Good evening and thank you for standing by, and welcome to today's conference call. I would now like to hand the conference over to Alnylam Pharmaceuticals. Please go ahead.
Thank you. Good morning, everyone, and thanks for joining us for this RNAi roundtable. Today, we'll be discussing progress with the delivery of RNAi therapeutics to the central nervous system and reviewing our ALN-APP program, which is in development for the treatment of Alzheimer's disease and cerebral amyloid angiopathy. I'm Josh Brodsky, Senior Director of Investor Relations and Corporate Communications at Alnylam. With me today are Eric Green, Senior Vice President of Development Programs, Kirk Brown, Senior Director of Research for the CNS program, and Tim Mooney, Director and Program Lead for the ALN-APP program. Today's RNAi roundtable is part of a series of roundtable webinars that we've been hosting to review progress across our various programs and R&D efforts. Previous roundtables are archived on the Capsule section of our website if you missed an event and wish to view the replay.
Today's event is expected to run approximately 60 minutes. Eric will moderate a Q&A session at the conclusion of the presentations, and if you'd like to submit a question, you can do so at any time during the event by typing your question into the Ask a Question field located in the upper right corner of the webcast window. Finally, as a reminder, we will be making forward-looking statements during this webinar, and we encourage you to read our most recent SEC filings for a more complete discussion of risk factors. And with that, I'll now turn it over to Eric. Eric?
Great. Thank you, Josh. Hello, everyone, and welcome to our final RNAi roundtable session of 2022. As Josh said, I'm Eric Green. I'm the SVP and head of our development programs here at Alnylam. Today, I'll just provide a brief overview of Alnylam, our overall pipeline before Kirk and then Tim dive into our overall CNS efforts and the ALN-APP program specifically. Alnylam was founded just over 20 years ago to turn a new discovery in biology, RNAi interference, into medicines to treat human disease and to hopefully help patients live longer, healthier, and fuller lives. In the past two decades, Alnylam has pioneered a new class of innovative medicines, leading to five approved products in less than four years.
With Onpattro, it was the first-ever RNAi therapeutic approved in August of 2018, and Amvuttra as our most recent, with approvals in June of this year in the U.S. and more recently in Europe, the U.K., and Japan. We are commercializing four of these medicines ourselves through the global commercial capabilities that we have established. Importantly, we feel our organic product engine is capable of delivering sustained innovation that will drive our future growth. Most of our programs are based on strong genetic validation, and through our continued platform innovation, we are now in the early moments of bringing RNAi therapeutics to new tissue types, as Kirk will discuss in a moment. Twenty years ago, Elbashir et al. published the results on the left showing the effects of siRNA in mammalian cells.
Since that time, Alnylam has focused on identifying genetically validated targets in specific target organs, primarily, but not only, in the liver, and figuring out how to modify the siRNA to have more drug-like properties that can be delivered to the appropriate cell type. The result, as for now at least, are the five products on the right to which I alluded to a moment ago. Here's our current clinical stage pipeline. While our initial programs were focused on rare genetic diseases, you can see that our earlier pipeline is more diverse, including several cardiometabolic programs, as well as our first CNS program, ALN-APP. That's the green dot near the bottom for the potential treatment of Alzheimer's disease and cerebral amyloid angiopathy. Obviously, this will be the focus of the latter part of today's presentation.
We have built an industry-leading pipeline based on products that were specifically designed for delivery to the liver to address diseases where a key protein is produced in the liver, such as ATTR amyloidosis. We are now starting to extend our delivery innovation beyond the liver to access new tissues. Today's focus will be on delivery to the CNS, where there are still many genetically validated targets for diseases with established biomarkers. In 2019, we entered into a landmark alliance with Regeneron, and that was, in part, about accelerated innovation for diseases of the central nervous system. The alliance leverages our expertise with RNAi therapeutics with Regeneron's world-leading capabilities in human genetics. From this collaboration, we have named three targets: APP, SOD1, and HTT, with many more unnamed programs possible in the future.
Indeed, earlier this year, we dosed the first-ever CNS-targeted investigational RNAi therapeutic in a human clinical trial. You'll hear more about this later. So please, before I hand over to Kirk, remember, please feel free to submit any questions you may have during the presentations. We'll keep track of those throughout the session and hopefully be able to answer many, if not all of them, during the Q&A session at the end. I'll now hand it over to Kirk to discuss our progress with CNS delivery. Kirk?
Thanks, Eric. Good morning. Currently, therapies to prevent or restore function in neurodegenerative diseases are lacking. We believe this is a very high unmet need area for treatments for CNS diseases. There are many genetically defined neurodegenerative diseases. Some of those include Alzheimer's disease, ALS, FTD, Huntington's, Parkinson's, and many more. Many of these have genetically validated targets, but there are no current disease-modifying therapies available. We believe this is a significant opportunity for RNAi therapeutics to target these disease-causing CNS expressed genes. Based on our preclinical studies, we expect the potential for differentiated potency, duration of activity, as well as systemic safety with these modified siRNA therapeutics.
