Arrowhead Pharmaceuticals, Inc. (ARWR) Earnings Call Transcript & Summary

I think for the last couple of years have been tough for everybody and coming back face-to-face and talking about all this new and exciting stuff going on at Arrowhead is really, really great. So thank you for joining us. So safe harbor. So the obligatory slide, what we're going to be talking about today includes forward-looking statements. So please refer to all the risk factors in our SEC filings. And here are our panelists. We've got a great group to talk to you guys today about our pulmonary platform and the 2 new pulmonary products, MUC5AC and RAGE and then our newest announced program today against MMP7 for IPF. So the external panelists are Dr. Mario Castro and Dr. Matthias Salathe, both from the University of Kansas Medical Center, both are experts in the field of muco-obstructive and inflammatory pulmonary disease. So we're very lucky to have them today. From the company myself, Chris Anzalone, our CEO; Erik Bush, our Group Vice President of Biology; James Hamilton, our Senior Vice President of Discovery and Translational Medicine; Javier San Martin, our Chief Medical Officer; and Anjli Warner, our Senior Director of Commercial. So here's the flow for today. Here's what we're going to cover. Chris will go into an overview of the company and kind of where we've come over the last few years. Erik will talk about some nonclinical pharmacology for the new programs that we're bringing into the clinic. James will talk about learnings from the ENaC program and nonclinical toxicology results. And this is important. So keep an eye on this. This is what has enabled us to reemerge with the pulmonary platform very confidently. And then Dr. Castro and Salathe, they will talk about mucins as well as the RAGE target in obstructive and pulmonary and inflammatory pulmonary disease. Javier will talk about our general clinical development plan for MUC5AC and for RAGE. We'll start with the initial Phase I studies and then go on to how we -- how we're thinking about Phase II and beyond. And then on Anjli will talk about some of the market research we've done over time about pulmonary generally, about route of administration and about what's really needed. Where is the unmet need in these diseases. And then Chris will do some concluding remarks, and then we'll open up the -- open up to your questions for the whole panel. So hold your questions up until the end. And now I'll turn it over to Chris.

Thank you, Vince. Thank you all for coming today. It's great to see all of you. It's great to be out again. So I'm the least important speaker today, and so I will be brief. About 4.5 years ago, we had an R&D Day here in New York to introduce the TRiM platform. It was a different time in our -- in the history of this company. The prior year, we had discontinued the DPC platform. Our stock was trading for around $3 a share. And I showed the following slides on the Hubble telescope. So when Hubble was developed, when it was launched, they decided to see what it could do. And to do that, they looked for some corner in the sky, they didn't seem to have anything going on. And so they found this little area shown here in the -- with the red dots right into the Big Dipper that seemed to be relatively devoid of objects. So as they looked closer, they found this little area here that was -- that looked truly devoid of objects. As they continue to look close, they were sure enough, it was empty. So perfect. Let's aim Hubble there and see what we see. And what they saw was not a void but tons of objects. And it's a mind-blowing concept, right? But they found this tiny little corner in the sky that appeared to be empty and it turns out it was not empty. And the point is, they had developed a tool to see what others could not. And so now NASA was able to see what others couldn't. And that was an important concept to us and really resonated with us. And so 4.5 years ago when we talked about the TRiM platform, what we were really talking about was developing a set of tools that allowed us to see what others could not see. These tools then enabled us to develop the TRiM platform, they enabled us to design, I think, the most potent triggers on the planet. They enabled us to make important new lung-directed drug candidates. And it held out the canalizing possibility that we could bring RNAi outside the liver. And so today's presentations are really an outgrowth of that concept of 4.5 years ago. We are expanding RNAi outside the liver. We are going to where diseases are rather than just going to the liver. It's also -- the presentations today are also an expression of our commitment to continuous innovation. We learned a ton from the -- from our first clinical pulmonary program, ARO-ENaC and we use those lessons to inform what we're doing now in the platform. And in fact, in less than a year since we stopped that program, we'll be treating human subjects in these next 2 programs. So today, I hope you walk away with a few thoughts. First, that the targets that we're addressing are important and potentially powerful. Second, that there is substantial unmet medical need that we'll be addressing. Third, that our nonclinical data suggests we've got a pretty good shot at success. Fourth, our clinical plan is appropriate and achievable. And fifth, that we're just getting started. This is a broad platform we see -- we can see within this platform, not just 2 or 3 drugs, but 8 or 9 or 10 drugs. And so we are looking forward to expand this platform to bring it to where patients really need it. So with that, I will hand it over to Erik Bush.

