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 huntingtin in mouse models of Huntington's disease. Next slide, please. As we've heard, Huntington's disease is an autosomal dominant disorder. It generally starts in mid-life to late old age, although there are cases that begin in early childhood or adolescence. 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 chorea form 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. We've heard that the mutation that causes Huntington's 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 or less CAGs, 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- 39 CAGs have an increased chance of developing the disorder.
This encodes a large multifunctional scaffold protein that is present in every cell, and the CAG repeat encodes a stretch of glutamine residues, and the mutation imparts a gain of function to this protein. Whilst the CAG repeat expansion is related to the age of onset of the disorder, it is not the only factor. As you can see from the graph on the left-hand side, those early onset cases beginning before the age of 20 or so do 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 at any time from 20s up to the 70s or later. There must be other factors involved, and we know that some of these are genetic modifiers. Next slide, please.
If we look at the neuropathology of Huntington's disease, here we are looking at the coronal sections through a brain from somebody who is unaffected with the disease and one of somebody who died from late stage HD. As Kevin said, this is a whole brain disorder. There is a generalized atrophy across the whole brain. On top of that, you have a selective neuronal cell death. This is very apparent in that region of the striatum or the caudate putamen, which here has been reduced to a rim of tissue because 95% of the neurons in that region, the medium spiny neurons, are ones affected by the disorder. But there is cell loss in many other regions of the brain as well. 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 N-terminus of the protein. That reveals these inclusion bodies. As you can see in this figure in D, there are inclusions in neuronal nuclei. Otherwise, they are scattered throughout the neuropil lining up in processes, and they are present in presymptomatic brains. Next slide, please. Kevin has mentioned somatic CAG repeat expansion, and we now think this is the first step in the disease pathogenesis. What is this repeat expansion? Next slide, please. As we work our way through this slide, the top left-hand corner, as I have mentioned, more than 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.
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 alleles of repeats of several hundred, up to 700 CAGs. We have known that for more than 20 years, that some of these very large expansions can be present in the brain. Genome-wide association studies over the last 10 years or so have produced high-quality, reproducible data. Irrespective of what the phenotype that they are 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. 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 somatic expansion from occurring. 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. If that's the first step in the pathogenesis, what might be the second? Next slide. Here again, we have the gene with 67 exons. This is transcribed to a pre-mRNA, processed, produced the mature mRNA as shown on the right-hand side of this slide, and then that encodes this large protein. We know that some proteolysis of that protein 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 huntingtin 1a, and this encodes the huntingtin 1a protein or exon protein. We've known for 30 years that this protein is very, very aggregation prone and highly pathogenic. Is it the huntingtin 1a protein that's the effector through which the expansion causes these pathogenic consequences? Next slide, please. Drilling down into this just a little bit more detail. What happens is that when you have a long CAG expansion, cryptic poly(A) sites within the first intron become activated and the transcription terminates at those sites. 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 or 7.3 kb into the intron, and in mouse 680 and 1,145 base pairs, and in both cases, it's called huntingtin 1a. Next slide, please. 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. These are knock-in mouse lines, and the mouse Exon 1 has been replaced with a human Exon 1 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 expansions shown in this table. There's a line with 20, 50, there's line 80, 111, 140, and the one called 175 actually has stabilized at 190.
We've used 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 huntingtin 1a transcript and protein levels. Next slide, please. 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. All of these mice were the same age at an early. They're young mice. They're all the same age. The blue bars show you the amount of huntingtin 1a in wild-type mice. The orange ones in heterozygous mice, you can see it's increasing with increasing CAG repeat length. The silver ones in homozygous, which is twice as much as the hets.
In the panel on the right-hand side, we have an assay that is specific to the huntingtin 1a protein. Again, we are seeing that increase with increasing CAG repeat length. Next slide, please. The huntingtin 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. We have used digital droplet PCR. We have developed digital droplet PCR too, specifically to the human huntingtin 1a messenger RNA, and this is showing the levels that have been detected in postmortem brains, in the sensory motor cortex and in the hippocampus. The CAG repeat length is on the bottom along the X-axis. It is moving, actually, I think. You can see there are increasing levels at the level. The amount of huntingtin 1a is increasing with increasing CAG repeat length.
