Hello, and welcome to the Denali Therapeutics webinar highlighting our therapeutic programs addressing amyotrophic lateral sclerosis, or ALS, infratemporal dementia, or FTD. I am Laura Hansen, Vice President of Investor Relations, and I'd like to thank you for joining us today. Please note that the press release we issued earlier today and the slide deck for this webinar are available in the Investors section of our website, denalitherapeutics.com. Before we get started, I'd like to note that the presentations given today and the responses to questions will contain forward looking statements regarding Denali's future plans, business strategy, product candidates, planned preclinical studies and clinical trials, among other things. Such statements are subject to numerous important risks, uncertainties and assumptions.
Should any of these risks or uncertainties materialize or should our assumptions prove to be incorrect, our actual results could differ materially from those forward looking statements. These risks, uncertainties and assumptions are more fully described in our filings with the SEC, including our latest quarterly report on Form 10 Q and our latest annual report on Form 10 ks. Any forward looking statements are based on information available to us as of today, and we disclaim any obligation to update any forward looking statements except as required by law. On the webcast today, I am joined by members of Denali's management team: Ryan Watts, Chief Executive Officer Carol Ho, Chief Medical Officer and Joe Lukak, Chief Scientific Officer. It is also our pleasure to have Doctor.
Jean Yeoh as our guest speaker. Doctor. Yeoh is a professor of cellular and molecular medicine at the University of California San Diego, UCSD, a founding member of the Institute For Genomic Medicine and a member of the UCSD Stem Cell Program and Moores Cancer Center. I would like to take a moment to review the agenda and Q and A logistics for today. We've scheduled approximately 1 hour and 45 minutes for the webinar, including the presentations and a Q and A session at the end.
If you would like to ask a question at any time during the presentations, you may do so by typing it into the Q and A box. We will do our best to answer as many questions as possible during the Q and A session. And now, I'd like to turn the program over to our Chief Executive Officer, Ryan Watts.
Laura, thank you for the introduction. It's very exciting to be with everyone today to share some of the progress in our key programs. Denali was founded to defeat degeneration. We have a broad portfolio of therapies across multiple degenerative diseases. We've spoken recently quite a bit about our lysosomal storage disease programs as well as our Parkinson's programs.
We have a number of programs advancing as well in Alzheimer's disease. However, today, we're going to focus on ALS and FTD. I'd like to set the context by reminding everyone of the size of the unmet need in neurodegenerative diseases. Alzheimer's, Parkinson's, FTD and ALS represent a huge unmet need with basically very limited treatment options and very few disease modifying therapies. We're applying very specific principles to increase our probability of success.
We follow 3 principles. 1st, what we call genetic pathway potential or using degenerogenes, genes that are mutated that cause neurodegeneration. 2nd is engineering brain delivery. We'll speak about engineering brain delivery about 2 modalities today, both our small molecule as well as our large molecule efforts. And then finally, biomarker driven development.
I'm pleased to announce that Carole will be presenting new data with our programs, clinical data using this biomarker driven development strategy. We've seen a lot of recent progress that creates momentum for our portfolio. We have 5 clinical stage programs, 15 transport vehicle enabled programs. Those are biologics that are engineered across the membrane barrier. And we're well resourced to go after this broad portfolio, including As mentioned, we've spoken recently about our LRP-two program as As mentioned, we've spoken recently about our LRP-two program as well as our ETV IDS program.
Today, we'll focus on 3 programs around ALS and FTD. The first program will be EIF2B, our lead molecule being TNL-three 43. The first indication that we're going after with this molecule will be ALS. We'll then speak about RIP kinase, which is also advancing in ALS in clinical studies. And then we'll end with a summary of the progress we've made on pro granulant for FTD.
However, I'd like to set the stage by mentioning the steps in neurodegeneration that we're targeting. There are multiple steps. However, we're focused on 2. The first is what we call the triggers. These are direct genetic links to disease.
The second target set of targets are mechanisms that speed up disease progression known as accelerators. Today, again, we'll focus on our pro granulant program as well as our EIF2b program, which squarely fall into triggers being directly genetically linked to disease and then our RIIB kinase program, which is modulating microglial inflammation. This being said, these 3 programs address fundamental pathways within neurodegeneration. Our focus again will be on ALS and FTD. However, there's broad therapeutic potential for each of these programs.
We'll spend time on each of the programs focusing on the rationale around ALS and FTD. However, there are direct genetic links for these various programs to other diseases including Alzheimer's disease. In addition to using genes to identify the targets that we're going after, 1 of the major challenges for us is the blood brain barrier. The blood brain barrier evolved to protect the central nervous system from toxins as well as creating a microenvironment for signaling. This has been a major focus of Denali as basically engineering therapeutics that can cross the blood brain barrier.
It's worth noting that both large molecules and majority of small molecules are kept from entering the brain through this endothelial barrier. I'll start first by highlighting our approach for small molecules. Our small molecule approach relies on a rigorous assessment of chemical starting points with BBB delivery potential. From there, we have a disciplined application of CNS amenable physical chemical properties and then derisk these programs with further in vitro and in vivo studies. Finally, we identify compounds that have robust trough coverage and approximately 1:one brain to plasma ratio.
Highlighted are a number of small molecules on the right hand side that have entered clinical studies. We have an excellent chemistry team who have invented these compounds over the last 6 years and we continue to bring small molecules forward to treat neurodegenerative diseases. And today, we'll focus on 2 of those molecules, DNL-three 43 as well as DNL-seven 88. With that in mind, I'd like to highlight the progress of DNL-three 43, which is an EIF2b activator, a novel mechanism for restoring protein homeostasis. It's designed to address hallmark TDP-forty 3 pathology as well as RNA stress granular biology.
We completed a Phase 1 study where we achieved our biomarker goals as well as our safety goals and Carol will summarize the data today presenting that for the first time. We've initiated a Phase 1b study in ALS and we'll continue to enroll this study over the next several months. Our RIF kinase inhibitor in collaboration with Sanofi known as SAR-four 40 three-eight 20 or DNL-seven 88 is designed to address TNF receptor 1 inflammatory pathway. We've also completed a Phase 1 study where we've achieved our biomarker goals and it's generally well tolerated. We'll be initiating a Phase 2 ALS study in Q1 of 2022.
This is being led by Sanofi and received fast track approval. Just a reminder, ALS represents a huge unmet need that affects motor neurons, ultimately leading to voluntary muscle control and movement disorder. About 250, 000 people are affected worldwide. DNL-three 43 and DNL-seven 88 are potential 1st in class treatments for ALS and have potential for expansion into multiple additional indications as I've already outlined. The last program we'll focus on utilizes our transport vehicle technology, which is a technology that's invented to get large molecules across the blood brain barrier.
Illustrated here is the transferrin receptor, illustrated here is the transferrin receptor, which is expressed at high levels on endothelial cells in the brain. Our technology utilizes this transferrin receptor, which is required for transporting iron to get large molecules in the brain. Now our lead program for FTD is PTV programmulin or protein transport vehicle programmulin. This is a TB enhanced brain uptake molecule, which is the most direct way to increase programulin in the brain. We are planning to file an IND or CTA by the end of this year and begin human study thereafter.
It's the 2nd TB modality and it represents the expansion of our platform. A summary of FTD, it's a deterioration of the frontal and temporal cortical areas of the brain, which results in language and planning ability to function, ultimately dementia and death. It's the most common form of dementia in people under the age of 60. It's affecting approximately 800, 000 people worldwide. There are 3 genetic subtypes caused by mutations in either pro granulins, C9orf or MAPT.
We will be going after the FTD granulin population with our PTV progranulin molecule. Next, I'm going to hand it over to Jinyo who's going to talk about RNA stress granul biology and disease specifically in ALS.
Thank you, Ryan, for the beautiful introduction here to the unmet need here in ALS. So let me see if my slides work. Okay. So I'm here to tell you about the some of the work in my group that touches on the biology of stress granules and how stress granules are important in the new generation with the idea that some of the insights we uncover may be helpful in industry and thinking about therapeutic modalities. So just to remind everybody, the central dogma here is represented as genes are transcribed as RNA from the human genome and the RNA is then processed and then translated into cytoplasm.
And all of these involve many RNA binding proteins that modulate the RNA lifecycle. Brian, could you go back a couple slides?
Thanks.
Next slide, please. And the importance of RNA binding proteins have been showcased can be showcased here by showing that many of these familiar mutations actually encode variants in RNA binding proteins and RNA processing defects are prevalent both in familial and sporadic disease. And just to remind everybody that a large fraction of ALS patients display typically 43 cytoplasmic aggregates that is now a clear hallmark of the disease. This slide is very clearly indicating that there are many RNA binding proteins, as I pointed out, that have mutations linked to AL, that's NSCD. But it's been very exciting to see that many of its mutations are actually found in intrinsically disordered regions, in the proteins themselves, in this glycine rich regions.
And these regions, next slide please, these regions are involved in homotypic and heterotypic interactions that cause the formation of liquid phase transitions and condensates. And so we call these condensates in the subplasm stress granules. And these stress granules contain RBP RNA complexes. And here you can see on the figure below that 1 of the hallmarks of stress granules is Ggp1 positivity. It's Ggp1 is our antibody protein that is typically diffused in the cytoplasm.