The goal of the CNS platform was to build, similar to what we built for the liver, a highly potent, widely distributed siRNA that is also producing a long duration of action with a favorable risk-benefit profile, and we're now introducing these, what we call our C16 CNS conjugates, which contains an internally modified C16 moiety, as well as other stabilized chemistries. We've recently published this work in the October issue of Nature Biotechnology. This work was also recognized as the Article of the Year for OTS, or the Oligonucleotide Therapeutics Society, in the basic research category. And this is a description of some of the things we've been observing with these C16 conjugates. Looking on the left is an image of a rat brain with an unconjugated siRNA looking for distribution. You can see very little distribution with an unmodified, unconjugated CNS siRNA.
However, the image on the right, 24 hours after a single intrathecal administration, we see remarkably distributed siRNA throughout all regions of the brain after a single intrathecal administration in a rat. Now, taking a look at the different properties of our traditional GalNAc siRNA on the left, as well as our new internal C16 conjugates on the right, the GalNAcs are for specifically hepatic uptake, and the CNS conjugates are essentially enabling a lipid-facilitated tissue distribution as well as stabilized siRNA for enhanced RISC loading. Both of these contain our traditional stabilization chemistry, as well as modifications to improve potency and specificity and also enable low renal clearance. When we set out to identify these, we wanted to essentially exhaustively optimize the siRNA lipophile, as well as the design chemistry and position with which we would place these modifications.
We also optimized the backbone chemistry to, again, maintain stability as well as specificity consistent with our ESC+ design. In addition, we enabled enhanced siRNA loading with a stabilized phosphate modification on the antisense strand. Now, the figure on the left is showing a comparison with different lipophilic moieties from C10 up to C18, and we're looking after a single administration into the intrathecal space in rats at 900 micrograms. This is now two weeks post-dose, and what we see is a really remarkable uptake and distribution throughout the spinal regions, as well as the different various regions of the brain, and we're looking at mRNA knockdown as a readout here. And what we can see is the C16 conjugate was essentially separating itself from the other lipophilic moieties.
On the figure on the right, we compared then some of these design features, including the five-prime stabilized phosphate combined with internal C16, one without the five-prime phosphate, one without the C16, and one without either, and what is quite clear is the combination of both of those design features enabled very nice and robust distribution throughout the spinal cord and brain of these animals after a single administration, and this is now one month post-dose. One other thing I would point out is if we take a look at the liver and kidney shown here in blue and pink bars, we do not see much knockdown with these designs and this CNS chemistry in the liver and kidney compared to the CNS tissues and the spinal cord tissues. So, next. We then wanted to ask a question. So that data was all with a single dose level.
Here, we're looking at a dose response now using intracerebroventricular, essentially a direct injection into a mouse brain, a much more common procedure for mice, looking across a dose range of 50-300 micrograms in a single administration. Now, we're looking at a toolkit siRNA targeting SOD1, and this is 14 days post-dose. What is clear is that you can see a tunable dose response. At 25-50 micrograms, we see partial knockdowns of 50-75% knockdown, and then once we get beyond 50 micrograms, we see pretty much a sustained strong maximum silencing anywhere from 100-300 micrograms in the dose, and that includes all regions of the spine as well as different regions of the brain.
We then often asked around cell subtype distribution, so we wanted to take a look at differences in uptake in neurons, astrocytes, and microglia, and we do see a strong uptake of the CNS conjugates in each of these cell subtypes with also subsequent target lowering in these cell subtypes, then importantly, we need to be able to move from mice and rodents to nonhuman primates, so using the optimized design features for a toolkit siRNA targeting APP, now we moved to nonhuman primates, and this is a single intrathecal administration of 60 milligrams into nonhuman primates, and now, instead of looking at tissue, we're looking at target engagement biomarkers or circulating biomarkers in the CSF, and when you target APP, one of those biomarkers is soluble APP alpha and beta.
What we can see is very rapidly after a single dose, we see a dramatic lowering of soluble APP alpha and beta target engagement biomarkers in the CSF of these animals, and we serially collect CSF over the course of nine months, and we can see that there's at least six months of sustained silencing of around 50% and some recovery between six and nine months after a single dose. This is one dose of siRNA against amyloid precursor protein. Summarizing the platform work at this point, we've seen potent and durable silencing throughout the CNS, throughout the spinal cord, and throughout the regions of the brain. We've optimized internal C16 conjugate with a five-prime vinyl phosphamate, which enhances our RISC loading and activity in extrahepatic tissues such as the CNS.
We see dose-dependent lowering in the spine and the brain, and we see this extended pharmacodynamic profile, which is consistent with what we see in the liver now moving into the CNS, with at least six months of silencing after a single intrathecal administration with these designs, and so these platform and ALN-APP CTA-enabling studies now support further development, which is how we've moved into ALN-APP Phase 1, which is ongoing, which Tim will tell you about more in a moment, so moving then from platform to clinical programs, we look for genetically validated targets. We look for those targets that have biomarkers for clear POC in Phase 1 and to the final path to approval, and for these reasons, we selected amyloid precursor protein. A little background on the particular target: it's one target with two distinct pathological processes. It's an 87-kilodalton membrane-associated protein.