Thanks, Chris. Do I have a pointer here? Or will I just have to gesture. Is there? Okay. Good. Make sure we have all that. Okay. Good morning, everyone. So a brief reintroduction to the TRiM delivery platform for pulmonary delivery of therapeutic siRNA triggers. At its core, the platform is comprised of a therapeutic siRNA or as we refer to it, an RNA interference trigger. Critical to the success of these triggers is an algorithmic approach that selects the most potent and specific triggers and avoids any off-target. Our approach is unchanged to what we've used in the past. But critically, we're now beginning to employ some additional modification chemistries that improve the duration and potency of our conjugates and this is what will be highlighted in our ARO-RAGE program. And then finally, the third critical element of the pulmonary delivery platform is a targeting ligand to the alpha V beta 6 integrin, a small molecule that facilitates internalization by epithelial cells after inhalation. One brief comment on the inhaled platform and the environment of mucus, we just want to highlight that the physical or chemical properties of the TRiM conjugates for inhalation are highly compatible with mucus transit. Critically, they are of a very small size, 3 to 10 nanometers in size, much smaller in order of magnitude or more smaller than the mesh size of mucus, which is 100 to 200 nanometers in size. By comparison, respiratory viruses or lipid nanoparticle formulations for mRNA delivery are much larger on the order of 100 nanometers. The TRiM conjugates have a negative charge, which is highly favorable in terms of anionic compounds, are minimized with respect to electrostatic interactions with mucus and they're soluble. We have, in addition, multiple lines of evidence showing efficient delivery through the mucus in vitro through mucus layers as well as in models of airway mucus hypersecretion delivered drug having a robust pharmacodynamic response. So with that, I'll proceed to our first program we'll be talking about today, which is targeting the receptor for advanced glycation end-products. Dr. Salathe will be giving us a much deeper dive into this target later this morning, but I just want to briefly introduce this receptor. This is a pro-inflammatory pattern recognition receptor that's highly abundant in the lung epithelium, expressed at very low levels outside the lung. And it's important in the lung because it has a very large repertoire of pro-inflammatory ligands that it's able to interact with, and we'll be hearing about that. But critically, signaling through this pro-inflammatory receptor culminates in canonical signaling through NF-kB and other pathways that produce cytokines, reactive oxygen species, mucin production as well as reinforcing expression of the RAGE receptor itself, which amplifies and perpetuates chronic inflammation in the lung in combination with other canonical signaling pathways and again you'll hear more about it. But what has made this an exciting target for respiratory inflammatory disease is a knockout phenotype of the mice. These mice offer nearly complete physiological and histological protection from various allergic asthma stimuli. But traditionally, this target, despite its great interest as an inflammatory target, has been quite challenging to drug with traditional small molecule approaches. And this is largely due to its structure. Its structure is not a canonical G protein-coupled receptor with a discrete binding pocket for a subset of ligands, it is more the structure is an antibody. It's an immunoglobulin structure, which allows many interaction moieties with a wide variety of ligands thus making it difficult to block with a discrete agent and making it ideal for a therapy like RNA interference. Finally, this target is quite interesting because it's abundantly expressed in the lung, and it also is shed into circulation in a form called soluble RAGE, which can be readily detected in serum from treated animals or patients, and it allows a circulating biomarker of target engagement for delivered lung inhibitors. So let's move directly into rodent pharmacodynamic data with a RAGE silencing trigger conjugate. These rats received a single inhaled dose of a RAGE conjugate at day 0. And we're at 0.5 mg per kg delivered dose in the lung. So just a single dose, and we're following these animals out for 2 months post dose. In blue, we're tracking whole lung expression of the RAGE receptor on mRNA. And what you can see is that by day 3, post inhalation, greater than 90% of the whole lung mRNA for RAGE is fully depleted, and it remains in that range for at least 60 days after dosing. We'll see more detailed recovery kinetics in a moment. Importantly, serum RAGE also falls. It takes time to deplete the protein pool from the lung. But by a month post dose, serum RAGE also reaches undetectable levels, showing that in the rats, the vast majority of soluble RAGE that can be detected originates in the lung. And this is confirmed by immunohistochemistry looking at RAGE protein expression in the lung at day 36 post dose in these rats. You can see that this pro-inflammatory receptor has been completely removed from lungs of animals dosed with this RAGE conjugate. So moving on from there, we were interrogating proinflammatory effects through RAGE signaling and seeking to phenocopy what's been observed in the knockout mice. We do this in rats with an allergic asthma model. These are rates that are sensitized to a fungal allergen Alternaria alternata and can be delivered to rats to provoke an allergic asthma response, inflammation and other sequelae. So what we see here is that rats received this Alternaria challenge, they get a robust inflammatory response. We can do alveolar lavage in those animals, retrieve samples and count inflammatory cells and measure cytokines. So what we observe is that as expected with this fungal allergen challenge, we get a robust eosinophilia. We see neutrophils as expected. But in animals that received that single dose of RAGE silencing conjugate, and we've depleted this inflammatory receptor, we see very robust reductions in eosinophils in lavage and neutrophils and reductions through a wide panel of pro-inflammatory cytokines that can be detected in the lavage as IL-13, MIP-1alpha and IP-10. So again, we have clearly phenocopied the mouse knockout phenotype now in a rat model, but using an inhaled RNAi conjugate. So on the basis of this work, we've moved forward with ARO-RAGE, our clinical candidate that we intend to take forward. This was a study done with a single inhaled dose in cynomolgus monkeys at a deposited dose of 1 milligram per kilogram. And what this slide simply shows is that after inhalation, we look at multiple regions of the lung, left lung, right lung, proximal, medial, distal, all throughout the lung, we see very robust greater than 90% silencing throughout the lung. Next slide. I guess I had this slide. So from there, we moved on to dose response studies in cynomolgus monkey. These monkeys received a single inhaled dose of ARO-RAGE ranging from 0.13 to 0.47 mg per kg deposited dose. We take the animals down a full 4 weeks post dose, where, again, we expect a depletion of the protein. And we see, indeed, here by Western analysis greater than or equal to 90% depletion of lung protein in cynomolgus monkeys at doses of 0.3 deposited dose or higher. So from there, we -- given the potency of ARO-RAGE and these RAGE conjugates, we wanted to explore alternative routes. ARO-RAGE is intended for inhalation. But we were curious to -- our GalNAc conjugates are so effective subcu for delivery to the liver, we wanted to explore the effect of these conjugates towards the lung. So indeed, we can see efficient delivery to the lung at higher exposures when given subcu. So in the lower left-hand panel, these are rats that received a dose response, a single dose of RAGE targeted conjugates. And what we observed at a week after dosing, roughly 70% to 80% whole lung knockdown of RAGE can be achieved via a subcutaneous route of these RAGE targeted conjugates. And importantly, this knockdown was entirely dependent upon the epithelial-targeting ligand. Without the targeting ligand, there was no delivery and knockdown of RAGE in the lung. So a little bit higher dose, of course, to achieve knockdown. We see about 70% to 80% knockdown at the 15 mg per kg dose level, what we recognized was that with repeat weekly doses, in this case, 3 weekly doses at 15-milligram per kilogram, we could start to build even deeper knockdown over time. So what this slide shows is the pharmacodynamics of silencing and recovery for inhalation and comparing that to what we see with subcu. So again, this is rats on the left, having a single inhaled dose of our RAGE conjugate here on day 1. As I showed you before, now we're monitoring serum sRAGE as a marker of target engagement in the lung. We have full depletion by a month post dose. It stays near the limit of detection for about 2 months post dose. And then the next 3 to -- 2 to 3 months or so, we have slow recovery. So this knockdown and Recovery Kinetic are roughly analogous to many of the GalNAc conjugates for liver targets. Now by subcu routes, we again have to give a bit higher doses. But in the upper right-hand panel, where we're looking at a weekly dose strategy where we observed that at 15 and 10 mgs per kg doses, not weekly, every other week, we could slowly build and achieve very deep lung silencing of our RAGE target that was maintained. Lower doses at 5 and 2.5 mg per kg were below the threshold to achieve the very deepest knockdown. But those 15 and 10 mg per kg dose levels could also achieve deep lung silencing of RAGE given monthly, but it took a longer period of time, perhaps up to 3 months to achieve that knockdown via a subcu route. So just demonstrating the possibility of subcutaneous. So one final slide before we leave RAGE, just to highlight the improved pharmacodynamic response of this next-generation RAGE conjugate versus our first-generation ARO-ENaC targeting conjugate that was designed to silence the epithelial sodium channel. Here, we're looking at rat ENaC mRNA expression following doses of ARO-ENaC on days 1 and 2, it's 0.7 mg per kg. We see a nadir of roughly 60% to 70% whole lung silencing of ENaC mRNA by 2 weeks and then a slow recovery over the next couple of weeks. For ARO-RAGE, the differences are quite striking. Again, deeper knockdown at a lower exposure with a slower recovery in connecting. So from there, I'd like to move quickly on to the next program we'll be hearing about today, which is a therapeutic strategy to silence a pathologic mucin MUC5AC for severe asthma. So MUC5AC is 1 of 2 genes that encode gel-forming mucins that are secreted by secretary epithelial cells in the airway. And these 2 gel-forming mucins together mix and form the mucus layer that rides on top of the airway surface liquid in the lung that is moved by cilia to create efficient mucociliary clearance. Now what's important about these 2 isoforms is that they're quite different. MUC5B is the predominant form of mucin expressed in lungs. It comprises about 90% of mucin at baseline. It's constitutively expressed and required for normal lung clearance and knockout animals, it's lethal. They do not have efficient mucociliary clearance. The form we're talking about today is MUC5AC. It's a minor component at baseline, comprising about 10% of the mucin protein in the gel. Knockout of this mucin as we'll hear about is completely normal, but is highly induced in settings of inflammation, like asthma, allergic asthma, COPD. Here, you can see this -- the mucin stain in inflammatory asthma models. And what that means is that mucin hypersecretion and particularly MUC5AC upregulation disproportionately contributes to the pathology of muco-obstructive lung diseases like severe asthma. So what we are proposing here is the very first therapeutic approach to directly and specifically silencing the -- silence expression of this pathologic mucin gene. So there's a great deal that's been written on the role of mucus in asthma, and we're very fortunate to have Dr. Castro here today to tell us more about it. And I will not sort of preempt much of his discussion, but the core themes here are that the disproportionate upregulation of this MUC5AC mucin creates a highly sticky mucus that's hard to clear by airways and leads to mucus plugging and airway occlusion driving airway dysfunction in these diseases. Touch briefly on the knockout mouse phenotype. Again, Mario will walk us through this data again, but it was a key insight into this specific mucins contribution to asthma. So these knockout mice were found to be completely protected from airway hyper-responsiveness in late-phase allergic airway response. So this is what happens with an allergic reaction in the hours or even days after an allergy and allergic asthma attack. And these knockout mice instead of having these muco-obstructed airways with high amounts of MUC5AC, there was no airway obstruction. And when these mice were challenged with a bronchoconstrictor to further provoke airway dysfunction, they were completely protected. So it shows that the combination of MUC5AC plus bronchoconstriction were a key driver of late-phase airway dysfunction in the mouse. So that's critical because we'll show you a little bit more data here on our program. So when we started on this journey for MUC5AC, we started with our own allergic mouse models. These are mice that are sensitized to various allergic asthma allergens, such as house dust mite allergen or IL-13. And at baseline, these mice express almost undetectable levels of MUC5AC mRNA. And then when they're subjected to this allergic stimulus, there's a very strong induction of MUC5AC mRNA. And if animals have been pretreated in the prior weeks with a trigger silencing MUC5AC expression we can block, to a very significant extent, much of that induced MUC5AC expression in the range of 70% to 90%. This is both in mice as well as cynomolgus monkeys. So what really comes through is if you look at the protein level by immunohistochemistry here, we're staining for MUC5AC protein in red. In normal airways, these are the airway epithelial cells secreting the mucins clearly, with an allergen, you can see how much of this mucin is being produced and stored in secretary granules and airways with the trigger on board, we're silencing much of that upregulated mucin expression. So where it really matters is in animal models of airway dysfunction. So what I'm showing you here is data in a sheep model of allergic asthma with ARO-MUC5AC, our clinical candidate that you'll be hearing more about today. So this large animal model, the sheep model is a standard model for the study of asthma drugs. These sheep are sensitized that are allergic to a nematode allergen, ascaris. And if the sheep get inhaled ascaris antigen, they have a classical asthma attack, an allergic asthma attack, which is comprised of bronchoconstriction, they have inflammation in an immune cell recruitment, there's robust secretion of mucus and airway occlusion, airway dysfunction, and these animals respond to standard of care therapy. So they highlight many of the key aspects that are seen with asthma patients. So what I'm showing you here is the late phase response of sheep in the 4- to 8-hour period after this allergic asthma attack. So first, focusing on the black line here, we're observing lung resistance. So this is lung mechanics of sheep, conscious sheep. And we're studying how occluded their airways are, how much lung resistance there is during this period. And what you're seeing here is a classic late phase response is that after this allergen challenge in the 4- to 8-hour period, you have an increase in lung resistance as inflammation and mucus come in, and you have this essentially doubling of lung resistance during that period. And what we observed in animals that received ARO-MUC5AC in the weeks prior to this ascaris challenge is a dose-dependent reduction in late phase response, read out by lung resistance. And then critically, we also studied these animals at 24 hours post challenge. Now at 24 hours post allergen challenge, lung mechanics have fully returned to baseline. These animals are breathing normally; for all intents and purposes, their lung mechanics seem to be normal. But you can superimpose a stress on those animals by giving them an inhaled bronchoconstrictor to tighten up their airways. So in untreated animals, we measure this by giving this inhaled bronchoconstrictor, carbachol, and measuring lung resistance again and seeing how many breaths of that bronchoconstrictor those animals need to take to get a fivefold increase in lung resistance. So a normal sheep that -- or in sensitized sheep that have not received that allergen challenge, it takes them about 22 breath units to get that airway resistance, okay? In the 24 hours after this ascaris challenge, you do the same bronchoconstrictor challenge, it only takes them breaths to get this airway resistance. So this is the classical definition of airway hyper-responsiveness. And what we saw at all dose levels with ARO-MUC5AC treated sheep is that there was no evidence of airway hyper-responsiveness in the 24 hours post dose period. So this is a clear phenocopying of the mouse data that I showed you previously with the MUC5AC knockouts. So this is strong evidence that MUC5AC expression is playing a critical role in airway hyper-responsiveness in the sheep model. We also did additional controls looking at, for instance, a version of MUC5AC, where we chemically modified the sequence to prevent its loading into the risk complex and gene silencing, and that was entirely inactive in this model and looked just like untreated. So before we leave MUC5AC, I just want to highlight the fact that beyond asthma, mucus hypersecretion underlies a wide variety of muco-obstructive lung diseases, such as COPD, non-CF bronchiectasis, cystic fibrosis. When we examined mucin concentrations in all these patient, sample of these patient groups, we see a disproportionate increase in MUC5AC highlighting its critical role in driving the central pathophysiology of all these muco-obstructive diseases. Now moving on quickly to our new program in idiopathic pulmonary fibrosis. This is the first time we've spoken about this program. So this program is designed to silence a secreted endopeptidase, matrix metalloproteinase 7 in idiopathic pulmonary fibrosis patients. Now Matrix metalloproteinase 7 and its relationship to IPF has been well established. It is one of a very large diverse gene family of proteases with many different functions. But critically, it is the most or one of the most upregulated genes in patients that have idiopathic pulmonary fibrosis. It is a validated or perhaps one of the best or the best biomarker for disease severity and progression in idiopathic pulmonary fibrosis. And critically, it's been understood for quite some time that it plays multiple roles in IPF pathogenesis as well. It's a therapeutic target. It promotes inflammation, aberrant epithelial cell repair, fibrosis; we'll see some of these data shortly. And it's been known for some time that knockout mice for MMP7 are robustly protected from bleomycin injury, which is a model of IPF preclinically. Again, why aren't there drugs to MMP7? Well, largely because it's hard to make isoform-specific MMP7 inhibitors. This class of enzymes are highly related in their structures of the catalytic domain and specific inhibitors to MMP7 alone simply don't exist. So moving forward into some of the pharmacology with silencing MMP7, we use a rat model of idiopathic pulmonary fibrosis. It's a standard model of bleomycin injury, which causes inflammation and fibrosis. In this model, we give -- how do I go back? In this model, we give a single inhaled dose here at 1.4 mg per kg of MMP7 silencing trigger. We wait 2 weeks and then give a bleomycin injury, in this case 2 doses of bleomycin separated by a week. After this bleomycin injury, there's a phase of robust inflammation that transitions to fibrosis. In IPF patients, this persists, of course, but in the bleomycin model, it spontaneously begins to resolve, and we'll see that in the data. But what's critical here is that with bleomycin injury, there's an induction of MMP7 expression here at a week after the last bleomycin dose on day 28. And by day 42, it's beginning to resolve a little bit in this dynamic model. But what's important is that dosing of the MMP7 trigger on day 1 is leading to significant suppression of that induced MMP7 expression at both day 28 and day 42 shown here. So what does this mean functionally for fibrosis and function? So the most important element to study in the preclinical models of IPF is evidence -- histopathological evidence of fibrosis. And this is done by assigning what's called the Ashcroft pulmonary fibrosis score. I'm showing you hear images of lung parenchyma from control animals, bleomycin injured with these fibrotic lesions and then as apparent, having the MMP7 trigger silencing in these animals, there's significantly less fibrosis. This was sent out to an external CRO that scores these slides blindly for us. You can see here this Ashcroft score assigned from 0 to 5 with bleomycin injury and no treatment, most of the animals score as severe fibrosis with MMP7 silencing on board, most of the animals here at day 28 are in the moderate-to-mild category. Furthermore, one of the key pathologic drivers of MMP7 in IPF is thought to be through promotion of inflammation. And critically, what we observe in this model, bleomycin injury, robust inflammation, and this can be read out by doing bronchial-alveolar lavage and inflammatory cell counts. Like with our asthma models, we see a very profound neutrophilia and eosinophilia in these samples. And with the MMP silencing trigger, very significant reductions in both neutrophils and eosinophils showing an anti-inflammatory effect with MMP7 silencing. I will have no time to show you the additional data here, but this was correlated with robust protection of lung function as well as increased animal survival in this model. So on the basis of this work, we have identified a potential clinical candidate, ARO-MMP7 for idiopathic pulmonary fibrosis. We've moved this into studies in cynomolgus monkeys. We're showing you here a terminal study with a single inhaled dose of ARO-MMP7 at 4 different dose levels and here at weeks post dose, we observed again, silencing of whole lung MMP7 in cynomolgus monkeys at doses at 0.6 mg per kg or higher for single dose. And we have confirmed MMP7 protein reductions in BAL samples from these cynomolgus monkeys, which we intend to employ as a target engagement marker in future clinical studies. And then finally, we've begun to employ a new model in-house of culture to human precision-cut lung slices, which allow us to take explanted human tissue, incubate them with our TRiM conjugates and measure target engagement gene knockdown. In these human lung slices, we see robust silencing of MMP7 with our trigger conjugates. And we have established that this is conclusively risk-mediated and internalization receptor dependent because using a negative control ligand, we see very little evidence of knockdown. And if we modify that trigger to block risk loading, there's no gene silencing. So a much larger story around MMP7 will be presented at the upcoming European Respiratory Society Meeting in September. So before we leave the nonclinical section, I just want to touch very briefly on respiratory virus discovery efforts at Arrowhead. Our HBV programs have taught us that the TRiM platform offers a very powerful approach to directly silence viral gene expression, opening the door to the possibility of programmable antiviral therapeutics for both existing viral disease and emergent diseases. We are currently working on a candidate for SARS-CoV-2 ARO-COV, lead optimization continues for that candidate, and it is promising. And behind ARO-COV, we are rapidly developing a pipeline of additional therapeutics for other respiratory viruses that we will provide updates in the future. So my last slide on preclinical pharmacology. I just want to highlight 3 key issues here. ARO-MUC5AC and ARO-RAGE are candidates for muco-obstructive and inflammatory lung diseases. Our new platform designs offer improved potency, duration as well as potential for subcutaneous delivery. And we're expanding our therapeutic area -- therapeutic area opportunities in respiratory into pulmonary fibrosis with ARO-MMP7 and respiratory virus with ARO-COV. And with that, I will turn it over to James Hamilton. Thank you.