And when cells are subjected to stress, GTPV1 and its partners condense into these liquid droplets that over time will become insoluble fibers in disease. Next slide, please. And so the way we think about the interaction between stress granules and neurodegeneration is that all cells require stress granules to form. And this in the context of stress and stress granules are typically transient and in wild type healthy conditions, stress granules dissipate over time. But in the context of ALS mutations, these stress granules do not dissolve and maintain a pathological state that becomes permanent and insoluble over time.
And next, we see that there have been many, many reports And so in the big picture perspective here, everything that stress granular dysfunction is a clearly shared feature in not just ALS and FTD, but also in Alzheimer's disease and many other neurodegenerative diseases. Next slide. And so I'm going to quickly go over how we have been thinking about stress granules and how we model stress granules and to clarify what is the composition of stress granules as well. Next slide. So, 1 of the key features of modeling stress granular biology is using human derived cell lines and that are the correct cell types.
So, we typically use iPS derived motor neurons that have genetic risk factors and mutations in RNA binding proteins or sporadic ALS lines and subject these to environmental stressors that can then perturb the system and the interactions and model in these IPS lines to create disease relevant phenotypes relevant to, for example, ALS and FTD. Next slide, please. And just to remind everybody again, GgPV1 positive full sign puncta are what we use as a working definition of what stress burners are. And G3V1 forms this core complex of proteins and RNAs are in the 1 in this particular model here RNAs can then recruit other RNA binding proteins that contain IDR regions. And this growth of these granules is what creates these very clearly visible puncta known as stress granules.
You can see from the arsenide treated samples across a variety of different cell types. Next slide, please. And so just to set the state for what we think of as a relevant model for how we can use stress to model ALS. We use IPS remote neurons here, and we can show that stress drives CDB 43 into stress granules in the cytoplasm on the far left, top left. And stress conditions actually perturbs TDP4 3 binding across the transcriptome.
And here we can also show that TDP4 3 binding is now lost from key biomarkers such as statin 2 exon, such as statin 2 exon inclusion can happen in the stress and of a disturbance in RNA localization. And we show that in mutant cells, stress leads to the mRNA species not recovering and sort of being static and presenting in a perturbed state. And finally, stress can exemplify disease delayed onset cell death in ALS mid to mid to mid to mid to mid. Whereas we don't normally see aberrant cell death, but in a stress condition ALS lines exhibit increased cell death. Next slide.
And so starting to answer the question, what are in stress granules? We have leveraged approximately labeling using the APEX2 enzyme here that are knocked in into IPIC, right, mononeurons and different cell types. And so this proximity labeling allows spicinylation of other proteins that are in proximity to ggbp1 in the stress state. And this allows us to then use mass spec approaches to identify the proteins that are found in stress granules. Next slide.
This represents the 1st comprehensive effort to look at the human stress granule composition in a variety of different cell lines and cell types. And so we've identified 300 over proteins that are clearly identified in human SGs. And as you can see in the middle here, majority of these are actually RNA binding proteins, as we expect, because the stress granules contain protein RNA aggregates. We find that in neuronal cells, they contain many similar components as in non neuronal cells, but there are cell type differences. You can see below that there are a variety of different RNA binding proteins that are identified only in the neurons.
And from this compendium of RNA binding proteins and non RNA proteins in the stress granules, we're able to then deplete these proteins and show that many of them lead to if depleted leads to a reduction in stress granule formation. Next slide, please. And just to amplify the importance of this stress granules protein, we can modify these stress granules novel stress granules proteins in fly models with collaborations with Mark Kanco at Biogen at the time and then just next slide with Fengdao Gao and Umass to show that if you alter the levels of these novel stress granular proteins, you can prevent and in fact reverse some of the degenerative phenotypes we find in ALS models, in this case, FLY models. Next slide, please. 1 of the other studies we have been conducting to think about how to manipulate stress granules is to utilize GgBP1 assays as high throughput high content screens.
And so we've also identified compounds meant to reveal to us how these proteins aggregates can form, how can we perturb their interactions. So here we're able to identify compounds that you can incubate with purified stress granule like aggregates. And these stress granules in vitro will fall apart with the intercalation of these compounds. Whereas upstream compounds like cyclohexamine and isrip may not affect the direct may not directly affect the purified stress granular aggregates. And that tells us a lot about the chemical structure and the physical components of stress granules.
Some of these compounds can also be incubated in primary neurons derived from mice models of ALS. And interestingly, they can prevent increased cell death and push it back to control conditions. So we believe that some of these compounds have clarified the mechanism of how some of these stress fractures can form, how we can cause them to fall apart, dissipate and recover in primary cells. This was highlighted in a F1000 report that suggests that this study is exciting. It lends credibility that you can identify drugs that could modify these membraneless organelles.
Next slide. This slide highlights another effort in the lab that thinks about what are the genetic determinants of stress granular abundance. And previously, we have articulated that RNA binding proteins are a major component of stress granules from our previous cell paper. Here we focus on RNA binding proteins and ask the question, if we deplete these RNA binding proteins genetically, which RNA binding protein depleted can cause a change in the stress granular abundance. And here we utilize 2 combined 2 different approaches here.
1 is put CRISPR screens with a library targeting RNA binding proteins. We've also combined a micro raft array assay, where there are 40, 000 micro rafts, 1 of the slides. And with a PUCUS per approach, we're able to get single cell or single clonal growth of cells with 1 RVP depleted in each raft. And then these rafts are then subjected to imaging to ask if the depletion of that RNA binding protein led to a change in the stress granular abundance. Next slide, please.
And these micrographs are unique because once you identify using imaging, if stress granular abundance is affected, we can physically pinpoint the position of the RAF, release the RAF from the slide and then sequence the barcode that represent what gene is being depleted or knocked down or knocked out in the cell and identify what are these novel genes that control stress granular And so we were able to, for example, show that surprisingly, there are multiple steps in the RNA life cycle, where proteins and in this case, RNA binding proteins can affect the abundance, including both initiation and resolution of stress granules. Here we show 1 specific example, snirp200 depletion using sRNAs in human cell lines can actually reduce the stress granular abundance in cells. And then excitingly, just this year, Christine van de Boal's lab showed that there are snub200 inclusions found in ALS patient samples, right? And this indicates that our ability to identify genetic determinants of structural abundance may actually be also relevant in patient samples. Next slide.
And so I hope I've tried to summarize here how we think about strategies to understand the manipulate stretch granules. We've been excited to identify proteins found in the formation of stretch granules. We believe that manipulating those can also be helpful in thinking about therapeutic approaches. We've identified genes that control stress granule formation, and a combination of imaging technologies and put CRISPR screens. And finally, we've been excited to think about ways to manipulate the protein RNA interface to prevent recruitment of disease associated RNA binding protein including CBP-four 83 blocking the transition of these trust granules into persistent insoluble targets.
Next slide. And with that, I'm happy to end my talk here and want to thank the lab for lots of exciting collaborations with folks at UCSF, UCSD, Montreal and of course, our colleagues in the industry. Thank you, Ryan.
Great. Thank you, Gene. We appreciate you going through the RNA stress granular biology and its link to ALS. I'm now going to focus in on EIF2b. We'll bring back in the stress granular biology as it relates to EIF2B and specifically to DNL-three 43.
So I'll start by summarizing where we are with EIF2B. This is a novel first in class approach to the treatment of ALS and other degenerative diseases. DNL-three 43 is a brain penetrant small molecule activator of EIF2B, and I'm going to describe a little bit the molecular cascade and pathway around EIF2B. Also happy to present preclinical data showing robust rescue of pathology as well as key insights into pathway modulation that we plan to translate into the clinical setting. Carol will present the Phase 1 data study, which has been successfully completed or achieved both biomarker and our goals around safety.
We've initiated the Phase 1b study in ALS, as I've already mentioned. And as we mentioned before, our first therapeutic area is ALS. However, there is a direct genetic link to other diseases such as vanishing white matter disease. We see a broad role for the integrated stress response in Alzheimer's and other degenerative diseases. It's important to note that this small molecule was discovered and is wholly owned by Denali.
Just highlight on the left hand side the broad therapeutic potential around EIF2b. So I'd like to start first with just a general description of the integrated stress response. You'll see throughout the slides that I'll link it to RNA stress granules and AppreciateGene showing a close relationship with various ALS genes and RNA stress granules, but I'm going to start first by describing integrated stress response. And importantly, the ISR is a consequence of stressors, cellular stressors that are actually 4 conserved kinases that sense cellular stress and signal the ISR to essentially shut down protein synthesis transiently. I'll highlight on the left hand side that in healthy cells, the EIF2b cascade is on.
And in fact, that's what our small molecule does. It restores cells to a healthy state. In the context of a healthy cell, you have normal protein synthesis, no stress granular formation, and the cell is in a homeostatic state. In the context of both acute stress and chronic stress, you see that EIF2b is turned down. You see stalled protein synthesis and the formation of these reversible stress granules that Jean had just described.