It is most apparent in those brains from people who have had longer repeats and have developed the disease in childhood and in adolescence. Next slide. Next slide, please. It is challenging to identify this in patient brains because we have to think about what the CAG repeat length is in most of those cells. Some of the outstanding work in this area has been done by the McCarroll Lab, who have taken postmortem brains, and in this case, sorted the nuclei from the striatum of those brains and then measured the CAG repeat length in those cell nuclei. If you are looking at a brain, if we look at that plot along the top, if you are looking at brains that start off with around 40 or 42 CAGs, as would be measured in the blood, the CAG repeat is expanded in most cells.
But in most cases, it is still in this range of less than 100, here with a median of around 70. It is only 2%-5% of the cells that have these very longer repeats, more than 100. Given that the huntingtin 1a is associated with the long CAG repeats, and that is where we are seeing the most generated, when we are looking at bulk RNA, this is going to be diluted out by the fact that most of the cells have shorter repeats. Of course, there are all of the cells that are not the vulnerable neurons in which no expansions have occurred. 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? We can see the protein in the fact that it has aggregated.
If we are looking at on the left-hand side, we have brain sections from someone with Huntington's disease, and on the right-hand side, it is a control brain. The top panels are immunoprobed with an antibody to the beginning of the huntingtin protein, and we can see these inclusion bodies. If we use an antibody that is specific to huntingtin 1a, it only detects that small protein. Again, we can now see those inclusion bodies. That means that within these cells, there was sufficient protein to aggregate and form these very large structures. Next slide, please. If we want to lower huntingtin, 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 huntingtin DNA.
Within the nucleus, we have the huntingtin 1a pre-mRNA and the full-length huntingtin pre-mRNA that still contains the introns. The full-length RNA is spliced and exported as a mature mRNA to the cytoplasm, and the huntingtin 1a is transported to cytoplasm. There, of course, they are translated to generate proteins. If we wanted to target the full-length huntingtin alone, as Kevin said, we can target the transcripts from Exon 2 downwards. 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 would work in the cytoplasm. If you target from Exon 2 downwards, you will just reduce the level of full-length huntingtin alone.
If, as Kevin mentioned, you target Exon 1, either with ASOs or small molecules in the nucleus or RNAi in the cytoplasm, you will reduce the levels of both huntingtin 1a and full-length huntingtin. If you want to target huntingtin 1a alone, you are really restricted to using RNAi in the cytoplasm against this intronic region that is left on that huntingtin 1a messenger RNA because those intronic regions are still left in the nucleus. If you target that in the nucleus, you will, again, knock down both species. Next slide, please. The question then is what is the relative pathogenesis of huntingtin 1a and the full-length mutant huntingtin? Should we target full-length huntingtin alone, huntingtin 1a alone, or full-length huntingtin and huntingtin 1a? Next slide.
I am going to describe three pieces of work that were all published this year. I am not going to have time to show very much of the data, so I have just selected a couple of results from each study. 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. The first approach we used was to try and prevent the huntingtin 1a protein being generated in a mouse model. For this, we used the Q150 knock-in mice and removed the cryptic poly(A) sites from Intron 1, so that transcription could not terminate in Intron 1. This was to prevent the generation of huntingtin 1a, and the mice with the deletion in the intron are called the Delta intron mice. Next slide, please.
These western blots show how much we have managed to reduce the huntingtin 1a protein. This is huntingtin that has been immunoprecipitated with an antibody to the mutant protein. The wild type lanes are all negative because there is no mutant protein. In the first panel, we have probed with an antibody that will detect all fragments. We have full-length huntingtin at the top, huntingtin 1a down below. Then there are two antibodies to huntingtin 1a that are specific to huntingtin 1a. You can see that in the Delta intron mice, there is very little left. The final panel shows you an antibody to Exon 2, which does not detect huntingtin 1a, and again, now we do not see it. Next slide, please. What is the effect of removing huntingtin 1a or decreasing the level so much on aggregation?
If you look at the top two rows, there is a 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. We are looking at aggregation from 2- 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. Within the nuclei, you get this diffuse aggregation accumulating, which resolves as a nuclear inclusion as the disease progresses. You can see in the Delta intron mice, this has been slowed down considerably. Next slide, please. 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.
One of these is neurofilament light chain, and the other is one called YKL-40 in humans, and here it is BRP39 in mice. Kevin talked about neurofilament as a safety biomarker, but it is also a biomarker that accumulates with disease progression in HD. Here, we are looking at. We have a wild type, the wild-type mice with the deletion in the intron, the knock-in mice, and the knock-in mice with the deletion in the intron. We are looking at 12 and 17 months of age. If you look at the blue dots, you can see that the NfL and the mouse YKL-40 both increase with disease progression. When you look at the red dots, which is for the Delta intron mice, there is absolutely no NfL or YKL-40 there.
