Ladies and gentlemen, our program is about to begin. Please note that during this program, we will be making a number of forward-looking statements. Please take a moment to review our slide on the webcast, which contains our forward-looking statements. These forward-looking statements involve risks and uncertainties, many of which are beyond Sarepta's control. Actual results could materially differ from those forward-looking statements as any and such risks can materially and adversely affect the business, results of operations, and the trading price of Sarepta's common stock. For a detailed description of applicable risks and uncertainties, we encourage you to review the company's most recent quarterly report on Form 10-Q filed with the Securities and Exchange Commission, as well as the company's other SEC filings. Ladies and gentlemen, please welcome to the stage Douglas Ingram, CEO, Sarepta Therapeutics.
Good morning, and thank you all very much in the room, and also on the web, for joining us for what is our inaugural R&D Day here at Sarepta Therapeutics. This will be a long day, but I think you'll agree with me, it'll be an insightful day, and we will be providing some preliminary results throughout the presentations today. The video that we just watched together was created some 15 or so years ago by PPMD, the leading U.S.-based patient advocacy organization for Duchenne Muscular Dystrophy, to show the natural course of this life-robbing and life-limiting disease, Duchenne Muscular Dystrophy, or DMD. As you can see here on this timeline, it was around 1987 when Dr. Lou Kunkel identified this protein dystrophin, which is the shock absorber-like protein that, because of genetic mutations, boys with DMD do not possess.
The amount of dystrophin that you and I have, those of us who don't have DMD, is infinitesimally small. It's about 0.002% of our muscle mass, and that is enough to be the difference between living a full life or dying at a very young age after muscle wasting, like the boys in this video right here. For the last 30 years, many people have dedicated their energy, their lives, their resources, their passion to finding some way to either restore or replace the dystrophin missing in these boys, and to give them back their future. Patient advocates, for instance, often parents, who by necessity have become experts in the science and the regulatory governance of DMD, have shined a light in this area and driven focus in this therapeutic area. Clinicians have found better standards of care for these boys.
Scientists, scientific luminaries, and the biotech ecosystem have spent enormous amounts of energy trying to find solutions. Literally billions of dollars of effort have gone into this over the last 30 years. As the result of universal use of steroids, the progress of the disease is slowed. As a result of things like exon skipping for DMD that was invented by Dr. Steve Wilton, and the advent of therapies like EXONDYS 51 and golodirsen and casimersen, for at least a subset of these boys, the disease is moderated over time. We have had progress. There has been progress. If we showed a video today with all of that progress, at each stage, those boys would be milder than they are here. Progress, yes, but the truth still remains that it's modest compared to our goals, right?
The harsh truth is that all of these boys, unless we can find some transformative therapy, will die still at a very young age after muscle wasting, and that is as true today as it was 30 years ago. Progress, we've made it, but we have not achieved our goal. We have not achieved our purpose. Sarepta's purpose, its goal, its mission, in concert with patient advocates, with scientific luminaries, and with patients themselves, is unambiguous. Our goal is to fundamentally transform Duchenne Muscular Dystrophy and give these kids back their futures. It's then to take that expertise and experience and apply it to other therapeutic areas to help other children with life-threatening and life-limiting diseases. In so doing all of that, to become one of the most meaningful genetic medicine companies in the world, literally over the course of the next few years.
Sarepta exists solely for boys like this boy on this video right here. This boy has DMD, and you will hear about that boy later in this presentation. Our strategy and our goals are certainly ambitious. In fact, some might think audaciously ambitious to suggest that we will be one of the most meaningful genetic medicine companies in the world in the coming few years. It's not arrogant, and you'll see that in the presentations today, because our goal is not to arrogantly imagine we can do this on our own, but rather to identify and partner with luminaries around the world to find solutions. On top of that, to take a multi-platform approach, genetic medicine approach, to conquering this disease and to giving these kids a better life.
Of course, one of our areas, and we've been there for a long time, is our RNA technology, and you'll hear about that today. We're focusing on gene therapy. We're focusing on gene editing as well, and you'll hear a presentation by Dr. Gersbach about our efforts on gene editing and what we're funding with Dr. Gersbach. We're obviously going to take all of that, and we're going to move into other therapeutic areas. In fact, we've already commenced that now. One of the presentations you'll hear today is about our five limb-girdle muscular dystrophy gene therapy programs with MyoNexus. There's a lot to listen to today.
One thing I would definitely suggest is this: If after listening to Dr. Louise Rodino-Klapac and Dr. Jerry Mendell talk about the preliminary results from our microdystrophin gene therapy program at Nationwide, one becomes excited about the transformative possibilities of gene therapy, that I would strongly suggest that you pay close attention to our discussion regarding our five limb-girdle muscular dystrophy programs because they are strikingly similar. Our limb-girdle muscular dystrophy programs relate to the same vector, RH74. In most cases, they relate to the same promoter. They have a common inventor in Dr. Louise Rodino-Klapac. They have a common investigator in Dr. Rodino-Klapac. They are progeny of the same institution, NCH.
This will surprise you, our lead program is not far behind in development from our microdystrophin gene therapy program because next month we will be dosing our first boy in our Limb-girdle muscular dystrophy lead program, and we'll be getting preliminary results by the first quarter of 2019. Over the last 12 months, we've begun to build, putting the building blocks in place for our strategy and to serve this purpose of ours. The first thing we did was increase our ambition and transform our pipeline with a sense of exigency, so that as we stand here now, we have 21 development programs between Duchenne muscular dystrophy and Limb-girdle.
We went out and we raised the funds to ensure that we could fuel our pipeline with the sense of exigency that we have, so that when we entered 2018, we had $1.1 billion to pour into this pipeline and make it a reality. We've also spent the last 12 months focusing on bolstering our infrastructure and our manufacturing capabilities. For instance, we've taken out additional lab space and other space in Kendall Square, in Cambridge, in Andover, and now in Columbus, Ohio. All right. On top of that, we've entered into our relationship with Brammer Bio pursuant to our hybrid gene therapy manufacturing strategy.
This is a gene therapy manufacturing strategy that allows us two things at the same time, speed to ensure that we can actually meet our development goals and treat patients to the extent that we have therapies that are successful, and also the ability to build a true gene therapy manufacturing expertise and platform. We chose Brammer Bio because they are the undisputed gold standard for gene therapy manufacturing around the world today. We've entered into that Brammer Bio long-term partnership with the goal and understanding that assuming we have an aggressive development strategy and an aggressive timeline, we will be able at launch to fully serve the physicians and their patients that are waiting for our therapy.
To put a fine point on that, at launch, pursuant to our Brammer Bio partnership, we will have more gene therapy manufacturing capacity than any biopharmaceutical company that exists in the world today, as of today. That's not simply bravado. That's necessary. The fact is that we need that sort of capacity if we have a successful program. That's actually what the community is going to need at that time. We've also started to build our gene therapy division, and you'll hear more about that later. Finally, we began to attract significant talent. I think this is probably one of the most significant things we've done over 2017 and in 2018. In 2018, from January 2 on, we've been hiring about 1.5 employees every single business day. Those are scientists, developers, pharmacovigilance professionals, regulatory professionals, toxicologists, and I could go on.
On top of all of that, we've attracted some really impressive talent. This is, of course, Dr. Louise Rodino-Klapac. You're going to hear more from her today, and I'm thrilled to say that she is now the head of our gene therapy division. Without hyperbole, I can tell you there is no one that would be a better suited leader for our gene therapy manufacturing division than Dr. Louise Rodino-Klapac. As you probably know, she was the co-inventor of our microdystrophin gene therapy program. She is the inventor of all 5 of our Limb-girdle gene therapy programs. She's got an extraordinary pedigree. In a time when people often say that there is a dearth of true practical gene therapy experience, Dr. Louise Rodino-Klapac is associated with 11 INDs in gene therapy, and she shares our passion and our mission, and our vision for the future.
I couldn't be more excited to have her here, and you'll hear much from her today, both on microdystrophin and also relating to our Limb-girdle programs. I'm also extraordinarily excited to talk about our Chief Medical Officer that we've been able to attract recently, who is Dr. Gilmore O'Neill. For those who are in the biotech ecosystem in Boston and Cambridge, you will no doubt know of Dr. O'Neill. Dr. O'Neill was immediately before we were able to get him to become our CMO at Sarepta, the head of all late-stage development at Biogen, and responsible for most, if not all, of the significant therapies developed and approved over the last 15 years at Biogen. He is a neuromuscular expert. He has an impeccable pedigree. Like Dr. Louise Rodino-Klapac, he obviously shares our passion and our commitment and our culture.
An interesting issue about getting Dr. Gilmore to Sarepta, when we identified Dr. Gilmore as the person that we wanted to go after because he seemed like he was a perfect fit for us, many folks who in the know in the sort of the biotech ecosystem in Boston, Cambridge said he would be brilliant. That would be a brilliant choice. Many people over the last few years have attempted to dislodge Dr. O'Neill from Biogen, and all have failed, and you are going to fail as well. To be honest, and I don't see this, I'm told by people that I'm a bit competitive. I created for myself a great message track. I was going to lobby him, persuade him, cajole him to get him in here.