It goes through a variety of processing and enzymatic cleavage with alpha, beta, and gamma secretase, including the most famous amyloid beta 38, 40, and 42 species. And this is a genetically validated target for both AD and cerebral amyloid angiopathy. So, we wanted to ask the question around APP mRNA lowering by siRNA in a mouse model of disease, and we looked both pre-symptomatically and post-symptomatically with an ICV or intrathecal ventricular dose of a toolkit molecule targeting APP. And in this study, we observed lowering of amyloid as well as reduction, statistically significant lowering of inflammation. We see durable silencing out for several months after a single administration. And importantly, looking in the open field test, we see animals upon treatment presented behavior consistent with that of wild-type animals, and we looked at rearing frequency as well as distance traveled.
What we found was the distance traveled in rearing frequency was consistent with wild-type animals after treatment, and that was just two months after treatment. We did not see any change in velocity. This was all extremely encouraging to us. Next, there's a growing body of evidence around the intracellular manifestations of these diseases, and one of which is around this endosomal and trafficking phenotype that is observed in mutants presenilin and APP patient-derived neurons. These observations have also been found in mouse models of disease and post-mortem AD brains. What is observed is a remarkable enlargement in trafficking, toxic trafficking phenotype of Rab5 positive endosomes in mutant patients' lines. We wanted to ask a question of targeting APP with siRNA, lowering that messenger RNA, do we see any change in this observed endosomal enlargement phenotype?
Upon administration, 48 hours later, we look at mRNA knockdown and RAB5 endosomes. We see both mRNA reduction and a statistically significant change in RAB5 endosome size. This is consistent with an intracellular correction of a disease manifestation as well as potential extracellular reduction with APP lowering. Next, we wanted to examine APP lowering in a rat hereditary CAA model. Here, again, we take an siRNA targeting APP, a single 900 microgram intrathecal administration. Here, we're looking at neurons and their detection of amyloid reduction in these models, and what we see is a very rapid reduction after a single month. This is a hippocampal section, and we're looking at a lowering of amyloid in APP.
And then we look two months out, and in this disease model of vascular amyloid production, we see statistically significant lowering of APP as well as amyloid beta 40 two months post-dose, which is encouraging toward the potential of using an APP-targeted siRNA for the lowering of both parenchymal as well as vascular amyloid in this mouse model. I'll now turn it over to Tim, who will share updates on our APP program.
Thanks, Kirk. The overview Kirk gave you gives some idea of why we had confidence moving APP forward as our first target for development of our CNS conjugates. My name's Tim Mooney, and I'm the program leader for ALN-APP, and it's my privilege to bring this innovation that Kirk has developed forward into clinical development.
If you go to the next slide, the work we're doing with our CNS platform and ALN-APP is really taking us back to our roots at Alnylam. Platform innovation has been at the heart of our success so far as a company. The ability of Alnylam to engineer solutions to delivery challenges with our LNP and GalNAc platforms has been pivotal to enabling our successful transition to a commercial company and to creating the innovation that Eric described with our liver programs. Now, with our CNS platform, we're back at the hard work of platform development.
As you've heard from Kirk, we believe that the data that we've seen so far with our CNS conjugate suggests that they may unlock significant opportunity for RNAi therapeutics in the CNS, and that data shows us that lowering APP appears to have a very encouraging profile in these disease states of Alzheimer's disease and CAA. Another way we're going back to our roots with this program is with this target, the amyloid precursor protein. As Kirk outlined, APP is a protein that's metabolized into peptides that can misfold into amyloid deposits that form in the brains of people with Alzheimer's disease and in the blood vessels of the brain in people with cerebral amyloid angiopathy.
With ALN-APP, our therapeutic hypothesis is that by lowering APP protein production in the CNS with RNAi, we can reduce the downstream fragments that aggregate into deposits in tissues and thereby also halt or ideally improve the clinical manifestations of these diseases. This therapeutic hypothesis probably looks pretty familiar because we've pursued a very similar therapeutic hypothesis with our work in ATTR amyloidosis. This gives us confidence in the potential of this application of our RNAi therapeutic to these disease areas. The amyloid precursor protein is best known for its connection to Alzheimer's disease. Mutations in the APP gene and in other genes involved in APP metabolism cause early onset Alzheimer's disease. Other APP mutations that reduce metabolism and A beta peptide levels are known to be protective from Alzheimer's disease.
These genetic studies provide validation for this target and suggest that using RNAi to reduce the production of APP in the CNS could be a useful therapeutic strategy for Alzheimer's disease. APP is also a good target to evaluate the pharmacology of our drugs, which is especially critical in this first test of our CNS platform. Soluble APP alpha and beta can be measured in CSF and provide a clear view of the level of target engagement we are getting. Lastly, this is an area that has well-defined clinical and regulatory precedent and high unmet medical need. The reach and devastating impact of Alzheimer's disease to individuals and the families and friends who love them has left few without a clear understanding of why continued innovation in this space is desperately needed.