It is completely rescued the levels of these translatable biomarkers in the CSF if you are not making the huntingtin 1a protein. These mice, of course, still have all of the full-length mutant huntingtin. Next slide, please. The next project I am 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. He used antisense oligonucleotides. He was able to target the full-length huntingtin gene, both the mutant and wild type, and compared that to the full-length mutant as well as huntingtin 1a. Next slide. Just looking at where these are targeted in a little bit more detail. 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 the knock-in allele.
I told you that mouse Exon 1 is replaced with human Exon 1. Part of the intron is also humanized. He had an antisense oligonucleotide to the humanized part of the intron, so that targeted huntingtin 1a because it was before the cryptic poly(A) sites, and also the full-length mutant transcript. His red ASO mutant was targeting huntingtin 1a and full-length mutant, and then he compared this to a pan- ASO, which is the purple one in Exon 42 that just down-regulated the full-length transcripts and not huntingtin 1a. Next slide, please. 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. The green is indicating this is huntingtin.
You've got, again, the diffuse aggregated huntingtin and inclusion bodies. The purple, in this case, are neurons. It's showing that the aggregation is in the neurons. The first panel is with saline. That's the level of aggregation reached in these mice by nine months of age in the striatum. The pan- ASO, which down-regulates full-length mutant huntingtin, really had negligible effect on aggregation. But when he looked with the mutant ASO that down-regulates huntingtin 1a and full-length mutant, there was negligible aggregation left. It was delayed by lowering huntingtin 1a, but not by full-length huntingtin. Next slide. Another phenotype that he looked at was transcriptional dysregulation. A large proportion of the transcriptome becomes dysregulated in Huntington's disease, and we know that this is caused by that aggregated huntingtin in the nucleus.
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. 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 saline-treated mice with the ones that had knocked down huntingtin 1a and mutant huntingtin, then there were a lot of gene changes. 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 huntingtin 1a and mutant huntingtin rescued or partially rescued about 50% of the transcription levels.
Again, now the targeting huntingtin 1a as well as full-length mutant huntingtin had profound effects as compared to just the full-length huntingtin alone. Next slide, please. The final study I want to tell you about is one that was performed in my lab, and here we use these Q175 knock-in mouse models. Here we were using siRNAs, and we were able to compare the effect of targeting mutant and wild-type full-length huntingtin alone with huntingtin alone. Next slide, please. This work was done with Anastasia Khvorova, who is the Director of the RNA Therapeutics Institute at UMass, and she had a very potent siRNA that targeted full-length huntingtin. With her lab, we identified some that targeted the intron before the first poly. Because these are acting in the cytoplasm, then they are specific to either huntingtin 1a or full-length huntingtin.
Again, the full-length one is very potent and will knock down up to 80% after one month in vivo. The one targeting huntingtin 1a was much less potent and really we never saw more than 50%. Next slide, please. We designed a fairly complex study with these. It was ICV, bilateral ICV injection. There was an untreated group, a group treated with a non-targeting siRNA, a cohort with the one targeting full-length huntingtin, and one with the targeting just huntingtin 1a. We had a baseline set start at two months, take at three, just to make sure that the siRNAs were working as they should do. Then an early cohort, so we injected two months and take at six months. The double cohort, two months, six months, and again at six months and at 10 months.
The late cohort at six months and 10 months. These siRNAs were most potent and durable in the hippocampus, so that is the data I am going to show you next. Next slide, please. What does this do to aggregation? This is a bit complex. I will work you through it. 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. That shows you what aggregation looks like in the pyramidal cells of the CA1 at six and 10 months. We have this diffuse staining again, and the inclusion bodies are more pronounced at 10 months than they were at six. The non-targeting siRNA had no effect. The one targeting full-length huntingtin had no effect.
But when we looked at the one targeting huntingtin 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. It is a bit more complicated than that, so I just highlight what we saw in the CA3 region. Again, if you look at the untreated region, we are looking at the untreated samples on the left-hand side. Early as 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 extranuclear aggregates alongside the nuclear layer in this region of the brain. Again, non-targeting had no effect whatsoever. But now targeting full-length huntingtin, 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 extranuclear aggregates. Once again, huntingtin 1a, although less potent, was more effective, but full-length huntingtin certainly did have consequences. Next slide, please. If we look at the transcriptional dysregulation here, this is a chart showing the number of genes that were altered. This is only looking at 10 months of age. The top two are the double cohort treated at six months and taken at 10 months. Sorry, at two months, then six months, then 10 months. The late at six months and 10 months. You can again see that although less potent, the huntingtin 1a lane in the double cohort was more protective than the full length lane.
