Viridian Therapeutics, Inc. (VRDN) Earnings Call Transcript & Summary

Good day and welcome to the KOL call on MRG-229 for the treatment of idiopathic pulmonary fibrosis hosted by Miragen Therapeutic, Inc. I'll now turn the call over to Dr. Bill Marshall, Chief Executive Officer of Miragen Therapeutics. Please go ahead, sir.

Thank you very much. Good morning, everyone, and thank you for joining us for this key opinion leader call on MRG-229. We're happy to be able to host this webinar call this morning and fortunate to have a couple of very important key opinion leaders in this area join us today for a discussion of some recent discoveries that we've made at Miragen Therapeutics that we believe are going to be important -- potentially important in the future treatment of idiopathic pulmonary fibrosis in the development of MRG-229. Joining us today is Naftali Kaminski. Naftali is the Boehringer-Ingelheim Endowed Chair -- Endowed Professor of Internal Medicine and Chief of Pulmonary and Critical Care and Sleep Medicine at Yale School of Medicine. Naftali has been studying the disease for his entire career, and he really is an internationally renowned expert in the genomics of pulmonary fibrosis and other advanced chronic lung disease. We are very fortunate to collaborate closely with Naftali on the development of microRNA-29 replacements as part of an NIH CADET grant. We've been working together for several years on this. So we're excited to have Naftali join us today to add some color to the importance of microRNA-29 in development of pulmonary fibrosis based on the seminal work that he's conducted in his laboratory. We're also very fortunate to have Teresa Barnes join us this morning. Teresa is a research advocate who has been working in Capitol Hill for many years, advancing the clauses for pulmonary research funding. She will be discussing sort of an introduction to the disease and also give the patient perspective on this disease and the high area of unmet medical need that we have in idiopathic pulmonary fibrosis. Finally, from Miragen, we have Rusty Montgomery. Rusty is our Director of Research, and Rusty has been working in microRNA-29 biology really from the very beginning of this program. Miragen discovered the -- in collaboration with the investigators at the University of Texas Southwestern Medical Center, the important role of microRNA-29 in regulating pathway control over the pathologic fibrotic process. From that point, we've been able to develop molecules that are able to replace microRNA-29 and really resulting in a reversal of the pathologic fibrotic phenotype that we've seen across multiple different conditions. While we first discovered this in the setting of cardiac fibrosis, subsequent to this, there have been multiple independent publications from all around the world that really point to microRNA-29's key role in regulating fibrosis. And we have subsequently created a first-generation molecule called remlarsen. That molecule is currently in a Phase II trial in dermal fibrosis settings, and we believe that remlarsen has an important role to play in local and topical administration applications for antifibrotics. Subsequent to this, Rusty will describe the work that we've done to be able to really push things forward and move to a next-generation compound. This is MRG-229, essentially a more highly chemically modified species that's been now enhanced for targeted delivery with a specific conjugate. And this has allowed us now to really contemplate moving towards systemic applications for microRNA-29 replacement in pathologic fibrosis. And again, Rusty will spend a fair amount of time introducing the topic as well as the path that we took to arrive at MRG-229. We're very excited. Today is -- the work that we'll present today is work that allowed us to fulfill some important milestones in our NIH CADET grant with the NIH and Yale, and we were able to receive some additional milestone funding that positions us to move the MRG-229 towards the next logical state of development. So with that introduction, I'm very happy to welcome Teresa Barnes. And Teresa, as I said, will be giving us an introduction to the disease as well as some patient perspective on idiopathic pulmonary fibrosis. So welcome, Teresa. We're happy to have you here.