While there has been extensive development in this area and some recent success, the unmet need for new treatments remains staggering. Even the recent clinical successes, while landmarks for the field in their own right, do not come close to fully addressing the progression of the disease and its impact to tens of millions of people worldwide. Targeting APP with an RNAi therapeutic is a new approach in Alzheimer's disease, which acts upstream of many of the mechanisms of action that have been studied previously. This approach is designed to reduce the APP protein at its source, reducing both intracellular and extracellular drivers of disease pathology. As Kirk showed you, our non-clinical work with APP targeting siRNAs has demonstrated the potential benefits of this approach.
In vivo studies in mouse models of AD have demonstrated that APP lowering can reduce amyloid in the brains of mice and improve the behavioral phenotype of the mouse model. In vitro studies in cells derived from patients with autosomal dominant Alzheimer's disease mutations have shown that APP lowering can improve the intracellular endolysosomal abnormalities that are characteristic of these human disease cell lines. Therefore, we believe that targeting APP at the mRNA level has the potential to act upstream of APP cleavage into A beta and other pathogenic cleavage products and enable the reduction of all A beta isoforms in assembly states, and thereby provide a more comprehensive intracellular and extracellular impact to the disease state. We believe that reducing the production of the substrate for amyloid deposits may reduce the formation of new deposits and also potentially enable natural clearance mechanisms.
And for all these reasons, we're excited to see what type of therapeutic effect using RNAi to reduce APP protein may have. In addition to the opportunity for ALN-APP to address Alzheimer's disease, we believe that lowering this target may also enable the treatment of another disease called cerebral amyloid angiopathy. Where Alzheimer's disease is characterized by the accumulation of amyloid plaques in the parenchyma of the brain as well as the intraneuronal accumulation of tau tangles and neurodegeneration, cerebral amyloid angiopathy is a disease where amyloid plaques deposit in the vessels of the brain, which can weaken them and increase the risk for hemorrhagic strokes. This is also a genetically validated disease for APP targeting. Specific mutations in the APP gene result in early onset cerebral amyloid angiopathy. Today, there are no specific treatments available to treat cerebral amyloid angiopathy, and there's very little activity in clinical development.
We believe that the high unmet medical need to reduce the risk of strokes in patients with cerebral amyloid angiopathy and the lack of treatment options for patients makes this an additional attractive area of development for ALN-APP. Like Alzheimer's disease, the plaques found in CAA are made up of fragments of the APP protein. In this case, typically the A beta 40 peptides produced when APP is cleaved by beta and gamma secretases. Both genetic factors and age can contribute to the accumulation of plaques in the vessels. In CAA, amyloid accumulation in the vessels results in progressive cerebrovascular disease. Over time, amyloid deposition results in a disruption of the vascular architecture and loss of vascular reactivity. As a result, CAA gets worse as time passes, amyloid deposits spread, and vascular damage becomes more severe. This results in the clinical manifestations of the disease.
The most common clinical manifestation is a hemorrhagic stroke, typically in the lobar region of the brain, which is a severe and life-threatening clinical event. As a result of this clinical picture, CAA is a disease with high unmet need. It is the second most common cause of intracerebral hemorrhage after hypertension. After having an ICH or intracerebral hemorrhage, patients with cerebral amyloid angiopathy are about three times more likely to have another stroke as people who have an ICH from a different cause. As a result, CAA is a significant driver of stroke risk, mortality, and disability. CAA may also be an independent driver of cognitive decline, as studies have shown that patients with CAA pathology have a faster rate of cognitive decline independent of other common comorbid pathologies. CAA is a disease of vascular amyloid deposition. Here, our therapeutic hypothesis is simple.
Use siRNA to reduce the production of the amyloidogenic protein. Again, this is a hypothesis that we have tried before. By reducing APP and thereby reducing A beta 40 and all other amyloid isoforms, we aim to reduce the progression of amyloid deposits in the vessels and potentially enable natural clearance mechanisms to clear the existing deposits and thereby address some of the vascular damage that occurs during the disease. Now, as we think about how to bring ALN-APP forward in development, Alzheimer's disease and cerebral amyloid angiopathy represent two significant independent opportunities for this program, both with genetic validation and both with a very high unmet need for new therapies. We have begun this journey of development with ALN-APP with our Phase 1 study in patients with early onset Alzheimer's disease.
This study is designed as a two-part study, a single ascending dose part A followed by a multiple dose part B. The primary endpoint of this study is safety and tolerability of ALN-APP. We also hope to begin to characterize the pharmacology of our CNS conjugates and assess the level of target knockdown we can achieve and the duration of effect. The study also includes a variety of exploratory biomarkers, which will allow us to assess whether ALN-APP is showing any impact to other biomarkers of disease progression. These include fluid biomarkers of amyloid, tau, and neurodegeneration, measures of synaptic health, neuroimaging, and exploratory cognitive and functional measures. For now, we continue to enroll patients in part A as we progress through the dose escalation portion of the study. We expect to share preliminary data from the study in early 2023.
We expect that the results of this Phase 1 study will help inform our next steps in development. These results will de-risk further development in early onset Alzheimer's disease and give us an idea of how to begin the journey of later stage development with ALN-APP for patients with Alzheimer's disease. In addition, they will also provide us useful insights into the pharmacology of ALN-APP and enable the initiation of development of ALN-APP as a potential treatment for cerebral amyloid angiopathy. In this way, we aim to explore both potential opportunities and allow the emerging data and evolving landscape in these indications to inform our later stage development and commercialization approach. ALN-APP is a representation of how Alnylam continues to expand our pipeline driven by our organic innovation engine.