This transient acute stress, especially when overtly large, results in transient protection. However, what we see in chronic diseases such as neurodegenerative diseases is that this stress cascade is elevated in its constitutive, which results in impaired protein synthesis, persistent stress granule formation and deleterious ISR. Importantly, I think as Gene already highlighted, mutations in genes that associate with stress granules that result in the stress granules being locked in place will also result in this chronic stress and ultimately cell death. So, there are a number of publications, extensive evidence around the ISR pathway modulation across many diseases. We're focusing today on neurodegenerative diseases.
But of course, what was already described by Gene and ALS, but there are many others, including cognition, related connection to the EIF2b signaling pathway. So again, I'd like to focus on our therapeutic hypothesis. So what does EIF2b stand for? It's a eukaryotic translation initiation factor 2b. This pathway is required for protein synthesis in healthy cells.
On the left hand side, we're illustrating that in neurodegeneration, EIF2B signaling is reduced. And our goal with DNL-three 43 treatment is basically to restore cells to normal function. On the right hand side is a molecular architecture of EIF2B and an approach for EIF2B activation. As you can see in the off state or in the stressed environment, you see 2 heterotetramers. These tetramers come together and form a dekamer or a heterododekamer, which can be activated with a EIF2b small molecule.
Intracellular protein complex that regulates protein synthesis is this EIF2b complex regulating the amount of protein synthesis. The CIF2B activator stabilizes. In fact, this CIF2B complex and it overrides the off state. In other words, in healthy cells, this complex is already this dekamer complex is already formed and cells are under stress, this is blown apart and we're able to bring it back together with small molecule. So DNL-three 43 is a novel therapeutic approach designed to restore STELIS to healthy state in the context of chronic stress, which is obviously relevant in neurodegenerative diseases.
I'm going to take it 1 step further and focus in on ALS as gene is already done, but start again with the genetics. So roughly 50% of ALS degenerogenes are associated with these stress granules or RNA homeostasis is already brilliantly highlighted by a gene. These ALS mutations have been shown to alter stress granule dynamics. You've seen some
of that data in fact.
TDP-forty 3 associates with these stress granules and can form insoluble inclusions. And you can see the graph on the bottom basically genetic discoveries over time using stress granule linking to stress granule biology as well as RNA homeostasis. So in summary, the ALS associated genes discovered to date highlight the importance of stress granular biology and TDP-forty 3 in ALS. I'm now going to focus in on DNL-three 43 specifically. So, as Gene has already shown, about 95% of ALS patients have TDP-forty 3 inclusions.
We see that roughly 50% of FTD patients and approximately 30% of Alzheimer's also have TDP-forty 3 pathology. On the right hand side is a cellular assay in which we induce stress to form RNA stress granules, again labeled by G3BP1, which Gene has outlined. We see a co localization with TEP43 as part of these protein inclusions. Importantly, when we form these stress granules and they're preformed, we can add DNL-three 43, turning on EIF2B and essentially rapidly dissolving these RNA stress granules. This also correlates with protection from cell death.
In summary, the EIF2B activator DNL-three 43 reverses these preformed stress granules and TDP-forty 3 inclusions. So, similar to the data that Jean had presented, we're just going to highlight here an iPS derived neuron assay of C9orf72. And here we're looking at propensity to form RNA stress granules in the context of stress with or without C9orf72 using an isogenic line, which we use CRISPR to knock out C9orf72. And what you can see in the image in the upper left is basically formation of stress granules quantified in the middle graph. When we add DNL-three 43, we can dissolve these stress granules in these human iPS derived neurons from an ALS patient.
In other words, C9orf patient derived neurons display increased stress granule formation compared to our isogenic control and DNL-three 43 rescues the formation of these stress granules. We next went to tissue samples from patients with ALS. And here we're looking at gene expression and specifically downstream of integrated stress response, there are a set of genes that are upregulated as part of the ISR. As you can see on the graph on the left hand side, there is an increase in expression in the ISR related genes from tissue samples from spinal cord ALS patients. We highlight that some of the top genes on the right hand side are these ISR expressed genes.
So in summary, gene set enrichment analysis of target ALS transcriptome database indicates up regulation of the ISR pathway. So now I've basically outlined the integrated stress response, the role of EIF2B and the connection to ALS and specifically to TDP-forty 3. I'd like to spend more time talking about DNL-three 43 itself and the actual molecular cascade and how DNL-three 43 works. I'll start by using this molecular diagram and I've already highlighted that there are 4 kinases that can be activated in the context of various stresses including nutrient deprivation or protein dyshomeostasis. When this happens, EIF2 alpha is phosphorylated and EIF2b is turned off.
What we're going to what I'm going to show you in the subsequent slides is that EIF2b can be turned on with in the we can decrease stress granular formation and a set of genes that are upregulated in response to stress such as ATF4 and JAK1 are reduced with DNL-three 43 treatment. So I'll start here with some of the in vitro work highlighting the mechanism of DNL-three 43. As shown here on the left hand side, we're looking at protein stability of BIF2B in the context of DNL-three 43. What we see is that in a dose dependent fashion, we increase the stability of this complex, which essentially turns EIF2B on and restoring cells to healthy protein synthesis. DNL-three 43 activates EIF2b by directly interacting with the EIF2b protein complex.
We next asked what happens downstream of activating EIF2b with DNL-three 43 and what we can see is ATF4 both at the protein level as well as CHAK1 at the gene transcript level are robustly inhibited in a dose dependent fashion with DNL-three 43. And then to tie this together with RNA stretch granules as we showed in human iPS cells and other cell lines including co localization with TDP-forty 3, we looked in these same cell lines, the ability of DNL-three 43 to dose dependently reverse RNA stress granule formation in the context of stress. And this is shown on the left hand side. So importantly, we have now understand the mechanism of DNL-three 43 in a cellular context and the molecular cascade, Vignesh wanted to study DNL-three 43 in vivo. Here, we generated our own mouse with a mutation in EIF2B5 known as the R191H mutation.
This mutation causes chronic ISR activation and this mutation is linked to vanishing white matter disease. This is an ideal model for DNL-three 43 proof of concept. So importantly, to establish the ability to engage EIF2B, we wanted to show a dose dependent increase in both plasma and brain of DNL-three 43 as shown here on the right hand side of the graph. Notably, DNL-three 43 exposure in plasma and brain were tightly correlated across dose levels suggesting essentially a 1:one ratio for brain exposure. We next ask 2 questions.
Could we engage the target and could we modify the subsequent pathway? On the left hand side is again looking at stability of the IF2b now in vivo both in spleen and in brain and again you see a dose dependent increase in the IF2b stabilization. This is critical. Essentially in this chronic ISR model, we're able to restore signaling, even with mutations directly in the EIF2B complex. This led to a dose dependent reduction in CHAK1 gene expression in brain.
So in summary, DNL-three 43 treatment led to an increase in the IF2b stability, attenuation of CHAK1 gene expression in the brain. And we've now asked the question of what about broader ISR pathway engagement. Similar to the data that we obtained from spinal cord of human ALS, we can look in brains of these mouse models and we see a broad set of ISR related genes expressed. And as you can see in this graph, with increasing doses, we return these genes expression level back to wild type levels with DNL-three 43. I'm now going to focus on the functional results using DNL-three 43.
So just to step back and talk a little about the dosing paradigm, here we dosed mice for 13 weeks at the following at increasing doses up to 10 mgkg. And what you see at the 2 highest doses both at 3 mgkg, we restore body weight in these mice in this vanishing white matter disease mouse model. Importantly, we also restore motor function, looking at time to cross the beam, foot slips or falls, we see a robust rescue with DNL-three 43. Chronic DNL-three 44 treatment dose dependently restored both body weight and motor function in summary in this mouse model of vanishing white matter disease, which is specific to the EIF2b pathway. So now I'd like to summarize in vitro and in vivo data.
We've shown that increase in EIF2b protein stability results in suppression of ISR gene expression and reduction of RNA stress granules in vitro. In vivo, we've seen a dose dependent target and pathway engagement as well as brain penetration rescue of both body weight and motor function. Summary, DNL-three 43 proof of concept has been demonstrated in preclinical models and supports studies in ALS and other neurodegenerative diseases. And with this, I'm going to turn it over to Carol to share some of our new clinical data.
Thank you, Ryan. And thanks for the overview of the preclinical data, which provide an excellent foundation for the biomarker driven development strategy that we have applied to the design of our Phase I healthy volunteer study. The study design and results of this Phase 1 study were presented today at the Northeast ALS Consortium Conference as well. The Phase 1 healthy volunteer study is a single center, double blind, placebo controlled, 1st in human safety tolerability, PK and PD study of DNL-three 43 in 95 healthy volunteers aged 18 to 50 years of age. The study included a single ascending dose portion, a multiple ascending dose portion and also a food effect study to enable this data to support moving into patients for the next clinical study.
The key endpoints were safety, PK and PD readouts of the integrated stress response pathway using an assay of ex vivo stimulated stress conditions in PBMCs or peripheral blood mononuclear cells that were collected from healthy volunteers dosed with DNL-three 43. The Phase I study is completed and met all safety and biomarker driven development goals and supports moving forward to Phase Ib in ALS patients. In the next few slides, we'll share the results of this study. So, I'll start with a safety summary. DNL-three 43 was generally safe and well tolerated across all dose level studies studied.