The issue is, as soon as Dr. O'Neill and I met, it became obvious that that was not the issue. In fact, it was clearly the case that Dr. O'Neill, independent of Sarepta, had developed the same vision, which is the vision of genetic medicine, and particularly the combining of RNA technology and gene therapy and gene editing together could become something that would completely transform healthcare in the coming few years. To me, it became obvious my goal was not to lobby him, it was simply to paint the picture of our shared vision. Of course, I'm very proud to say that worked. I'm also very proud to call Dr. O'Neill my colleague at Sarepta, I'm also very pleased to invite him to the stage.
Dr. O'Neill, who is our new Chief Medical Officer, will act as our host today for this, which is our inaugural R&D Day. Please join me in welcoming Dr. O'Neill to the stage. Thank you.
Thank you.
Thank you, sir.
Good morning, everyone, and thank you very much, Doug. I was always taught that one should set expectations low and then exceed them. Thank you for breaking that rule in the first week of my being here on board at Sarepta. I'm incredibly excited to be here with you today, and I hope that In fact, I know that as you see the data unfold at our R&D Day, I think you will join in our excitement. I think what we're doing is standing at the threshold of something that is going to be genuinely and truly transformative. Doug is right. I have been looking for an opportunity to move beyond just simply transforming therapeutics. I've had great good fortune to be associated with success in that prior to joining Sarepta.
I'm actually interested in actually taking something that isn't just going to use cutting-edge science to transform therapeutics, but that could ultimately have the same effect on healthcare delivery and that ecosystem that the mobile phone had on landline telephony. Truly disrupting and moving us away from a sort of very heavily developed infrastructure that delivers multidisciplinary palliative care to, and obviously this is the ultimate forward-looking statement and sort of that great dream, that we could actually get to single or occasional dose curative therapeutics. That was the first thing that brought me to Sarepta. I think the second thing is that using or doubling down on monogenic diseases, rare diseases, is a very good way to really develop that approach, validate that approach, and move the system forward.
Thirdly and finally, which is really the reason I went into medicine and the reason that I wanted to go into therapeutic development was those boys. The idea of going after and changing the lives of people who have devastating and fatal diseases. I think the important thing about that video is that you see a disease that takes away so much before it then actually takes away your life. That's something that really I think we can achieve or that we can actually deal with and fix. It's great to be sitting on the threshold of something that is so big and to be a part of something that's going to be so big and so ultimately could change the whole approach to healthcare delivery. I'm not going to bore you with philosophy.
That's really something that one does in a pub later in the day over a drink. I will tell you several strong core beliefs, which is one should continuously test and understand the biology of the diseases that you're treating and try to imagine all the possibilities should your biological hypotheses be right. You should also place the patient first and foremost in your thinking, not least of all by asking them what do they want? What do you need? What is required from us to help you? Then following the path as I've outlined there, I won't take you through it bit by bit, but what I'm trying to say really in summary is that we should always put patients first. We should use the best biology, and we should use the best development regulatory science to deliver to patients that which they need.
I've learned many lessons over the years, and some of the really positive ones, again are, and I can't say it often enough, focus on patients. Focus on the patients who are in the clinical trials, focus on the patients who are going to use the medicines that we develop in future. Always understand the biology, not just of the target, but of the patients who have the diseases, so you could control the confounding variables as much as you can in your clinical trials. Always aspire for perfection, then compromise around what the real world will allow you to do. I think most fundamentally and importantly, remain humble before biology and disease. They are complicated foes and merciless foes.
I think the other final piece is you can never underestimate the importance of ensuring that you're delivering the right amount of biology and therapeutic effect to the right cell in the right tissue at the right time for the appropriate duration of time. All of these things, if you get right, are basically what are going to get you an efficacious component or compound or treatment associated with an appropriate risk ratio. What I want to do is introduce you to our pipeline, and I think, as Doug said, the striking thing about it is that it is broad and deep. The one thing I want to make sure you understand is that it's focused. We're not actually losing our focus. We are focusing on those things that Doug's outlined, and that I've already said at the beginning of my few remarks.
We're focused on muscular dystrophy. While we are modality, if you will, agnostic, the key concentration of our pipeline is on RNA-directed chemistry and gene therapy. What I'd like to do is tell you a little more about how Sarepta is positioned to deliver and execute on such an ambitious pipeline. The first thing is, if you look at our chemistry, our PMO and our PPMO technologies. That's a platform that we own, that we're continuously improving, and you'll hear a little bit more about that later. The important thing to understand is we are using, for PMO or PPMO, we are using the same chemistry. We're using the same bases. We may be changing the ordering of those bases within the morpholine structure, but they're the same chemistry. The second thing we're doing is we are going after the same target.
In this case, we're going after the dystrophin target. Finally, we're using the same mechanism of action. Thus, enabling ourselves to keep doubling down on an evolving expertise in which we are the leaders. That is, in the development or pharmaceutical development of muscular dystrophy therapeutics. It actually also enables us to leverage the regulatory path, which is similar across the compounds. Ultimately, what we'd like to do is develop those compounds in the more common groups or subgroups of DMD patients to validate our technology. Ultimately, as we go into rarer types, develop or explore pathways that will enable us to use or use the previous experiences as we seek regulatory approval. The next element is our gene therapy element.
What you can see is we're using several approaches where we're actually going to develop or deliver surrogates or replace dystrophin or other components of the dystrophin-associated protein or glycoprotein complex, or indeed edit the dystrophin gene itself. Just to help you again illustrate in a different way how we are retaining our focus with this approach and with this deep pipeline, I want to take you to this cartoon. Right at the bottom of the cartoon, you can see dystrophin sort of in tan, and that is kind of the core of our strategy. It's the core of this dystrophin-associated protein or glycoprotein complex. As you know, it serves as a shock absorber that essentially ties the cytoskeleton of the muscle fiber to the membrane, or sarcolemma of the muscle fiber, and then to the extracellular matrix.
When that is disrupted, we all know that it leads to damage, severe damage, degeneration of the muscle, which phenomenologically results in progressive disability and ultimately death. Dystrophin is one component of a chain of multiple proteins. It's one link in a chain, and we already know from the videos of these young boys with Duchenne how breaking that link in the chain disrupts the efficacy and the potential for that chain to maintain the integrity of the muscle fibers. If you move to the top right-hand corner, in pink you can see the sarcoglycan and dystroglycan complexes, which are a critical component in the sarcolemma for bridging from dystrophin through the membrane of the muscle fiber into the extracellular matrix. You will actually see that we really, in our targeting and R&D strategy, are literally moving to adjacencies, molecular adjacencies.
As you'll see during the presentations, you will see how the phenomenology of those disruptions in those adjacent biologies really generate a phenomenology that looks quite similar to Duchenne muscular dystrophy, if not as severe, although certainly some cases are very severe. Today is really a very special day because we are truly privileged to be joined by global experts in the field of muscular dystrophy and in gene therapy. These are not just global experts, these are also our partners, and it's one of the things that really excites me about coming to Sarepta, the opportunity to partner with such phenomenal visionaries and experts. What I'm going to do is actually use our cartoon to sort of lay out how the R&D Day is going to go. You will see just how the day goes and how it ties to our ultimate R&D strategy.
To kick off the R&D Day, Dr. Kevin Flanagan is going to talk about his work on GALGT2 gene therapy and give some preliminary human data. From the point of view of genetic strategy, his use of GALGT2 as a modifier is really going to focus, and he'll talk a bit more about this, on the use of a surrogate gene therapy approach to dealing with a dystrophinopathy. Owing to its, dare I say, potent biology, it actually has the potential to directly impact or indirectly impact some of the target proteins that are disrupted in limb-girdle muscular dystrophy, and in fact, merosin- deficient CMD, or congenital muscular dystrophy. That's MDC1A.
Following his presentation, Dr. Serge Braun from Genethon is going to talk about his work on microdystrophin gene therapy, and he will tell you, as will Dr. Rodino-Klapac and Dr. Mendell, how the microdystrophin can actually complement or replace dystrophin. He's going to actually demonstrate across multiple models the expression success he's had and the functional outcomes and improvements that one can observe with that. After the break, building on that, Drs. Rodino-Klapac and Mendell will tell us about and disclose preliminary data from their phase I and II microdystrophin gene therapy trial. Building on this, and you can see the focus remains on dystrophin, Dr. Charles Gersbach is going to talk about how he has used an exon-skipping approach within the genome, not the RNA. We validated that with our PMO technology. We're leveraging that with our PMO and our PPMO technology.
He has taken to the next level to see how can you leverage that proof of concept to a genome level editing using CRISPR technology. We move to the adjacency, that's when Dr. Rodino-Klapac will return to the stage to talk about a number of different Limb-girdle muscular dystrophy programs, you can actually see highlighted in the sarcoglycan complex some of those targets. There will be some other targets relevant to muscular dystrophy on which she will touch, but this is the key element, again, demonstrating our focus around dystrophin and moving to adjacencies. Finally, after lunch, our resident experts in PMO or PPMO technology will present. Dr. Hanson will talk about the chemistry of the peptide-linked PMO, Dr. Pacini will talk about the biology that we're seeing with this new evolving technology from our PMO platform.
At that point, we will hopefully have delivered to you a picture of our R&D strategy, how we're going after a very serious set of diseases, developing a deep portfolio which combines RNA-directed therapeutics and gene therapy to ultimately transform some really serious and terrible diseases, we hope ultimately transform our healthcare ecosystem. Having had the opportunity to share some of those thoughts with you and outline the day, it's my great pleasure to invite Dr. Kevin Flanagan to present his phase I/II clinical trial of intra-arterial gene transfer of the AAVrh74-MHCK7 GALGT2 for DMD, outline some preliminary observations and the initial safety profile. Before he comes up, or as he prepares himself to come up, I'd like to just tell you a bit more about him.