This program represents our continued commitment to platform innovation and our commitment to bring forward new genetically validated targets for high unmet need diseases. As such, we hope to achieve many firsts with ALN-APP. The first first that you see on this list has already been achieved. Earlier this year, ALN-APP became the first siRNA delivered to the CNS by intrathecal dosing. This is a major milestone not just for Alnylam, but for science and medicine in general. ALN-APP is also the first therapeutic to target APP mRNA, the sole precursor of all APP cleavage products, including A beta 40 and A beta 42, and a genetically validated target for Alzheimer's disease and cerebral amyloid angiopathy. ALN-APP would potentially be the first therapeutic to comprehensively lower intracellular and extracellular amyloid proteins and the first therapeutic to lower other potential non-A beta drivers of Alzheimer's disease.
As we know, being first is not always easy, but as we have shown with our marketed products in our liver platform, platform and target innovation can lead to important advances for patients. And so, just as we have seen occur in the liver, we hope that initial success with ALN-APP can help de-risk a whole new pipeline of potential targets for our portfolio. Two targets that we have already announced include SOD and HTT. ALN-SOD targets SOD1 and is in preclinical development as a potential treatment for ALS in patients with a SOD1 mutation. ALN-HTT targets huntingtin, the mutant protein that causes Huntington's disease. From our experience in the liver, we know that RNAi therapeutics can be a modular and reproducible platform, so we expect this to just be the beginning of our opportunity to develop new candidates for diseases in the CNS.
And as with our liver program, we will not stop working to improve our platform. We will continue to pursue advances in our technologies that can enable further improvements to our CNS conjugates and continued expansion to other extrahepatic tissues. In summary, our CNS platform and programs represent an important expansion of our innovation engine. We hope these advances that we are making can help patients who so desperately need new options for diseases of the central nervous system. And with that, I'll hand it back to Eric to close this out.
Thanks, Tim. I appreciate it. And thank you, Kirk, for your presentation on all the CNS delivery. We have lots of questions coming in, so please, if you have others that you'd like to submit, please do so online. Maybe I'll jump in first with one for you, Kirk. Just kind of a broad question.
You kind of hit it on a little bit earlier, but how do we explain such long durability that we're seeing with these CNS-directed siRNAs?
That's a good question. We've seen really remarkable distribution. I think we have very good PK across the different CNS regions as well as CNS cell subtypes. And then what we are seeing is what appears to be consistent with what is seen in the liver is essentially a sustained, durable subcellular depot that is consistent with what we're seeing in the liver in that we get sustained delivery to the cytoplasm of the axis site of action, which produces then and in turn a sustained amount of target mRNA lowering in that region. And we believe it's because we get very good exposure and highly stabilized siRNAs that are both able to load RISC well, but also able to resist nuclease degradation.
And so consistent with the liver platform, very metabolically stable also within the CNS.
That's great. Any work yet where we could start to estimate what a clinical dosing regimen could look like? I mean, you showed an NHP over six months, still pretty significant knockdown. Any thoughts on maybe translatability in humans?
Yeah, I mean, that's a great question. We've seen duration go in certain preclinical experiments, single doses almost completely clamped for six months, so it's difficult to assess much beyond. We could see nine months of sustained knockdown, potentially even a year depending on how well we translate to clinic. I would expect at some level, maybe twice a year dosing in clinic is probably consistent with what we're seeing in preclinical.
Okay. And of course, the Phase 1 will obviously help us to start to understand.
It will inform on the correct thing.
Yeah. Maybe I'll stick with you, Kirk, for a little bit more. So you showed some of the distribution to the brain, various brain tissues and parts of the CNS. Are there any regions of the brain where we're not getting substantial uptake?
Yeah, good question. With intrathecal administration, we're going right into the CSF space, and with the doses we're using and the dose volume, it enables a pretty robust and even distribution throughout the spinal cord and into the brain. We see robust uptake in the frontal cortex, temporal cortex, and hippocampus, and we're even seeing uptake in the deep brain regions to some degree, 25%-50% silencing in the deepest parts of the brain, such as the striatum. And so we're quite encouraged by the profile that we have right now.
Okay. I'm going to stick with you a little bit longer, Kirk, but specifically for APP as a target, is there particular regions or cell types within the brain where APP is expressed?
I mean, certainly we're accessing the key cell subtypes like neurons and astrocytes. And importantly, we're hitting it to the level that we feel is enabling both intracellular phenotypic changes as well as the target engagement biomarker reductions that we think are important for potentially modifying the disease. And what we've shown at least preclinically is that we're able to tune that silencing based on dose. And so we can control the lowering of APP quite well just based on dose and distribution.
Okay. One last one before I start to spread the words around a little bit. Any non-CNS expression of APP you may be worried about?
Even with IP dosing, if this gets out more systemically, is there any other concerns there?