There were no serious adverse events or discontinuations due to study drug. Of those participants receiving DANIEL-three 43 that experienced treatment emergent adverse events, all were mild in the single ascending dose and mild or moderate in the multiple ascending dose. Moderate AEs in the multi ascending dose portion of the study were also more frequent in placebo than DNL 03:43 treated participants. No clinically important findings were observed in vital signs safety labs or other safety assessments. The most common treatment emergent adverse event experienced in DNL-three 43 participants are reviewed in detail on this table.
So, this table provides an overview of treatment emergent adverse events reported in 2 or more participants in the active or placebo groups. There were no dose related increases in overall treatment emergent adverse events observed across the studies. As you can see, all treatment emergent events in subjects receiving DNL-three 43 resolved without intervention except analgesics for some headache and procedural pain. Notably, postural dizziness treatment emergent adverse events were mild and not associated with clinically meaningful changes in systolic blood pressure with a similar frequency observed across 343 and placebo participants. In summary, 343 was generally safe and very well tolerated in both the single ascending dose and multiple ascending dose cohorts.
Now, I'll review the pharmacokinetic profile of DNL-three 43 in this first in human study. DNL-three 43 exhibited well behaved clinical pharmacokinetics. Following once daily administration for 14 days at steady state, DNL-three 43 exhibited prolonged oral absorption, low fluctuations in plasma concentrations and a long terminal half life of 40 to 50 hours. Extensive distribution into the CNS was observed based on a mean CSF unbound plasma ratio of approximately 0.65 to 0.89. This pharmacokinetic profile supports once daily dosing of DNL-three 43 and extensive penetration into the CSF.
The well behaved pharmacokinetic profile of DNL-three 43 was also associated with robust peripheral inhibition of integrated stress response biomarkers as measured by ATF4 protein and CHAK1 gene expression. Data from the single ascending dose portion of the study were used to select dose levels for the multiple ascending dose study that were anticipated in stress induced peripheral blood mononuclear cells to reduce the integrated stress response pathway. And this is exactly what we saw. On the left graph, you can see that peripheral blood mononuclear cells collected from healthy volunteers treated for 14 days with DNL-three 43 demonstrated a reduction in ATF4 protein after ex vivo stimulation of PBMCs at all multiple doses tested. After completion of the dosing after 14 days, a recovery of stress response is observed after day 15, reflecting the long half life of DNL-three 43.
Similar effects are seen on the right graph where PBMC is collected from healthy volunteers treated with DNL-three 43 demonstrated a reduction in CHAK1 protein expression after ex vivo stimulation of PBMCs from treated participants. These data support that robust inhibition of the integrated stress response pathway at all multiple doses tested were achieved and the data support dose selection for our Phase Ib in participants with ALS. With Denali's biomarker driven development strategy, we always aim to utilize preclinical model data and human data to understand and predict effects in the CNS and the brain. Ryan earlier shared data from the vanishing white matter EIF2B mutant mouse, which exhibits overactivity of the integrated stress response pathway. In the mouse model experiment, a dose dependent effect on brain pathway engagement as measured by CHAK1 was observed with near normalization of ISR biomarkers at the highest dose.
The relationship between exposure and pathway engagement from the DN0343 treated mutant mouse is shown in the orange and green lines here, demonstrating that both plasma and brain PK similarly predict mouse brain integrated stress response pathway admission as measured here by CHAK1. We did a similar experiment in humans where we can only obviously collect peripheral tissues. But as you can see here, the data from the Phase 1 healthy volunteer study demonstrates a similar relationship between exposure and integrated stress response pathway inhibition in the blue line. Together, these data support the translatability of human peripheral PK and pathway engagement for rationally predicting inhibition of the integrated stress response pathway in the central nervous system. These data enabled Denali to select the correct DNL-three 43 dose levels to test in humans in our Phase 1b study.
We hypothesized that DNL-three 43 dose levels in humans that maximize the peripheral ISR biomarker response will also inhibit the integrated stress response in the activity in the brain and spinal cord. So, here is the design of our Phase Ib study in ALS. This Phase 1b study is a randomized double blind placebo controlled 28 day study to evaluate the safety tolerability PK and PD of DNL-three 43 in adult participants with ALS. This is followed by an 18 month open label extension for collection of additional biomarker data. The key endpoints include safety PK and similar pathway engagement biomarkers that we studied in the Phase 1 healthy volunteer study, along with exploratory biomarkers of integrated stress response and neurodegeneration.
Key inclusion and exclusion criteria include a diagnosis of ALS less than 3 years from symptoms onset and a slow vital capacity of greater than equal to 50% predicted. We'll study 2 dose levels selected based on our Phase I data that I shared and we'll select 1 of these doses after the 28 days based on biomarker data for the open label extension. Enrollment was initiated in Q3 of 2021 at global sites with an anticipated readout of Part 1 in Q2 of 2022. In summary, the data shared here validates the DNL-three 43 CNS distribution and activity on the integrated stress response pathway in healthy volunteers. Based on this data, we have initiated a Phase Ib study in ALS, which is currently enrolling.
While our first clinical indication is ALS, CNL-three 43 has broad therapeutic potential to address the ISR pathway that is active in multiple neurodegenerative diseases. I'll now segue to share an update on the progress of our Rip K1 program, which also has broad therapeutic potential across multiple neurologic diseases. Denali identified the Rip K pathway as an important therapeutic target back in 2015, recognizing the importance of microglial RipK1 inhibitor into the clinic and also generated peripherally restricted RIPK1 inhibitors that have also advanced to the clinic in autoimmune indications with our partner Sanofi. In late 2018, Denali partnered with Sanofi on a strategic co development and co commercialization across a portfolio of RYP K1 inhibitors for both autoimmune RIKK1 inhibitors for both autoimmune and neurodegenerative indications, including ALS, multiple sclerosis and Alzheimer's disease. Sanofi is leading the SARS820 or DML 788 development in ALS.
The joint development team successfully completed Phase 1 healthy volunteer testing for SAR820 this summer. The first in human single ascending dose and multiple ascending dose study demonstrated a good pharmacokinetic safety and tolerability profile, excellent central nervous system penetration and strong target engagement as measured by phosphoserine166 ripk1 kinase activity. We now have plans to advance to Phase 2 in participants with ALS. As a reminder, the genetic rationale for RIPK1 inhibition in neurodegeneration is linked to the microglial risk in Alzheimer's disease and familial mutations in ALS that interact with RIPK1, like TBK1 and optaneurone and trigger disease. We hypothesized that accelerator of disease whereby RIPK1 activation via TNF receptor 1 results in microglial dysfunction astrocytes and neurons.
RipK has broad applicability in neurodegenerative diseases, but consistent with the theme of our webinar today, we will focus on the development in ALS. Along with Sanofi, we asked the question of whether RybK1 activity was up regulated in the spinal cord of sporadic ALS. Data shown here demonstrates that RIPK1 expression as well as kinase activity is elevated in ALS tissue. Based on this and preclinical data, we have initiated and SAR820, a CNS penetrant inhibitor of RIPK1. Leveraging our experience with our portfolio of RIPK1 inhibitors, including a prior CNS penetrant inhibitor that was evaluated in a previous Phase 1b study, we plan to advance to a Phase 2 clinical endpoint study in adult ALS participants.
Sanofi is actively planning this Phase 2 study and the study will evaluate safety and clinical efficacy. Here is the design of the Phase II ALS study, which has been named the HIMALAYA trial with SAR820 or DNL-seven 88. This is a multicenter, randomized, double blind, placebo controlled, 24 week study to evaluate efficacy, safety and tolerability in adult participants with ALS. It is followed by a 2 year long term safety extension. The key primary and secondary endpoints are clinical in nature, including a primary endpoint of change in ALSFRS R score and secondary endpoints, including a combined assessment of function and survival, respiratory function, muscle strength, quality of life, and also biomarkers of neurodegeneration.
Key inclusion and exclusion criteria include a diagnosis of ALS less than 2 years from symptom onset with standard of care medicines permitted. Slow vial capacity of greater than equal to 60% predicted and ability of the ability to swallow tablets is also required. Enrollment is anticipated in Q2 of 2022 with 50 to 60 global sites in North America, Europe and China planned. In summary, the joint development program for the only CNS penetrant RipK inhibitor in the clinic, to our knowledge, has demonstrated target engagement and a well tolerated safety profile. Sanofi is leading the development in ALS with plans to begin the Phase II Himalaya ALS study in Q1 of 2022.
DNL-seven 88 has broad potential in neurodegenerative diseases and evaluation and planning for other indications, including multiple sclerosis and Alzheimer's disease are also in progress. And with that, I would like to turn over to Joe Lukak, who will take us through the development program in FTD for the PTV program. Thank you.
Thank you, Carol. I'm excited today to provide an update on our PTV pro granuline development program, also known as DNL-five 93. DNL-five 93 is a CNS penetrant pro granuline replacement therapy. As such, we're initially targeting pro granuline deficient FTD as the initial indication, but we do think that this program has potential for expansion into other indications. Based on our exciting preclinical data, suggesting we're able to achieve broad CNS biodistribution of PTV progranulin as well as rescue both lysosomal phenotypes and disease related pathologies present in these preclinical models.