Treating late, actually, we had a lot of transcriptional changes were exacerbated in those mice. Essentially, lowering huntingtin 1a was, although a less potent siRNA than the one targeting full-length huntingtin, was generally more effective. But the one targeting full-length huntingtin was very potent and did result in some improvements. Next slide, please. In summary, from the work that we have done in mice, we think that the targeting strategies for lowering huntingtin should include huntingtin 1a. But right at the moment, we do not know whether or not targeting full-length mutant huntingtin as well gives any added benefit to targeting huntingtin 1a. I will 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 have described in this presentation. Thank you.
Thank you, Dr. Bates, for sharing your insights from your research on the molecular basis of Huntington's disease with us. My name is Toby Ferguson. I am a neurologist, a neuroscientist, and the leader of neuroscience therapeutic area at Alnylam. I will now take you through our initial phase I 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 Ib study. It is a randomized, double-blind, placebo-controlled study designed to evaluate the safety and pharmacology of one dose of intrathecal brezosiran. The trial enrolled people with HD between ages of 25 and 70 with over 39 CAG repeats. They also had to be diagnosed with Huntington's 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 U.K. Here we show a more detailed overview of our study. As Kevin had noted, understanding the safety of huntingtin knockdown has been confounded by technical issues, and therefore, this study was designed to evaluate two important questions in parallel. How much huntingtin 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 six months, followed by a variable duration follow-up that allowed us to characterize the durability of the molecule. 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 plasma PK profiles, as well as importantly, change from baseline and CSF mutant huntingtin levels, the key biomarker of target engagement. Additionally, we are measuring exploratory endpoints such as clinical scales like the TFC 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 functional capacity and CAP100.
CAP100 is an estimate of the cumulative exposure in mutant huntingtin, 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 huntingtin levels in the CSF has been a longstanding 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 huntingtin as we increase the dose. 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 huntingtin lowering. Given the sensitivity limitations, it felt important to confirm more robustly our measurement of CSF huntingtin. To that end, we also measured CSF huntingtin with a new, more sensitive mutant huntingtin NULISA assay. This assay was developed in collaboration with CHDI, Alamar, and Evotec, and we helped 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, enabling us to measure mutant huntingtin across all study participants, even those with very low levels.
We saw similar dose-dependent reductions in mutant huntingtin with a mean reduction of 60% at month three and individual reductions up to 83%. Thus, these results provided orthogonal 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 mutant huntingtin, supporting a key objective of our study. We also highlight that the other key goal was to determine is this huntingtin knockdown tolerated. 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 included in the analysis, we observed only a limited number of serious and severe adverse events, and importantly, they did not increase with frequency with increasing doses. There were two serious adverse event, or excuse me, two severe adverse events in dose group four, a radius fracture and a likely bacterial meningitis. The two severe events were also reported as serious, and there was one participant in group two 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 four: an acute myocardial infarction that has resolved with sequelae, and a resolved post-lumbar puncture syndrome. We did not observe any deaths, and there have been no study discontinuations due to adverse events.
It is 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 huntingtin and wild-type huntingtin has been well-tolerated so far. We have seen from the initial clinical and safety observations that reduction of huntingtin appears to be well-tolerated. However, it is important to understand whether biomarkers of CNS inflammation are altered, given that in some other huntingtin-lowering programs, evidence of CNS inflammation was observed, and it could be potentially related to either huntingtin lowering or the platform or study drug tested. With CNS inflammation, you can observe moderate, and 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 were 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. Let's turn to the question of whether or not we see increases in neurofilament with huntingtin lowering. As both Kevin and Gill have mentioned, neurofilament is an important biomarker of both safety and, in some cases, efficacy. To remind, neurofilament is a cytoskeletal structural compound that in many CNS neurons is 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. As Kevin discussed, several previous huntingtin lowering programs have observed increases in neurofilament within the first six months after dose initiation. Here we show both the absolute and full change in CSF neurofilament over the course of the ongoing study. We've also demarcated 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 neurofilament in individuals receiving brezosiran that exceeded the range observed in placebo, nor do the absolute changes differ meaningfully from placebo. Furthermore, even with increasing doses, we observed no change in neurofilament levels.