Not really. I mean, I think I showed in some of the early preclinical work that our intrathecal siRNA CNS conjugates, they do get out to the systemic circulation, but we do not see a dramatic amount of knockdown in those systemic tissues. Again, we get uptake in the liver, but because they're not GalNAcs, we don't get hepatic silencing to the degree that you might be used to seeing with the GalNAc siRNA. So these conjugates, although we do get distribution, we don't see a lot of silencing systemically.
Okay. So I guess I would imply from that there is some APP expression outside of CNS, but we don't think we hit it in any substantial way.
Correct.
Got it. Okay. Thank you.
Maybe, Tim, turn it to you for a couple of questions on the current Phase 1 study. So a couple of people have noted that last week we did make a slight modification to our guidance for when we expect data from APP. Maybe you can just provide a little bit more of an insight to the reason for that change.
Sure. As I said, we're continuing to make progress enrolling patients in the part A of our Phase 1 study in patients with early onset Alzheimer's disease. The change in the guidance really just reflects the pace of our continued progress through the dose escalation cohorts.
We are taking a measured approach with this first CNS conjugate that we're bringing, and we want to make sure that when we do bring forward the initial data, we have something to say about some of the key endpoints that we're looking at. In this Phase 1 study, again, remembering this is our first kind of venture into the CNS, we're really focused on the safety and tolerability as the primary endpoint of the study and the pharmacology that we're able to see. So I think we have a lot of great data from animals that suggest that we can get target engagement and a durable effect.
And Kirk, I think, showed a compelling set of data on that front, but we want to start to see that in humans to see what doses are going to give us the type of target lowering that we're looking for and what does that duration look like. So we're continuing to enroll patients in our Phase 1 study, and we're continuing to move through the dose escalation. And when the data are ready, we'll be excited to share them.
Excellent. Maybe broadly on the disease, there's some questions coming in about the role of APP, obviously, in normal brain function. So is there any concern or any data we would expect with significantly lowering APP expression that we may or have seen anything in maybe preclinical models even? Maybe, Kirk?
Yeah, good question.
In our non-human primate work, we've seen maximally 80%-85% silencing that was sustained for at least six months with some degree of knockdown between six and nine months post a single administration with no issues or clinical signs or any surprises that would be concerning. We've also shown that with lower doses, we can control the silencing in the window of 40%- 60%- 70% silencing just based on dosing. So we feel like we're able to relatively well direct the degree of silencing throughout the spine and the brain in these preclinical species, which we expect to be able to do in clinic.
Okay. Which maybe is a related question somebody had asked. So is there a level of APP knockdown we're looking at? And your point of being able to tune to that kind of 40% or 60%?
Yeah, we're aiming in that 50% silencing range, which we, again, because we're addressing it from the inside out, we're treating it in a way that hasn't been essentially addressed before with, say, like an antibody-based approach, right? So being able to target or tackle the intracellular manifestations of the disease, we believe that 50% is going to give us a view into that potential benefit.
I guess we believe we can tune it, but until we get it tuned quite right, is there any risk of going too high on knockdown?
I think Tim mentioned we're taking this a very measured approach. Phase 1 is all about safety, and we're walking it up as you might expect to be cognizant of that.
Yeah. Okay. That's a fair point.
Obviously, on our single ascending dose, we start at very low doses, and we'll move up along the way and see what knockdown of the biomarkers are as we go. So we'll be very thoughtful about that. That's fair.
With a goal of around 50%.
Very good.
Okay. Kirk, jumping back to when you talked about the C16 lipophilic conjugate, and I think there was a mention of GalNAc as we did that design work. So maybe you can explain a little bit more about that?
Sure. Yeah, that's a great question. I believe it's in the Nature Biotechnology publication, but not shown here. One of the things we did in the early days was look for placements of that lipophilic moiety along the sense strand of the siRNA.
We essentially walked along every position of the sense strand to look for which positions would be most tolerant to enable still loading into RISC and still functional activity against the target messenger RNA. And so what we found in those walks is that there were certain positions that were essentially intolerant of maintaining activity. So that's what we mean by walking. We looked for placements where we would get a benefit of distribution and activity while maintaining that sort of high degree of activity. So we essentially moved around those positions that were intolerant, maintaining those positions that were most tolerant and most beneficial to the siRNA activity. So that's what we mean by walks. We literally walked the ligand along the strand.
W here the ligand was as well as how long the ligand was.
Correct. Yes. Yeah.
Sensitive amount of work to get to this particular conjugate.
Yep. Yep. Yep. Relatively big final undertaking.
All right. Looking through some of these questions, there's quite a bit about how do we place ALN-APP in this mechanism of action relative to antibodies and other BACE or gamma-secretase inhibitors. So maybe, Kirk, you can help us think a little bit about how that mechanism may differ. And then maybe we'll get a little bit to Tim later on the kind of recent clinical data in one other program.
Yes, sir. I mean, as I've mentioned a little bit earlier, our approach is truly an inside-out. We're targeting all intracellular peptides and fragments, not just the amyloid beta. And we're doing so within the cell and also anything that would be outside of the cell by essentially shutting off the APP.