He is the director of the Center for Gene Therapy at Nationwide Children's Hospital and a professor of pediatrics and neurology at Ohio State University College of Medicine. I think those of you who have looked at the literature will know that he is a leading expert in neuromuscular disorders. He is a board-certified neurologist in both neurology and neuromuscular medicine, is the director of the Nationwide Children's Hospital NIH-funded P50 Center of Research Translation in Muscular Dystrophy Therapeutic Development. He's a member of a physician team at Nationwide that delivers world-class multidisciplinary care and coordinated care in the pediatric program, or sorry, neuromuscular program, he also leads the Nationwide Hospital's pediatric neuromuscular fellowship program. His primary research interest is in the genetics and molecular characterization of inherited neuromuscular disorders and the development of therapies directed toward these diseases.
The major focus of his laboratory, in fact, you can see how this led to what he's going to talk about, is the identification of genetic modifiers of Duchenne and other muscular dystrophies. Another is in developing viral-based exon skipping strategies for muscle diseases. I have to forgive him for this, he trained in neurology and neuromuscular disease at Johns Hopkins. Those of us who trained at Mass General do have to articulate. There's a little tension there. I'll deal with Columbia later. That's where he trained, then went on to fellowship training in human molecular biology and genetics at the University of Utah, joined the faculty of Nationwide Children's in 2009. I'm really delighted to welcome him to talk to you about his program. Dr. Flanagan.
Thank you very much. There you go. Thank you. Thank you very much for the invitation to come and speak about this program of a surrogate gene therapy approach, really spearheaded by work of Paul Martin, who I'll mention many times, who's a member of our faculty and investigator at Nationwide Children's Hospital, and with preclinical development of a team over time, including Dr. Mendell and Dr. Rodino-Klapac, who are here. For the little overview of what we'll do, I'll talk first a bit about GALGT2, what this protein is and what this protein does, and share some of the preclinical data that led to this trial. Give just some really preliminary data on the first patient that we've treated with it, and then close with some conclusions and next steps as we head forward with this.
GALGT2, as mentioned earlier, is a surrogate gene therapy approach. The idea is instead of replacing the dystrophin gene, the missing dystrophin gene, we provide a gene that can substitute for the function of it. It has some potential benefits, or we should say scientific rationale for this is supported by a couple of ideas that are important. One is that by, instead of replacing the dystrophin gene but offering a different protein, we have the potential to treat really essentially all patients, or almost all patients, with dystrophin deficiencies, irrespective of their mutation class or irrespective of the location of the mutation. As long as dystrophin is missing, this is an approach to therapy. Because we're delivering a gene that is already expressed, an endogenously expressed protein, we don't expect any immune responses to the transgene itself.
That is, the protein encoded by this transgene is already recognized as self by the body. As I'll point out at the end, it has a potential for treatment for multiple forms of muscular dystrophy, including early animal-based preclinical evidence for potential benefits in one form of Limb-girdle muscular dystrophy and also for Congenital Muscular Dystrophies. This is a rather simplified version of the cartoon that you just saw, an older version of this cartoon representing the dystroglycan, a dystrophin associated complex, which, as you noted, prevents muscle damage by linking intracellular actin to the extracellular basal lamina. This critical linkage is done by the beta-dystroglycan protein that's spanning the membrane there, where the linkage occurs.
That links to an extracellular protein, the alpha-dystroglycan protein, and then linkage to the ligand laminin alpha-2, or merosin, as it's also known within the extracellular matrix, is dependent on glycosylation of alpha-dystroglycan. Glycosylation of that protein is specific mutations in genes that are deficient in glycosylation, cause different kinds of Congenital Muscular Dystrophies. For our purposes, the important thing is that this linkage is dependent on glycosylation. It turns out, although we always draw that cartoon one way, there's two dystroglycan complexes in muscle: the dystrophin associated one, which you just saw, you can see here on the left, which actually occurs in almost all of the muscle membrane, and then a different version of the complex that is actually present at the synapse, the synaptic version. At the synaptic version, there's a couple of differences.
One is that the binding partner is not dystrophin. The shock absorber is not dystrophin, but is instead the paralog utrophin, which sits at the membrane. The second difference is the presence of the expression of a protein, this beta 4 GalNAc transferase GalGT2. The presence of GalGT2 makes a specific glycosylated epitope recognizable. We often refer to it as the CT antigen. This different sugar pattern, glycosylated epitope, can identify the synapse, but it marks the difference between these two complexes. In the absence of dystrophin, such as in the MDX model of muscular dystrophy or in patients with Duchenne dystrophy, the actin cytoskeleton can still be linked to the basal lamina via utrophin expression at the neuromuscular junction.
If we add the transgene, as I'll show you data from this in a moment, if we add the GalGT2 transgene, either by a transgenic approach or by delivery with AAV, we can actually express GalGT2 at the membrane, across the entire membrane, and express utrophin across the entire extrasynaptic region of the membrane as well. This is the principle behind GalGT2 therapy. The preclinical data supporting this goes quite a ways back to 2002, when Dr. Martin's laboratory first began to work on it. This is a graph from an early paper showing activity of the serum creatine kinase assay or CK assay. Creatine kinase is an assay that's elevated in the presence of muscle damage. It's a clinically important assay.
It's often the first signal in a biochemical testing that a boy has problems with his muscle, and that a boy has Duchenne Muscular Dystrophy. In the animal model, the MDX model, here marked by the tan squares, you can see that in mice, the MDX mice, they have an elevation of serum CK activity, just like boys do here through six months' time. The wild-type mouse is marked by the blue circles at the bottom of this graph. What one can see is that both in the GalGT2 overexpressing animal marked in purple, and in the MDX mouse that is crossed with an overexpressing GalGT2, the black squares, there's normal CK levels. This shows us that a GalGT2 overexpression normalizes the serum CK through the life of the animal.
A more direct measure of muscle damage is to look at it under a microscope, this is a measure of what's called central nucleation. When muscle degenerates and remodels, there's centralization of the nuclei from its usual peripheral distribution in a mature myofiber. We can use central nucleation as a measure of muscle damage. As you can see here, again with wild-type mice marked in blue, and the MDX mice marked in the tan squares, that at five weeks of age, about 50% of fibers show central nucleation, and by 18 months, about 90% of fibers show central nucleation in the MDX mouse. In contrast, the MDX mouse overexpressing GalGT2 shows essentially no central nucleation through most of its lifespan, with only a mild elevation toward the end of its life. Muscle pathology is essentially absent in MDX mice that overexpress GalGT2.
We also look at muscle physiology. This is work done, again, from Dr. Martin's laboratory. There's a measure called eccentric force drop, the force drop to eccentric contraction. The tetanic force is applied to a stretched muscle. What we normally see on the left side here, marked by the blue circles, is a force drop with repeated contractions. After the first two cycles of contraction, there's a clear drop in force. This is accentuated in the MDX mouse marked by the tan circles on the left. The GALGT2 mouse, just an overexpresser of GALGT2, doesn't show this force drop. In fact, shows some climb in the force with repeated contraction. The important thing is the black squares on the left graph here that says with GALGT2 overexpressing mice on an MDX background, that force drop disappears, suggesting that there's correction of this physiologic abnormality.
On the right, you can see an animal treated with an AAV construct with the same AAV construct that is used in the human studies. That overexpression by AAV delivery shows a similar pattern of correction. Not quite the same degree of correction as the transgenic animal, but a similar and quite significant increase, or I should say diminution of the force drop that we typically see. Although we do know that utrophin is upregulated, we know that this is not solely a utrophin-dependent process. In addition to binding of the dystrophin-associated complex, there are other binding partners in the muscle membrane, such as the integrin complex, that also stabilize the muscle membrane. When this virus is delivered to non-human primates, to macaque monkeys, there are multiple membrane-associated proteins that are expressed. What you see on the left is a Western blot.
This is from a normal monkey, it's already expressing dystrophin. One can see that the treated animals in the four lanes on the right portion of that blot, they show expression of GALGT2, as seen in the top, an upregulation of utrophin, as we've mentioned, but also upregulation of other proteins, including plectin, laminin alpha-5, agrin, and other components, including dystrophin itself. Other multiple proteins that have individually been associated with stabilization of the muscle, and in fact, overexpression of plectin and agrin and laminin alpha-5 have been shown in MDX animals to, by themselves, show improvements in muscle pathology. This is seen at both the protein level on the left and in gene expression levels on the right for many of these proteins.
The approach I'm going to share with you is using isolated limb infusion via the femoral artery. I'll show you more details about this in a moment with a clinical study. This is an example of the dose escalation studies. The physiology that I showed you showed correction with levels of about 20% of fibers expressing GALGT2, measured here as staining for that CT antigen, the glycosylated epitope around muscle. In these studies, which were undertaken in part to establish the dose for the clinical trial, you can see an animal treated in the vastus medialis at 6e to the 12th vector genomes per kilogram or 2.5e to the 13th vector genomes per kilogram.