Thank you, Bill, and thank you, Miragen, for the invitation. I really appreciate it. As you can see on the slide, I really believe that we need to -- in every step of the way in drug development, we need to think about the true end point, which I believe is the patient. I think that everyone would agree that it is. One of the -- what I believe the most important things in the process of drug development has been sort of this sort of new thing that companies and the FDA and also they've come together in order to advance the process and also to understand the end user better. I was a fellow of the Drug Information Association, the DIA, about 6 years ago, and I remember when that was a fairly new concept. So it's great to see that taking hold and to see that the patients are involved in -- at least in the minds of those that are developing new therapies, at every step of the way. I'll get the next slide. Here we go. So I put this slide up because it really says to me what I've known for years, and that is that there's a gaping hole of need in the IPF space. And truly, there needs to be a lot of advancement in drug therapies and other ways of treating patients, and I don't think anyone would argue that looking at the numbers of deaths from the disease. I think given this current, really incredibly difficult time in our world, there has been a little bit more attention towards fibrotic lung disease as a result of the complications of COVID. The SARS portion of the COVID complications is an important part of it. There have been many published papers that show the direct impact on the lungs and lung fibrosis. So there's a question in my mind of will there be more IPF interest in not only short and long-term survivors but in drug development. And perhaps, the NIH and others may consider looking at doing survivorship studies so that we know if the fibrotic component that we're seeing continues and if we can follow those patients in order to better develop therapies in the future. I want to talk a little bit about the current state of affairs. So right now, as most of you know, there are 2 drugs indicated for IPF that are approved and on the market. Both of these, we're grateful to have. I actually spoke at one of the meetings where the FDA was reviewing one and was very clear about the desperate need that we have in this space. Where there -- as you know, also, there are no devices cleared for treatment of IPF, which is really sort of shocking given that there are many devices that are used for different heart diseases and so forth, that we still have nothing like that either, and that lung transplantation is currently the only life-saving therapy that's available to patients. And of course, it carries with it extremely high risk. Most patients who have IPF are not candidates for that procedure, and about -- only about 1% or so of those patients are -- or of any patients are considered for transplant even at all. So transplant is not necessarily the answer for the disease. And if you look at the numbers over the last few years of transplant -- lung transplant in this nation, it's definitely rising. 2019 was an all-time high of lung transplantation, with 2,700 -- just over 2,700 patients transplanted, which is a huge step in the right direction. In 2018, it was just over 2,500. And just in 2017, it was 2,478. So with a population of patients though that are -- 200,000 diagnosed, and there's a huge argument that there are many more that are not diagnosed, and 50,000 patients who die each year, they were grateful for transplant as an option for our patients. It's not an option that's available for those different patients. So we've got to consider all options, have every possible option on the table for these patients. So I'm happy to hear about the data that Miragen has and the promising future of this disease. I also want to talk a little bit about my personal perspective on the disease. In 1996, when I was in college at UNC Chapel Hill, my father was diagnosed with a disease that I've never heard of. I was already a working news reporter, and I've done a couple of stories on patients who've gotten lung transplants. So immediately, I started looking to see if my dad was a candidate for transplant. He -- they told us that because he was 59 years old and the cutoff at the time was 60, that he would not survive long enough to be transplanted and that he would be too old by the time that a lung became available. Fortunately, he was transplanted. An organ was found, and he was transplanted, but he did not survive it. And we thought his case was isolated. There was no indication that it was anything other than maybe his history of smoking. In 2000, my -- I helped start a new medical device company with a transplant surgeon. And at the same time, I helped start a nonprofit for the IPF community and was very involved in its organization and served on its Board for 6 years. And then in 2001, my dad's brother was diagnosed with IPF and later died within about a year. That was the first indication to us that the disease may have a genetic impact. So we joined a research project at Duke University, looking at familial IPF. And through that study, my dad's only sister was diagnosed with IPF as well even though she was nonsymptomatic. She had surgery about a year or so later for a knee replacement, and she ended up going into respiratory distress and died. In 2005, my dad's youngest brother, who was in his 50s, was diagnosed with IPF, and he died within 2 years. And then in 2007, my dad's oldest brother died of IPF. That was 100% of my dad's generation. So that same year, I decided to join the nonprofit that I'd helped create as the Vice President and to work full time on this disease because of its impact on my family and also because of the impact that I was seeing in patients that I've gotten to know because of my work. In 2012 and just prior to that, I joined the Board of the American Thoracic Society. I was the Chair of their Public Advisory Roundtable, their patient voice for 5 years. During that time, the ATS decided to take an idea that I had talked to them, asking them to study the disease and to bring researchers together across organs, looking at various forms of fibrosis in heart, liver, kidney, skin, lung. And they did that, and it was a huge effort forward. In 2014 -- actually, in 2007, I joined the Board of the Westie Foundation of America. It's a group of dogs you may be familiar with, the West Highland White Terrier, and it turns out that the terriers were high risk for naturally occurring form of the disease. And so that's a sort of look at my past and why I'm involved in the space, and I spend an awful lot of time still working to try to increase research dollars so that the researchers have what they need and also helping to support the companies and people that are trying to change this. We -- if you add up the numbers of the deaths we have per year and you look back for the last 20 years since I've been involved in this space, there have been 1 million patient deaths. That's too many, and we've had 16 or more IPF-related Phase II and Phase III drug trials that failed. Last year, at the American Thoracic meeting in Dallas, Dr. Gary Gibbons, who is the Head of the NHLBI, announced a drug discovery milestone, and that was that IPF would be a moonshot at NHLBI. I know that still -- they're still looking at ways that they're going to bring that to fruition, but that was a huge signal to me that the world is paying attention. And so the fibrosis across organs, fibrosis across species efforts, I think, have really helped move the needle. And having the researchers, including Dr. Kaminski, involved in those efforts has been pivotal. I believe that those early -- the earlier we can look at naturally occurring disease in domestic animals, potentially the faster that we can translate these efforts into humans. And I believe that the multi-organ impact, as NIH stated, it held an IPF workshop in 2012, 6 months after we held the Fibrosis Across Organs meeting with the American Thoracic Society, and NIH was part of that meeting. They held a workshop, and they pulled out our efforts in fibrosis across species and fibrosis across organs as ways forward as priorities for NHLBI going forward for the next decade, and we're 8 years into that decade right now. I think the biggest, most important thing to me that we've made progress in is not only the excitement of drugs like Miragen, but also that there's a patient-focused drug development effort. And thanks to the FDA's efforts to understand the patient's process and the path that the patients take and how difficult it is for them just to breathe, just to walk, just to function every day is a true challenge, and they are heroes. And to have them involved in the development is something that potentially could change their lives is incredibly important. I think the ability to fail or achieve success faster is critically important to these patients and to generations to come. And I can tell you, I don't sleep at night. I have a 9-year old. My goal is to make sure that my child never has to worry about this disease. So I want to thank all of you and Miragen for inviting me and for allowing me to speak to you today. I appreciate your time.