And so other things that I wasn't able to get to today were the relationship between one of these more recent fragments of interest, beta C-terminal fragment, seems to be associated with this endosomal lysosomal phenotype. And we've shown that we can actually lower that fragment as well, which also might go some way to explain why we're seeing that potential benefit on the endosomal lysosomal phenotype. And that is an area where an antibody just simply cannot address the disease. Essentially, it's clearing everything that's extracellular, but everything that would be intracellular, all those same intracellular toxicities would essentially continue on in the presence of an antibody-based approach or not. And again, similar to the question with BACE, BACE does some of that, but not nearly to the degree with an siRNA approach can because we're taking out all intracellular fragments. So there's some differentiation there quite clearly.
Okay. It'll be very interesting. Maybe, Tim, recently, monoclonal data came from lecanemab. Maybe your thoughts on what that may mean for the field and maybe particularly on our development program?
I think it's really exciting to finally see some progress in this space. I think we started to see it even with Aducanumab, and now we're seeing more convincing data sets with lecanemab that lowering amyloid with an antibody does seem to have some potential therapeutic benefit for patients with Alzheimer's disease, and obviously, as we talked about, those patients are desperately in need of new therapies. We think that this update is supportive of our therapeutic hypothesis for APP. As Kirk just described, APP acts upstream of the amyloid deposits that are targeted by lecanemab. We're targeting APP mRNA. We're reducing the production potentially of the APP protein.
But in so doing, we expect that we would potentially have an effect on the continuing deposition of amyloid. And that's really where the antibodies are sort of having their effect is on the amyloid deposits that are extracellular. In addition to that, by knocking down APP intracellularly, we're also expecting to have an impact on the intracellular drivers of the disease. And we believe that could have some important role in addressing the clinical progression of the disease. I think we're still watching very closely to see how lecanemab is going to impact the overall disease landscape in Alzheimer's disease. We'll be following closely to see how broadly lecanemab is used if it's approved. We know that there were access hurdles for previously approved therapies for Alzheimer's disease, and it's not clear yet to what extent lecanemab might be able to make a more significant impact for patients.
But in any scenario, we believe that there's still going to be residual high unmet need in patients with Alzheimer's disease for treatments that can continue to more fully address the disease progression. Even with these antibodies, which, to be clear, this is a major advance in the field to now have some therapies that have convincing evidence of disease modification. Patients still continue to progress in their disease even with these treatments. So we believe that there's still opportunity for us to help address some of that residual unmet need that remains.
Thank you, Tim. A couple of questions coming up on the Phase 1. And maybe you can give a little bit more color about what type of data.
I think you mentioned a little bit earlier, but a little bit more color on what type of data we expect coming out of this early Phase 1 study and when we do talk about it publicly.
Yeah. So as I mentioned, the primary objective of the study is safety. And this is actually really important because this is the first time we've brought these CNS conjugates into clinical development. So we want to understand the safety and tolerability profile of these molecules. And I think in many ways, those data will help de-risk not just ALN-APP, but additional molecules that we would like to bring forward for other targets as part of our CNS platform. In addition, we'll be looking to see what level of target engagement can we get.
So we are blessed in this disease area to have a soluble biomarker that we can measure that gives us a clear idea of how much target engagement we're getting. So we're looking at those soluble APP alpha and beta. We'll also be looking at biomarkers that specifically look at amyloid beta, 38, 40, 42, to see whether we're actually seeing the downstream effect, meaning the protein lowering soluble APP alpha and beta and the A beta subspecies to see whether we're seeing that therapeutic benefit. So as we think about our initial data, it's really going to be focused on safety and tolerability and what are we starting to see as far as that pharmacological profile. What are we seeing as far as target engagement? Okay.
Tim, I might add in our preclinical work, I saw a question around amyloid beta silencing and how does that relate to soluble APP lowering. So in preclinical work in non-human primates, I showed soluble APP alpha and beta lowering, but really what we see is essentially a matched lowering with all three common amyloid beta species, 38, 40, and 42. So if you see 50% soluble APP alpha and beta, we also see 50% lowering of the three most common amyloid beta species in the CSF. So we're quite confident we have a clear view essentially on target engagement in this study.
And that's what you would expect. Exactly. When we talk about hitting all of the downstream fragments of APP, what we're talking about is all of these different isoforms of A beta, the beta C-terminal fragment that Kirk mentioned, soluble APP and alpha.
We're expecting that this mechanism of action will reduce all of the cleavage products of APP by reducing the production of APP itself.
A single target way upstream is they're trying to hit.
Exactly. And that's the point of the differentiation from antibodies in that they have a very rapid clearance approach of amyloid, right, where we are going to be expected to have a more, I would say, natural clearance because you'll be producing less amyloid, and we won't be accumulating any new amyloid. So it allows the cell and the brain to naturally clear as opposed to more rapidly dropping out in a very short amount of time.
Which is a great segue to another question that we had had. So that natural clearance piece, is that mostly theoretical?
I mean, because I understand not putting more amyloid out with APP being shut off, but do we have data on that natural clearance, or is that more theoretical?
Yeah, we're building that data set out. I mean, we have some into that with the CDN mouse as well as in the CAA rats. We see clearance of the amyloid after a few months of dosing. And so continuing to look at that in longer studies now.