We can see that this helps to establish the dose that the animal at the 2.5E to the 13th, taking sections throughout the vastus medialis muscle, one of the quadriceps muscles, that we can see expression that ranges between 22% to above 80% of muscle fibers. Suggesting that this would be sufficient based on the preclinical mouse studies to give clinical benefit. With this data in hand, we've actually initiated a clinical trial. This trial is supported by a R21 grant from the National Institutes of Health, from NIAMS. This uses rAAVrh74 with an MCK promoter and the GALGT2 transgene. This is an isolated limb infusion approach to deliver it to both legs via the femoral artery. To do this, as I'll show you in a moment, the femoral vein and artery on each side are cannulated and catheter localization is confirmed via angiography.
In either leg then, actually we begin on one leg, the blood flow is interrupted via inflation of the venous and arterial balloons, and the vector dose of approximately 300 milliliters is delivered over a brief period of 90 seconds. The blood flow is then interrupted and remains interrupted for precisely 10 minutes, a dwell time of 10 minutes. The arterial and then the venous balloons are deflated and blood flow reestablished. The procedure is then repeated on the contralateral leg. This provides time within the arterial circulation of the treated limb. When the balloons are deflated, of course, the vector is not withdrawn from the limb, so it is into the systemic circulation at that point.
This was initially designed as a dose escalation study with a low dose of three patients planned to receive 2.5E to the 13th vector genomes per kilogram per leg, or a total of 5E to the 13th vector genomes per kilogram, and a high dose cohort of three additional patients receiving double that dose, 5E to the 13th vector genomes per kilogram per leg, to a total of 1E to the 14th vector genomes per kilogram. The primary objective is really to be designed as safety, to assess safety of the expression for the first time of this vector in humans, with the primary efficacy outcome defined by expression of the CT antigen by immunofluorescent analysis.
Clinical outcomes are included in the study, of course, including the 100-meter walk time, the amount of time it takes a boy to walk 100 meters, and the North Star Ambulatory Assessment Score, which is a measure of it's a Duchenne-specific ambulatory assessment that's been shown to track with disease progression over time in ambulant patients. The first patient was dosed right at the end of the year. He was enrolled at 8.4 years of age and had a duplication of DMD exons 22 through 41, and at the time of enrollment was somewhat underdosed by standard dosing protocols. He was dosed on prednisone, 10 milligrams per kilogram per day. This is just some baseline values to show he entered the study with a value for his six-minute walk time, about 59.9% of that value predicted for boys of his age, normal boys of his age.
100-meter walk time, also about 59.6% of that predicted for his age, and a North Star Ambulatory Assessment score of 18 out of 34. We also have some measures on the right of what's called MVICT, maximal voluntary isometric contraction testing. It's a measure of force against a transducer in kilograms of force of a limited number of muscles that are addressed by this therapy, knee extension and flexion on both sides. This is a mid-thigh MRI, a gradient echo image of, actually, I'm sorry, the distal thighs. Just to give you a picture of what the muscle looks like at the time we did this treatment. You can see the two components of the quadriceps marked, the vastus lateralis and vastus medialis muscles.
You can see in the posterior compartment of the thigh at the lower side of the image, some of the variability that we face in delivering gene therapies with the bright signal marking an increase in the amount of fat in the muscle, the fatty replacement that goes along with progression of disease. We've marked here on the right, the muscle that was biopsied for his initial pre-treatment biopsy. This is the procedure itself. As you could see at the beginning of this, perhaps we can run it through one more time, I'm not sure. At the beginning of that, we can see that the balloons that are inflated and the distribution of the angiographic dye all the way down the leg, showing that in fact we have delivery of this throughout the leg. The expected adverse event was bruising at the site of catheterization.
This is similar to a coronary artery catheterization. If you've known anyone who've had that done, that was all resolved by day 30. There was a transient decrease in the absolute lymphocyte count. Here you can see that marked. The absolute lymphocyte count is at the lower end of this. I'll note that we started the patient on prednisone at one milligram per kilogram the day before therapy, carried it out, and began a taper about day 45 after therapy, day 50 after therapy, and continued down to baseline steroids at the end of this time. We saw no significant increase in CK at the time of dosing, suggesting we did no injury to muscle, as we would expect. This is his serum CK measured with normal activity, his normal voluntary activity. We know that this changes in patients with Duchenne muscular dystrophy based on their physical activity.
Overall, there seems to be a decrease in the serum CK by a decrease of about 43% at the last point at which it was measured, and that we had only a transient increase of serum transaminases, the AST and ALT, as commonly seen with viral gene therapies. Right now, they remain at the level that we saw at enrollment. I'll point out that all boys with Duchenne muscular dystrophy have elevated ASTs and ALTs, not as markers of liver injury, but because those same transaminases are released by muscle. As we can see, compared to when he enrolled in the study, he's had no increase of AST and ALT.
We look for cellular immune responses by interferon gamma ELISpot tests. We saw in this boy a transient response at 14 days to the capsid peptide pools, to all three of them, with our definition of positive as 50 spots per million cells. This was only transient. I'll show you, this is really very preliminary. I'll note that our actual staining by WFA to identify CT antigen is actually the assay for this particular test is undergoing validation. The blinded quantitative immunofluorescence analysis that we've predefined is not done yet. This is preliminary analysis done in the same way we did pre-clinical data in the laboratory of Dr. Martin.
Compared to the pretreatment specimen, which is the first pair, column of these images, you can see two randomly selected images in which there is, in fact, widespread expression of the CT antigen in the, thus GALGT2, at levels that vary in intensity among fibers, but approach 80% of all fibers within these regions. Again, comparable to that which we see with the pre-clinical physiology studies. Functional measures show at day 120 stability of function. We believe this is plotted as a percentage change from the baseline. On the left, the only measure that decreased somewhat was the six-minute walk test. It decreased by 37 meters, just outside of what we consider to be the sort of variability within test-retest of 30 meters. Although we'll point out that this is widely accepted now.
We know all the challenges about reproducing this measurement over time. In fact, measures such as the 100-meter walk time improved by 15%, as did the time to rise, and the 10-meter walk time. On the right, measures of isolated muscle strength certainly show we've done no harm in measuring kilograms of force. We saw three of the four actually improved in strength through the four muscle tests, showed an improvement in strength. What can we conclude, and what are our next steps? Certainly, we conclude that this isolated delivery by femoral artery at this dose is well-tolerated. We can conclude that this vector clearly is expressing in muscle. There's stability of functional and isolated muscle strength measures observed at four months.
Based on these results, we're actually moving to an IND amendment and seeking to move, instead of doing our original design of three patients at the lower dose cohort, to move on to do directly to the higher dose cohort in patient, now that we have proof of concept that the vector expresses safely. Based on this, the next step include this current plan to seek IRB approval to increase dose now. My colleague, Paul Martin, is completing dose-finding studies that will allow us, we believe, to proceed beyond that directly to systemic delivery for an intravenous trial of this. That data should be complete shortly. I mentioned again that there's the potential for broad therapeutic potential.
The figure you've seen some version of here on the left with MDC1A being merosin deficient, congenital muscular dystrophy with mutations in the laminin, the gene encoding laminin alpha-2, LGMD2D, and DMD or Becker muscular dystrophy, in which a partially functional version of dystrophin expresses. We'd expect all of these to show improvement or have the potential for improvement. For each of the three circled here, Dr. Paul Martin has shown preclinical evidence in animal models. Again, we're keen to move forward because of the fact that it's a therapy independent of mutation class and independent of mutation location. Because of this issue, we think that the transgene immunity risk is really quite limited. I'll close just by highlighting this is 15 or more years of work by Paul Martin, the preclinical work that's published. That includes work done by Dr. Louise Rodino-Klapac and Dr. Mendell.
It represents work from an entire team at the NCH, and funding, again, from the NIH through our R21 mechanism. Thank you very much.
Thank you very much, Dr. Kevin Flanagan. I think that was an interesting view of how one can use a surrogate gene therapeutic approach which actually upregulates a paralog, and ultimately can upregulate other components of the dystrophin-associated protein complex. It is now my great pleasure to invite Dr. Serge Braun to present his work on microdystrophin gene therapy approaches to the treatment of Duchenne muscular dystrophy. Currently, he serves as scientific director of AFM-Téléthon, the French MDA, or muscular dystrophy association, where not only is he a scientific director, but he tells me he actually contributes to the fundraising efforts by running marathons around the world. In that role, he basically develops innovative therapies for rare diseases. In addition, he is president of GenoSafe SAS, a clinical research or contract research organization.
He is a veteran in the field, having had 10 years of experience in the academic center, or academic sector, excuse me, in addition to 10 years in the biotechnology sector. He earned his PhD in pharmacology from the Louis Pasteur University of Strasbourg, France, and experienced or had his postdoctoral fellowship at USC, University of California, Los Angeles. In 1995, he joined Transgene SA, the largest French biotechnology company, where he became vice president of research and developed his career in the field of gene therapy for genetic diseases and in the immunotherapy of cancer. He is a member of the Académie nationale de Pharmacie. It is my pleasure to call on him to present to you today.
Thanks very much. Thank you. It is my pleasure to be here. Yes, several years ago, I ran the New York Marathon, and I realized how lucky I am to be able to run, even to walk or to breathe. I know that there are many, many people who are unable to do that, so we will do our best to help those people. I am representing a rather peculiar organization, which I am going to present very briefly in my presentation. I will move on to our microdystrophin gene therapy program, with some data in small animals, but also in a large animal, namely the golden retriever muscular dystrophy dog, and then a summary and the next steps. AFM-Téléthon is, as I said, a peculiar patient organization. It is run by patients and parents.