Thank you, Teresa. It's such a personal story, and we really appreciate all your commitments to these patients. This is an area of large unmet medical need, and we're very excited as a company to bring forward what we hope will be a differentiated approach to patient treatment. Thanks again for participating today. Now we're going to move on to a presentation of the most recent observations with MRG-229 and a little bit of a background on microRNA-29 in general. And we'll introduce Rusty Montgomery, who's our Director of Research and the -- really has been working on this program from its inception. So welcome, Rusty.

Yes. Thank you, Bill, and thank you, again, Teresa, for that story. Clearly, anything we can do in the space to contribute to the advancement of therapeutics in this disease is of most importance. So what I'll try to do in the next few minutes is give an overview of what Miragen has been doing in this space as a way to bring microRNA therapeutics to the indication of idiopathic pulmonary fibrosis. I'll give a couple of slides on the introduction of microRNAs as they're a little bit different than conventional therapies and how we believe that these approaches might be applicable for a disease like IPF that has been so devastating to date. So microRNAs, from an evolutionary perspective, are selected to regulate a specific network of genes. And miR-29 is one of the poster childs for this. What we've seen to date in the last 20 years of research or so in microRNAs, in general, is that they appear to be dysregulated in many diseases. At steady state, they control homeostasis, and the dysregulation of microRNAs themselves in these disease states results in alterations in downstream biological pathways. And miR-29 is kind of a highlight for that, which I'll talk about in the next few slides. And so because of this, the ability for microRNA-targeted therapy to be focused on disease modification itself, we believe that these are particularly suited for complex, multigenic disorders. So on the right, where you see conventional therapies, whether they're small molecules or antibodies or siRNAs against one given target, microRNAs from an evolution perspective bind multiple targets within a similar pathway. And for miR-29, it's really up and down the collagen synthesis pathway. So instead of binding a single target, it really binds every -- almost every aspect, up and down the collagen synthesis pathway. So as such, miR-29 is considered one of the most antifibrotic microRNAs. What we and others have shown is that its expression is reduced in nearly all pathological fibrotic conditions from ocular fibrosis to pulmonary fibrosis, cardiac fibrosis, liver fibrosis and NASH. Nearly any indication where there's a fibrotic response or fibrotic condition, miR-29 expression is reduced. And what we and others have shown over time is that it does this by inhibiting a number of different mechanisms that includes TGF-beta activity, epithelial-to-mesenchymal transition, fibroblast-to-myofibroblast transition and extracellular matrix production. And with this, it kind of inhibits every step of the fibrogenesis pathway. And so the simplest way to think of this is that miR-29 has downregulated in the disease, this results in an increase in TGF-beta production, extracellular matrix production and results in fibrosis. And so for us, putting miR-29 back into the system, it intersects this feed forward loop of TGF-beta and extracellular matrix production and thereby reduces the fibrotic response. So Bill referenced remlarsen, which is our first-generation miR-29 mimic, and I'll highlight just a couple of pieces of data associated with remlarsen because, as Bill mentioned, as you think about where micro-29 replacement therapy could intersect a fibrotic response, it's quite vast. There are topical ones like skin as well as a number of systemic ones like lung and liver as well. And so what we did is we did a proof-of-concept Phase I trial using remlarsen in incised skin in normal healthy volunteers as a way to look at microRNA therapeutics and mimics as a new modality. Can we translate the pharmacodynamic effects that we observed in preclinical models? Do those extend in the human setting as well? And so what we did is we gave an incisional wound on the lower back of normal healthy volunteers, and we basically asked do these targets that we know miR-29 regulates, the collagens, the TGF-betas, the MMPs of the world, are they dysregulated in this incisional wound system? And so what you're looking at on the blue bars on the left there of the graph, you're seeing that if you make an incision in the lower back compared to unwounded skin, you'll get an increase in these targets, which through all preclinical data, we knew miR-29 could regulate. And so then we put remlarsen into the system as a single dose following an incision, looking at day 5, and we could see that putting remlarsen, this first-generation miR-29 mimic, around the incision could actually engage the target of these collagens and these TGF-betas and downregulate their expression. This is at the mRNA level. What we could see is that not only could we see pharmacodynamic activity at the RNA level or the mRNA level, but this actually resulted in a significant reduction in fibroplasia. That's what's quantified on the right that was performed by a mass pathologist. What was interesting here is that there was no effect on normal wound healing, which is great to see, nor effects on epithelialization. It was mainly effects on the fibroplasia, which is considered an early indicator of scar formation. So because of this, as Bill referenced, this is now moving forward into a Phase II clinical trial for keloid formation. So then what we did, as we said, we asked the question, do these effects that we're seeing