Given the emerging importance of neurofilament as a tool to measure neuronal injury, these data clearly suggest over these time points and with this analysis, that neither brezosiran nor huntingtin lowering increases neuronal injury or degeneration. We feel this is an important observation given the prior concern that huntingtin lowering may exacerbate neuronal injury, or that potential treatments may increase neurofilament levels independent of target reduction. Finally, we measured brain ventricle volumes using MRI. While the importance and 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 huntingtin lowering as a therapeutic approach have observed more pronounced dose-dependent ventricular enlargement over time in participants with huntingtin lowering agents.
There is a concern in the field that this could potentially reflect either increased atrophy or inflammation that disrupts the CSF flow, with resultant increase in ventricle size. In contrast to those other huntingtin lowering programs, we did not observe any dose-dependent increases in the size of the ventricles after brezosiran treatment, as the ventricle 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 huntingtin lowering. I'd like to quickly recap a few key points. First, brezosiran, unlike most other huntingtin lowering agents being investigated, aims to reduce both full-length mutant huntingtin and the shorter huntingtin 1a isoform.
The emerging preclinical data and the human data suggests that huntingtin 1a could be an important contributor to HD pathogenesis, as you heard from Dr. Bates. Finally, initial data suggests that brezosiran has the potential to deeply and durably reduce huntingtin in people with Huntington's disease, and so far is well-tolerated. These results give us confidence to advance brezosiran in two complementary studies in parallel. The ongoing phase Ib open-label extension will be continued and will allow all participants to receive brezosiran every six months. This will enable us to continue to characterize the longer-term safety of sustained huntingtin reduction, 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 for whether or not brezosiran can slow Huntington's disease progression. To the best of our knowledge, it will be the first placebo-controlled test for the huntingtin 1a lowering hypothesis. Advancing these studies in parallel reflects both our confidence in 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 huntingtin lowering, supported by three key findings.
First, our inclusive Exon 1 targeting strategy is designed to lower all forms of huntingtin, including the huntingtin 1a isoform, which may be quite important in disease pathogenesis. Second, our initial phase Ib results, we've seen patients on brezosiran experience deep and durable huntingtin lowering after a single intrathecal dose.
Across the first five cohorts, dose-dependent reductions of CSF huntingtin were observed with a mean reduction of 60%-64% at month three 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 brezosiran-related safety events, no evidence of dose-dependent increases in either markers of CSF inflammation or markers of CSF injury. Finally, we saw no evidence of dose-dependent change in ventricular volume. Our findings to date do not establish clinical efficacy. We believe longer, larger studies are required for that. Together, they give us confidence in the molecule, the targeting strategy, and the path ahead. Now that we have highlighted our molecule, emerging clinical data, and next steps, I'd like a moment to comment on the unmet need and the opportunity in Huntington's.
The unmet need in Huntington's 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 presymptomatic 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 brezosiran 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 would 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 are going to share a brief version of her video now for those who may have missed it.
I am 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, and I was proud of him. Watching him decline over the past decade with Huntington's disease has been really hard. I decided to get tested for Huntington's disease in 2020. I had seen multiple family members deteriorate. I found out in November of 2020 that I was gene positive for Huntington's 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 we ultimately decided to proceed with implantation of a Huntington's disease-free embryo. I am currently 18 weeks pregnant with a baby boy due in June. We are super excited that our baby is, in fact, Huntington's disease free. It is 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 brezosiran with urgency, rigor, and in close partnership with the broader HD community to determine if brezosiran can fill the need for a meaningful therapeutic for people with HD. I will 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 brezosiran to help patients with Huntington's disease. We are 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 will 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 is the neuroscience therapeutic area head, and Christi Kelsey, who is the Senior Vice President of Value and Pipeline Commercialization at Alnylam. As well, we are very privileged to have Dr. Gill Bates, Professor of Molecular Neuroscience and Co-Director of the Huntington's Disease Centre at University College London.
She will specifically speak to the preclinical biology of Huntington's disease and research on the huntingtin 1a isoform. We have gotten a number of questions, a lot of interest, clearly, in this program. I think probably the number one question that I have now seen a few times is, what level of HTT knockdown is necessary or optimal to be clinically meaningful in this disease? 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? Then maybe, Kevin, you can speak to what we have understood internally from some of our preclinical data and supplement what Dr. Bates has to say.