Thank you, Erik. So I'll cover our learnings from the ENaC clinical trial and the nonclinical tox studies, and we'll discuss why we think what we've learned from ARO-ENaC will enhance our probability of success as we progress these new programs, ARO-RAGE, MUC5AC and MMP7 in the chronic tox and forward. So first and foremost, I'd like to state that in the ARO-ENaC clinical study, there was no safety signal identified. And just as a reminder, this study was a Phase I/IIa clinical trial. We enrolled 24 healthy volunteer subjects who received drug on days 1, 2, 3, so a 3-day cycle. And then we additionally enrolled 4 CF patients who receive drug on day 1, 2, 3 and then again on days 22, 23 and 24. So they received two 3-day cycles. Importantly, ARO-ENaC showed no evidence of adverse effects on lung function as measured by spirometry, no adverse changes in oxygen saturation or chest X-rays and no predominance of AEs or SAEs in the treatment arm. Adverse local lung effects were seen in a chronic 6-month rat and a 9-month NHP GLP toxicology package and to review how we dose these animals, the monkeys received drug on days 1, 2, 3 every 2 weeks, so similar to how drug was administered in the clinic, but more frequently. And then the rats received drug day 1, 2, 3, every two weeks in 3 of the dose groups. So there was a low, mid and high dose group that received drug on day 1, 2, 3. And then there was a fourth group that received drug only on day 1 every two weeks. And this was actually our lowest exposure group. And at this level, we were able to achieve a NOAEL or a no-adverse-effect level. However, due to the histopathologic changes noted in the chronic rat study, the ARO-ENaC clinical trial was placed on a voluntary clinical hold. As you might expect, we've spent quite a bit of time thinking about and investigating the underlying mechanism of the tox findings in the rat and the monkey studies. And it appears that the underlying culprit is a process described in the literature as lung macrophage overload. This is a condition essentially of impaired clearance where the macrophage mediated clearance mechanism of inhaled particles in the lung following prolonged exposure to otherwise nontoxic particles overloads the macrophages and it induces recruitment of other inflammatory cells that can eventually lead to lung parenchymal injury. Importantly, these inflammatory effects are due to a general particle response. This is not something that's specific to a sequence or even specific to siRNA or oligos, this is found with other drug classes with particles used in dry powder inhalers and other inhaled particulates. And importantly, there are thresholds that are described in the literature for where adverse effects are seen. The histologic findings from the chronic rat and monkey studies are consistent with the literature description of macrophage overload. And also importantly, our lung histopathologists, one of the leading KOLs in the field, reviewed these slides and took a look and said that these are consistent with classic macrophage overload. So here on the left in the green panel, I described the classic literature findings associated with macrophage overload. And then on the right 2 panels, described the findings from both the rat and the monkey studies. We did see enhanced transfer of particles to lymph nodes in both species. We also saw increase in lung weight in both species. We saw evidence of alveolar macrophage accumulation in both species, which in itself is not adverse. We did not see neutrophilic cellular infiltrates, which would have been considered adverse. We saw evidence of alveolar epithelial hyperplasia but no metaplasia. And then if you really push the dose at the highest dose levels in some of the rats, there was evidence of fibrosis but not in the monkeys. Interestingly, this was not called adverse because it occurred at a rate that was only slightly different from the background rate in the animals. Importantly, we did not see any evidence of preneoplastic lesions in either species. So here are the thresholds I was describing, and this is from the literature. This plot shows lung tissue concentrations of inhaled particles, this could be inhaled drug. Based on the literature, the upper threshold of 1 milligram per gram of lung tissue, this threshold is associated with adverse findings. So if you push the dose or the dose frequency and your lung tissue concentrations are above this, you're likely to see adverse effects on histopathology. If you can stay below or close to the 0.1 milligram per gram of lung tissue concentration, this is the region where the histopath is typically not adverse, things like increased alveolar macrophages just evidence of normal clearance mechanisms. And then in between, as you'd expect, this is a variable region where if you push the dose and get closer to the higher threshold, you're more likely to see adverse findings if you can stay closer to the 0.1 milligram per gram threshold, less likely to see adversity. And so what did we see with ENaC? These are lung tissue concentrations from the rat 6-month chronic tox study. Again, we dosed in 4 different groups. The first 3 groups across the bottom are the day 1, 2, 3 cohorts, both the low, mid and the high-dose groups. And then on the far side is the mid dose -- same dose level administered only every 2 weeks. So no day 1, 2, 3 cycle, just day 1, every 2 weeks. There was no NOAEL seen in any of the day 1, 2, 3 cycle groups, but we were able to achieve NOAEL when we drop the dose frequency from day 1 to 3 all the way down to day 1 every 2 weeks. So the take-home message here is essentially a less frequent dose administration is critical for avoiding adverse effects and keeping those long tissue concentrations low, this can be achieved by avoiding the daily dosing and spacing out the doses. And as Erik showed with the data from MUC5AC and from RAGE, we think we can space the dosing out even wider to either every 28 days or even every 3 months which should further allow us to avoid adverse findings on histopath. This slide shows the cumulative dose levels over the course of a 6-month rat study. Again, the blue dots represent -- this is data from ENaC here on the left, the 6-month ENaC rat study. The blue dots show the cumulative dose level where there was no adverse effect. And then anything at or above this green line that represents the threshold for adverse findings on histopath, and this is at about 100 milligrams per kilogram; any cumulative dose levels at or above this in a 6-month rat study demonstrated adverse effects. And so if you extrapolate that to what we are expecting to administer in the MUC5AC and the RAGE chronic tox studies, even at the highest expected exposure, the cumulative dose in a 6-month rat study stays well below this threshold for adversity. Here are the top line results from the ARO-MUC5AC in ARO-RAGE, Phase I enabling tox study. Importantly, the take-home here is that no adverse clinical or histopathologic findings were seen at any dose level and the top dose was the NOAEL in both studies. On this slide, this is more of a pharmacology study. This was not a tox study, but the intent of the study was to show that the knockdown of RAGE for a prolonged period of time could be well tolerated in the rat. So this is with a rat surrogate trigger administered via inhalation 0.5 milligrams per kilogram. So a pharmacologically relevant dose, not a tox dose. The first thing of note is that we are achieving great knockdown with great duration administering drug every 70 days and this goes out through about 1 year. We're able to maintain knockdown at better than 90% in sRAGE, so serum measurement of sRAGE. And then on the right, you can see we did a takedown at 6 months, day 170. We're getting great knockdown in mRNA, tissue-level mRNA. Importantly, over the course of this study, which is still ongoing, the treatment was well tolerated in these animals. There were no significant adverse changes in labs, including hematology or chemistry. And then importantly, there was no significant adverse changes on histopath at the day 170 takedown and no evidence of macrophage overload. So in summary, we believe the ARO-ENaC experience has helped us to derisk the future chronic toxicology studies for programs such as ARO-RAGE, MMP7 and MUC5AC. We believe this because we have new clarity around the mechanism of toxicity and how that mechanism correlates with the exposure levels, we have a better understanding of the exposure levels where we are likely to see adverse findings. We also believe that we have better triggers, both ARO-RAGE and ARO-MUC5AC acute tox studies compared very favorably with both GLP and non-GLP acute tox work we've done in other programs, and we think the better depth of knockdown and the duration of knockdown that we can achieve with these newer triggers will allow for overall lower exposure, and this can facilitate spacing out the doses in tox studies, but also in the clinic. And then lastly, but very importantly, we have available biomarkers for all 3 of these programs that can be measured in the sputum or in BAL fluid or even in the blood, and this will allow us to better understand dose response and set dose levels and dose intervals in the clinic, but also in chronic tox studies. And this was not something that was available with ARO-ENaC. So next, I'd like to hand over the discussion to Dr. Mario Castro. He'll be describing mucins in obstructive lung disease.