We are planning an IND or CTA filing for this program at the end of 2021. And a reminder that this program is a part of our strategic partnership with Takeda for joint development and commercialization. As Ryan mentioned in his introduction, the PTV represents an expansion of our TV platform designed to deliver biologics to the brain. PTV stands for Protein Transport Vehicle and its ability to increase the uptake of these proteins into the CNS. And in this case, we're using a full length pro granuline molecule conjugated to our TV.
Much of the data that I'm going to show in my update here today was recently published in a manuscript in Cell shown on the right side of the slide. And so, although I'm only going to be able to show highlights today, if folks are interested in more detailed information, I would refer you to this manuscript. So first, let's dive a little bit into the biology of pro granuline deficiency. So, what has become well accepted now in the field is that multiple different mutations in the granulin gene result in decreased levels of progranulin protein, both in the plasma as well as the CSF. And some Denali data to show that is shown on the bottom left side of the slide here.
This results in a number of disease related phenotypes in these patients and that includes significant microgliosis or glial activation as well as TDP-forty 3 pathology and significant neuronal atrophy that correlates with increases in neurofilament light in the CSF. What data from our group at Denali as well as other labs in the field has recently identified is that it seems like the mechanistic underpinnings for these disease related phenotypes are a result of lysosomal dysfunction that is caused by pro granulant deficiency. And this lysosomal function then relates into disease related phenotypes that can be observed. And I'm going to go into a little bit more detail on some of these lysosomal phenotypes today and how we feel like they can be rescued by our molecule. So, before I dive into the data, I wanted to start with the schematic to talk people through exactly how we feel like pro granuline functions in the cell.
So pro granuline is present in the extracellular space and is taken up into cells through cell surface receptors such as sartiline and targeted to the lysosome. When it reaches the lysosome, the full length progranulin is broken down into individual granulin peptides. Once these granulin peptides are in the lysosome, they seem to bind specifically to a relatively lysosome specific lipid known as BNP and pro granulant binding to BNP stabilizes BNP. And this becomes important because BNP is a major vesicles in lysosomes. And these interluminal vesicles are important areas to enable proper function of lysosomal enzymes.
Therefore, when you have a pro granuline deficiency, your BNP becomes destabilized, You get less functional interluminal vesicles in the lysosomes and you get lysosomal enzymes that then become less functional and therefore you get accumulation of lysosomal substrates and broader lysosomal dysfunction within the cell. And this core mechanism is really important to understanding what we think about how pro granuline contributes to disease and therefore what we want to do about it therapeutically. Of cells and now labeling the lysosomes in green here. Of cells and now labeling the lysosomes in green here shown with the LAMP-two stain as well as BMP shown in red and pro granuline shown in aqua. And what you can see as mentioned in the introduction is that both pro granuline and BNP are localized to these lysosomal compartments.
In cells lacking pro granuline, what you see is not only of course a reduction in pro granuline itself, but a significant reduction in the levels of BNP, this lysosomal lipid. We can then add our PTV progranulin molecule to these cells. And what we're able to see is that we can first direct the PTV Progranuline appropriately to the lysosome. And this lysosomal localization of Progranuline is able to rescue levels of BNP within the cell. And quantification of both of those metrics is shown on the bottom side of the slide.
So, what does this mean for lysosomal function? When we look more closely at lysosomal normal function. When we look
more closely at lysosomal
function here using a DQ BSA assay in order to measure the amount of lysosomal proteolysis, What we can see is as compared to granuline or wild type cells on the left side of this slide that the granuline knockout cells have reduced lysosomal proteolysis and therefore abnormal lysosomal function. And that's shown in the pictures on the left and quantified in the bar graph on the right. Encouragingly, when we add our PTV pro granulan molecule, but not a control IgG, we see that this is able to rescue normal lysosomal function within these cells. So therefore, we could show that not only can we deliver pro granuline BNP, but then also rescue normal lysosomal dysfunction. So now let's move in vivo.
And what I'm showing here is Ryan, can you go back 1 slide, please? Thank you. So now we're looking in vivo. And what I'm showing here is that PTV Progranulen following systemic administration is able to achieve broad biodistribution within the CNS. So that our TV sequence not only enables enhanced BRAIN uptake of our PTV pro granuline molecule, but also effective delivery to multiple of the key CNS cell types.
And that's shown on the image on the right hand side of the slide where if you focus in on the pink that is our PTV molecule that's been effectively delivered to multiple cells within the brain, including neurons which are co stained in green as well as microglia that are co stained in aqua. And both of those are indicated with arrows on the right hand side of the slide. This is in contrast to dosing of a control molecule, which is an Fc progranuin. The only difference is it lacks our TB sequence. So you can see in this case, systemic administration of a progranulin molecule alone is insufficient to achieve BRAIN uptake or biodistribution and targeting of these multiple cell types in BRAIN.
Then we went on to look at the ability of PTD pro granulant to rescue some of the phenotypes that are associated with granulin deficiency in vivo. We did a number of experiments to address this and I'll show 1 of them here, which is involves the first method of separating each of the CNS cell types from BRAIN into discrete into ability to look at them independently. So that includes neurons, microglia and astrocytes. And then we went on to look at the presence of some of these lysosomal lipids in each of these cell types. So first, I'll show that we were able to in microglia, the microglia showed the most prominent changes in lysosomal lipids.
And so what you're looking at here is a heat map of multiple lysosomal enzymes. So shown on the left hand side is a wild type animal and comparing that to a granuline knockout animal, you see changes in multiple different lysosomal lipids, including BNP as well as some other lysosomal substrates. And so this is consistent with significant lysosomal dysfunction in these cells. However, through weekly or biweekly treatment with PTV Progranulin, we are able to rescue these lysosomal accumulations that occur in microglia. We see a similar story in other cell types.
So shown below is in first in astrocytes as well as in neurons that although these cell types have less significant lipid changes, which is consistent with the function of these cells in the CNS, but that we are able to rescue these lipid changes in all cell types consistent with the rescue of lysosomal function throughout all cell types within the brain. So now let's take 1 step farther and look at the impact of granuline deficiency on the function of certain lysosomal enzymes. And I'll focus on 1 lysosomal enzyme here GKs, which is encoded by the GVA gene and well known for its links to neurodegeneration. And what you can see is in pro granulant deficient animals shown here that there's significant reduction in the amount of GK's activity in BRAIN as compared to wild type animals. Although this cannot really be effectively rescued by an Fc granuline alone, when we treat with PTV pro granuline, we're able to rescue the amount of GK's activity back up to healthy wild type levels.
Correspondingly, we see an accumulation in knockout animals of glucosyl sphingosine, which is 1 of the major substrates of GK's enzyme in granulant deficient animals. And we're able to rescue the accumulation of these defects through treatment with PTV pro granulin. Measuring these types of lysosomal enzymes is not only an encouraging way to measure a functional effect in preclinical studies, but may also have the potential to correlate to clinically relevant biomarkers for the program as well. And I show that here on the right side of the slide now looking at BRAIN samples from patients with pro granular deficient FTD. And what's very encouraging is these patients show a similar increase in glucosal sphingosine levels in BRAIN, arguing we may not only is this biology that we're looking at quite relevant to the clinical situation, but we may be able to use this biomarker as a metric of drug function in our clinical studies.
So then moving downstream of this lysosomal dysfunction, what is the impact on disease related phenotypes? And I'll show an examples of a couple of those here today. The first is looking at lipofuscin accumulation that occurs in neurons, both in FTD patients as well as in the granular knockout mouse model. And that's shown in the sort of gray inclusions here that can be seen in the middle panel of the pictures. And these accumulate in age dependent fashion and granular knockout animals, but through either weekly or every other week treatment of these animals with our PTV pro granuline construct, we are able to rescue this lipo but the downstream impact of these defects.
Then let's move on to look at the microglial activation that's present in the brains of these animals. And as I mentioned in the introduction, this is also 1 of the hallmark pathologies present in FTD pro granular patients. And what you could see in these 3 images is now looking at IBA1, which is marking microglia, GFAP marking active astrocytes and CD68, which is a marker specific for activated microglia. In the granulant deficient animals, you see significant gliosis, both microgliosis and astrogliosis compared to the wild type control animals on the left. However, like with the other phenotypes, dosing with our PTD progranulin molecule is able to result in significant rescue of both of these phenotypes, as shown in the panel on the right.
And then I'll just include some quantification of that data here looking at IBA-one on the left, again, a complete rescue, a significant rescue of both CD68 and GFAP levels as well, suggesting that PTV is able to rescue these gliosis phenotypes that are present both in patients as well as in the granular knockout animals. So just to wrap things up from here then, I wanted to bring it all together in 1 schematic that tries to capture all elements of how pro granular impacts disease biology and how different therapeutic approaches may affect this biology. So first, when we look at the healthy situation, we see normal levels of pro granulant both in the blood compartment on the left hand side of the panel as well as in the brain on the right side. This results in normal lysosomal function in CNS cell types and a lack of any neuro inflammation. However, in the case of progranulin deficient FTD, the reduced progranulin levels in these patients, both in the periphery and in the CNS, result in altered lysosomal function and eventually accumulation of lysosomal substrates that results in neuroinflammation, neurodegeneration and other phenotypes.