Yes, thank you. I think you would have seen from our mouse studies that knocking down huntingtin 1a by about 50% was having quite profound effects. I think one of the things you have to also remember is that the mice we are using have CAG repeats that are very long in all cells. This is an effect we are seeing in mice in which the disease's onset is going to be early and very progressive. I think our preclinical data is very encouraging. I think perhaps one other thing to say is, though, it is also that in our experience, when we have looked at agents that are targeting Exon 1, the level of knockdown of full-length huntingtin and of huntingtin 1a is relatively comparable. Although I have to say, we have not looked at your agent. I think that is our preclinical experience.
Great. Kevin?
Yeah. Look, I think the short answer is that no one really knows for sure. At this stage, a clinically meaningful threshold for huntingtin lowering has not been established. This has been a hot topic of debate in this 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? With a monogenic disease like Huntington's disease, we assume greater lowering of mutant huntingtin would lead to better outcomes. We know from animal models, like the work that Dr. Bates has talked about, that 50% seems to give a good effect. On the safety side, we also know from human genetics that heterozygous loss of wild-type huntingtin appears to be tolerated from birth, which may be similar to 50% lowering.
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 50% lowering would be a good place to start. We do not think this is a floor it kind of target, 90%, 95% is necessary. Then I will just also highlight the preclinical work that I spoke to earlier. We really, really deeply interrogated the safety of wild-type huntingtin lowering in preclinical studies in non-human primates to really ask, is there any phenotype associated with very deep wild-type huntingtin lowering in primates? We have not seen one. That gives us confidence that we can maybe start trying to target 50% lowering, and maybe there is room to push beyond that.
Great. Thank you, Kevin. Then 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 huntingtin 1a. But question saying, look, if huntingtin 1a is central to your therapeutic hypothesis, what do you know about how much you are actually lowering 1a? Are we inferring that? Are there preclinical data? Why can't we measure it? So maybe Dr. Bates, I think you did touch on this, but you could spend a little bit of time in terms of, it seems like we are able to measure this in certain mouse models, but there is maybe some limitations you highlighted in terms of being able to measure this in actual clinical studies. So maybe you could speak to that.
Then Kevin, you could also then expand on what we understand about whether brezosiran is in fact lowering the 1a species.
Right. Yes. When we are working with these mouse models, again, as I say, we are working with very long repeats in every cell. As you saw from the presentation, as the CAG repeat increases, the amount of huntingtin 1a produced is increasing. In the mouse models, we have huntingtin 1a levels being generated in every cell. We are able to measure huntingtin 1a easily in these mice, and we can tell very easily and accurately what the level of knockdown is. This is much more difficult when we come to people. Most of the work that informed us about huntingtin 1a in people at the moment is coming from postmortem brains.
When you look at the level of the CAG repeats, if you remember the work I showed you from Steve McCarroll's lab, when they are taking a postmortem brain and sorting cells and looking to see how long the repeat is, there is 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. The amount of huntingtin 1a produced in the whole brain, it is not like the mouse where you have a lot generated from all cells. It is going to be very small because it is only being produced in quantity from these small number of cells, and those are only the vulnerable cells with the long repeats.
We can see it because we know the protein is there because we can see the aggregates, but actually being able to detect the protein in a clinical sample would probably be quite difficult. As the mice age and get more aggregates, the amount of soluble protein you can detect decreases because it is all getting sucked into the aggregates. Measuring soluble huntingtin 1a in the CSF, I think is very unlikely to be possible. I would be very happy if it was seen, but I think it is unlikely to be possible. You can measure full-length huntingtin decrease in the CSF. As I mentioned earlier, in our experience when we have worked with an agent that targets Exon 1, the level of knockdown of huntingtin 1a and full-length huntingtin is equivalent.
You would expect that to be able to infer in a person the amount you are knocking it down by measuring the knockdown of full-length huntingtin. In the mice, we can measure it precisely, but it is going to be a challenge in people. 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 brezosiran to target a conserved sequence in that Exon 1 that is present in all forms of huntingtin, including huntingtin 1a. We have confirmed target engagement both in vitro and in vivo in mouse models, but huntingtin 1a is not present in normal primates, so we can't do that work in the monkey work. We've not yet tried to measure huntingtin 1a in CSF from our ongoing phase I trial, and so far, as we just heard from Dr. Bates, 1a has only been detected in postmortem brain tissue, in aggregates tied up in cells, and co-localizing with cells that contain the elongated CAG repeats. It's not yet been detected in the CSF of living patients. 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 huntingtin 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 a subset of cells with those elongated CAG repeat expansions that are very short-lived, or may also remain trapped in aggregates within cells, leaving very little or no protein 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 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 may give it a shot and look in the future.