Thanks, James. So now we move on to the clinical part of the discussion this morning, and I will be talking about the role of mucins in obstructive lung disease, really not talking about any of the drug issues just really setting the stage for the opportunity that is available here in our patients. Just to give you a little bit of background, I'm from Kansas City at the University of Kansas there. This is a shot from our Plaza, which is in the middle of Kansas City. We're known for our fountains. We have more fountains than any other city in the U.S. and second in the world only to Rome. And these are my disclosures. So in regard to asthma, it's one of the most common chronic diseases that we encounter and over 25 million Americans are impacted by this. And this results in substantial healthcare utilization in terms of exacerbations that these patients are experiencing, leading to emergency room visits and hospitalizations. And unfortunately, even though this is a completely preventable disease, there still remain over 3,000 deaths per year. So what is the unmet need in asthma? So despite our work in this field and [ really came up ] with great therapies for our patients, we still are not achieving asthma control and about 50% to 60% of patients when we've looked at this in longitudinal epidemiological studies, this results in a substantial number of hospitalizations, as noted, significant morbidity and mortality and a high economic burden and estimated over $25 billion or -- $25 million per year. There's a subset of these patients that really drive the healthcare cost, and this represents somewhere up to 10% of our patients with asthma that failed to respond to the conventional therapy. And these are patients that are using the standard of care therapy, but they're really not responding to that therapy. And so I'll subsequently share some light into why we think they are not responding to that standard care therapy. But first, I'd like to talk about where we've evolved in terms of our understanding in regard to how do we approach asthma. It's such a common chronic disease, it can be misdiagnosed in a substantial proportion of patients, and therefore, we need better tools to identify these patients and segregate them into various categories. So we have grouped patients based on clinical characteristics. But what we need to know is not one drug or one therapeutic approach is going to fit all of our patients with asthma. And therefore, where we've evolved to is really understanding what are these clinical characteristics that segregate the various patients of asthma and I wrote an editorial, we call this the many buckets of asthma because there are certainly -- it's not one disease. And because of this phenotypic clustering that we've done in cluster analysis and unsupervised learning analysis in cohorts, we've come up with understanding then perhaps what is the underlying pathobiology that's driving those clinical characteristics. And therefore, we could then go to a much more precise kind of precision medicine approach in regard to asthma. So when we look at these many buckets of asthma, we have grouped them into 2 large categories, T2-high asthma and then the other bucket is basically everything that is non-T2 disease. And what do we mean by T2 and non-T2 is -- for those that are not familiar with the immunology, it's based on that the immune system the T helper system drives a lot of the production of cytokines in response to allergens and other stimulants in the airway. And we now understand that it's not just T helper cells but also other lymphocytes that are important in this immunologic response. So we have broadened that category to T2 high asthma. We have made a lot of progress in the left-hand side of this in terms of early-onset disease and as demonstrated here in childhood onset asthma tends to be highly allergic type of patients and then they develop eosinophilic asthma in this particular subcategory. However, the challenge right now is to the right hand of this. We have a substantial proportion of patients that have T2-low asthma that we really don't have good therapy for these various groups of patients demonstrated here. And these patients are taking the standard of care and the medicines are not working for them. So how can we tease this out and dig a little bit deeper into the pathobiology of this. And certainly, our understanding for this has evolved over the last couple of decades. Classically, in the past, we just focused on the role of allergens in regard to asthma. But now we understand there are certainly a number of other stimuli to the airway epithelium, including pollutants and microbes that stimulate the immune response. So in the classic allergen-driven model, you can see these allergens encounter antigen presenting cells, such as Dendritic cells, trigger downstream production of immunoglobulins that are important in terms of IgE production and activation of Mast cells and other cells. This has been targeted with an agent called omalizumab, a biologic agent that has now been in our armamentarium for the last almost 20 years. So we have a lot of experience in that regard. Now when we look at the other side of this, these T2 cytokines in high T2 asthma, they can produce IL-4, IL-5 and IL-13. These are what we call the T2 cytokines. These are the signature that we use to identify T2 asthma. IL-5 is one of the important T2 cytokines because it attracts eosinophils into the airway and it lets them survive longer and unfortunately, do the damage they do to the airway. Fortunately, now we have 3 drugs that block eosinophil infiltration into the airways by blocking IL-5, benralizumab, and reslizumab and dupilumab here is a blocker IL-4 and IL-13. The other one that's not listed here is mepolizumab, which is IL-5 blocker. And so this dupilumab is the most -- one of the more recent entries into the market, it blocks both IL-4 and IL-13 by blocking the IL-4 alpha receptor. The latest entry into the biologic world for asthma is tezepelumab. And tezepelumab has a unique mechanism by blocking upstream this TSLP, which is one of what we call the alarmins. So you can imagine when you're inhaling either an allergen or some other insult to your airway, the airway epithelium is the kind of first signal to the body immunologically and releases these alarmins IL-25, IL-33 and TSLP. But what you can see here is in terms of targetable things, there are a number of other non-allergic non-eosinophilic pathobiology that we haven't really tackled yet in regard to asthma. So we believe that this represents 40% to 50% of patients that really don't have any of the type 2 biomarkers that we use in the management of our patients with asthma. We typically do a blood count and look at the differential. We look for elevated eosinophils in the blood, we do IgE levels and we look at exhaled nitric oxide. So those are the 3 biomarkers that we have available in the clinic to identify this type 2 asthma. However, a lot of my patients that I see on a regular basis really don't have these markers of type 2 inflammation. And these are what we currently have in the clinic, tezepelumab might work in the T2-low asthma because of that proximal upstream effect that has in their epithelium. Recent data just released this last year suggest that there's about a 30%, 40% reduction in exacerbations; macrolide antibiotics and bronchial thermoplasty have also been used in relatively small subset of patients with mixed effects. And so the question is here, could we develop new therapies that would tackle this subset of patients with asthma? Now the other obstructive lung disease, which represents a fair amount of my practice is COPD or chronic obstructive pulmonary disease. It is a major public health problem and a leading cause of disability. It represents, over 20 million Americans in the U.S. have COPD that is identified. There is a prevalence around 6%. And it's the third leading cause of death and now surpassed stroke as a cause of death. Second leading cause of disability and third leading cause of death worldwide. So this does represent a substantial impact in terms of morbidity and mortality worldwide. Another thing I often say to my medical students is that all of our COPD patients aren't the typical older -- elder patients. In fact, the majority of our patients is you can see here, 70% are less than age 65. Now certainly, when we look at therapies for COPD, we often follow what's called the gold guidelines or global obstructive lung disease guidelines. These are international guidelines we have for the treatment of COPD. And we categorize our patients into these 4 different categories: A, B, C and D, based on their exacerbations and based on their symptomatology. But as you can tell from this, the agents that we have are all, mostly inhaled therapies, bronchodilator, a long-acting muscarinic agent or long-acting bronchodilator are a combination of these and sometimes we throw in inhaled steroid. These are really very limited. They have very limited impact on airway inflammation in these patients and really are just helping us symptomatically and do not reverse the progress -- the disease progression that occurs in these patients. So certainly, there's opportunity for improvement in terms of treatment for both of these diseases, asthma and COPD. And we believe that mucus-directed therapies represent really an opportunity to advance our therapies for the treatment of these diseases. We know that biologics do not resolve the disease process. When I stopped the biologic, in about 12 weeks those eosinophils come back and the disease recurs in these patients. We, as I mentioned, really have ineffective approaches to COPD. So one of our thoughts is, one of these targets in that non-T2 population is really addressing the role of mucus hypersecretion. And we've known this for a century now, all the way dating back to this quote by Huber in 1922, looking at the outstanding feature of obstructed lung diseases, the failure of clearance of these bronchial secretions, these are postmortem cast of the airways, and these are just basically bronchial casts from these airways. And what we've learned in terms of comparing a normal individual that dies compared to a fatal is that the airway basically becomes pruned. You can think about the airways as a tree. And you can think about the branches in that tree allow air circulation and ventilation and gas exchange. And you can see in asthma patients, there's basically a pruning of those branches that occur. And so we need better ways to restore this process, and we believe most of this truncation is being due to the mucus that's obstructing those airways. And is nicely outlined a little bit earlier, when we look at those mucins in the airways, there really are only a few targets. There are a number of mucins that are important in terms of the pericellular layer here, but in this mucus layer, there's really 2 targets here, MUC5AC and MUC5B. And what's demonstrated here in terms of disease progression is that we believe there's this normal kind of homeostasis of the ENaC channel and other channels here that maintained the hydration that is important in the airway and you have this normal mucus layer here and cilia beating along to progress that mucus normally. Well, in the muco-obstructive lung disease, what happens is that often there's a failure of this hydration that occurs, there is a thickening of the mucin layer that can occur because of what's referred to as goblet cell hyperplasia. And therefore, then mucociliary clearance is impaired markedly in these patients, leading to the symptoms that our patients experience in terms of cough, airflow obstruction, leading to increased risk of infection in these airways. So we believe that the mucus really that is predominantly in these airways, MUC5AC is one of the key players in terms of the causation of muco-obstructive lung disease. So let's delve a little bit deeper into MUC5AC. And what's demonstrated here in COPD patients on the far left, is that if you have increasing severities of COPD, you have increased expression of MUC5AC in the sputum that you can detect in these patients. And there's really no difference here in terms of MUC5B. And so as we'll talk about, we believe that this is, in essence, kind of a muco-protective mucin, whereas MUC5AC is an inducible mucin protein in these patients. Now when we look at patients with asthma. Again, very common thing we'll do is we'll look at the ratio of MUC5AC to MUC5B, this actually is inverse here. So they're looking at 5B to 5AC. And you can see in asthma and in the setting of an exacerbation, it's significantly reduced. Now look at cystic fibrosis, another muco-obstructive lung disease that Dr. Salathe will be talking about subsequently. You can see here in terms of patients with cystic fibrosis, again, have an elevation of that MUC5AC and in the setting of an exacerbation, again, elevation in the MUC5AC. Lastly, another opportunity for muco-obstructive disease are patients that have bronchiectasis, bronchiectasis is basically dilatation of the airways that's due to recurrent pneumonias. In the past, we used to see this a lot from tuberculosis. But nowadays, we still see this in patients that have recurrent pneumonias, and it's not related to cystic fibrosis, but again, showing this elevation in MUC5B -- MUC5AC. So again, this ratio, we believe, is very common across this muco-obstructive lung disease, and it's showing this increased production of MUC5AC in relationship to MUC5B. What about genetic predisposition? There have now been several GWAS studies or genome-wide studies that have looked at various genetic causes potentially of asthma. We have not found one thing. It is likely a very complex multigenic causation of this disease. But in this GWAS study, which was recently published in Lancet, it showed in a large population, this was in Europe, 5,000 cases, 25,000 controls, there were 3 different targets that met their criteria for significance, which was set at a p-value of negative 10 to the eighth. And 1 of them -- 1 of these 3s was MUC5AC, suggesting that there are a subset of patients that have a genetic predilection to development of muco-obstructive lung diseases like asthma. So when we think about this muco-obstructive lung disease, one of the drivers behind that is changes that we're seeing in the airway epithelium in the airways of our patients, in this case, a fatal case. And one of these cells that we have identified is the goblet cell. This is turned on by IL-13, one of those T2 cytokines that we're talking about. And when that goblet cells turned on, it basically caused us increased mucus production in a plug in the middle of that airway. Now fortunately, I don't have a lot of these cases of fatal cases, but I can use the tool that every clinician has in a hospital here in the U.S., which is a CAT scanner. And so a CT of the chest allows me to identify a mucus plug. So this is in the right lower lobe. In this patient, we enrolled in the severe asthma research program that I'm part, of sponsored by the NIH. And you can see this plug here in this distal airway. And now looking at this patient 3 years later, again, persistence of this plug in that same airway. So as we'll show you next, we believe that this is very important in terms of the disease causation and progression. And this is work by Eleanor Dunican that we published in JCI a few years ago, again, showing these mucus plugs in the airways. And the way we did this is we selected 5 chest radiologists around the country to identify these mucus plugs and they went through every CAT scan. We had over 200 of them enrolled in our program, and we had them score if -- a segment of the lung had a plug, that was a score of 1. While there are 20 segments in the lung, 10 on the right, 10 on the left. And so you can imagine this is a pretty tedious work in order to do that. But as all things go, now there's a software system that does this automatically for you. And one of the key findings from this publication was plugs matter in that if you had a high mucus plug score, 4 -- just 4 out of those 20 that correlated with a marked reduction in lung function. This reduction in lung function. This mean change in lung function is considered severe airflow obstruction by a clinician. And so you can see that we believe that, again, in this cross-sectional analysis that there is a potential role for the causation of airflow obstruction in our patients with asthma. But recently, this was just published this month by Monica Tang. Looking at this now over time, so we've been fortunate to follow this severe asthma cohort over a 3-year period of time. And you can see the mucus plug score really hasn't changed over a 3-year period of time. This is what we call a Sankey plot where you look at these patients and how they change over time. About 1/3 of patients had these high mucus plug scores. Again, trying to get to that precision medicine approach of what are the subset that we can really target. And you can see a majority of these patients, 1/3 still out here 3 years later. There's a little bit of a change back and forth here, but the majority of the patients are staying in that category. And what we demonstrated here in Monica's paper was that this does matter because when you look at the change in lung function here by 3 different parameters and you look at the change in mucus score, there is a direct correlation between an increase in the mucus score and a decrease in lung function, an inverse correlation. So suggesting, again, causation in terms of progressive loss of lung function in our patients and some other more advanced CT metrics that we're using in quantitative CT, we are also demonstrating relationships to air trapping, which makes a lot of sense as well in regards to mucus scores. What about COPD? Is the same thing true in COPD or not? And again, work led by Eleanor Dunican in our group in the severe asthma research program. Again, in COPD, you can see here the mucus score, again, from 0 to 20, a pretty interesting group here in terms of these scores in comparison to the healthy individuals here. And then there's another group here, which I think is very interesting and potential target is smokers that have not developed airflow obstruction. And just in recent years, we've identified that these patients are actually quite symptomatic. They have frequent cough, shortness of breath and chest tightness and these patients really are not abnormal in terms of lung function testing yet have significant mucus plugging. Again, that same Sankey plot that you can see in this particular study coming from SPIROMICS, showing just a year later that there's persistence of these mucus plugs and about 1/3 of patients out there out to 1 year. And again, this appears to matter in terms of the mucus score being the highest and the most severe patients by that global obstructive lung disease categorization of 4, they're mostly GOLD 4 type patients. We also use this data to segregate out patients. As I mentioned from asthma, there's many buckets of asthma. On COPD, there's many buckets of COPD as well. You can imagine the spectrum from chronic bronchitis patients all the way to emphysema type of patients. And so what the mucus score allows us is to really select that subgroup of patients where mucus is driving that disease process. So there is another important role about mucin proteins in the airways. And that's how -- what role it has in regards to host defense. The most common cause of an exacerbation in both asthma and COPD is a respiratory tract infection, a cold. And most often, these are driven by viruses. Now when you look at COPD patients, you can see at baseline and in the setting of an exacerbation, there's a marked up regulation of MUC5AC in the sputum. And this was just a natural observation study where they took 40 patients with COPD and a subset of them, they were able to identify a virus and then follow them over time. So again, we have this biomarker that appears in the sputum in the setting of an exacerbation. In the middle here is a knockout mice of MUC5AC. And you can see here in comparison to the wild type, there is decrease in neutrophilia in these patients, a decrease in inflammatory cytokines, IL-1 beta and IL-6 correlating to the knockout. And when you use UV radiation treatment of the rhinovirus, which is the most common colds of the common cold, it basically aggregates that inflammatory response. So this indicates to us that there is a role for mucin in terms of attenuating that inflammatory response that we see in the setting of a cold. Again, the most common colds of an exacerbation. And then on the far right, you can see here is that when you give MUC5AC, as demonstrated here in the setting of the rhinovirus infection in the blue bars here see a marked up-regulation of inflammatory cytokines, both IL-1 beta and IL-6. So clearly, mucin is -- MUC5AC here is playing a role in this inflammatory response in the setting of an exacerbation. So like all academician, we like a model. And so this model helps me understand where does MUC5AC play a role in muco-obstructive lung disease. I do believe that there is this chronic basal secretion because MUC5AC is a common constituent with protein in the airway. But we know that in a chronic model, a chronic disease model that increase in MUC5AC now appears to be correlated with chronic airflow obstruction demonstrated in those CAT scans that I was showing you by increase in mucus plugs and leading to symptoms in my patients. We know that there's another side of this that you can stimulate increased MUC5AC production in the setting of a viral infection, leading to exacerbations of the disease and airway inflammation basically accelerates this process in our patients, leading to their progressive loss of lung function and progressive disability in that regard. So how do I think about this in terms of looking forward in terms of targets that are targetable drug targets in regards to the disease. Well, I think we can utilize a tool that we have in the clinic in every location, which is the CT scan of the chest in order to identify the subset of patients that have a high burden of mucus, okay? So I refer to this as the mucus high phenotype. Another way we can use this is looking at mucus on this access here versus inflammation. And we know that type 2 inflammation, we have a number of targets in asthma at least. We have 6 biologics that I mentioned. But in COPD, we have no biologic that is currently approved for the treatment of COPD. And so we have a number of drugs that potentially we can use in this type 2 high inflammation. We don't have a lot of information about these biologics and what impact that has on mucus obstruction, but certainly, this category here, where you have high mucus score but low T2 inflammation we have no candidate drug currently in this place. So this is certainly a key opportunity for advancement of drugs in this population. But certainly, even in this group, there may be a role for co-treatment of both T2 inflammation and mucus plug related inflammation. So just to end, I think, as we summarized in this data nicely before in terms of the knockout mouse, certainly, this preclinical data suggestive now a genetic information in the GWAS data suggesting a causal role for MUC5AC. This clinical tool that we have in terms of measuring plugs that every clinician could use and their software now developed to give this information to us based on the CT, the role that MUC5AC has in a very common cause of exacerbations, which is viral infections. And therefore, I believe that this is a key opportunity to really interrupt this pathway that has quite a bit of potential. And as demonstrated, we're certainly focused on asthma and COPD but there are a number of other diseases like cystic fibrosis, non-CF bronchiectasis and primary ciliary dyskinesia that would represent an opportunity. So thank you. I'd like to end there and turn it over to my colleague. I'd like to introduce Dr. Matthias Salathe, who is the Chairman of Medicine and Interim Vice Chancellor for Research at the University of Kansas, actually he is my boss. So I got to turn it over to him.