Now there's multiple ways to approach this biology and 1 that's become popular in the field is through blocking the internalization of progranulin and therefore increasing circulating levels of progranulin. And this can be done through blocking cell surface receptors such as sortilin. And it has been proven both preclinically and clinically that blocking sortilin does increase extracellular programuline levels, both in the blood and to some extent in the CNS as well. However, the potential concern with this approach is just by the fact that you're blocking and the direction of pro granuline to and the direction of pro granuline to the lysosome where it's thought to be most functional. So we feel like our program, our PTV progranulin approach is the most direct way to increase progranulin levels in the periphery, then using our TV technology facilitate uptake of this molecule from the periphery into the brain.
And then conveniently through both sertilin receptors and some of the native pro granulin receptors as well as through TFR, it facilitates uptake of this molecule and targeting to the lysosome where it can improve the function of lysosomal enzymes and rescue the lysosomal dysfunction present in these patients. So we're quite excited about this program based on its ability to enhance the uptake of peripherally administered pro granuline and the preclinical data suggesting that we're able to rescue a range of lysosomal defects with this molecule. In addition to that, we're able to rescue both microglial dysfunction and neurodegeneration in progranuent deficient mice. And some of the mechanistic work we've done preclinically, we feel like it sets us up well to have a biomarker driven development strategy based on being able to measure lysosomal function, gliosis and neuronal health using biomarkers that are translated from our preclinical studies. So overall, we feel like our TV enhanced BRAIN uptake is the most direct and effective way to increase pro granuline in BRAIN And we're excited to advance this program into human studies in 2022.
And so I will stop there and transition back over to Ryan to go over some of the near term milestones.
Thank you, Joe. Appreciate the update on the programs. Let's now highlight some of the milestones that we should expect coming forward. I'll just focus first on our first our 2 programs, DN L310 and DN L151. So these 2 programs are most advanced programs.
Our focus is essentially initiating late stage trials for both of these programs, including to continue to collect data from our ongoing Phase III from the DNL-three 10 or ETV IDS program. Today, we focused on 3 programs: DIF2B or DNL-three 43, the RIB kinase program and PTV program, NUELIN. In terms of next set of data for these programs, we will expecting Phase 1b top line results in mid-twenty 22 for EIF2b and we'll get into some of the questions and discussions around this program. Rip K program, we're initiating a Phase 2 study with Sanofi. Sanofi, of course, taking the lead on that study and that's slated to begin at the beginning of 2022.
Then we'll be filing the IND and or CTA for DNL-five 93 by the end of the year. Just highlight that also ATB TREN2 is on its way to filing 9D or CTA. Both of these programs are opt in programs from our partner. Beyond that, we continue to expand our portfolio, especially our ETD portfolio with a focus on building clinical manufacturing and commercial capabilities. We recently had Katie Peng join the team and Katie will be leading the build out of our commercial team around rare diseases.
We continue to partner with our collaborators on Alzheimer's and Parkinson's and look forward to advancing those programs as well. And as you can see, today we really focused in the center here on ALS and FTD where we have both partnered programs but also wholly owned programs. So, with that, I'm going to ask our presenters to come back and we're going to go into Q and A. We have a number of questions here and look forward to addressing these questions. Excellent.
Welcome back. All right. So I'm going to start with a set of questions. And I think, Gene and Joe, you're going to be I'm going to be asking you a few questions at the beginning here. And then Carol, definitely some questions on the clinical development as well.
So the first question is, what is an intrinsically disordered region? And I think this is actually a great question because it's really important when we think about the mechanism of these ALS mutations. So Gene, why don't you tell us what an intrinsically disordered region?
Joe, maybe
you can follow-up with what you see the relevance of these regions are?
In the proteins that we study and also DNA protein, these are typically very glycine rich regions that often are sort of floppy and unstructured. They enable a lot of protein protein interactions with themselves or with other proteins with similar domains. And so they are interested in this order only because they tend if you express them, recombinantly, they will aggregate and they will then phase separate from the rest of the constituents in the test tube. There are probably better definitions for the working definition for these. But in online, they are unstructured regions.
And Joe, maybe the relevance of these intrinsically disordered regions in ALS.
Yes. And I think Gene hit on it really well, which is most RNA binding proteins contain both an RNA binding domain and 1 of these intrinsically right? And so that's why most of these RNA binding proteins have them. Right? And so that's why most of these RNA binding proteins have them.
But as Gene alluded to briefly in his talk, most of the mutations that are associated with ALS also occur in these intrinsically disordered regions and make them more aggregation prone, right? So it's some of the best data linking some of these RNA binding proteins functionally to disease is really showing that when you have disease associated mutations, your stress granule dynamics are changed. And that is really impacts that equilibrium that Gene mentioned in his talk.
Great. Thanks, Joe. So I think I love this next question because it's like the fundamental questions I think in most neurodegenerative diseases. So in the presence of TDP-forty 3 inclusions in ALS and FTD patients, is it a driver of disease or a marker of the disease? Is it a driver of pathophysiology?
Is it sufficient on its own? How far downstream or upstream in the disease pathology is it? And so I would love to answer this, but I think, Joe, maybe I'll hand it to you and just comment that TDP-forty 3 itself can be mutated and as a degenerate gene essentially cause neurodegeneration. But Joe, I'm going to hand it to you.
Yes. No, and I think that's the strongest evidence, Ryan, is that mutations in TDP-forty 3 can cause disease. Now that's not, it's only a small subset of patients. But when you look at the RNA binding proteins in aggregate, mutations again in these disordered regions of multiple RNA binding proteins are sufficient to cause disease. Functional contribution to this.
And I think there's growing work looking functional contribution to this. And I think there's growing work looking downstream of TDP-forty 3 and showing a functional impact of disrupting TDP-forty 3 biology through these aggregates on downstream biologies. And I don't know, maybe Gene can comment on that. He's done a lot of work in that area.
Yes. I mean, I think there is a consensus in the field now that CDB403 loss of function, because it's being sequestered and trapped in these granules or in the cytoplasm as infusions, may lead to severe downstream consequence, right? So for example, I think Don Cleveland's and Kevin Eggman's manuscript on statin2 being a downstream target, where the exon inclusion and this cryptic exon then leads to a loss of presence of the FETN2 protein. It's a contributor to disease, right? And in our data and also consistent with our data, if you lose Tb403 from the cells, either by knocking it down and showing that knockback experiments or in our case, by stressing the situation in the cells, Tb4a3 is no longer binding the exon to prevent it from being suppressed, right?
So when it's no longer there, is that included and then causes a down regulation of the F7-two. So I think we are leaning towards eventual loss of function of the protein. Now, whether it is the only driver of the disease, I don't think that's really clear yet. And is it the is there something else upstream? And I think that evidence in the field will probably show up over time to indicate there are other upstream misregulated events.
I think I'll just add a simple point, which is it's a question of necessity and sufficiency. And definitely TP43 is sufficient, and I think the genetics implies that. In the context of patients that don't have TDP-forty 3 mutations, the question is, is it necessary? And I think the fact that it's 95% of ALS patients have these inclusions suggest that it's playing a role and it's certainly sufficient on its own. Okay.
So, what is the evidence that breaking up stress granules can actually reverse disease pathology in ALS and FTD? And maybe Gene, I'll start with you and Joe, you can comment.
Right. So there have been experiments in the field that have shown that if you alter some of the key proteins that are implicated in structure information and stability, you can see reversal of phenotypes in cells and in mice models, right? And 1 of those experiments have come from the attacks into depletions from Aaron Gittler's work. Attacks into is an important structural protein or at least part of this structural interact film. And there's been also work showing that you can create stress granules using optogranules or technologies, which with light induction creates these aggregates.
And that can cause these results in the phenotype. And then you can turn off the light and let these things dissolve and then the phenotypes go away. So these are good in vitro models suggest that these have consequence and removing them can have reversion to a normal state.
Yes. Great. Joe, any thoughts on stress granules around reversing pathology and its association with cell death in particular?
Yes. No, and I think that the Jean described it very well. There's data from a number of labs suggesting that if you can't dissociate stress granules, you'll have toxicity, but then being able to dissociate them rescues that toxicity. I think he gave a couple of good examples. Paul Taylor has done some excellent work in this area as well.
And then I'll also comment that the ISR itself, so the chronic activation of the ISR will lead is sufficient to lead to neuronal apoptosis. And this has been seen experimental situations where even if you look at stress granules or not. And we've published a paper not so long ago showing even in acute neuronal injury that inhibition of the ISR is able to protect neurons from degeneration. So it does seem to be a broadly relevant pathway in controlling neuronal survival.
Which is Actually, in 1 of our early papers, a couple of years ago now, right, and we showed that some of these planar compounds, which are 2 compounds, do seem to disrupt stress granule stability and recover to B4three localization back to its normal state. And in cells with mutations, when we treat these cells, these cells survive longer and actually have much, much decreased cell death, right? So I mean, these are, again, maybe not they're completely direct experiments, but they do point to SGs being an interesting therapeutic modality that can be corrected, yes.