It started very small in 1958, it became very big thanks to the Telethon in France that we imported from the American Telethon, but with a French touch. Together with the 30-hour TV show, the first weekend of December, we are organizing more than 20,000 festive events throughout the country. Even in the smallest villages, you have Telethon, people organizing sports, food events, of course, music, whatever, to collect funds. This helps AFM-Téléthon to raise every year around $100 million. Every year. Most of it is invested into research and development. This is our R&D budget. So far, AFM-Téléthon has invested more than $1.6 billion in the rare disease field, not only neuromuscular disease, but even though our commission is on neuromuscular diseases.
We do fund around 300 programs, not only in France, everywhere, in Europe, in the U.S., in Japan, anywhere where there is good science. We have co-ownerships on some key patents and key know-how, we are running currently around 35 clinical trials in 30 different diseases, half of them being neuromuscular. What is also peculiar, we have our own labs, our own research laboratories. The largest one is Genethon. Genethon is a nonprofit biotech dedicated to gene therapy. Originally, Genethon was launched to work on the human genome, actually, the first human genome map was published by Genethon in 1992. Four years later, the team switched to gene therapy. You have around 200 people working in the gene therapy field at Genethon.
We have other institutes, I-Stem, a stem cell lab, the largest in France, working on embryonic and iPS cells, on drug screening as well. The Institute of Myology, also the largest in France. It is the largest reference center dedicated to neuromuscular disease in France, where you have patient care, basic science, clinical trials. There are currently 20 clinical trials going on at the Institute of Myology, including in the gene therapy field. Very recently, we launched a company named YposKesi. YposKesi is the GMP manufacturing facility of Genethon. YposKesi is producing AAV and lentiviral vectors. It is a 50,000 sq ft facility, probably the largest in Europe, but already too small. It produces for our own needs, but also for customers, including Big Pharma. The needs are expanding, the facility needs to expand.
We, together with our partner, the public bank of investment, the French Public Bank of Investment, we have a $150 million program that will triple the size of this GMP facility. This is a snapshot of Genethon programs, some of them late-stage clinical trials, some in the neuromuscular disease field, including Duchenne dystrophy. You may have heard about myotubular myopathy recently with the fantastic data released by Audentes , the American biotech. Actually, it's a Genethon product that was licensed to Audentes , and it's an AAV8 gene therapy that makes a big difference in this disease, which is very severe, even more severe than Duchenne. We have programs in SMA. You also heard about the recent acquisition of AveXis by Novartis, $8.7 billion, mainly based on one product, SMA gene therapy. Guess what? It was a Genethon product licensed to AveXis.
We have several partners, and one of them is Sarepta. This is YposKesi. I mentioned the 50,000 sq ft facility. This manufacturing was commissioned several years ago, and it's running pretty well. 2 types of gene therapy products. Four independent suites, so we can produce different types of vectors simultaneously. Now back to the AAV microdystrophin program. We heard about Duchenne dystrophy. We now know what this disease is all about. You see on the picture the staining for dystrophin in a few muscle fibers, in a normal muscle on the left, in a diseased muscle in the center. There is an ongoing process of degeneration, regeneration that takes place, and which is little by little exhausted. This explains the progressive muscle weakness in the patients, which leads to paralysis, progressive paralysis.
You have seen movies, but what we know at the end of the day, this is a 100% fatal disease which affects both skeletal muscles but also the heart. Cardiomyopathy is very frequent in those patients and is life-threatening. There are several strategies to tackle Duchenne dystrophy. I'm going to focus on microdystrophin. We took a rather straightforward approach. There is a defective gene, dystrophin, and we'll try to replace this defective gene with a normal version of the gene. The problem with dystrophin, it's the largest human gene known, and the cDNA sequence is too big to fit into an AAV vector. The virus is too small.
There was a need to squeeze the size of the dystrophin gene, which led to the so-called microdystrophin version, a minimal version of the gene, which retains the function, the essential function of dystrophin, and is then able to be carried out by AAV virus and injected into models. This is a picture of a drawing of the full-length dystrophin sequence, and the microdystrophin construct, which is much smaller, was designed by George Dickson in London and was also optimized in order to increase the level of expression. This is being expressed under the control of a muscle and heart-specific promoter. We increase microdystrophin expression in the targeted tissue, whereas we decrease the level of microdystrophin in non-muscle tissues. This product was tested in three animal models, the mouse mdx, the rat model, and also in golden retriever muscular dystrophy dogs.
First of all, the small animal models. We use an AAV8. That's the same serotype, the same AAV, that the one that is currently used by Audentes in myotubular myopathy. We know how it works, including in human, and that's exactly the same serotype that we'll be using in Duchenne patients. Transgene being different, of course. What you see on the left here is microdystrophin expression, so shorter than the full-length dystrophin on the Western blot. This is in the tibialis muscle. You see on the right part of the left Western blot, the black band corresponding to microdystrophin. On the right hand, the level of dystrophin-positive fibers in the heart. We inject the vector intravenously, just perfusion, and we have expression both in skeletal muscle and the heart. The microdystrophin gene is functional.
There is not only protein expression, but also functional improvement in the animals. First, the muscle turnover is almost back to normal, and that's very important. You need to stabilize the tissue. I mentioned early on that skeletal muscle undergoes a continuous necrosis regeneration process. This has to be stopped, and this is being stopped as we see in mice on the left graph. The middle graph shows improved muscle strength and normalized resistance to eccentric contraction on the right graph. Same type of functional analysis that Kevin Flanagan showed you previously. We have patients, and we have different animals that display similar disease, but with more or less differences. The golden retriever is a spontaneous model. We do not induce Duchenne dystrophy in those dogs. Some of them display the same mutation as the mutation in humans.
They display a very similar disease, even though with some cardiomyopathy, but milder than humans. The MDX mouse is almost normal. It has no dystrophin. It has some decrease in muscle strength that we are able to measure, but it has almost a normal lifespan. Dogs have a shorter lifespan. There was a need to generate a new model, which could be an in-between model, and that was done by a team in Nantes, which we are collaborating with. They generated a rat model of dystrophin deficiency, which happened to recapitulate the human disease very well, including very severe cardiomyopathy, and that was very important to us. What we see in this model, in the rat model, is dystrophin expression, microdystrophin expression. You see the pictures on the upper panel, a wild type on the left, so dystrophin is present.
The middle picture shows the lack of dystrophin in the animals in different muscles, biceps femoris, as well as diaphragm. Diaphragm is very important muscle for breathing. The red color highlights fibrosis, and fibrosis is being reduced upon gene therapy. The two pictures on the right show both dystrophin expression, microdystrophin expression, and reduction of fibrosis, and this can be measured as seen on the graphs. Actually, the percentage of microdystrophin-positive fibers is really high. It's between 50%-100%, and that's exactly what we want to achieve in humans. In order to be very efficient, in order to stabilize, to stop the necrosis regeneration process, you need to be very, very efficient. The objective, the threshold for gene therapy is really high for Duchenne dystrophy, and you need to be very, very efficient.
This is also dystrophin expression and reduction of fibrosis in the heart of those animals. So very similar data than in skeletal muscle. That was also a prerequisite for us before moving on into the clinic. The heart function is improved, so this treatment is really making a difference in this very severe model. You have three different functions measured here. Diastolic free wall thickness, early and late ventricular filling velocity ratio, and relaxation time on the right panel, which are normalized by gene therapy. You see the dark dots as compared to the blue dots that correspond to normal animals, and the light dots corresponding to non-treated rat MDX. Strength is also improved. On the left panel, the maximum four-limb grip strength.
Even if you repeat the test, the four-limb grip test every 30 seconds, what you see in those animals is a sort of fatigue which takes place. There's a decrease in the four-limb strength, which is normalized by gene therapy, the black squares. Now in the golden retriever dog. We have treated many animals. This is a table that recapitulates a small dose range study, two doses, low and high, IV administration, so just IV perfusion in animals that were two months old upon treatment, and that we followed over time, the oldest one being now more than four years. As I told you before, those animals, if you don't treat them, have a very short lifespan. I'll come back to that. What you see here on the left is dystrophin or microdystrophin expression in healthy dog, dystrophin expression in a healthy dog.
The upper picture as compared to untreated dystrophin-deficient dog in the biceps femoris muscle before injection. Following IV administration of vector is similar type of data as what we have seen in mice and dogs. Very high level of microdystrophin expression throughout the muscle mass. With very significant percentage of microdystrophin, more than 50%, our objective. We analyzed many muscles and different conditions, but we have really a dose range correspondence between the level of virus that gets into the muscle and the level of microdystrophin expression. Roughly, you need one vector genome, copy genome, as compared to one copy of the diploid genome, so one genome copy of the dog muscle. One-to-one ratio leads to 50% of microdystrophin expression throughout the muscle. There is a histological correction of the muscle following treatment.
You see on the left hand the histopathology, healthy control, the two upper pictures, so normal muscle, both right and left biceps femoris muscles. Untreated muscles of a GRMD dog, as expected, very severe strong modification of the histology of the muscle. You have necrosis regeneration going on in those muscles. The two bottom pictures are the same type of muscles following treatment. There is correction of the histopathology of those animals. The greater the percentage of microdystrophin-positive fibers, the lower this process of necrosis regeneration. Actually, the same holds true for fibrosis. On the right hand, the level of fibrosis that is being corrected, and this is also dependent upon the percentage of microdystrophin-positive fibers, which also shows that you need to be at least around 50% in order to make the difference in those animals Kevin showed you some MRI analysis in humans.