Thank you. Well, thank you, Mario. As the bosses always do, right? He set everything up. So I have to do very little at this point in time. But I wanted to talk a little bit more about RAGE and how we really believe this could be a great target for asthma. Now Mario told you a lot about the pathophysiology of asthma and where we have potential interesting drugs and where we don't. And there is a huge number of people, obviously, that still have no treatment in that space. And then I'm ending up that I don't believe this is the end, asthma is not the only one, like muco-obstructive disease RAGE plays a role in those as well. So here are the disclosures and we're jumping right into this very complicated receptor that Erik already introduced very well. So it's an immunoglobulin, it's a superfamily of immunoglobulins and very difficult to target in terms of inhibitors. You know that there are some developments, but none have really been successful, at least on a clinical basis and even in animals, it's not a great target to go for. But oops, at was not good. I need the right -- here we go. There are lots of agonists for that receptor as well. So it's not an easy receptor that is just like stimulated by one target. It's targeted by a lot of agonists that then create a signaling pathway in the cell that is complicated. And depending on the cell can really induce a lot of inflammation in all kinds of pathways. [indiscernible] was mentioned, but there are lots of other pathways. And the important piece here is that RAGE is really upstream of a lot of the other inflammatory targets. So the idea is if you can really take RAGE out, you will be able to suppress inflammation in other areas dramatically. Here is just another example of a RAGE, how complicated this signal can be. And you can imagine that really, if we can block up here, we don't have to deal with all the complication that comes below RAGE activation. So how is RAGE activated? You've seen all the alarmins, the DAMPs and PAMPs and however, these agonists are called. But originally, it was high sugar that was the classic trigger for RAGE activation. So the sugar molecules that created these advanced glycation end products that activated the receptor levels discovery. But we know now that pollution, cigarette smoke and allergens, the classic house dust mite and cockroaches and other allergens are stimulating RAGE as well. And here are the classic muco-obstructive diseases, again, CF, COPD and asthma that we have all these cytokines following the RAGE activation. So again, if we could take RAGE out, we can decrease inflammation in a lot of these different diseases. As you have already seen, this is a sort of a normal airway with the lung around it, and here is an asthmatic airway and you're not needing to die from the mucus plug, right? But you have a lot of mucus, but the idea here is not only having mucus, it's this whole inflammatory cascade around the airway that will stimulate the mucus. So it's not only that you target mucus here. It is potentially able to decrease the inflammation that will lead to the mucus obstruction as well. Mario walked into -- so through this a lot. We're always thinking about type 2 high asthma and T2-high asthma is really the one we think we can reasonably treat, that's not true either, not everybody in T2-high asthma is actually treatable, and we have a significant number of patients that are not. And as he mentioned, the low T2 asthma, we don't really have therapies for them. And that's a huge issue and a large number of people in COPD are obviously 2. So this is just another depiction here is the T2 high. We understand these pathways. We have created -- we -- I take a role we here. I have nothing to do with that. But here are people that created these antagonists for these pathways, we really don't have anything meaningful in T2-low asthma. Now how is RAGE playing a role here? What you're seeing here are biopsies, and these are not quantified, but these are representative biopsies from non-asthmatics, myelo-asthmatics, moderate asthmatics and severe asthmatics, and these are stained for RAGE. So what you can see easily is that in the more severe asthma you have, the more RAGE expression is there. Now expression itself obviously doesn't mean it's signaling. So that's always a tricky piece. But here is an experiment in a mouse lung. You see here toluidine as an allergen and it increases RAGE and then this not great inhibitor of RAGE FPS, which cannot be clinically used is slightly decreasing the RAGE expression. But more importantly, if you look here at PAS staining, there is clear mucus production in the allergic mouse model, but you can block it this production of mucus with this RAGE inhibitor. So if you look at these pieces here, you can say the information is reduced and thereby the consequences of the inflammation through RAGE inhibition is actually beneficial. When you look at these alarmins and other antagonists of RAGE, you see there are increased in healthy -- I mean, mild-to-moderate asthma, it's going up and severe asthma is going up, this is HMGB1. This is a classic agonist of RAGE, but it's also true that these that HMGB1 is increased in COPD and in cystic fibrosis and even worse in cystic fibrosis related diabetes. So this is an agonist that is not unique to asthma, but it's increased. And so is S100A9, this is one of the other agonists of RAGE, increased in asthma compared to healthy controls. And you see here also in neutrophilic meaning T2-low asthma, it is also increased, at least again suggesting that if we can block RAGE, we may be beneficially influencing T2-low asthma. You see here sputum neutrophils again, a correlation between this agonist of RAGE S100A9. There are lots of other S100s, by the way. And TNF-alpha goes up. And here, this is the hyper activity. This is upside down, meaning the lower numbers of inhalations of methacholine, you need the higher -- the hyperactivity. That means higher S100A9 is associated with worse hyperactivity. So again, a correlation between the RAGE activation and hyperreactive airways. And this is just to show you how complicated this is and how I really don't understand anything about it. This is sort of a cartoon that was put together from all the inflammatory pathways we know that are activated in asthma. But what you can see, you can point out all these RAGE agonists right through this. So in these pathways, RAGE agonists and therefore, the perpetuation of inflammation is basically given through these known inflammatory pathways. Making it just a little bit simpler. This is RAGE. We believe it's really upstream of all these T2-high, but also T2-low asthmatic responses. Now we don't have good pharmacological treatment, but we can use knockout mice, and Erik already showed some of that. So when you look here at knockout mice, they were stimulated with allergens. Again, most of these animal models are really allergic asthmatics, right? It is very difficult to do a T2-low model. I'll show an attempt at that. But when you compare that to the wild type, there is a huge blunting of IL-33, other cytokines with Alternaria other models. So every time you take RAGE out with a knockout model, you will actually reduce the cytokine levels that are typically activated in these allergic responses. The same is true here. This is a little bit more complicated because TLR4 can play a role in this allergic asthma as well. But when you look at the responses from IL-25, IL-13, here is the famous TSLP that Mario talked about and IL-1 alpha, you can see that RAGE knockout will decrease not completely blunt, but will decrease these responses basically at relevant time points of all these cytokines. And it doesn't need TLR4 knockout either. So it is seemingly upstream. And what you see here, it's not only upstream in this cartoon. As long as RAGE is there, that's IL-13 activation, you see STAT6 can be sustained, meaning it is activated in a sustained fashion. If you take RAGE out, there is no permanent activation. So RAGE seems to be important to actually sustain the inflammation in these airway diseases. It's not only to initiate it. And this is seen here. Again, you treat with IL-4, for instance, in a RAGE knockout mouse, you don't see the eosinophil, P-STAT6, phosphorylated STAT6 activation. This is IL-5 and IL-13, same event. So using these cytokines to create an allergic model like T2-high model. If you knockout RAGE, it is blunted or eliminated. Now T2-low, that's a hard one. The only thing we found is if you have cigarette smoke exposure, so it's not typical asthma, but again, an attempt to do a T2-low model, you're going to see hyperactivity that is actually eliminated by RAGE knockout and you see neutrophils decreasing and the relevant cytokines decreasing as well. This is not a perfect model. But again, it suggests that there is an avenue to potentially approach T2-low inflammation as well. Now these are the current guidelines by the NIH and the different societies, how to treat asthma. Looks pretty simple and straightforward, and that has been true for about 20 or 25 years. But interestingly, this was published in 2020. Like there's this little box down here, and it says consider adding asthma biologics, not clear in 2020, that was 2 years ago. And yes, they're lagging behind. But if you go a little bit further into this, and this is just another depiction of what Mario already showed. Well, there are these biologics, right? But they're really targeting very, very specific pathways and to select the patients that will benefit from them becomes, therefore, somewhat complicated. And I guess this box explains that, they didn't really want to go into the complications of who will benefit or not. And this is the algorithm that they think you should follow. Now if you see a complicated algorithm like this, then you know this is going to be very difficult to actually do. So I guess we have to all send our patients to Mario to make the decision here, which blocker you need and which biologic, but this usually translates that a lot of our patients will not get correctly treated or optimally treated, I would say, because it's not simple to make these decisions. One of the biologics, and I should have given that to Mario too because he was the first author on that paper, right, in the New England Journal that was one of the Phase III clinical trial for dupilumab and you can't read this. So I blow it up here a little bit. To take then a biologic and bring it into the asthmatic population, even though some selection occurs, you need to get a lot of patients enrolled to actually show positive outcome that as depicted here. If we have a more broad inhibitor of inflammation that would reduce that significantly. I'm not a statistician, I'm going to tell you how many patients will have to be in this trial, but I'm going to refer to Javier on that. But that is the problem with more specific biologics. On the other hand, if you select the right people, and this is dupilumab as well, if you do it right, then you can show significant benefits. And this is in this large population, there was actually a significant improvement in lung function. So yes, using the right population works with the biologics to actually pick the right population for these biologics. That is the very difficult point. So coming back to a patient profile and Mario had a patient that was on multiple, multiple, multiple medications and still just never got as good as he or she should. This is just a regular sort of presentation of a patient with asthma. We're going the regular route, what the recommendations are. Then after all of this failed there was a biologic introduced, did a little bit of something, decreased prednisone daily, but you know what that actually means, having prednisone every day for patients and their consequences of that. Then that biologic was stopped, another biologic was tried, but it never really in the end, control the patient. So what are we doing with those? And I believe there or maybe even for other people, RAGE would be a great target to have. So why do we use RAGE? I think I walked through this every part here that we have good evidence, at least in silencing models that this is upstream of inflammation in general, and therefore, helpful it might be really also helping in T2-low asthma that there is no treatment at the present time. But beyond asthma, right, we talked about COPD. We talked about cystic fibrosis, all of those have inflammation that is related to RAGE. And therefore, these are diseases that can target -- be targeted with RAGE inhibition as well. And now I don't know. That's the end of my talk, whether we have a break or whether Javier San Martin is going to come and talk about the clinical development.

We're actually -- we're going to take a break for about 15 minutes while they bring lunch out. So let's reconvene at 11.55 Eastern. Thank you. [Break]

I will give it a minute so we can be ready. Okay. So next up is Javier San Martin, our Chief Medical Officer, and he will talk about the clinical development for the new pulmonary assets.