But as the point that Dr. Bates made, it's unlikely we will ever be able to find it in the CSF.
Thanks, Kevin. Toby, maybe there is a question for you. I think people commenting on the reproducible pharmacology and the dose-dependent pharmacology across the two assays. There is a question about are we reaching a ceiling effect as you look across these two assays, and they are not seeing incremental additive knockdown. Why are we continuing to escalate the dose as we look at this? Maybe you could comment on how you are thinking about the pharmacology that we are seeing.
Thanks, Pushkal. I think it highlights overall our goal really is to identify, as we have highlighted, a regimen that can really achieve deep and durable knockdown to target what I have heard, at least as a target, 50% lowering of huntingtin. In the data we shared today, we saw with both assays, reduction in the mean range of about 60%, particularly at those two top doses. I think it is important to note that we really have not yet begun to explore the overall durability of these data. At those higher doses, we think we will both explore not only depth of knockdown but durability. If you put together the data we have seen in the clinical studies, the data we have seen pre-clinically, I would note we highlighted in multiple doses the non-human 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. I think maybe a related question that is coming up around the dosing is, you are measuring this in CSF, but we think we need to actually get the siRNA into the striatum, into the putamen and caudate. What do we understand, and is that part of the reason for escalating the dose, to get into those deeper brain structures? What do we understand about the biodistribution? Maybe you can touch on this, and then Kevin, you can add on top of that.
Sure. I'm happy to highlight.
Where this molecule's going.
When this molecule is administered in the intrathecal lumbar space, what we know, we've added the C16 moiety that allows that robust biodistribution. 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. Conceptually, we do know that we need to really achieve this knockdown in the key regions of interest, which are the striatum. We're not driving for a 50% knockdown per se in CSF, but we want to drive for that knockdown in those deeper tissues, and that may be another reason to explore these higher doses.
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 non-human primates where we can measure tissue lowering and measure CSF signals, and then we can build modeling across all of those. What we've seen consistently is dose-dependent lowering in brain tissue. As we push the dose up, we are able to get target engagement in various deep brain tissues, including the caudate and putamen and the striatum, which are relevant for this indication, and other deep regions may be relevant for other indications. Then, we really use that modeling data across all the programs to think about how to extrapolate from CSF lowering to tissue lowering.
But in clinic, 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. 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 huntingtin 1a, but also the wild type. 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- type lowering.
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 mutant and wild type. So treatment from 2- 10 months of age, we didn't see any adverse effects, but the knockdown's 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 is a fascinating question. When we first got into this field around 2018 and we were attending conferences, a lot of people raised this issue. What is the safety? We are not sure. Does the strategy make sense? I think there is probably some nuance here about the safety of wild type huntingtin lowering during development of the brain versus in an adult setting, after the brain has largely become developed. So there may be multiple answers to this question. When we look at human genetics, there really is not any evidence of humans walking around with complete knockout, like homozygous huntingtin 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 do not appear to have a phenotype. So we think that is probably equivalent to, let us roughly say, 50% loss of function. That gives you some confidence that 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 have just been talking about.
In our primate studies internally, we have done quite a lot of work. I think we have conducted now at least five individual primate studies over the last several years. Those have been for various purposes, various depths of knockdown over various periods of time. But, for as deep as 90% or more tissue-level huntingtin lowering for as long as 12 months in duration, in very rigorous toxicology studies where we are working up the data and really looking at the tissue at a histological level, we are not seeing any adverse effects. So that is really given us confidence that there may be a path forward, specifically with this siRNA-mediated huntingtin lowering approach.
Super. So really, the best we can see from the extensive non-human primate work we have done, we can get to pretty deep knockdown of wild type, we believe, and we are not seeing any adverse consequences even with that over an extended period of time. So let us flip to a little bit of the path forward. Toby, you highlighted sort of the ongoing phase I and the open label extension. I think a number of questions about when might we see some updated data from that might give us an inkling around efficacy. When will we see either clinical data, imaging data, more NfL data, et cetera? 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. I think overall, we have highlighted the design of the OLE is such that participants of the study can roll over. They get dosed every six months. We have also highlighted, as you noted, some key measures will include clinical scales such as the TFC and the cUHDRS. We will include important biomarkers like neurofilament. You have heard that can both be a safety biomarker, also possibly an efficacy biomarker. So these are going to be useful over time. I think particularly the longer time. What we have seen 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 Huntington's to really understand if there is potential efficacy.