Here are some MRI pictures of GRMD dogs following limb perfusion administration, not IV. Just to demonstrate that if we treat only one leg, we can see the difference as compared to the contralateral leg. You have a picture showing the light intensity, which corresponds to fatty infiltration in the untreated GRMD dog on the left hand, and the right hand, MRI of a healthy dog. The bottom picture, the left is an untreated leg, and the right is the treated leg of the same dog. You don't have to be a radiologist to see the difference. Muscle strength is also increased in dogs like in mice and rats. This is proportional, not only to the weight of the animal, but also to the level of dystrophin expression. Also it increases with time. That's what we see on the right.
There is improved muscle strength at 45 days following treatment, which is almost back to normal at 90 days, as shown by the dark squares. The clinical score, there is a composite score that assess the clinical status of the animals. The clinical score, the overall score, is also normalized by treatment. This is following IV administration in GRMD dogs. Also dependent upon the dose or the load of virus that you perfuse in the animals, both at six months and nine months. The timing is important. Nine months is pretty late, which suggests something that I'll just briefly comment in a following slide. Gait analysis also shows that those animals have normal gait. They regain normal gait following treatment, as shown by the dark curves, which meets the normal curve, the light blue, as compared to the untreated animal gait analysis, the bottom curve.
We have long-term expression, and this is despite some humoral immune response. We do see antibodies against AAV. Very little against microdystrophin, but the protein is inside the cell, so it's not targeted by the antibodies. But more importantly, we don't see cellular response. We don't see tissue necrosis due to some immune response, either to the transgene or the vector. That's crucial. It means that IV administration of AAV carrying the microdystrophin gene is pretty safe and doesn't lead to tissue rejection or rejection of the transgene. You have seen a movie, very emotional movie, earlier on. It's emotional, but it can be also very informative. This is a movie of an untreated golden retriever dystrophic dog. He can barely walk, he cannot jump, he cannot run. This is a dog, seven months old, treated with the AAV microdystrophin product. Another dog treated with a high dose.
This is an untreated dog. Even the camera is suffering. Those are two dogs, seven months and nine months old, so they were treated two months. We are either five or seven months later, pretty healthy. The oldest one is now more than four years, still alive, very well, which encourages us to move on to the clinic. To summarize my presentation, I hope I convinced you that we have a good product, which is safe. We are currently doing some dose range study in the rat model in order to make sure we do frame very well those that we're going to use in patients, in young patients. We know it works in the different animals that we have tested, small or big.
We have a good idea of the optimal dose of this product that we're going to use. We are currently putting all the things together to move on to the clinic pretty soon. Thank you very much.
Thank you very much. Thank you very much for that lovely presentation, which demonstrated across multiple models, molecular biology, pathology, and functional outcomes. What you've seen so far, as promised, is a surrogate gene therapy strategy which leverages a modifier to improve function that was actually demonstrated in humans. We've seen the move now towards a microdystrophin genetic therapy with the lovely data that Dr. Braun has demonstrated. We're now going to take a brief break and reconvene at 9:20 A.M. precisely. At which point, we'll be joined by Doctors Rodino-Klapac and Mendell for discussion of the preliminary results from a phase I/II clinical trial of the treatment of Duchenne muscular dystrophy with a microdystrophin construct. Thank you. Okay. Welcome back.
I think we have just a few stragglers coming in. I think just in the interest of time, it's a long and ambitious agenda. I think we're about to hear some very exciting data. It gives me great pleasure to restart our R&D Day by introducing Doctors Rodino-Klapac and Mendell. First, let me start with Dr. Rodino-Klapac. It's really exciting not only to introduce an expert in the field, but actually also a new colleague, because I get to work with Dr. Rodino-Klapac at Sarepta going forward. She joined Sarepta from Nationwide Children's Hospital, where she had served as a principal investigator in the Center for Gene Therapy and as an associate professor in the Department of Pediatrics at The Ohio State University. She is a co-founder of MyoNexus Therapeutics and has served as CSO there since the company's inception in February 2017.
Her laboratory has developed several, as you've already heard, gene therapies for Limb-girdle and Duchenne muscular dystrophies. She has moved those with great success from bench to bedside, or into clinical development, with multiple INDs under her belt. Her dedication to innovation has been recognized by recent awards such as the 2014 Junior Faculty of the Year Award for Innovation and Columbus Business First 40 Under Forty Class of 2017 awards, which I find incredibly intimidating. She's a member of the Musculoskeletal and Membership Committees for the American Society of Gene & Cell Therapy, and she's a standing member of the MDA's Research Advisory Committee. She received her Bachelor's in Science in Biology from King's College in Pennsylvania and her PhD from The Ohio State University in Molecular Genetics, and was a Ruth Kirschstein NIH F32 Postdoctoral Fellowship mentored by Dr. Mendell.
She will be telling you a lot more about herself by implication as you see the research that she discloses today. Dr. Jerry Mendell is somebody who casts a long scientific shadow in the field. He holds the current Peters Chair of Pediatric Research at Nationwide Children's Hospital and holds multiple professorships in neurology, pediatrics, pathology, physiology, and cell biology at Ohio State University. He established the Center for Gene Therapy, emphasizing, not surprisingly in view of his background, muscle and nerve diseases. His work there included the development of a vector manufacturing core and recruiting a team of wonderful researchers that have made an enormous difference to the field over the past 14 years, you will see the results of that work today.
He has worked over a lifetime in the laboratory using experimental models of muscular dystrophy, and at the bedside, translating those discoveries into patients. He's a founding member of the study group known as the Clinical Investigation of Duchenne Dystrophy, CIDD, that defined testing methodologies for clinical trials in boys with DMD and delineated the natural history of the disease. He was a lead investigator on breakthrough corticosteroid therapy for DMD. Really has experience in those first steps in developing therapeutics for muscular dystrophy. He is the first to perform gene therapy for DMD and also started gene therapy studies in limb-girdle muscular dystrophy. In 2004, he has received multiple awards. I'm just going to highlight a few.
In 2004, he received the Scientific Achievement Award, the highest honor given by the Muscular Dystrophy Association. It gives it to scientists and physicians who have made major contributions to muscular dystrophy research. He received an honorary lifetime membership of the AAN, or sorry, excuse me, the American Association of Neuromuscular & Electrodiagnostic Medicine. In 2006, was recognized by the American Academy of Neurology for clinical and scientific contributions to the muscular dystrophy field. He was, in 2009, presented with the Distinguished Scholar Award by the president of The Ohio State University. In 2018, just this year, received the Science Breakthrough of the Year from Science Magazine for SMA gene therapy. He's also received an award from CR Forum as one of the top three clinical trial investigators or clinical trials of 2018 for SMA.
Today, he will share with you the preliminary results from the early clinical trial evaluating safety and tolerability of therapeutic doses of microdystrophin gene therapy in children with DMD. I'm going to call first on Dr. Rodino-Klapac to actually prepare you with the non-clinical data. Doctors Rodino-Klapac and Mendell. Please help me welcome them. Good luck.
Thank you, Gilmore, for that very kind introduction. Let me just start out by saying that I'm really privileged to have joined Sarepta to lead gene therapy. It feels like a very natural progression. I've been working on DMD gene therapy as well as Limb-girdle muscular dystrophy gene therapy for over a decade, alongside my mentor, my colleague, and glad to say, my friend, Dr. Jerry Mendell, for that time. We've really worked hard to establish this group of therapies. I have an amazing team, many of which have been with me for 10 years. Now it's my great privilege to transition to Sarepta, which just feels like the next logical progression to be able to take these to the finish line.
We're really at the cusp of transforming patients, this really feels like the next step, so I feel very honored to be able to take this role. For the purposes of this presentation, I'll really be focusing on the rationale behind our microdystrophin program, why we designed our vector the way we did, and the pre-clinical data that supported the clinical trial, which Dr. Mendell will show you about. We had a very rational design for our engineering of our microdystrophin construct, I'll show you the data that supports that. Dr. Mendell will take over and really talk about how SMA gene therapy provided a stepping stone to the intravascular delivery trial that we're going to show you. Lastly, focus on the clinical data, the much awaited clinical data from the microdystrophin gene therapy trial.
I think it's very clear from all of the presentations that our motivation is Duchenne. This is the most devastating childhood form of muscular dystrophy, and we've been really focused on an approach that treats not only the skeletal muscle aspects of the disease, but the entire disease, focusing on the respiratory complications and the cardiac complications as well. Ultimately, patients die from these complications. At the age of five is the average diagnosis of patients, CK elevations range from about 20,000 to 40,000. As we've mentioned, CK is an enzyme that really resides inside the muscle cell. Upon chronic muscle damage, it leaks into the serum. A normal individual has CK levels of about 150 units, whereas these Duchenne boys have much higher levels. We've heard a lot about the dystrophin-associated protein complex.