in the skin of these healthy volunteers of our preclinical models, do they extend to internal organ fibrosis as well? And so this is a heat map looking at biomarkers where red has increased in expression and blue has decreased in expression. And we created a subset of genes where remlarsen has a certain signature in mouse skin, anti-miR-29, which would have the opposite effect has a certain signature in mouse skin. And we said, how do these -- how does this signature, which we know our compound can regulate, how does it translate across organs? And so we looked at the day 9 to day 16 human incised skin, albeit it's much stronger, but you can see there's a pretty good overlap on what happens in human incised skin compared to what happens if you just inhibit miR-29, and what we know there is that remlarsen can regulate that signature. And as we move to available data sets to look at human disease tissue versus normal tissue from systemic sclerotic skin to lungs of systemic sclerotic patients to lungs of IPF patients, you can really see that the overlap of the genes from the incisional wound study we did in Phase I clinical trials, the inhibition of miR-29 and incised skin, the overlap on gene signature is quite strong between the lungs of IPF patients and what we were able to identify as pharmacodynamic biomarkers from our preclinical models as well as our Phase I clinical trials. So this gave us support that the ability to regulate these, which we know we could do in the Phase I clinical trial, might extend to those internal organ fibrotic diseases. And as we've discussed already, the real focus of that is, can we have an effect in pulmonary fibrosis. In collaboration with Naftali, early on, we did quite a bit of studies looking at miR-29 expression itself. There are 3 miR-29 family members, miR-29a, b and c. And what you can see here is that compared to nondisease controls, all 3 family members are significantly downregulated in the lungs of IPF patients. And Naftali is going to discuss this type of detail and disease progression a little bit more following my talk, but it's really a hallmark that miR-29 is downregulating in these patients. And can we put miR-29 back in the system and see an effect on the disease progression? The other thing that we've done, and this is unpublished data, with Naftali as well is we looked at circulating expression of miR-29 and how it correlates with survival of IPF patients. And this was some studies that were done back when Naftali was at Pittsburgh, but what you can see in the graphs on the right is that if you look at the above cut point on a Kaplan-Meier transplant-free survival curve that if you have increased or higher expression of miR-29, it correlates with survival of IPF patients. And on the right, you're looking at serum MMP-7, which is one of the most well-known at least targets for circulating, a correlate for survival in IPF patients, and there's an inverse correlation there. MMP-7 is not a direct target of miR-29, but this really highlights that it's not necessarily a global dysregulation of RNAs, that it really is miR-29 increase or higher detection really does correlate with survival moving forward compared to what we know or the downregulation of MMP-7. It correlates with poor outcomes. So as we move forward to think about how these therapies for miR-29 mimics could translate to therapeutics for pulmonary fibrosis, we started with remlarsen, obviously. Remlarsen worked as a very good miR-29 mimic. However, the data to date show that we needed very high doses to get it to the lung. Remlarsen does demonstrate antifibrotic activity in preclinical models of bleomycin models as well as a number of other models that Naftali has run in his lab. But what we really came down to is that this is really not amenable for systemic administration. Getting it down to lower doses did not really result in the efficacy that we needed to move forward. So because of that, we developed additional miR-29 candidates that really kind of highlighted the systemic bioavailability and delivery to the lung. And as we think about microRNA mimics, this required quite a bit of medicinal chemistry optimization for enhanced stability while still maintaining its ability to act as a microRNA once in the cell. And then with that, we've done a number of different targeting conjugates to get these to the delivery -- to deliver these to the tissue and cells of interest that we believe are most relevant. So this is a schematic on how we did this. If you're looking at the next -- this is the schematic for the next-generation miR-29 mimics for systemic administration. You can see remlarsen is the parent compound. The more color basically references more modifications. Remlarsen has a cholesterol conjugate to it, and this compound demonstrated mechanism of action and validated this method of microRNA-29 mimicry in human clinical trials. So we took a step back there and said, how much can we modify this compound where we can increase its stability? Can we improve its potency without making it too rigid that it's no longer functional as a microRNA once in the cell? And so we did is we took the conjugate off and we made all the stabilizations and said, can we optimize this for stability and activity and maintain activity? And then once we achieve that, can we then add specific targeting conjugates to it and now get optimized biodistribution and delivery in vivo? And we'll cover that here in a second. So this is a slide that kind of highlights some of the first steps we do when we look at these next-generation compounds, and it really highlights that MRG-229, and that's what we call the next-generation compound here, is a potent inhibitor of direct and downstream targets in normal human lung fibroblasts. So these normal human lung