I mean, I think the ultimate proof will come in our treatment studies where we're looking at disease patient survival and various biomarkers. So, which is more important, reversing preformed stress manuals or the inclusion of TDP-forty 3 or both? Maybe, Joe, I'll have you answer this 1. I think it's going to be a tough question to answer, but I think the question is really around the relevance of the stress panel or TDP-forty 3 or both or are they just intimately linked?
Yes. I mean and Gene's done some work on this in terms of 1 answer to this question is when does TDP-forty 3 appear in stress granules, right? And I'll let Gene comment in more detail, but the data suggests it's not a core stress granule component, but you have chronic stress granules and then you get TDP-forty 3 accumulation, right? And before I let Gene kind of go into more detail there, I will say, I mean, I think we do also have to think about ALS on sort of a like cell by cell level, right, where it's not like you get TDP-forty 3 pathology in your entire nervous system all at 1 time. I mean, it's well known that ALS moves spatially through the likely still it's likely still forming in other areas.
And so I think that there regardless of what stage you intervene, I think that there's potential for benefit in terms of progression in these patients based on that mechanism.
Yes. When we treat IPF derived motor neurons with a long term chronic stressor, what we see is that we have stress granules that form and then CDP-forty 3 begins associating this transgranule. So it's not a core transgranule partner, but associates. And then in wild type cells, when you remove the stressor, Tb4a3 recovers and leaves. But in cells which are from ALS patients, they don't recover.
And this stickiness, I guess, is retained, right? And so we do think that if you can find ways to perturb the interaction between stress granules and CDP fracture accumulation that will enable stretch granules to or CB42 to then depart and return to its normal state. So there I think there are different ways to think about that problem. I think resolution is, I think, a key part of how we're thinking about from a therapeutic opportunity.
Great. Okay. Now a series of questions on DNL-three 43. I think, Joe, probably you and I on the mechanism can cover these. So do you have any evidence that DNL-three 43 is effective at targeting inclusions containing phosphorylated or ubiquitinated TDP-forty 3?
Yes. It's a great question. And I think the reason for asking, I'm assuming is because phosphorylated or ubiquitinated TDP-forty 3 is associated with more pathological granules, right? And the answer is we've done some work there. It's been hard to generate phosphorylated TDP-forty 3 or ubiquitinated TDP-forty 3 that we really believe in some of our cell models.
And so we're attempting that, but it's something that we haven't looked at as much as we could because of technical reasons at this point.
We'll do some rapid fire. We have a large number of questions here. So can DNL-three 43 prevent the formation of stress granules? If so, is there a path of development for 343 in early stages of the disease to prevent progression? And I think I would just comment that, yes, it can prevent stress granule formation.
What we show is a higher bar, which is dissolving preexisting stress granulars. The answer is both. Can DNL-three 43 activate all types of EIF2B alleles or is there a specific mutation for which DNL-three 43 doesn't work? And obviously, we showed in the mouse model 1 mutation in EIF2B in which it obviously works robustly. Most of the time, we're targeting wild type BIF2B.
So, I don't know, Joe, if you've done any other work on sort of these very rare mutations of BIF2B beyond R-1 hundred and 91 H.
What I can say is it should it would be predictive to work on all disease related alleles. We've tested some of them in the lab, but others based on the prediction of the mechanism of how it works, we'd expect it to be equally effective in all cases. There are specific mutants that we engineered with EIF2b that disrupt the binding of our molecule. And so we can identify what residues are key for its activity. But none of those are the same as the disease associated mutations.
And for all of those, we see equal activity in the ones we've addressed so far and can predict for others as well.
Does DNL-three 43 mimic a naturally protective mutation in EIF2A. So I'm reading it exactly. So I want to make sure that I get this correctly. So the question is around are there naturally occurring protective mutations? Does DNL-three 43 mimic what would be a naturally occurring protective mutation?
So, I guess what I could say exactly is we've never done that exact experiment, but theoretically it would be very similar. So if you were to take the phosphorylation site in EIF2 alpha that's phosphorylated upon stress and you mutated that so it couldn't be phosphorylated, that would probably act pretty similar to our compounds. Again, that's not that mutant has been published in some groups. We haven't done a side by side comparison, but the prediction would be the case. And what's important mechanistically there, if we dive a little bit deeper into that question is and you spoke about it a little bit, Ryan, as we call the molecule an EIF2B activator because it's a convenient way to talk about it, right.
But most accurately, it's blocking the repression that it gives upon stress, right. So when in a healthy cell, the molecule is essentially inert, right. It doesn't further activate EIF2b upon more than normal levels. But when you have this stress induced repression of the system, the molecule is able to block that stress induced repression and bring it back up to normal levels. And so, I think that's important and can get to some of the safety related elements of the molecule itself as well.
And that's a perfect that actually answers the next question. Is there any theoretical risk of over activating EIF2B? And I think I'll just highlight what Joe said is that it's not your traditional activator, right? It's essentially restoring normal function. When function is normal, you're obviously not over activating it.
So I think you nailed that 1. So Carol, I'm going to turn to you on some development questions. So are there any on target expected AEs?
So based on our GLP studies in both mouse and non human primate, our definitive studies, we did not have expectations of adverse events that we were looking for in the Phase 1 study. But I think notably, given that this is only 1 of 2 EIF2b activators that have gone into the clinic and there's been no published safety data, we absolutely were looking in our single ascending dose and multiple ascending dose for any adverse events of interest, which as noted, this was very well tolerated and we don't have any adverse events that we clearly feel are mechanistically related.
It looked like the PD data showed for 343 in healthy volunteers, showed that the doses were essentially overlapping in pharmacodynamics. How do we think about or how do you think about the high and low dose for Phase Ib relative to levels of MD-one-seven shown in the data we just presented?
Yes, great question. And we do when we look at single ascending dose data, particularly at the lowest dose, see evidence of dose response. And if you look at the exposure response, we see a relationship between exposure and response. But I think essentially what we're seeing with those multi dose levels is that we're nearly maximally inhibiting the peripheral ISR response even with the lower dose. So I think the take home for me from that data really is that it has a fairly wide therapeutic window.
And so looking at the Phase Ib study and ALS participants, we'll study 2 dose levels that allow us a broader window for safety, if there is any safety issues that should emerge, but then also allow us to look at other endpoints, exploratory endpoints in that study that may differentiate with different dose levels.
Thanks, Carol. So why is the Phase 1b ALS study placebo controlled?
Yes. So I think this is always a question particularly because we're very cognizant about participants with ALS enrolling in a study and the lack of a desire to be randomized to placebo. However, given our biomarker driven approach and collection of CSF, looking at biomarker changes that really is essential for looking at these changes that you have a control given that actually the instrumentation of a lumbar puncture, as you can imagine, does create inflammatory cytokines and other changes in biomarkers that are very difficult to interpret without a control. And then I think with any study, having particularly for the first time, a mechanism is going into a population, having a placebo for controlling safety issues or cohort related clinical issues is really important.
Great. All right. Series of questions. Let's cover each 1. And I think, Carol, probably you and I can cover these.
What short term data would come out of a Phase Ib that could give you significant insight?
Yes. So the key endpoints that we're looking at are the markers of the integrated stress response pathway and demonstrating that in a patient population where the integrated stress response pathway is actually naturally, we assume to be elevated, whether we can see that we're still able to reduce that integrated stress response pathway as we have in the healthy volunteer studies. In addition to that, we will be looking at exploratory biomarkers of neurodegeneration and other biomarkers to give additional confidence that we're having an effect on the disease pathology. Now some of that may come after the longer term extension. So you notice that Part 1 is only 28 days, which enables us to look at those pathway biomarkers.
But for some of the exploratory neurodegeneration biomarkers, for example, we would be looking at those in the open label extension portion.
Yes. I think you answered the second question essentially related. So can you measure ISR genes or stress granules in CSF or elsewhere in ALS patients after treatment with 343, what time period would be necessary to measure significant changes? And Carol, you can comment here. I'll comment as well.
Yes. So we can't really measure the ISR response directly in CSF, and that's why we have done a very rigorous comparison of the effects of inhibiting the integrated stress response pathway and effects in brain in animal models. So we're really using the animal model to demonstrate to ourselves and to you that the exposures are very much correlated with inhibition of the integrated stress response pathway that's really independent of tissue. And so for us, when we can demonstrate that we have good exposure in the CSF, we can then use that relationship to predict BRAIN target and pathway engagement.
I'll just add to that. We have work on proprietary biomarkers in CSF. And I think to be fair, this is a highly competitive program. It was showing very robust data in both the animal models, but in particular the Phase 1 data target engagement. But we continue to invest heavily in these biomarkers.
And I would just say stay tuned. We have to consider that it's a long development path with a lot of competitors. In fact, along those lines, the next question is how do you compare and contrast your EIF2b agonist against your competitor also in Phase 1? And there will be obviously, there will be a desire to And we haven't we're And we haven't gone into great detail of our actual structure of our compound likewise unaware of the structure of their compound and their mechanism, but we're guessing that it's going to be highly competitive with a similar mechanism. And so again, I think this gets back to the sort of biomarker answer, which is there's going to be some data that's going to remain confidential to some period of time as we advance the program.