We are certainly interested in this over time, but we do think we will need a relatively long period of time, 24 months- 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 it will be some time until we really share any potential look at efficacy based on the OLE.
Toby, I guess on a related point, some questions. People noted that uniQure might be pursuing a potentially accelerated approval using a natural history comparison. Do you think that the data that you are generating out of this OLE might offer that same opportunity?
It is really an excellent question, but I think simply at this point, it is really way too early as to whether or not the data from the OLE could support an accelerated approval. The appropriate path regulatorily will really depend on the totality of the data, the discussions we have with health authorities, the evolving regulatory landscape. That said, we have highlighted we are pursuing two parallel tracks. The OLE, as I have noted, is intended to provide long-term safety and exploratory efficacy information. That is important. We have highlighted tools like the clinical scales and neurofilament. That being said, we have also highlighted we are investing in a large placebo-controlled pivotal study that we plan to start in 2027, pending regulatory discussions. It is really that study that we view as the definitive test of whether or not brezosiran can slow disease progression.
Ultimately, our real goal here is to advance brezosiran in the most efficient way possible while definitively testing the efficacy and safety. Our current belief is that the adequate and well-controlled pivotal 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. This is actually a perfect segue to the next set of questions that we are getting, which is really around that pivotal trial design. You spoke at high-level terms. Are you able to provide any more color in terms of the population? I think there are questions around whether we will be 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 have certainly engaged with regulators early and often around our phase I data sets, and that is ongoing. But fundamentally, as we said, we plan to initiate the placebo-controlled study in 2027, but that really is pending on regulatory discussions. So really, once we have those discussions, we can share more detail on the study design and the key features such as the inclusion criteria, the populations, and the key endpoints.
I think it is 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 clinical endpoints as the primary endpoint, and then a series of secondary endpoints. Is that all fair?
That is quite fair. I think, in the context of the field, it has coalesced around a number of key endpoints, such as the cUHDRS and TFC, and as I already highlighted, the longer durations are likely needed.
Good. 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, for example, TfR shuttle, to administer agents systemically, rather than going through the intrathecal route. I guess the question about, A, is that necessary to move forward in this kind of an approach, and where we are with our own shuttle approaches? Maybe, Christi 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 brezosiran for patients with Huntington's disease. Then, maybe Toby Ferguson, 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. Maybe the question behind the question there is, do we see the intrathecal administration as a key commercial challenge? 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 disease, 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. We've heard so far that an infrequent, potentially twice-a-year 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 Ferguson to talk about our efforts related to shuttle. Toby , over to you.
Thank you, Christi. 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. 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 siRNA to? 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? I think there are a number of factors, but I think you've heard from Christi Kelsey 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 brezosiran in a strategy that targets both huntingtin lowering of huntingtin 1a and the full length can slow Huntington's 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. Dr. Bates, one for you. I think you showed us some really interesting data on preclinical models. There was 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 preclinical evidence that you've generated, or you and your colleagues? What might be the ideal time to intervene 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. Again, if I come back to the fact that the mouse models 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. The data we have suggests that if you administer the therapeutic very early, then you have a more pronounced effect than if you go in a little bit later. I think that's a message that people are very receptive to generally with these treatments for Huntington's disease. But I think we'll only find out really with time, with clinical studies, because we really don't understand the trajectory of these molecular changes in the patient brain when this somatic expansion is occurring in a stochastic manner throughout the brain and throughout life.
Within the mice, it says earlier is better than later, but I think 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. Maybe just the last question for this morning. Christi, 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? 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? Obviously there are none now.
It'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 100,000 gene carriers. If you just do the math, that shows a very low diagnosis rate. Unsurprisingly, patients are hesitant to get diagnosed if there's no therapies available. You heard Alyssa talk about her situation and seeing what her dad went through. She was one of the few patients who did get diagnosed early, but as our agents have brought forward to the market, we do expect this to change. Patients will get diagnosed earlier, as we saw with Alyssa, and also more patients will seek diagnosis. There's a lot of potential upside here as obviously new treatments come available.
There's a high unmet need, high efficacy aspirations of our profile, and what's very encouraging is the data that we saw today. This just gets us to one step closer to bringing a sizable opportunity forward to patients, and it's fully aligned with our Alnylam 2030 strategy of additional transformative medicines beyond TTR. Very happy to have the opportunity to see this data brought forward today as it gets us one step closer to a very sizable opportunity.
Well, I think that's a fantastic way to end this roundtable. Thank you, Christi. 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 Alnylam's website. Thank you all for joining, and have a great day.
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