Dystrophin really functions as that shock absorber in the cell, it's important for reassembly or assembly of the dystrophin-associated protein complex. If we can restore dystrophin, we can restore function of the dystrophin-associated protein complex. The very elegant way to deliver dystrophin still remains using gene therapy, using a virus called adeno-associated virus. I'd like to take a step back and just talk a little bit about why we use AAV. AAV viruses have evolved over millions of years, really because they've been able to infect cells, they've been able to enter the body and target cells that we want. We're really just elegantly harnessing the power of that vector delivery to take out the viral payload and put in the payload that we want, which is the dystrophin gene. The AAV particles enter the cell via a receptor on the outside of the cell.
They translocate to the nucleus. The viral capsid the outside of the vector particle is dissolved, you're left with a single stranded DNA. Over several weeks, this single stranded DNA is converted to double strand and converted into what's called an episome or a miniature chromosome within the cell. It persists like this as a little miniature chromosome. It does not integrate into the genome, it's not affecting any of the genes within the cell negatively, it persists like this for the lifetime of that cell. It's ideal for muscle, which is a non-dividing cell, and it will persist through the lifetime of that cell. When we design AAV vectors, as we call them, these are single stranded DNA viruses, in the viral genome itself, there are just two genes.
This is one required for replication and one required for the capsid formation. We've simply removed these two genes and replaced them with our gene of interest, driven by what's called a promoter. The promoter is important for driving expression within the cell. That says we're going to put this virus into the body, but we only want it to express in a certain area. In this case, muscle, skeletal muscle, and the heart. That's the objective of adding the promoter. It's a very elegant and simple process. The packaging capacity of this AAV virus, which has never been associated with human disease, is about 5 KB, which poses some challenges, we'll talk about how we got around that. As we've heard, the DMD gene is the largest gene in the human genome.
It's 2.6 megabases, Translocates to about a 11kb cDNA, which obviously poses a critical obstacle. The protein itself is composed of four protein domains, the N-terminal domain, which is shown in red, this is important for binding to the inside of the cell, the actin cytoskeleton. A very large rod domain, which is shown by the 24 spectrin-like repeats, those four purple hinge regions. The hinges are required for flexibility of the molecule, which you can imagine is important for shock absorption. The cysteine-rich domain, shown in green, this is important for binding to components of the dystrophin-associated protein complex, the C terminus. How can we take this very large gene and decrease it to fit into AAV? Really the whole rational design for mini and micro dystrophins began with a very elegant case study by Kay Davies almost 30 years ago.
She observed a 61-year-old non-ambulatory Becker muscular dystrophy patient. Becker muscular dystrophy is a less severe form of Duchenne Muscular Dystrophy. It's still caused by mutations in dystrophin, these patients either have in-frame deletions or low levels of gene expression that allow them to have a less severe disease course. This patient was unique in that he had half of his dystrophin gene deleted, yet he had a very mild phenotype. His resulting natural mini dystrophin really consisted of the N-terminal region, spectrin repeats one, two, and three, as well as 20 through 24, three hinges. If we take a closer look at this mini dystrophin, it includes rod domains, spectrin repeats two and three. We've shown over the years, as we've compared our micro dystrophin to others, that these two spectrin repeats are critical for force production.
It's also been shown in recent publication by others, that these spectrin repeats are critical. We also see that spectrin repeats 16 and 17 are not included in his microdystrophin, which is where the nNOS binding site is for dystrophin. That microdystrophin was about 6kb. How can we further modify this very large gene to retain the flexibility of the molecule and the essential regions for force production? In thinking about the design of our construct, we were certain to include spectrin repeats one, two, and three to enable force production, and three of the hinge regions to enable maximum flexibility. If you look at our molecule, by deleting spectrin repeats four through 23, we really kept dystrophin in the most natural form within the molecule. We were not chopping it up so that we were creating these novel junctions within the microdystrophin.
It's in the most natural form that we could imagine. As I mentioned, cardiac complications are critical for this disease, we wanted to use a promoter that specifically had high levels of expression in skeletal muscle, the diaphragm, and the heart. We utilized a promoter called MHCK7. This is a promoter that includes the muscle creatine kinase gene, which expresses in muscle, but then also something called a myosin heavy chain enhancer, and this allows for optimal expression in the heart. We have very high levels of expression in skeletal muscle and the heart, so we're trying to protect the heart for long term. We chose to use an AAV serotype called RH74. We identified this at Nationwide Children's almost 15 years ago in a non-human primate model.
In comparison to many other AAV serotypes that are known to express in muscle, we found that it gave us the broadest distribution of muscle, including the heart and the diaphragm. In our resulting construct, we have it driven by the MHCK7 promoter, microdystrophin, and then it's packaged into the RH74 AAV serotype. Just a few more words about RH74 and why we chose it. I've already told you that we've compared it to other AAV serotypes and found that we had the broadest distribution of expression across muscle. We've now shown it to be safe and effective in multiple phase I clinical trials, by both intramuscular delivery as well as intravascular delivery. Importantly, we have very low pre-existing immunogenicity to this serotype. This is a non-human serotype, so many patients have not been exposed to this.
As a result, we see less than 15% of patients are positive for AAV antibodies to RH74. This is an important point in terms of the treatable population. We've looked across both DMD and LGMD patients and found this to be consistent across all of these forms. We've demonstrated safety with this serotype. We've done multiple toxicology studies in both mice and non-human primates. We've never observed an adverse effect level with RH74. We've now collectively dosed 14 human subjects, and this includes intramuscular delivery and IV delivery, with no vector related adverse events. There are six approved INDs using this serotype. Based on that data, we performed a definitive study, dose escalation study, using this cassette that I mentioned, looking at both safety and efficacy in the MDX mouse model of the disease. We chose three different doses.
If we look in the middle, this is our proposed clinical dose, which is 2 x 10^14 vector genomes per kilogram, which equates to 6 x 10^12 total dose in the MDX mouse model. It was performed two bracketing doses, one lower and one higher. This study was aimed to look at both efficacy and safety across multiple measures, histologically as well as functional. Following three months of treatment, we see robust levels of microdystrophin expression. Just to orient you, these top panels on this image in red, this is showing wild type stains. This is what dystrophin looks like in a wild type mouse, in skeletal muscle on the left, in the middle is heart, and on the right is diaphragm. The MDX mouse model is deficient for dystrophin, so we're seeing no staining in the second panel.
The third panel is microdystrophin expression. We're seeing widespread microdystrophin expression properly localized at the muscle membrane. Importantly, in the last panel, what I'm showing here is beta-sarcoglycan staining. As we've heard, restoring the dystrophin-associated protein complex is critical for function, and we've shown that we're able to restore the beta-sarcoglycan to the membrane in skeletal muscle, heart, and diaphragm, which is deficient in the MDX mouse. Not only were you expressing the protein, but we are restoring its functional benefit at the membrane. This is just to show that we have widespread distribution, not only in select muscles, but across all muscle types. This is just a survey of the heart, the diaphragm, psoas muscles. Both proximal muscles, distal muscles, we're seeing very widespread expression localized to the membrane.
We've quantified that, and this is the percentage of muscle fibers transduced with microdystrophin. At all three dosing levels, we see very high levels of expression. At our clinical dose, which is 6 x 10^12 in the mouse, 2 x 10^14 vector genes per kilogram in patients, we see greater than 60% of muscle fibers across all muscle groups and 100% of gene expression in the heart. Importantly, this was consistent across all animals that were analyzed. We also wanted to look at fibrosis or infiltration of the muscle by collagen. This is a staining called picrosirius red, which stains for collagen I and II, which are critical components of fibrosis. The panels that I'm showing you, the wild type image, this is healthy muscle shown in green. Fibrosis is shown in pink.
We're looking to see a dose-dependent response following treatment. At both our clinical dose and higher dose, we're seeing significant reductions in the amount of fibrosis. The graphs on the right, on the top right-hand panel, is showing that we see a significant reduction in the collagen content in these mice. We also wanted to look at force in the diaphragm. How does this improvement in the muscle result in force? We looked at the diaphragm as well as skeletal muscle force, and we saw a significant improvement in force output in the diaphragm following treatment. To further look at histology, we performed hematoxylin and eosin staining. What you notice qualitatively from the pictures is that the microdystrophin images are very similar to what we see in the wild type images. The microdystrophin is on the left, wild type is on the right.
In the middle is the MDX panel. Here we see infiltration of the muscle by immune cells as well as fibrosis, and we also see inconsistency in the size of the muscle fibers. Following treatment with microdystrophin, we see this fiber size is normalized, and that's what's quantified in the three panels on the right. This is fiber diameter measurements taken in the diaphragm muscle, the tibialis anterior muscle, which is skeletal muscle, and the triceps muscle. At all 3 dosing levels, we are seeing significant improvement in the muscle fiber size. This is just another way to say that microdystrophin is not only expressing it is functional, it is proving the health of the muscle fibers. We had an independent pathologist evaluate these slides for both efficacy and safety.
He concluded that we substantially reduced the muscle pathology in a dose-dependent manner, and that the microdystrophin did not induce any anatomic lesions within the muscle. How does this microdystrophin expression really translate into function, and how can we predict how it will work in patients? One of the ways to look at this is by looking at creatine kinase. We know that this is a marker for muscle damage. In the MDX mouse, as we've heard, and we've shown here by this tan bar on the left, we're seeing significant amounts of CK leaking into the serum. Following microdystrophin treatment, we see a significant reduction in the amount of CK following a single systemic administration. We also then looked at force in skeletal muscle. This is specific force in the tibialis anterior muscle.