Great. Thank you, Kirk. Just looking at these questions, we've had a lot come in. This is excellent. Maybe we should take a little bit of time as we're coming close to the end. Let's think a little bit more about CAA. So Tim, maybe tell us a little bit more about how big we think that opportunity is.
Sure. Thanks, Eric. CAA today is really an under-recognized cause of stroke.
Oftentimes, people, they have a hemorrhagic stroke, but people aren't necessarily looking to understand what was the underlying cause of that stroke. What we understand from autopsy studies is that CAA pathology, meaning the actual deposition of amyloid in the vessels of the brain, is quite common in older people. And this is why this is believed to be an age-related phenomenon. In neuropathological studies in older adults, moderate to severe CAA pathology is seen in upwards of 20% of the elderly population and in an even higher percentage of patients who have comorbid Alzheimer's disease. Only a subset of that group is actually going to have the sort of imaging evidence and clinical manifestations of CAA. But from a pathological perspective, this is a pretty widespread issue. We believe that the most significant unmet need in patients for CAA is to reduce the risk of strokes.
As I mentioned, CAA is the second most common cause of intracerebral hemorrhage after hypertension, and patients with CAA have a high rate of recurrence. So if they've had a stroke, they're more likely to have another stroke than patients that have a hemorrhagic stroke from another cause. So for us, we really feel like there's a significant unmet need to address the recurrence risk of strokes in patients with CAA, especially because there are no specific treatments to address the underlying disease, and there aren't any real alternative approaches that are in development today.
Thank you, Tim. And then for CAA, presumably APP knockdowns have to occur in endothelial cells. The C16 conjugate allowed for endothelial cell uptake. So maybe, Kirk, is that one first statement correct? It has to be in endothelial cells, or?
Well, the question is the source, right? Where are the fragments coming from?
And the lion's share of the fragments are neuronal and astrocytic, but we do get some uptake into endothelial cells as well.
Yeah. Okay. But I think what we believe from what's known about CAA natural history is that actually the vast majority of the amyloid that's deposited is made in the CNS itself.
Yeah. Okay. I think that makes sense. All right. Looking here for maybe the last couple of questions. There's a couple of interesting questions on siRNA design, actually. A lot of people are interested if you were to put multiple C16s at different positions on the sense strand, would that make a difference? Do you think the location or the length of the C16 is different by disease? Is there any reason to believe that? So maybe, Kirk, your thoughts on the general thinking on our design.
Yeah. It's a good question.
I mean, we set out to find a design that would enable very even uptake and distribution across the spine and the brain, as well as somewhat agnostic uptake across the different cell subtypes, right? So the question is, if you look at alternate designs, shorter lipid moieties or multiple ligands, does that improve or change that? And we've done a little bit of work toward understanding differences and also around clearance, right? If you have a much larger or bulkier design, does it stay in the CSF longer? And so we are exploring some of those, but at the moment, the design that we have now is hitting the cells and the regions that we're aiming to hit.
Yeah. I think this is similar to what Tim said earlier, right?
We're at the start of our work on platform innovation, and as we've seen and shown with our liver platform over the years, we'll continue to work, and I know you guys in the lab continue to do that. I think, can you scroll up a little bit too? If there's any other questions, I think we have probably time for maybe one more question. See if any jumps out. I think we've got just about all these answered. Maybe one last question. It looks like there's a question on Huntington's, a couple of different ones. And what can we say a little bit about that program? It's early days, obviously, so we haven't said much previously, but we've talked about it at R&D day in the past. Maybe a very general strategy for targeting, Kirk?
Yeah. I mean, it's consistent with our APP design.
We're still using the C16 conjugate, vinyl phosphonate on the antisense strand. We're still looking to distribute very well to the brain, but in particular for HTT, in addition to hitting the cortex, we're trying to hit the deep brain very well. And we've been able to do so with that program so far.
Excellent, so stay tuned. We'll have more and more information as we continue to move that program, hopefully, forward towards the clinic, so thank you, everyone, for the questions. I think we're just about at time, so maybe last slide just to kind of summarize what you've obviously heard over the last hour. We're really looking forward to generating data, but hopefully, with success of CNS delivery, we could see a significant expansion of our Alnylam product engine.
We could really continue to drive the sustainable growth through innovation and through organic pipeline growth, which is pretty unique and rare, I think, in this industry. We continue to move forward with our ALN-APP. It's our first CNS program, obviously. It will be very informative for this disease, early onset Alzheimer's, potentially CAA, but also as a view into the broader platform applicability for siRNAs. So I think with that, I will close out and hand to Josh for a few closing comments. Thank you.
Perfect. Thanks so much, Eric. And thanks also to you, Kirk, and Tim for participating. This concludes today's R&D roundtable. As always, you can access the replay of the webinar and download the slides in the Events section of Alnylam's website.
Before we go, I'd like to remind you that we'll be hosting a virtual R&D day on December 15th for a deeper dive into other pipeline programs and platform activities, including advancements in our TTR franchise. So we hope you save the date and can join us for that. Thank you all for joining today, and have a great day.
All right. Thank you, presenters, and thank you, participants, for joining us. You may now disconnect.