fibroblasts are treated with TGF-beta, which you can see in the black bars. Whether that's untreated or mock, these are transfected. And then if you add increasing amounts of MRG-229, you could see a dose-responsive downregulation of collagen 1A1, which we used as an initial readout for activity. On the right, you're looking at alpha-smooth muscle actin, which is not a miR-29 target. So this is a readout for downstream biology. And so not only stabilizing these compounds can we see effects on direct targets, but we are able to see downstream effects on alpha-smooth muscle actin and other profibrotic compound -- or profibrotic molecules as well. So we took a step back at this point and said, can we deliver these passively, so without active transfections, and kind of take an unbiased approach? So this is looking at a fibrotic gene array, again, where orange or red is upregulated, blue is downregulated. TGF-beta on the left, that's a signature that you get with these gene arrays. In normal human lung fibroblast, you can see a number of genes go up, a number of genes go down. And then on the right is comparing -- that is comparing that signature normalized to the TGF-beta-treated signature. And you can see that treatment with the miR-29 mimic, these next-generation compounds can completely reverse the profibrotic signature induced by TGF-beta. So this is really exciting for us to see that beyond just direct targets, if you look at these unbiased profibrotic arrays, we could actually completely normalize the TGF-beta signature. Again, that's at the RNA level. So then we went on and asked the question, how does this affect actual collagen production, how does it affect collagen secretion in these cells? And so we've done this in normal human lung fibroblast as well. But what you're seeing here is looking at pathological fibroblast from a patient that had IPF. And what you can see here is if we add TGF-beta to these pathological lung fibroblasts, you do get an increase in secreted collagen. This is assessed by looking at the supernatant or the cell media, and we have a method to look at collagen production or secretion in this method. So you can see an increase in collagen secretion with TGF-beta, which then can be dose-dependently downregulated with MRG-229. So not only can we normalize the TGF-beta signature at the RNA level, this results in functional effects on oncology and secretion and not only normal human lung fibroblasts but pathological fibroblasts as well. And then together with Naftali Kaminski at Yale and Maurizio in his lab, we then looked at how this affects fibrosis in human precision-cut lung slices. And so what we did here is we treated human precision-cut lung slices with a 4-compound cocktail, profibrotic cocktail, which you can see in red there, at 120 hours, this is collagen content by histology. You can see a robust increase in collagen content by histology in these precision-cut lung slices with this profibrotic cocktail. And here, you're looking at 2 different compounds, MRG-229 being compound 2 here. As you can see at this -- in this graph, you're seeing that 2 different compounds of miR-29 mimics that we tested, including MRG-229, have a nice -- a robust response in blocking fibrosis in human precision-cut lung slices, which is really great to see. So not only can we see blockage of the TGF-beta-induced signature, we can see it at the collagen secretion aspect, but we can also see it in these human precision-cut lung slices treated with profibrotic cocktail. So as we move in vivo -- again, these are -- all the data on the previous slide are with the targeting conjugates. So these have a targeting conjugate on it. So we asked can these next-generation compounds with a targeting conjugate, can we have an effect in bleomycin-induced fibrosis? So what you're looking at here is a readout of a therapeutic dosing regimen where bleomycin was given at day 0, MRG-229 initiated at day 10, was given twice a week. This was necropsied at day 21. And we have a way to assess a whole slide scanning quantification of collagen. And you can see on the graph on the left, bleomycin, you get a significant increase in collagen by whole slide scanning imaging. And then you can see in the green bar there, MRG-229 is able to greatly and significantly reduce total collagen quantification. On the right is a zoomed in histopathological image. This is false-colored for collagen content. And what you can see here is that the blue really represents the collagen staining compared to the red of the normal alveolar tissue. And you could see that there's -- you get a lot of dense cellular infiltration. You get a lot of loss of alveolar space, which is very typical of what you would see in bleomycin-induced fibrosis, and you can contrast that with the bleomycin on the bottom where we treated that with MRG-229. And you can see that you still get normal alveolar structure, albeit there is quite a bit of cellular infiltration, which is you would expect with bleomycin. But even in those denser regions of cellular infiltration, there's not near as much blue as you see in the control image above. So we really do believe that this is a robust inhibitor of the fibrotic response in vivo as well. So those studies were mainly done by intravenous administration. We then went on to say, are these doses relevant for a distribution at lung and moving forward? And can we give this by subcutaneous administration? So what we did is we compared subcutaneous administration to intravenous administration and what you're looking there on the left is distribution of the compound itself. And you can see from 2, 5, 10 to 20, we get pretty equivalent distribution to the lung whether it's subcutaneously administered or intravenously administered. And on the right, as you're looking at