Okay. So next question here. So for the 343 Phase Ib, you'll try to focus on sporadic ALS patients. It's a question, are you completely agnostic of the genetic driver of disease? How do we view the population for this study, Carol?
Yes. So in this case, we are looking at sporadic ALS. And I think that largely comes from the fact that the mechanism, more than 95% of ALS patients have TDP-forty 3 inclusions that we think are linked to the stress granules that Jean and Ryan described today. And so we don't feel that patient selection from a genetic perspective makes sense for this target. We are looking at in terms of as we think about future development, selecting for patients in terms of progression rate and other clinical features that may be important in seeing effects on clinical outcomes.
Okay, great. So, any reason to think there may be more or less activity in certain ALS phenotypes such as vulvar onset? And how do you and how about fast versus slow progressors? Also, how does the patient focus of the Healy program impact your trial? And do you apply to be involved in that program?
So really around patient phenotyping and then a separate question around the Healy program, Carol?
Yes. So I don't know that we have specific scientific data that would tell us about responsiveness in some of these subtypes of ALS. And in this study, just in terms of looking at more homogeneous patient population, we are looking at people that have a diagnosis of ALS and are within a particular time frame within 3 years of onset of ALS to try to have a slightly more homogeneous population. In terms of the question about the Healy platform trial, great example of, I think, a collaboration with academia and industry across a number of programs. We did not apply for that study.
And I think largely our biomarker driven development and the approach that we're taking may not easily fit into that structure of that trial at the current time.
Great. Okay. So for 343 Phase 1b, let's see. Looking down the line, where do you think is an appropriate place for 343 in the treatment paradigm of ALS and FTD? So, this is in the future here, Carol.
Yes. I think as always, we want to start with treatment of patients that have definitive diagnosis of the disease based on current clinical criteria for the clinical trial. But we would see this being used as early as possible in terms of the diagnostic paradigm and actually also could be used in combination with other therapeutic agents for ALS. So, I think just in terms of the ability to combine with other mechanisms, this may be very helpful. And so, we don't see this as being something that would be mutually exclusive of other therapies.
Okay. We're going to switch our attention to program for a minute here, and I'm sure we'll get back to EIF2B. So can you comment on the intra extracellular pro granular and its impact on disease? And I think I'll just add a second question to that, Joe, as you're preparing. Given the other pro granulant targeted agents in development, what do you see as the most critical points of differentiation for DNL
593? Yes, great question. And maybe to hit the first 1 in terms of intracellular versus extracellular there. Some of the best data to describe that in my view is in our published work, but I didn't get a chance to present today, is showing that the half life of our PTV pro granuline molecule in a mouse is on the order of hours, yet the pharmacodynamic response is on the order of weeks. And what this really suggests is that it's not the circulating progranuline that results in the biological activity, but it's those individual granulins present in the lysosome.
And so you can do this both in a mouse model or in a cell where you add pro granuline and you can get a lasting effect just by that lysosomal component. And I think that argues to us that the major function or the predominant function of pro granuline is actually impacting the lysosome rather than the extracellular pro granulant and interaction with cell surface receptors. And so I think that that's a core element of the answer there from a biology perspective. In terms of how we differentiate from our competitors, I think it comes down to the elements of the molecule itself. The first of which being effective CNS uptake in biodistribution.
I think that that's critical. It does seem like they're pro granulant the most of the pro granulant in the CNS is likely to be local, such that getting the pro granulant to the right place is priority number 1. 2nd is that we're really directly addressing what the genetics tells us about the biology through replacement of Progragnulin. Targeting other mechanisms that indirectly change pro granuline levels runs the risk that you're impacting elements biology that you are independent of pro granuline as well. And I think because we have confidence that we're not doing that with this molecule, we feel like it's the most direct way to bring ProGranulin to the right place in brain.
And in the long run, that's going to differentiate us in the clinical level as well.
Great. Thanks, Joe. I think related to that, can you comment on the cell biology data suggesting that sirtilin is not the key receptor for functional program and uptake into the lysosome? Do you find it robust? Have you been able to repeat these experiments?
Yes. It's a great question. And so, we have done some of that. I think the data is most consistent with sertilin is important for uptake of progranulin to the lysosome, but it is not the only way progranulin can get into the lysosome. I think there are other And so when thinking about modulating that path, And so when thinking about modulating that pathway, 1 has to think about really what's the impact on neurons and microglia and astrocytes and those key cell types within brain.
So I do think there's multiple mechanisms, but I do think sertilin is a major contributor. And actually preclinical and clinical data bear that out, where if you weren't blocking uptake of Progranulin through sortilin, I wouldn't expect to see that extracellular progranulin increase.
Okay. So we're down to 5 minutes. This is where we do the rapid fire and we have at least I mean, we have a bunch more questions. So let's see if we can rapid fire through some of these. So have you thought about developing vanishing white matter as a primary indication for EIF2b activator?
Yes. This is something that we're obviously very interested in. We've engaged with the vanishing white matter disease community as well around this and we're currently continuing to evaluate this.
Okay. So for DNL-three 43 and DNL-seven 80, they're expected to address an overlapping ALS population? Any differences between patient inclusion criteria for those enrolled in the DNLA Phase Ib study versus the SanofiDenali Phase II study?
Yes. So the Sanofi Denali study enrolls slightly less severe patients in the sense that they were diagnosed within 2 years as opposed to 3 years. I think just notably, the endpoint is very different where the Sanofi Denali Rip case study is really looking at a clinical endpoint. And that study, that fairly large Phase 2 study was designed also based on the fact that we have had additional clinical experience with that target across both AD and ALS in a Phase Ib study.
Do you have any data indicating the nuclear function of TDP-forty 3 is restored by treatment with DNL-three 43, Joe? We're just moving around now. We're just going forward.
No, that's good. We haven't looked at that directly. But I think as Gene mentioned, the 1 thing that we do see is that when we block the granule formation that includes TDP-forty 3, we restore normal TDP-forty 3 localization. So we haven't looked at downstream genes like staphmin, the ones that Gene mentioned yet. But based on that cell biology, 1 would predict normal function would be restored.
And I do think that those have a lot of potential as potential biomarkers down the line for measuring this pathway.
Okay. We have a goal to answer 8 questions in 2 minutes. Are there any biomarkers of ISR dysfunction that can distinguish between cells at acute stress versus chronic stress? This is a great, great question.
That's a really great question. And I'm supposed to be fast. So, what I'll say is we don't know yet. But what I will say is the signature of the ISR differs between cell types as well and between the types of stress. And so it's just going to take some effort to make sure we identify the right pattern there.
But I'm hopeful that we can find something.
Yes. This is a good question. I don't know if we can answer it, but our stress granules present in pre symptomatic ALSFTE patients? How closely does their appearance and or quantity track with onset of symptoms and disease progression? So gene, is there any evidence that stress granules appear in pre I don't know how 1 assesses that.
I would argue stress granules appear in most cell types, whether or not you have disease, right? Because they happen when you have a fever or a virus infection. So these are they should appear before. I don't think anyone's actually tracked in presymptomatic tissue or patients, whether they are appearing or disappearing. I mean, that's very hard to do.
Yes. But I think the point I think you're exactly right. The point is that it's the propensity of these stress granules to essentially become persistent, right, that is linked to disease. Okay. Will ISR restoration be sufficient to reverse ALS progression or should it be combined with other approaches?
Do ALS patients with different subtypes, genotypes have different degrees of dependency on the ISR? Can you talk about the expression of the IF2B in cells outside of the CNS? Any concerns about overactivation of the IF2B? I love like these are 4 questions all in 1. And maybe I can just comment.
We've already discussed overactivation that EI2V is expressed in all cells, right? And in healthy cells, it's essentially at normal healthy levels and we're restoring those broadly. For a reason, CNS cells are most susceptible to degeneration and probably because of their long lived cells. So I think the first question is really 1. So will ISR restoration be sufficient to reverse ALS progression or should it be combined with other approaches?
Who wants to speculate on that?
I think my view is that we want to start to see efficacy in this disease area. And then if we're in the luxury of being able to combine, there's no reason we shouldn't or wouldn't. I think we've seen an oncology combination therapy can be very efficacious.
Okay. So I think we're almost at time. I want to answer 1 more. So what is the baseline stage of ALS? You'll be enrolling the Phase Ib trial for EIF2b.
Line stage of ALS you'll be enrolling in the Phase 1b trial for EIF2b, Carol?
So those are patients that are within 3 years of diagnosis.
How do you think about timing of intervention with regard to disease severity? These are all linked together?
I think there I'll give the more generic example in neurodegeneration, but I think we want to go as early as possible, but also need to be able to recruit the trial and show generalizability of the findings that we see in a trial to a broader patient population.
We have a number of other questions, but definitely enjoyed the engagement from everyone. Fantastic questions. And we're very excited today to share some new data on our programs as our portfolio continues to advance. And with that, we thank everyone for joining.
Thank you.
Thank you very much. Thanks, Gene, for
joining us. Thank you.