All right. Thank you, Vince, and I hope you are enjoying your lunch, and I'm not going to be a huge interruption, but I think I have very interesting stuff to share with you. I will present our initial thinking on the clinical development program for both ARO-RAGE and ARO-MUC5AC, focus maybe on the first Phase I study that are about to start very soon. But also, I wanted to share with you our current thinking for the overall drug development for both molecules, thinking about different indications and different paths to regulatory filings. In every single drug of disease that I've been working over the last 25 years, I always was interested about the history of those diseases and the drugs that were developed to treat them. And so with asthma, it's very interesting that this disease was described over 150 years ago and the focus was always a bronchoconstriction and the intermittent aspect of this disease patient worse and then improve. And that was kind of the concept of how this disease was known by. So the first treatment, of course, was the ability to reduce the bronchoconstriction with beta 2. And this started maybe 40 or 50 years ago. Over the years, they improved the beta 2. They make it long duration in order to improve compliance and to some degree, the approach took care of the bronchoconstriction part of the disease. And it wasn't until about mid-80s, where the inflammation was considered a key component of the disease and perhaps the cause of the bronchoconstriction. And in fact, now we know that that's true. So inflammation at that point in the 60s and 70s was treated with glucocorticoids and initially was treated with systemic corticosteroids. And of course, as you know, there is no sustainable long-term high doses. So the industry developed inhaled steroids, and that was a significant improvement in terms of care. But of course, not all patients respond to it and compliance has been always an issue with daily -- by daily dose of inhaled steroids. So about, like I said, 30 years ago, inflammation was a big deal, and it wasn't until about 20 years ago that we had the first biologic treatment with Xolair and then another 10 years really without any other significant no biologic treatment. So the last 10 years or so different companies start to identify right targets that you can block with antibody therapeutics, and that's the new revolution of biologic treatment in severe asthma, something that happened in rheumatoid arthritis, for example, 30 years ago. So that's -- it's a very interesting moment, I think, in asthma drug development. We just went to the ATS meeting last week and I was really impressed with the energy, with excitement around all this biologic, the clinical trials, the result, the subgroup analysis, the very good combined work between the academic group and the industry, I did feel that energy when a field is progressing to the next steps. So that's why I think we're in the right time to initiate the clinical development program for these 2 molecules because the field is ready to keep learning and keep working on it. But again, one other point is it was about 40 years since people recognize inflammation is a key component of the disease and proper treatment was developed for it. Now mucus. Mucus has been describe as part of the symptom and the phenotype in asthma and COPD and many other muco-obstructive lung diseases. But it wasn't until recently that was considered a part of the program, not just a symptom or evidence of the program, but actually part of the underlying pathophysiology mechanism that increase the severity and the risk of asthma. So that's very interesting because that was really in the last 3 or 4 years. And here, we are 3 or 4 years later developing the first target therapy to address this component of the underlying pathophysiology in asthma. So here, big picture, how we're thinking about the 2 molecules that we will develop. One is ARO-MUC5AC. And of course, this is a new class of drug. It will block the production of mucus and by that mechanism hopefully will improve air flow and decrease exacerbations. Also another concept that is relatively new is that mucus is not just a consequence of inflammation but also is a cause of inflammation. And again, that's a very new concept. And of course, one of the reasons why we're developing this particular target. And the next concept with regard to MUC5AC is that yes, the #1 target will be severe asthma because it makes a lot of sense because it follows the pathophysiology and where the pathophysiology meets the mechanism action of the drug. But it also will allow us to study this drug in many other different muco-obstructive lung disease, including the asthma that is not well served today with the anti-inflammatory therapies. And on the other hand, we want to target inflammation and we do want to develop the best possible anti-inflammatory therapy, and we think that, that will be a RAGE. Why for everything that's been said before, Matthias really explained very well the role of RAGE with regard to the inflammatory response that you see in asthma is upstream of essentially everything else, but also may affect or intervening in the non-type 2 high, which is another very interesting concept, maybe by being such a high level inflammatory cascade it may prevent the fact that patients sometimes need to switch from one treatment to the other, the right example of the patient that Matthias presented today. So this broad class of anti-inflammatory plus this is a drug that will be given in an inhaled as opposed to systemic likely monthly, perhaps every 3 months. And again, back to a typical common problem in chronic diseases is adherence or compliance with treatment. So the fact that we will space this month apart, hopefully will translate into improvement in adherence compliance and that usually translate into improvement in clinical outcomes. So we'll start with our RAGE. And of course, this is the drug that we have to address inflammation in severe asthma. Airway inflammation is at the front of all the rest of the pathophysiology process of asthma, inflammation increase mucus, inflammation, of course, affect muscle contraction. So the idea is you block inflammation, and you improve airway flow, improve airway flow. It's the goal of therapy here. It should translate into improvement in FEV1, functional capacity, symptoms and hopefully prevent exacerbations. So this all has been presented today by my colleague, Erik and also by Matthias, which is the very significant level of evidence that exists to justify this as a very good target. The studies that look at association of the expression of this gene or increasing concentration of the protein. In both cases, its correlation associated with severe asthma. So that's one piece of information. The other piece of information is all we learn from the knockout mice models. And again, this has been described today. But as a drug developer person, when I see that we have drug or antibodies that essentially target each of these cytokines that are downstream of RAGE, I get more and more excited about RAGE being the right target that may take care of many of these. So -- and I had to say people know about RAGE for a while, but of course, it's not a druggable target with a systemic treatment like an antibody. So we believe that this is the base of -- so far, the only way to address RAGE and again, addressing RAGE, cascade down, we're going to tackle most of the cytokines and alarmins that are responsible of the pro-inflammatory process in patients with asthma. Also, the RAGE knockout mice provide some evidence that can also be effective in preventing inflammatory response in those with type 2 low asthma as well. So regardless of what is the initial inflammatory pathway being more on the type 2 high, eosinophil-driven or more like neutrophile-driven. It seems to be the blocking RAGE will improve or may improve in both type of circumstances. And finally, Erik shows what happened when you do the experiment by inhibit RAGE using an RNAi such as RAGE that definitely recalculate all the stuff that we learned in the knockout mice. So this is, I think, is a huge level of evidence that as someone working in drug development, I'm very happy to work with from now and take this to the clinic. Dr. Castro already present some of the issues that we had today with regard to the treatment of asthma. We have much better treatment in the last 10 years. It's been this biologic revolution, but there is a lot of room for improvement. When I think about and I've been doing the parallelism between the asthma biologics revolution, compared with the rheumatoid arthritis biologic revolution 30 years ago and at the beginning, there was 3 drugs in RA: Remicade, Enbrel and Humira and people thought that's it. Today, there are 15 biologics with indication to treat rheumatoid arthritis plus another number of small molecules that follow similar pathway. So I think there is room for a lot more better treatment for asthma and also different patients may require different approaches in terms of administration and in terms of the specific type of phenotype of disease that they have. As I said before, the type 2 low asthma is another opportunity that may be this target [indiscernible] will solve. And again, systemic subcutaneous administration, some people may prefer that, but inhale or infrequent nebulizations could be a very attractive approach that, again, the goal will be to enhance adherence, compliance and by that means a clinical outcome. All right. So let's jump into the clinical trial. This is the schema of the Phase I clinical trial for ARO-RAGE, number 1 goal is assessed safety, and we're going to say safety but, of course, collecting AEs, adverse event, serious adverse event, laboratory parameters in FEV1 or spirometries including the DLCO. So we have a very robust safety assessment included in this study. The other goal from the therapeutic perspective and the more important is the target engagement. It's really the ability to describe the pharmacodynamic and we are lucky that with this target, we can do that in a noninvasive fashion. And I will say a few more things about that later. The way that we study the patients and the normal healthy volunteers, the normal health is particularly we have a bronchoscopy baseline and another 1 at week 4, this patient, the normal healthy -- sorry, in all patients will receive 1 dose of ARO-RAGE and the reason that will receive 1 dose is because the duration of effect is expected to be really long. So we don't see the need to do multiple dose in the normal healthy volunteer patients. And then we collect sputum almost weekly at the beginning and then biweekly, we will enroll the normal healthy in 4 different cohorts with ascending dose from 10, 20, 40 and 80. When we get to the 40-milligram cohort, that cohort after it's complete, and we will enroll 8 people in each cohort, 6 [ in active ], 2 in placebo. That cohort will enable after the 22-day safety assessment by the DLC, they are asthma cohort. So the patients with asthma will have a broad range of severity of disease. We decided to go with patients with GINA 1 to 4 that will facilitate enrollment. But we will ask for a patient with type 2 high, not going to the 300 -- 300 is not [Indiscernible] we think that 200 is good enough. And again, with the intention to say this patient with this phenotype will represent the type 2 high and will be relatively easy to enroll. The patient with asthma will be dosed twice at baseline and at 29, and we will follow them for 113 days. Again, we're going to enroll 8 patients, 6 of them will receive ARO-RAGE; and 2 will receive placebo. You can see the doses here, the maximal dose will be 80 milligrams, and this dose represent the dose loaded into the nebulizer. Another reason to be excited about this particular program is that we have the ability to measure a biomarker that will speak to the pharmacodynamic effect of the drug in a noninvasive fashion. We're collecting, as I said, for sputum in both normal healthy volunteers and patients serum, of course, in everybody and in the normal healthy we're also doing a bronchoalveolar lavage assessment. In all these 3 specimens, we are going to measure soluble RAGE. And of course, important will be to correlate all these measurements because the goal is eventually to have just set on RAGE as a soluble RAGE as the biomarker that we can easily use in large-scale studies such as the Phase II, where we're going to do the dose selection or even the Phase III. And it's one of those things that when you fast forward, it might even help clinicians to select what's the best patient for one therapy or the other. In the normal healthy volunteer, we're also -- when we do the bronchoscopies, we have bronchial brushing and that will be used to measure RAGE mRNA expression. And I think the combination of all these 4 parameters that assess in RAGE will give us a good idea of how to assess pharmacodynamic effect in this condition. And I think this is one of the key benefits of this particular program, and we did not have this opportunity with ENaC. So this is something that we're welcoming from the drug development perspective, I think it's having a lot of value. I just wanted to show an example of one successful drug development in severe asthma, which is the dupilumab example. To give you an idea of how many patients, how many phases you need to go through to go to the FDA and file this drug, so this is a good example of a regulatory path forward. And in the case of severe asthma, there is very well established. So this after the Phase I study is in normal healthy to assess PK and PD, they did the first study in severe asthma, the Phase IIa study in patient with moderate severe asthma and with T2-high. They only enroll 104 patients. It was a relatively short study of 12 weeks and this approach about taper standard of care and when the taper is done, then evaluate acervation and pulmonary function. And that's a clever approach to do that because it allows you to have fewer patients. This was also done recently, I think it was in the anti-IL-23 program that was published in New England a few months ago. So that's the way to enrich the population when you are at the beginning. Get the patients that are more likely to respond, type 2 high and create protocol design that will prompt them to have exacerbations and eventually demonstrate the efficacy. So I thought that was a very smart approach for the first study that achieve a proof of concept, but not necessary a dose selection. In order to go dose selections, they needed to do a Phase IIb study, and this was a much larger experiment with 269 patients, moderate severe asthma and any level of eosinophils. So growth of the patient population with regard to the type of disease, but still model to severe asthma, which is the target population for which this drug could be used. ENaC in this type of study, again, you cannot be very creative. FEV1 is a classic endpoint that displayed improvement in airway flow. Annualized rates of exacerbation of course, is the key approvable endpoint. And it's important that in this field, you start to see those endpoints as early as Phase II, but of course, you need Phase II studies of this magnitude over 700 people. From there, they move over to the Phase III study. And here, again, patients with severe asthma, moderate and severe asthma, any comment with regard to the type of asthma. And that I think it was drove the very large sample size of 1,900 patients because I'm not a statistician, either Matthias, but I'm very sure that they power the study, you've seen an assumption for an effect size on the type 2 knowing that the type 2 low may not contribute too much to the FX size and therefore, I think they had to do this type of design, which is perfectly fine because in the end, you want a broad label, and this is the way you try to do a study to be able to have a label that is broad and included patients with de different phenotypes. Again, the endpoints are very much the same. And the last registration study has to do with patients, of course, severe asthma that they were receiving oral steroids. And it's a sizable number of patients that still receive oral steroids. And the idea is, can you treat this patient with a more effective anti-inflammatory and taper that oral corticotherapy. Because, of course, oral corticotherapy has a lot of consequences that you don't want to see. So this is a guidance. Tezepelumab program is very similar, maybe a couple of different features, but generally speaking, this is how the FDA expect you to show up when you file a treatment for asthma. This is our initial thinking in our Phase II study. We will include patients with high and low. We want to be inclusive, but why? Because we think that this molecule actually have the capacity to be effective in both type of asthma. The endpoints will be the one that most people use, exacerbation, FEV1 symptoms of course. And the goal of the study will be dose selection. So this will be a Phase IIb study. We don't know still the size of the study, but it will be to enable a Phase III study. On a Phase III study, I think it will be, again, patients with severe -- moderate-to-severe asthma. Those are the ones that need more help. But we will be inclusive of the different type of asthma more likely. And again, the endpoints will be similar to what has been done so far. We think that this therapy area range could play a role in COPD, could play a role in cystic fibrosis, and we will start to consider how to move from severe asthma to COPD. As you know, dupilumab is now running a study in Phase III. So I think the concept of a better than glucocorticoids anti-inflammatory could be helpful in COPD. I think it's out there. People believe, people are working on it. So we may need to consider whether we need a Phase II in COPD, what we need to know to have the confidence to run large Phase III COPD study because that will be a large study and clinical outcomes will be critical for approval. All right. Let me switch over to MUC5AC. Airway obstruction is what in the end cost disease, airway obstruction will cause symptoms, airway obstruction is what causes exacerbations. Therefore, it's at the center of the disease. And what I think is interesting, again, as I said before, is the idea the mucus is part of the mucus obstruction is relatively new as a targeted abiotic. And it's not just because there is a mechanic obstruction component as Dr. Castro show in those C CT scans, but also the excess mucus increased inflammation. So this is a cycle and applies to both drugs. When it's inflammation, increased mucus both together impaired muscle and airway function when you have mucus drive inflammation as well. So -- the way I see this is you can attack this problem 1 way or the other. And I hope at 1 point, we will be able to distinguish who are the patients that might benefit more from one or the other. Again, all this has been said already, but I think it's impressive the level of evidence that we have from observations with regard to the increased expression or concentration of MUC5AC and severity of the disease, severity of the numbers of mucus plugs that you observe and exacerbation. So the 3 components of the disease that you really care about, they're all in a way, drive, but the increase in MUC5AC that you see in patients with both severe asthma and COPD as well, and another disease that I will comment on later. In the case of MUC5AC, again, already was presented by Dr. Castro. There is a clear association, discovering more than one. She was studied. So that level of evidence add to the to the evidence to justify this target. The knockout mice again replicated the same concept. And once again, Erik showed that we -- when we do the experiments, we replicate, again, the same country that is able to decrease airway resistance. So again, we hit every single level of observation and preclinical evidence to justify these targets. Dr. Castro very nicely illustrated this content, which is relatively new, the idea to show the segments of the line that is completely obstructed by this mucus plug. It's so interesting to say that, that correlate with disease severity. And it can be quantified easily. So we're thinking about this as #1 tool to identify patients that may be tailored to this immune therapy, but it may be is an endpoint as well, and we can incorporate that into our Phase II studies and perhaps see whether there is a decrease in a number of segments that are mucus plug or preservation of prevention of new mucus plug. So this is new staff is developing as we speak, but I think it's another opportunity to have another biomarker of surrogate to learn more about how this throw affect the disease and the underlying pathophysiology problem. As we showed before, normal lungs have no mucus plug whatsoever and patients with asthma have a large proportion of them, a substantial number of mucus plug. So here, I think the correlation between this and severe disease is very clear. So again, when you think about MUC5AC, it seems to be a very, very appropriate target to address these issues. Phase I study with MUC5AC. Again, safety is the first priority and the first assessment, and we're going to do that exactly the same way as we do it in the RAGE study, which is clinical laboratory and spirometer. Here, we start with a normal healthy single dose who are sending single dose in which you can see 24, 56 and 108 milligrams, one single dose. And then once we're done with the first cohort that will enable the multiple dose normal healthy volunteer cohort, the same will happen with the next cohort of 56, going from single to multiple and at the same time with patients. Importantly, in this case, we selected moderate-to-severe patients with asthma -- with severe asthma. You can see the airway obstruction 40% to 80%. So we're looking here at more severe patients to really be able to see the signal of MUC5AC reduction. Again, these patients with part of the biomarker assessment will be sputum. As you can see, this is in black. And so we're asking the patient to do this in sputum very frequently, weekly or biweekly. And also the normal healthy volunteers in the multiple dose, we have a baseline bronchoscopy and a [Indiscernible] approximately 1 or 2 weeks after third dose. The multiple dose cohorts for normal and patients will be 3 doses, separated by 2 weeks each. And then bronchoscopies a week or 2 later, the total duration of the study will be approximately 85 days. So how we envision the long-term clinical development program for this molecule, moderate and severe asthma, again, the pathway is being written, but I think we have a couple of new answer here, particularly regarded to the CT scan and perhaps the ability to select patients based on the number of new construction in order to understand whether we can see an effect there or no that per se, select population that more likely will respond to this therapy, because it's reasonable to think that those patients who have more mucoid obstruction or mucus plaque are those who have more mucus in general, mucus is a cause of obstruction and mucus is cause of inflammation. So again, this is very interesting when the drug development process. At the same time, the academicians and the science is moving forward the same concept, and we're both learning together. And I've been in that place before and I think it's very -- it's very different when you are working on the synergies approach with the academic and the science. Eventually, will do a Phase III moderate and severe asthma study. Again, the details how we're going to go about, what will be the patient population. It's something that we need to learn more as we go along. Also, it's very important for this target. We're thinking in a number of other diseases where the mucus obstruction is the key feature. One of them is COPD, of course, cystic fibrosis. Perhaps it's another opportunity, non-CF bronchiectasis and primary ciliary dyskinesia, I would say a few things about each of these. This slide was shown before, but just conceptually, again, to say, normal mucus layer is important. It's important to enable the ciliary movement to get the mucus out of the system, but then when you had too much of the -- and sticky type of mucus and then it makes the work of the cilia a lot more difficult. So when you think about, this is like PCD, which are patients who have the highest concentration of mucin and they already have an issue with the mechanism which they get rid of the mucus then it seems to me that this is a very incredible target. Now it's a very rare disease, but I make a few comments about that later. But I see this as a great opportunity. CF is a similar issue, mucociliary clearance in pair. Now some patients are doing really well with the current therapy, but the new -- new patient had no choices and they have a lot of mucus production, mucostasis, mucostasis increase the risk of infection and you know the cascade of clinical events in those cases. Non-CF bronchiectasis, same deal. When you think about those patients, it's all about [Indiscernible], sputum, phlegm and very symptomatic diseases. COPD, particularly in the bronchitis subset or subtype of COPD, one of the key problem is the accumulation of mucus and how that impact quality of life. So this is how we see COPD and why I'm really excited about this. One is that I wanted to contrast the picture I saw at the American Thoracic Society Meeting last week between asthma and COPD. And I went to a number of presentations, particularly some of the industry seminars, and symposium. And in the severe asthma, it was all excitement and subgroup analysis, all kind of interesting stuff. When I went to the COPD, it was about compliance. And who can do a longer acting beta to longer-acting [Indiscernible] -- longer-acting inhaled corticoid, how you can combine in 1 part, make is easy, especially which is definitely important and don't get me wrong, I work mainly in chronic diseases and regardless the disease compliance, this is a problem and it's a real issue. So make it more friendly -- the current therapy is a good idea. But really, it wasn't any news -- well, like, okay, the COPD field is moving this direction to take it to the next level. And it's been -- so this is my area of expertise as a clinician, but that was 25 years ago, and I did not learn anything I didn't know already. So it is a huge opportunity when you see a target that may address an underlying problem of the disease. I think it's one of those critical moments in drug development. And as I said, with a lot of morbidities, as you probably all know. And all those morbidities are easily assess and quantify clinically. So the development program for a drug in COPD is relatively straightforward, but a lot will need to learn to select the right population, to do different correlations and so forth. But I'm really very excited about this opportunity. And one of the reasons is this, these are 3 different PRO instruments that people use in clinical trials in COPD and they're all driven by the burden of mucus in these patients or the clinical daily living, quality of life in part of the excess in mucus. So I see this an opportunity only to hopefully improve pulmonary function, prevent exacerbation and hospitalization, but also to really improve the quality of life. And it looks like the PROs are [Indiscernible] to really investigate how much the mucus hypersecretion translating to clinical event that impair these patients' quality of life. So this is from the drug development perspective is really a good place to be to say you are addressing an issue that you can clearly measure and impact the quality of life of patients. And lastly, I would say there is no treatment that prolonged survival in COPD for the most part, perhaps on oxygen. Imagine if this change the underlying condition, it is disease modifying aging and that's prolonged survival. So I'm dreaming here, but I will not exclude the notion that as we progress in drug development, one day, we started to study the really severe patients that had a phenotype that this intervention might be helpful. This is very preliminary, but this is how we see the COPD clinical development. Perhaps the new stuff here is the CT scan, again back to the gastropresentation, CT scan today is not part of the normal evaluation of a patient with COPD. Why? Because there is nothing to do about the pain of the phenotype, there is not really a lot of different decision making as a clinician. Now if you have a therapy that specifically targeted company of the disease, you can enrich the patient population and perhaps affect the disease a lot more. So this will be the first time that in COPD -- despite for many people know that there was a more emphysema phenotype or bronchitis phenotype, but now we can think about there is a tool to identify that population better, and there is a potential treatment that will address the underlying condition. Right. And I also have some background in rare disease, and I get very excited when I think about this. Molecule and these 3 conditions. I already mentioned primary ciliary dyskinesia, the treatment is antibiotics, airway hydration, bronchodilators, so very much symptomatic treatment. 3,000 patients in the U.S., so that gives us the opportunity for an orphan application, the mucus is at the center of this disease, the mucostasis is increase the risk of infection, infection produce more inflammation, inflammation more mucus, mucus make the severe more difficult to really do what they need to do. So the idea to reduce mucus in this disease, it seems to me a really interesting approach. Non-CF bronchiectasis from the clinical development will be challenging because it's a very heterogeneous population, but it's not small. And again, this is a condition where mucus plays a fundamental role in the daily living and the quality of life of this patient. And when you have that connection, it's also a very important aspect to develop the drug. And finally, as I said before, cystic fibrosis. We think that there is an opportunity there. Again, mucostasis impair and mucociliary clearance is what drive the disease. Of course, there are a number of patients with the new treatment that are doing much better, but still, there are room for improvement. And still, there are a population that the new CT are not able to address. So we're considering this as well. So to conclude, we're developing 2 molecules, MUC5AC with a complete novel mechanism of action, having a potential new tool to identify patients that might benefit from it, and that is a unique benefit in the clinical development program. And that can give us a broad pipeline. This is a molecule with the pipeline itself, and I think that is very unique. ARO-RAGE, our vision is hopefully to be the best anti-inflammatory therapy, local administration, no systemic administration, very long interval between doses and is in itself a benefit. And again, a broad pipeline because hopefully, it can be used with both type of inflammatory asthma and perhaps with other conditions such as COPD as well. So as a drug developer, it doesn't get any better to be in a place like this. The idea to be development in parallel, 2 drugs for the same disease is a unique opportunity and it will need -- we will need to be really creative to differentiate these clinical programs to eventually differentiate this product the day they see the market. So that in itself is a very interesting concept. The same applies to COPD. We have the 2 molecules, so the 2 pathway MUC5AC and RAGE that we could develop for this disease. Now these 2 will follow a relatively well-known regulatory path, and it will take time. But at the same time, I do believe and we do believe that these drugs may have other use and opportunities in rare diseases and that could be something that we'll do in parallel. So the regulatory clinical development will really be divided into the large indications and the smaller indications in which we will need to be creative to move this really fast into the development program. So I would pause here, this is like a very special moment for us, and I want to invite Anjli Warner to provide an initial reaction to how we're thinking about the market and these molecules. Thank you.