profibrotic gene markers that we use as readouts for activity, and IV on the right, which we know works really well in these bleomycin models, 10 mg/kg is kind of the cutoff for IV, we asked how well the subcutaneous administration affect these profibrotic markers, which contain the collagens, again, TGF-beta 3, MMPs as well and these other growth factors that we know are really relevant for the fibrotic response. And as you could see, we see a nice dose response as you move from 2 to 10 mg/kg, where you're starting to see really nice effects at around the 5 mg/kg range via subcutaneous administration on the ability to downregulate a lot of these profibrotic markers in the bleomycin setting in the mouse. One of the things that we began to look at, at this time was really -- is can we have -- is there any potential to look at biomarkers as we think to move forward into the clinic and readouts there. And so we began to look at a number of different potential biomarkers, both in bronchoalveolar lavage fluid as well as in serum, and these are 2 of the ones that have come out so far, IGF-1, which is a miR-29 target. And we've shown this in previous data with first-generation compounds. It's that miR-29 mimic administration is able to downregulate IGF-1 in BAL fluid, which is really nice to see. But in addition, we're also seeing effects in circulated serum biomarkers as well. And this is pretty exciting because TIMP-1 is not a miR-29 biomarker. So this is really looking at downstream effects and the potential to look at biomarkers not only in lavage but also in the circulation. And so since these data originated, and I think it was something that Teresa highlighted as well from clinical trials is the safety concerns associated with some of the compounds to date. And so what we did is looked at a couple of different toxicity studies. We did a rat study where MRG-229 was dosed intravenously to achieve the highest Cmax, and we gave these rats MRG-229 at 3, 10 or 30 mg/kg, 5 doses over 15 days. And the results of this were no test-related gross findings, no organ weight effects, no findings associated with MRG-229 in hematology, coagulation, urinalysis or any serum chemistry parameters. There was a little bit of minimal test article-related basophilic granularity in the tubular epithelium of the kidney. That was in one high-dose animal and is not atypical for oligonucleotide therapeutics. But beyond that, there were no test-related article -- test article-related findings in other tissues. We then did a mouse study where we looked at it even further at the high dose, 30 mg/kg, where we dosed it up -- for a month or 4 weeks. And in this study, we saw no test-related gross findings or organ weight effects. And again, in 4 weeks of 30 mg/kg dosing twice a week, we saw that no significant changes in hematology or serum chemistry parameters compared to vehicle control. So for us, in these initial rodent toxicology studies, even up to 30 mg/kg at frequent dosing, we're really seeing no effects from a toxicity standpoint. Because of that, we are currently initiating a nonhuman primate dose range-finding toxicity study. And because of the effects that we observed -- or the lack of effects we observed in the rodents, we chose to increase the dose here. So for the nonhuman primate study, we are dosing 5, 15 and 45 mg/kg IV, again twice a week for 2 weeks, so 5 doses over 16 days. And like we said, we are currently initiating that study. One of the things we looked at for outcomes from the toxicity study was try to gain some insights into PK and lung distribution of MRG-229. On the right is a 10 mg/kg MRG-229 dosed IV, plasma concentration first dose versus last dose. It has similar plasma half-lives compared between MRG-229 and remlarsen. However, because of the targeting conjugate, we believe, it has superior tissue uptake compared to remlarsen. And as we think about targeting to the lung, we really tried to assess how well the distribution to the lung is for MRG-229 compared to remlarsen, and it's about a threefold increase in the lung compared -- for MRG-229 compared to remlarsen, the difference here being the targeting conjugate for specific cells is different than remlarsen. So even there's -- the data to date suggests even a threefold increase has resulted in at least a tenfold increase in potency for MRG-229 in mouse bleomycin studies compared to remlarsen, and it's an area we're still actively pursuing. So where we are now, again a summary for the preclinical data of MRG-229 or miR-29 replacement for IPF. Again, miR-29 expression itself is reduced in lungs of IPF patients. Its circulation correlates with survival. These next-generation stabilized mimics, they demonstrate potent target pathway regulation in normal human lung fibroblasts. It regulates collagen secretion in pathologic lung fibrosis as well as shows an antifibrotic effect in precision-cut lung slices. And as we move from those in vitro studies and ex vivo studies, we've seen numerous times that it blocks fibrosis in bleomycin-induced pulmonary fibrosis with increased potency over remlarsen and can be given by systemic or by intravenous or subcutaneous administration. And then the other area of interest that we think is very exciting is the biomarker discovery as we move it forward into development. And then again, one of the highlights here is that the tox studies to date has shown really no adverse effects on organ histology, hematology, clin chemistry, coagulation, urinalysis even when dosed up to 30 mg/kg twice a week for 4 weeks. And so we are still, as we said, advancing to nonhuman primate studies now and increasing the doses there as a maximum tolerated dose was not achieved. So that is a summary of the preclinical data for MRG-229 in IPF. Now we can turn it over to Naftali Kaminski.

Thanks, Rusty. I appreciate that nice overview of the MRG-229 program and sort of the story behind how we moved from sort of concept into what we think is a very exciting potential product candidate in IPF. Now we're very happy to welcome Naftali Kaminski to give us some additional color around microRNA-29 and its role in the development and progression of pulmonary fibrosis. Welcome, Naftali.

Thanks for inviting me. For me, this is a really exciting story. Around I think 7 or 8 years ago, a little bit more, I decided to be more involved in increasing shots on goals and identifying therapies and creating active and dynamic collaborations with industry. And I still remember the first time that I sort of show a Miragen presentation. And looking at my own data and seeing that miR-29 was the most increased gene in the IPF microRNA, the IPF plan initiated this collaboration, and we were lucky that NIH, NHLBI was willing to fund us through the CADET II program and really excited to see how this program has increased. What I want to speak today is something that's a little bit more complicated. One of the problems is when we look at regulatory molecules like miR-29, it's really hard for us to understand where do they fit in. Do they contribute? Are they just a marker of destroyed lung? Or are they regulating processes, active processes in the human lung? And in the next quick few slides, I'll basically mention some of the evidence that we've generated. So basically, do we really know -- Rusty has shown you data about mice and animals and ex vivo and also the fact that miR-29 is indeed decreased in the IPF lung, but do we know if it has a role during disease progression? And what we -- there's no way you can do 2 biopsies on a patient. It's very rare that we do even one. So the approach that we took, and this is a small part of the work in my lab, but sort of interesting is we actually took a unique feature of the IPF lung, which is that normal lung is interspersed with fibrotic lung. And actually, when you take an IPF lung and slice it out, you could actually see areas that look like normal lung. You could see areas that look like more progressive disease and areas that look like end stage. And what we -- and actually, this was John McDonough and his team in Leuven and then in British Columbia that developed a method that you could actually quantitate these micro-CT images and come up with a measure that anti-correlates with the degree of fibrosis. And basically, when you take -- this is called alveolar surface density, and when alveolar surface density goes down, it means that fibrosis is up. And then what John did and my team did is basically looking -- we basically obtained samples from the same lung from multiple people on which we had multiple samples of different regions affected, non-affected and we had also normal controls. And then when we look at the distribution of alveolar surface density, we saw some clear things. One is some people were -- the normals, normals without IPF, all clustered together. The samples with the most severe fibrosis also clustered together. Then there was a group of intermediate fibrosis, we called them IPF2. But there was also a group that was fascinating because they looked exactly like normal. So if you look to their histology, you couldn't see disease, and they overlapped with normal. So what we hypothesized is basically if we could look at these 4 groups, control and then IPF1, IPF2 and IPF3, within the same line, we could hypothesize that this represents disease progression, so basically going from control to non-affected lung to moderately affected lung then to severe lung. The next thing we looked at then we asked ourselves, is the normal lung in IPF really normal? And when we looked at it, we saw that 900 RNAs are basically differentially expressed in all stages of IPF. So even when the lung is completely normal, if you look at it, you cannot tell, it's already changed, and there's around 459 genes that are decreased and 450 genes that are increased. The interesting thing there is actually miR-29 is among these decreased transcript. In the normal lung, it's already decreased. And the other interesting thing is that within those genes that are increased in IPF, there's many miR-29 targets. We then applied the systems biology model using -- in collaboration with Ziv Bar-Joseph at Carnegie Mellon, and basically, the idea was that, again, we can use control as sort of the starting point, IPF1 at the progression point, as the initiation point in the disease; IPF2 as sort of disease progression; and IPF3 as model. And as you can see, and it's not really important, we saw this was published in JCI Insight a few months ago, you can basically see that there is distinct patterns of gene expression, genes that go up early in the disease, genes that go up later in the disease, genes that go up constantly and the same thing for the genes that go down. So by looking at that, we could start looking at regulations. So are there molecules that regulate distinct events? And if we look here, basically, the pathway that you see highlighted is actually pathway A and B. It contains most of the fibrosis genes that you know. It is upregulated early in the disease and continues to go up. And it is dominated in the nodes won by regulators. And if you look at the slide, the sort of the highlighted green area, actually, miR-29 is identified by the software by our algorithm in an advanced way as a key regulator of tracks A and B, both early and late. And what has been -- what are -- and then -- and you can look, this is the expression of miR-29. It's significantly decreased in all stages. But what is interesting, it regulates the gene to join the progression of disease and just -- if you want to know what are the gene families in tracks As and B -- so basically, this is track A and B, and you could see that a lot of this is extracellular region, heparin binding, chemokine activity, extracellular matrix organization, proteinaceous extracellular matrix. So basically, whatever you think of -- what you think about fibrosis -- and I'm just showing a few molecules. And the cool thing that this data is also extremely overlapping with what we found in some culture experiments in the animal experiment. So here, you see data of the dynamic progression of fibrosis within the human lung that suggests that miR-29 is regulated. If you -- and basically, based on this and that paper and the -- in JCI Insight that you can look up and you can also find the data in our IPF Cell Atlas that you basically screen the QR codes, you can find it. If we hypothesize that what we have is basically several patterns of regulation of gene expression, and miR-29 is among these regulators that actually starts being abnormal early in the disease and then continues to be abnormal. And this is important because this would suggest that an intervention will not only have an impact in the areas that are already destroyed, but an intervention may protect the normal lung. And that's important because the way IPF is, it starts with a very mild changes in the periphery of the lung and then continues. So again, just to summarize, both are -- much of our mechanistic work through the CADET, the things that were presented by Rusty, and this sort of more systems biology suggests that miR-29 is really a key regulator of fibrosis, probably the main antifibro miR we could think about and thus supplementing it in the lungs that are missing it may have strong antifibrotic effects. Thank you.