As my colleagues have mentioned, ARO-RAGE has the potential to target underlying inflammation while ARO-MUC5AC has the potential to target mucus obstruction. A number of pulmonary diseases are addressable with these mechanisms of action from broad indications like asthma and COPD to rare diseases such as cystic fibrosis and primary ciliary dyskinesia. For today, I will focus on the unmet need and potential of these products in asthma and COPD. Currently, approximately 25 million patients are diagnosed with asthma. The funnel on the left represents patient segmentation by the global initiative of asthma guidelines, which categorize patients based on the level of therapy needed to control their asthma. Patients start on inhaled corticosteroids and step through add-on LABA and LAMA therapies. It is estimated that there are roughly 6 million patients on steps 4 and 5 whose disease is considered moderate-to-severe. These patients are uncontrolled due to lack of efficacy and the need for daily compliance with inhaler use. Of the patients who are uncontrolled, 1 million are considered to have truly severe asthma. Currently, biologics are indicated for these patients, but have low penetration due to the need for subcutaneous administration, access and reimbursement restrictions and efficacy issues. Additionally, Th2-low patients represent an underserved population. I will go into the unmet need for severe asthma patients on the next slide. In June of last year, we surveyed almost 40 pulmonologists and allergists regarding the unmet need experienced in both severe asthma and COPD. The right-hand side of this slide indicates some quotes we heard. Physicians expressed the need for therapies that are convenient for patients to use or that will reduce the need for daily therapy. They also identified non-Th2 patients as an underserved patient population. They told us they are looking for therapies that can help address the underlying cause of asthma and not just treat symptoms. The story is very similar in COPD. There is a large patient population that is diagnosed with COPD who continue to experience low FEV1. The funnel on the right segments patients according to the global initiative for obstructive lung disease or GOLD guidelines. There are almost 7 million moderate to severe COPD patients with FEV1 below 80%. Uncontrolled COPD patients continue to experience multiple annual exacerbations and emergency room visits, putting stress on the health care system in regards to costs and resources. When discussing the unmet needs for these patients, allergists and pulmonologists echo similar sentiments as they do for severe asthma. The left of this slide indicates pulmonologists reported challenges in COPD and their ranking for importance in solving those challenges on a scale of 1 to 10. The right-hand side are quotes from our market research. Physicians are looking for therapies that can slow the progression of COPD and not just treat symptoms. They also believe that there is a major inflammatory component of COPD that is not being addressed by current therapies. They described the daily burden of therapies, especially on COPD patients who may have cognitive and dexterity issues or low inspiratory rates. It is clear that current therapies are not adequately meeting the needs of asthma and COPD patients or their physicians. ARO-RAGE has the potential to be a better anti-inflammatory that can work upstream in the inflammation cycle to inhibit cytokines. ARO-MUC5AC has the potential to be a first-in-class therapy targeting mucus depletion and mucus obstruction. This is an underlying component of many diseases, including asthma and COPD. We look forward to exploring these therapies in the clinic and assessing their ability to reduce exacerbations and improve airflow, thereby addressing underlying disease progression and improving patient quality of life. These therapies also have the potential to offer biologic-like targeting and effectiveness with the convenience of a less frequently dosed inhalation therapy. This can lead to improved patient acceptance, compliance and ultimately, better disease control. We are excited for the potential of our pulmonary portfolio to address a broad range of lung diseases by targeting a variety of mechanisms of action. As new targets are identified, we are continuously prioritizing indications based on unmet need, market opportunity and the scope of clinical development required among other factors in order to approach the development of our pipeline thoughtfully. We look forward to keeping you updated on our progress. And I will turn it over to Chris.

Okay. That was a long day. Thank you, everyone. Thank you to all the speakers. I think that was a really helpful overview of where we are and what we're thinking. Of course, thank you to our guests, Mario, Matthias for increasing the IQ of the room by an order of magnitude or so. So quickly. So this slide, Anjli's last slide is a really good one. I was going to steal it, but I didn't. It's just such a nice representation of how many people we can help, I think, with just these first few programs, and we're so excited about this. This is just for the first few ARO-RAGE ARO-MUC5AC and MMP7 and of course, respiratory virus is, we'll see where that goes. So when we were going through our slides last night, someone from our team had said, that's a whole company. And it's right. In fact, that's a whole company and not an average biotech company. That's a whole broad and diverse and deep biotech company, that's only one part of what we're doing -- any event, I'd egress. So when I introduced the presentation earlier, I talked about this R&D Day we had about 4.5 years ago. At that time, we had 0 clinical programs, 0. And now fast forward to now, by the end of this year, I expect that we're going to have 13 clinical candidates in clinical studies, 7 wholly owned, 6 partnered. And by any -- by any measure, that is a successful track record over the last 4.5 years. So when you are handicapping our chances of success for this next platform for this next franchise, think of that track record. This is our pipeline right now, spanning cardiometabolic disease, a burgeoning pulmonary division, of course, the cadre of liver diseases, and we think also by the end of the year, possibly our first muscle candidate in the clinic. So over the past 4.5 years since introducing the TRiM platform, we've built what I see as a robust and scalable and substantially derisked hepatocyte-directed set of drug candidates. This has provided, we think, an enormous amount of hope for millions of patients and has created a substantial amount of value for us. And I think that we are on the cusp of doing that same thing for pulmonary. I think this is a good first step in that direction. I look forward to keeping you updated on our data this year and throughout the life of this franchise. So thank you again for coming. And I guess we have questions. Is that how it goes? I'll go to one more. Here we go Q&A session. All right. So do we want the speakers to come up here to answer questions? Let me rephrase that. May the speakers come up here to answer questions.

Great. Thank you. Can you hear me okay? Ted Tenthoff of Piper Sandler. So firstly, thank you, this is really informative. And one of the things that I walked away with, I really appreciate the value -- potential value of soluble RAGE, but also CT as potential biomarkers. So I guess, firstly, for the doctors, do you see the opportunity of a nebulized therapy that target upstream of select cytokines, likely to be ahead of the biologics, almost in a similar case as what we've seen with Otezla and orals in RA. And that's the first question. And I guess related to that, is there a higher safety bar for that kind of profile and/or maybe even a lower efficacy bar. So I don't know if that makes sense. But I'm wondering whether you see the potential, and obviously, this is going to be data dependent and we're still very early here. But do you see these potentially being used ahead of biologics?

I can answer first and Matthias can add to it. So when we think about the spectrum, at least in terms of asthma, and we can also address it on COPD, there is this gap right now between inhalers and biologics. So I was part of a drug development for VP France, which is an oral agent blocking prostaglandin D2 and it failed in its primary endpoint, and therefore, was not taking forth, but that was a perfect example of an oral drug that would be a predecessor to phase or 2 biologics, stepping up the biologics. In the pulmonary role, the allergy role that takes care of the vast majority of asthmatics, we are very comfortable with inhalers and nebulizers. Our patients are very comfortable in inhalers, they use them every day. And so it's a very easy segue to move to a nebulized treatment like this. The jump to subcutaneous therapy or intravenous therapy has not been as easy. And certainly, that requires patients to come into the office for their initial injections. The field has now transitioned to self-injection, which has helped, but still there's a pretty substantial gap in patients that are uncontrolled asthmatics that we just wish we had one more kind of step up before we stepped up to an injectable therapy.

I would support everything that he said about that. I mean it seems easy to inject somebody, right? But the truth is most patients don't want to do that. The question then is inhaled therapy, they're used to this, most of these patients, right? We're not treating people who have never had an inhaled therapy. So that is easy to do. The distribution of the ventilation will be interesting to study and you'll see how that goes. Is there a higher -- you asked, is there a higher safety...

Higher safety, lower efficacy.

Yes. So lower efficacy, I can't speak. I'm not the FDA. This is at the end of the regulators that like make that decision. In terms of safety, the worry -- and I'm not saying that is a terrible worry, but if you block a lot of the inflammation, is there still sufficient beneficial inflammation if you need it? And the answer to that question is I think we will find out. If you look at, for instance, RAGE knockout mice, again, this is mouse, it's not humans. If you look at knockout, mice, they can deal with pneumonias, et cetera, just fine. So it seems that if you have TLR4 still working, you get the initial inflammation, and that will resolve the problem with the infection but you don't get the prolonged not so good inflammation. At least from that data set, I don't believe that there is a higher level of safety, but because these things have been looked at.

Good. And then I guess, kind of following up on that a little bit, and this is sort of a conceptual question. But with respect to the dynamics around ARO-MUC5AC, how is this working? Does it just kind of hang around there and then really silences the MUC5AC when a trigger causes it to be overexpressed? Is that the way to think about it? Or is it really constitutive knockdown? And it remain suppressed during a trigger and an overexpression episode?

Well, so you need to look at 2 different situations, right? There is the situation where MUC5AC not been overexpressed as yet. And there, you would let the trigger sit there. I mean there is evidence that the trigger sits there, and will not allow MUC5AC to go up. If you already have a situation where you have a mucus plug, then that's another situation. There you need to get the trigger in the vicinity of those producing mucus cells, and then it will reduce the production, you still need to clear at 1 point in time that plug, right? Because this [iRNA] does not do that. But there are techniques to at least help doing that. And that will be interesting to see how much of existing mucus plugs are removable. As Mario showed, there are even spontaneously remove -- there is spontaneous removal of plugs in certain patients without doing much about that. So the question is, how does that go forward, but that's the idea.

Yes, I suspect clinically, this goblet cell metaplasia that drives the mucus in our patients is an inducible phenotype and that we can reverse by suppressing that the mucin production. When I take a biopsy in my severe asthma patients that they're just loaded. This is despite taking systemic steroids. They're just loaded with goblet cells. And so the idea here is we can reverse that phenotype and suppress that MUC5AC production. And we've had a lot of discussions about the mucus plug. And ultimately, think that the smaller airways are -- our path -- the pathobiology there is primed for this mucus to form there and that there's a fair amount of turnover that's going on. So even though those plugs persist, they are likely plugs that are forming and reforming over time and that with this technology, I think we can reverse that process.

Lisa Walter here, Senior Research Associate on Luca Issi's team at RBC Capital Markets. My first question is, may be for Chris Anzalone, are you ever going to show the human clinical data from the ENaC study? I believe you dosed about 24 normal healthy volunteers and 4 patients with cystic fibrosis. Will you share that in a medical meeting sometime this year or later on? Just wondering about that.

Yes. So we don't have plans of that right now. It's been difficult to interpret to be honest, just given the complexity of interrogating that. So we'll see. Right now, we don't have plans, but that may change.

Okay. And then just one more question. Just maybe more on your strategy here, from following Arrowhead, the strategy in the past has always going after -- has been to go after genetically validated targets or pharmacologically validated target. And then with these lung programs here, this seems to be a little bit of a divergence of the strategy and maybe perhaps opening up yourselves to a little bit more clinical risk. Just wondering what kind of made you change your thinking here? And what made kind of want to go after these targets that don't have a well-defined human genetic knockout component that you can rely on?