Thank you, Naftali. That's really fascinating observations and really linked together then some important observations in the human with the work that we've done preclinically and adds to our level of excitement for moving forward with microRNA-29 replacement as an opportunity for treatment of pulmonary fibrosis. So with that, we will -- that ends the formal presentation portion of this webinar today. We're -- operator, if we could go ahead and queue up any questions from people, we have the -- everyone available to answer any questions.

[Operator Instructions] Our first question comes from the line of Liana Moussatos with Wedbush Securities.

Towards the end, I thought it was intriguing about miR-29 having an effect early and late in the fibrosis process. How far into fibrosis do you think a therapeutic like MRG-229 could have a therapeutic effect maybe not where the tissues are already totally fibrotic, but how far into there? And can you talk a little bit about -- this is an unusual treatment. We have 2 approved drugs. How do you see it being used relative to those 2 drugs, if at all?

Thanks, Liana. Naftali, I was hoping that you may kind of weigh in on Liana's first question.

So the main thing with -- the main problem with IPF is that the disease progresses, right? So it has this almost stereotypic progression from the periphery into the center of the lung. So many of the people we see now, especially with increased awareness, their lungs are relatively normal. They lost 10%, 20% of their diffusion capacity. So in theory, if we can stop the process then, they will never feel a real disability. So being able to specifically stop extracellular matrix deposition in fibrotic processes will help. And remember, even the drugs, the 2 drugs that we have now, have been approved based on the fact that they reduce the progression of the disease by -- slow down the progression of the disease by around 40%, but there was no reversal of fibrosis. There's no halting to fibrosis. So I think if we could reduce extracellular matrix deposition substantially, we probably block progression of the disease. With regard to injured areas, can you reverse fibrosis there? That's a harder question. I think that our dogma is that you probably would not reverse with modeling completely. However, if you reduce extracellular matrix deposition, you may allow recovery of some areas. We do know that there is a particular regeneration in lung, and there's a chance that you will actually see some reversal of fibrosis. In the animal models of disease, we do know that scarring is actually, to some extent, reverse -- we've never shown it in humans. So the simple answer is in the less affected areas by stopping extracellular matrix deposition, you'll have a big impact on the patient.

[Operator Instructions] Our next question over the phone line comes from the line of Trevor Allred with Oppenheimer.

Two for me. Can you guys talk a little bit about what the length of expression you've seen for MRG-229 in the mouse model has been? And then also, you talked about the basophil granularity. Can you talk a little bit about that and what the implications of that might be?

Thanks, Trevor. I'll hand that over to Rusty to go ahead and answer that.

Yes. Can you repeat that first question? I had trouble hearing that one.

Yes. What kind of length of expression have you seen for MRG-229 in the mouse model?

For the compound or the microRNA itself?

For the compound.

Okay. Yes, yes. So we do see some detection. Those studies are ongoing currently. So we should have a better assessment of tissue resonance in the next -- soon. That's definitely something that we're actively investigating. As it relates to the effects in the kidney, these are very typical effects that we've seen in oligonucleotide therapeutics for kidney. They are very low. The doses that we're seeing in that are likely ten to twentyfold higher than what we expect to see in therapeutic -- or doses at. However, these are very classical and recovering effects in the kidney that are very typical of oligonucleotide therapeutics.

We have reached the end of our question-and-answer session over the phone lines. So I would like to turn the call over to Mr. Dan Ferry for any web questions.

Excellent. Thank you, Devin. So Bill, we have a couple of questions from the audience or at least one at the point. Does miR-229 (sic) [ MRG-229 ] impact cellular senescence in IPF?

Rusty, can you address that and what we know?

Yes, that's a great question. We have not looked into that specifically, but there is quite a bit of literature out there. I'm looking at this between not only fibroblast themselves but also alveolar epithelial cells. We have not directly assessed this with this compound itself though.

Okay, great. That is all the written questions I have. Devin, I believe we might have an audio question in the queue.

Our next audio [Audio Gap]