Yes. So I'm going to defer to these guys. But I'll tell you, I don't think it's -- we have -- I don't think it represents a departure from our strategy. Our strategy has always been to minimize target risk as we -- as much as we can and I think that these targets do that. If there are no target risk? Well, no, because no one has been able to intervene in these ways. But I think the evidence is compelling that should we safely and effectively knock down these targets, positive phenotypes will result. But again, I will defer to the experts on that.

At least in the common chronic lung diseases, we've been investing milliions, probably billions of dollars in genetics, and we have no treatment based on genetics at this point other than my colleagues, cystic fibrosis patients. So it is a tough part to, I think, at least in the pulmonary field to do it a medically-based targeting strategy. But Matthias?

Well, I agree. But what you also said, even in the GWAS, there was MUC5AC coming up, which is actually interesting. I'm actually surprised about that. But so it's not necessarily what you genetically validate, and you see that at COPD, if we're waiting for a genetically validated target, we're not going to get ever a treatment that changes COPD, for instance. But I think the targets are very well validated through programs, both clinically and preclinically that show how -- especially MUC5AC, but also Rage play very central roles in mucus plugging and inflammation in the airway. I'm not sure exactly what genetic proof that would require, right, to say this is a good target. I think preponderance of evidence shows that that's really important. And the MUC5AC knockout mounts, for instance, shows that mucociliary clearance is established or continue but these mucus plugs are gone. So it's not just a target of a mucus-producing cell because that will probably be detrimental. You need mucociliary clearance to clean your lungs and you need MUC5B for instance, to be there because otherwise, you're not going to do something good. Burton Dickie always says MUC5AC was "invented" for catching the worms that come out if you got -- and go through the lungs to come out, ask Chris [Indiscernible] basically, catch them and let them be there. So that has some reasoning that "half" MUC5AC, but in disease states like asthma, it's detrimental because you have these mucus plugs and in CF as well. So I think it's validated, maybe not genetically, but the preponderance of evidence shows that this is really highly relevant targets.

This is Sahil Kazmi From B. Riley Securities. First question is, can you comment a bit the relationship between MUC5AC and MUC5B. Is this a cooperative or additive relationship? And does the knockdown on MUC5AC have an impact on MUC5B?

So this is a highly complicated question. So we think about mucins, especially the gel forming mucins in airways specifically MUC5B and MUC5AC as mixing in the airways and provide you some sort of a layer of mucus that you can then remove. That is not as simple as it sounds because the major mucin that is on the normal condition produced is the MUC5B and that will maintain your mucociliary clearance. MUC5AC comes up in disease states. And there were data shown, I mean, I don't know whether they were clear, but MUC5AC is that sticky mucus, like I come back to the [Indiscernible] , to catch things, it's actually even tethered to the airway surface and therefore, very difficult to remove. So the MUC5AC is not a [Indiscernible] to think to remove things from the airway. This seems to be to hold things there. And so they don't perfectly mix. And genetically, they're not -- if you knock on down, the other 1 will not be knocked down. Now -- can you have too much 5b? I don't know, but this seems a new thing that you can then cough out and do it better, whereas the MUC5AC is making these rafts not going anymore. I'm not sure that, that answered the question that you really had.

No, no, that's very helpful. Very helpful context. And then maybe just one more question. In the context of what we saw in their ENaC program, acknowledging it's early days in IPF, but we're seeing some pretty good durability out to day 42 initially. How do you -- how long do you think that can go with a single dose?

You asking me? I have no answer to it.

Sure. We can...

Maybe you're asking about the MMP-7 or just in general?

Yes, exactly.

I think it's unclear in -- we actually have a duration study ongoing right now in monkeys with that trigger. If you look at the RAGE data, I mean, I think that's best case scenario where we're getting a single dose at 3 months of effect and then maybe less so durability with something like ENaC, which was in the order of weeks, call it, somewhere probably better than ENaC out to what we're seeing with RAGE. So that probably gives you a way to handicap it, but we'll see. We'll know soon.

This is [Indiscernible] from Cantor. Firstly, for the KOLs, is there a good evidence on what's the right cutoff for high and low levels of MUC5AC similar to what we have with eosinophils. And for the company, how do you plan to incorporate that strategy in your early Phase I trial to get a signal on efficacy similar to what you're doing with RAGE with high Th2, and I have a follow-up.

Well, I don't know if there's an absolute level because none of them have been clinically validated yet what the optimal level is. So I don't know if the company has any thoughts on that.

Well, yes. I don't think the -- but what is known is that the higher the severity, the higher the mucus plug, the higher the MUC5AC. So I think I will go more from the clinical phenotype as opposed to the biologic to identify patient might have more expression or overexpression, over pollution of the protein that we can probably affect. So I think that's the beauty of that program that you don't need to go through the detail. There is clinical features associated with higher exploration of MUC5AC and I think we're going to go after those initially.

And the ratio, right, the ratio of MUC5AC to MUC5B seems very important. So you cannot just say, "I want that amount of MUC5AC, it's the ratio. And if you reduce that and you rely on the 5B, that's where the transport of mucin or mucus gel is. In that sense, the less of 5AC the better.

And for the company, the number of doses, if I'm not mistaken, for the Phase I trial, they're more in MUC5AC so just curious if you would expect durability between the 2 targets, the number of doses that's been given in the Phase I trial.

Is 3 doses, 2 weeks apart. And what was the other part of the question?

Would you expect differences in durability between MUC5AC and RAGE?

Well, based on the preclinical data, yes, rate is about 3 months and counting, for MUC5AC is more in the weeks, 3 to 4 week span. So that's why we're dosing every 2 weeks, but one single dose. And in RAGE we're doing one dose, and we're going to learn clinically how long it lasts.

Right. So we'll follow those patients [Indiscernible], 113. Is that right?

Right. Then we follow RAGE, will follow-on beyond that. There's a threshold, they have to come back to a certain level. I would also measure -- mention that pre-clinically RAGE is very easy to follow because we have a serum measurable biomarker. It's been a little more challenging to track duration with MUC5AC in animals, specifically in the monkeys, I think we'll have a better luck with that, easier to measure MUC5AC in sputum in humans, even in healthy volunteers.

This is Madhu Kumar from Goldman Sachs. I guess the first one during dynamic range. So this is really for the company and the KOLs. What is the effective level of RAGE and MUC5AC knockdown that's needed to recapitulate complete genetic loss of function. And I guess kind of a corollary question to that is, do heterozygotes for RAGE or MUC5AC see kind of impacts in model systems to date?

I want to start with the -- so I am not aware of heterozygosity in MUC5AC or RAGE, whether anybody looked at I have no -- I can't answer that question. I don't know the answer on either.

Yes. As we showed preclinical models for MUC5AC, we're triangulating on-- we think we will need to achieve greater than 50% silencing for MUC5AC in order to have a protective benefit. We don't need to completely ablate it. For RAGE, the dynamic range is much higher. Right now, there isn't a single threshold that we would say that is necessary for an anti-inflammatory effect. We believe it's deeper than 50%, and we'll be aiming for as deep as possible because it's well tolerated.

Okay. Great. Second question relates to infections. You mentioned that certain types of pneumonia are not really affected by RAGE knockout, but other types of infections are, like RSV infections are affected by RAGE deficiency. Gram-negative bacterial infection are affected by RAGE, so kind of practical kind of clinical development respective, how do you think about kind of monitoring those types of infections in the real world as compared to laboratory, those animals aren't constantly exposed hopefully to RSV and gram-negative bacteria versus people in the real world are?

Do you want to answer that first?

Okay. Well, we didn't specifically think about that. But I would say something that to me clinically make sense. These are a chronic therapy, patients will be treated chronically with the intention to prevarication exacerbation likely can be due to RSV. So I don't see this as an acute event, let's treat this patient. I think this is like the biologics, like inhale steroids, you modulate the immune system with RAGE and modulate what happened with mucus with MUC5AC. And when the insole comes, that lung will be ready to response more properly. So that's the model of how we think this will work because there is a bunch of model that shows improving of prevention and exacerbation, you need to have background therapy to do that.

I do think it's a good question. I do envision that we're going to have to carefully monitor these patients in the early Phase II studies to look for that as a possibility. Fortunately, they're pretty rare in my adult patients. We see tons of RSV in infants, but we don't see it as often in our COPD population. So I think, unfortunately, it's going to be rare. And then the question is you knock it down to a level that still sufficiently protects you is the key question.

And we saw this question coming up in other biologics, right? I mean there -- this is not a unique one in terms of that. And they have been successfully put through by monitoring specific infections and making sure that there are protective ways in place. So I think this is a -- like you said, it's a good question, but it's not an unmanageable one.

Ellie Merle, UBS. Just in terms of thinking about the initial timing for getting some of this data, just given the fact that there is serum biomarkers, could we potentially see some of this healthy volunteer data such as the single ascending dose from the RAGE program perhaps this year? Or just any color on the timing clinical data disclosures, albeit and help us just even for the reads in terms of in the delivery and platform there, as well as could you update us on the timing of the COVID programs, just another interesting program as well as thinking about [Indiscernible] in terms of delivery? And then I have a follow-up question as well.

So the short answer is, yes, you could see data this year. But no, I can't give you guidance on when that will come. I think we'll start dosing healthy volunteers in couple of weeks. But until we start dosing, healthy volunteers and the patients and until we see how that recruitment is going, it's hard for us to give a guidance. But I think your sense is right. Our hope is that since there are circulating biomarkers that could have data on the early side and certainly this year's possibility. On COVID, I'd tell you, our data continue to be promising. Our choke point here is experimental models. They're difficult to come by any stage. And so that has slowed us down, unfortunately. But we're still excited about what we are seeing, and my hope is that we'll have some more data later this year that we could talk about and see where we are with respect to potentially nominating the candidate. We also didn't talk about other respiratory viruses. But as you can imagine, we are deep into others as well. And so we're not prepared to talk about those yet because we don't have guidance on timing yet, but those are half a step or still behind where we are with in [Indiscernible].

Great. And then just a question on the macrophage overload and the safety that you discussed and some of the learnings from the nonhuman primate data, as well as some of the rat studies. So given these would potentially be chronic dosing. Can you help us understand both the dose, as well as the time relationship to some of these inflammatory effects being seen and your confidence in terms of longer-term dosing even at, say, much more potent chemistry or lower dose levels and longer intervals between dosing that this wouldn't be something that happens over time?

Yes. Thanks. James, do you want to take?

Sure. Yes. I think -- and we'll see once we get the chronic tox study, but I think based on what we've seen in acute tox study and what we saw in the ENaC chronic tox study, where we were eventually in the rat able to achieve [Indiscernible] at a single dose every 2 weeks using the mid-dose for ENaC, we can spread that out quite a bit for both of the candidates for RAGE and for MUC5AC. So I think as long as we can stay below that threshold, the normal clearance mechanisms can do their thing to the extent that they need to, that we should be okay in chronic tox.

Yes. I think there are 2 really powerful slides to speak to that, well, at least 2. One is that one slide where James showed the various combined doses of ENaC superimposed on top of where that threshold is and it really sort of met expectations, right, where those where we had combined exposure that was higher than threshold. We saw this overloading for that group, where we're dosing just once a day -- 1 time every 2 weeks, we are underneath that threshold, and we had an OAL. That was compelling. And then compare that to what we for the dosing for MUC and for RAGE, we're substantially below it looks like that anticipated threshold. And so the modeling looks pretty good. We're excited about the -- and then -- sorry, and then the second slide that kind of speaks to that, that James also showed was the 2 plots of activity over time with ENaC versus RAGE. ENaC came back after whatever a few weeks as RAGE just clamped down. And so that gives us the ability to dose much less frequently. Certainly, we think -- certainly monthly, but maybe every 3 months, we'll see.

Joel Beatty from Baird. For RAGE, could you help reconcile the importance of dosing locally where -- while also being able to look systemically at serum ranges a relevant biomarker.

James?

Sure. All of the circulating sRAGE is coming from the lung. RAGE can be produced in areas outside of the lung in the setting of local inflammation, but in the healthy volunteer and in the asthmatic patients, first of all, when we administer via inhalation, we're only getting knockdown in the lung and essentially all of that sRAGE in the blood is coming from the lung. So there are no other significant extrahepatic sources.

Great. For MUC5A, what's the ability of the drug to make it through the mucosa and will presumably be quite common in these patients?

Sure. I think I have a slide that sort of addresses the overall physical chemical properties of the TRiM conjugates, and the MUC5AC drug is no different than the stereotypical 1 we're showing, very small size, net negative charge, soluble. When we go into models of new construction, we do a neutrophil elastase model, for example, where we invoke significant mucus secretion, we can dose our drug and still see pharmacodynamic response and not down. So we're confident that delivering to a higher mucus environment, we can still get mucus penetration and not debt. All right. Just one more question.

[indiscernible] from Jefferies. So is the macrophage overload an issue when administering subcu? Or is also the same thing? And then do you see differences in the overload based on the targeting ligand or just on all of the RNAi? That is for James.

James. So the question is the macrophage overload relevant to subcu?

Right. There still is some macrophage uptake of the drug in the lungs, in other tissues when administered subcu, it's really more of an issue when it's administered via inhalation. So that's really when it's the easiest just given the high concentrations to overload the macrophages.

Got it. And then for the KOLs, one big picture question, like you're targeting upstream. So I think obvious risk for infection, rather down the line when the knockdown of RAGE?

Can you repeat the question?

Like if you're targeting very upstream with the RAGE and then are the obvious risk downstream, for opportunistic inflation.

The answer is we don't know. I mean we know that at least in the animals that Erik presented for a year, there was no specific increase, but these were animals and they were in a laboratory, not kept germ-free but not that. So we are not certain. There are doubles discussed mouse knockout experiments with pneumonia, for instance, that didn't influence the outcome actually, if anything, in a beneficial. There might be other infections. So I think it's -- is there a potential risk? Yes, but that's true for any biologic that you use, TNF-alpha inhibitors classically, but they have similar risks. You just need to -- you need to understand them and then deal with that.

Okay. Well, thank you all for coming.