We see at both our clinical dose and a higher dose, we see no difference between those two doses. They are both significantly improving muscle force in the tibialis anterior. Importantly, this therapy also prevents damage from eccentric contraction induced injury. The graph on the right is showing that we see a significant improvement by both dosing levels, which are the two purple bars, compared to the MDX mouse, which is the tan bar on the bottom, following microdystrophin treatment. Next, we also wanted to assess where the vector copies were going. We know that microdystrophin using AAVrh74 has a broad distribution in muscle, as well as expression in other organs or location in other organs.
The important point to remember is that we included the muscle specific MHCK7 promoter to restrict expression to only muscle and heart tissue, and that's what we're showing by Western blot. We have widespread expression on the top in various muscle groups. You can notice that we have significant levels of cardiac expression like we saw with immunofluorescence. Importantly, the bottom panels are showing that the MHCK7 promoter does not express in non-muscle tissue, we're not seeing microdystrophin express in the tissues that we don't want it to target. To further evaluate the safety of microdystrophin, we generated a modified construct that we could express in non-human primates. There's no non-human primate model for Duchenne, but what we can assess is distribution of expression and safety.
We modified our construct to include what's called a FLAG-tag, which is just an eight amino acid epitope that we can stain for to differentiate from the endogenous dystrophin. This image is just showing that we can express a microdystrophin with its FLAG-tag in a normal non-human primate and still see expression. On the left in green, this is dystrophin that is endogenous to the primate. In the middle panel, this is microdystrophin FLAG. By overlaying them, we can see that they co-localize, so they're both properly expressing at the membrane, and that way, we can accurately assess safety within the primate. Following a single administration of 2 times 10 to the 14 vector genomes to a non-human primate, we are just showing that we have widespread expression by both immunofluorescence as well as western blot analysis.
By H&E, we see that there was no pathology induced by this microdystrophin. These slides were formally reviewed by pathologists as well and saw no adverse effects related to treatment. We took great care to monitor safety within the non-human primate, and we did this by looking for an immune response using both T cell assay, which is an ELISpot assay. Look for T cells against both the AAVrh74 capsid as well as microdystrophin. We see, not unexpectedly, is a transient rise in a response to the AAVrh74 T cells, which rapidly comes back to baseline by eight weeks. This is something that we see similarly in all of our AAV clinical trials. Importantly, there was no immune response to microdystrophin.
We also note that there was a rise in AAV antibodies following treatment, which then plateaus by 12 weeks. This is a similar paradigm to what we see in all of our AAV clinical trials. We see a rise in RH74 antibodies following treatment. The conclusions for this part of the talk is that we've shown you that the RH74 capsid efficiently transduces all muscle types. We have very low pre-existing immunity to this virus, and the MHCK7 promoter is very efficient at expressing in both cardiac muscle and skeletal muscle. What I hope you have seen is that we have very good widespread distribution of microdystrophin across all muscle types. We have a reduction in CK and improved functional measures and no toxicity, which all bodes well for the clinical therapy that Dr. Mendell will then tell you about.
Thank you for the opportunity to be here before you today. My passion for Duchenne Muscular Dystrophy began 15 years or more before Lou Kunkel discovered the gene, and that was in the early 1970s when I was at NIH. Those boys that you saw this morning, I thought were very significant in my life. They were Josh, Ryan, and Mitchell. J.R.R. Mendell, I don't think that was a coincidence. I also have had merging opportunities that have brought me to this point, where I have an opportunity to make a big difference for Duchenne Muscular Dystrophy. The opportunities go back to my relationship with Pat Furlong, which is at least 30 years old or 30 years before us. My previous experience with Sarepta on the eteplirsen trial and my recent experience with AveXis on making big differences in spinal muscular atrophy.
All these opportunities have merged to fulfill a lifetime of expectation for me. With that in mind, let me describe a little bit about what we've seen so far in our clinical trial. Let's go back to the beginning here. Did we change the order of the slides? It doesn't matter to me. I'm very happy to start with this. This is the experience we've had with spinal muscular atrophy. With that experience, what you see on this slide are two basic cohorts of treatment that were at different dosing levels. The top 3 patient or cohort 1 at 6.7E to the 13th vector genomes per kilo. That was a dosing range that was decided, again, as Louise showed you from our pre-clinical data in the SMA mouse that usually has a lifespan of 15 days.
We also had a higher dose that showed better efficacy at 2E to the 14th vector genomes per kilo. With the lower dose, we were able to have mice survive for 35 days, and with the higher dose, we were able to have the mice survive the full length of their expected survival at over 200 days. Combined with a natural history of SMA, where there is an 8% survival at 20 months, at 13.6 months, a 25% survival, and at 10.5 months, a 50% survival. What you can see on that top arrow there is that all of our patients have survived, whether they be in the low dose or the high dose cohort, beyond the 20-month survival expected, where there was only 8% survival. Even beyond that, we've had all patients in this trial.
With our IND for SMA, it was a 2-year plan with the FDA. All patients, whether low or high dose, have survived beyond the 2-year point. Now we're actually up to 4 years post gene therapy in the SMA trial, and we have patients who are now in the upper cohort who were initially treated. We have patients surviving for 4 years. In the other 12 that are in the high dose cohort, we have patients surviving for 3 years. This has really developed a path for what Louise and I have put in place for the Duchenne trial. Am I messing this up, or are you? Let's see. Okay, there we go. Thank you. This shows the functional result of the SMA gene therapy.
What this shows is that in the high dose, we were able to achieve incredible results with all kids. Now, SMA is a disease, as I said, that doesn't really survive any beyond 2 years of age, and with much lower expectations for many patients. Now, all kids in the high dose can bring hands to mouth, have good head control, are able to roll over, and can sit with assistance, with the exception of one, and that one patient is Patient 8. The reason Patient 8 did not make it through those milestones is because we mistakenly treated that patient at a very late time in their course, and that brings up the whole point about trying to treat these diseases at early time points. Early intervention does make a difference. Beyond the sitting with assistance, we have patients now using World Health criteria.
Eleven of the 12 can sit for over 5 seconds independently, 10 of the 12 can sit unassisted for 10 seconds, and nine of the 12 can sit unassisted for 30 seconds. Two of the children are able to crawl, pull, stand, and walk, I'll show you videos of those. Essentially, we have normal survival. The patient you see on the right here, I like to call my American Ninja Warrior in preparation for his TV appearance. He was also able to take his briefcase and walk off to his office, as you can see here, and turn around and go up to the second floor so he can get to his office.
That's the SMA trial, it was the 2e to the 14th vector genomes that really laid the foundation for what we decided was the right dosing for the gene therapy trial. Let's move on to actually what we've seen in our phase I, II clinical approach. This was a trial that we went to the FDA with. It was an open label, single site, two cohort study, there was a strategy for these two cohorts. Cohort A was six subjects that were designed to be treated at three months to three years of age. We knew that patients could be identified with Duchenne dystrophy from an over 40,000 patient newborn screening study that we did in the state of Ohio.
We have patients who are born with CKs of over 2,000 right at the time of birth, we could identify these then move on to formal DNA diagnosis. We wanted to treat this group because customarily, patients with Duchenne dystrophy over four years of age are treated with corticosteroids, one preparation or another. We naturally had two cohorts. We had six subjects who were three months to three years of age for treatment without steroids, another cohort, four to seven years of age with steroid treatment as part of their standard of care. The FDA wanted us to start with the older patients, we were happy to do that's basically what I'll present today. All of them had confirmed diagnosis. We selected the patients on the basis of another prior gene therapy study.
This is one of the potential advantages of being in this field for so long. I did my first gene therapy trial back in 1999. Patients we found in a 2006 trial that we did in Duchenne, which was the first one ever done. This microdystrophin was slightly different that we used in the first trial, but had the same principle and had the same spectrin repeats or most of the same spectrin repeats gone. When you express these spectrin repeats in an area of the patient's deletion, the patient will see it as a new protein, new peptide, and develop an antibody to it, that's exactly what we saw except for patients who had exons 18 to 58. Patients who had 18 to 58 were all included in this current trial, that represents about 70% of the Duchenne population.
We can discuss that more because I have some feelings that we could overcome that, there's several ways to do that to potentially treat the entire DMD population. Patients we also learned that we should omit patients who had high antibody titers to AAV. In one of the previous studies, again, this is the advantage of being in this field for a long time, in an LGMD study that we did with alpha-sarcoglycan that was very successful, we found that patients with antibody levels that were one to 3,200 had no gene expression. It served as a blocking antibody for AAV when we delivered it. We worked with the FDA and came up with a very conservative approach, as the FDA is often inclined to do.
We're down to not admitting patients in the trial who have AAV antibody titers above one to 50. That frames the study, the four to seven-year-old patients are the ones that I can tell you about today. It was, as most gene therapy studies are, first in human, a safety trial by FDA criteria. It's a phase I/II, the two stands for also doing safety plus efficacy. One of the principal outcome measures that we were interested in in this first trial were two biomarkers. One was did the gene get in? Could it express? We looked carefully at pre-gene expression in a biopsy that we did before we delivered the gene, then one at 90 days post gene delivery. We were also interested in CK.
I'll come back to the importance of CK, but we know that this is a very important biomarker. Kids have CKs that range between 20 and sometimes as high as 40,000, or even higher. We looked at functional outcomes, the 100-meter time test, which we prefer to do after many clinical trials. We prefer that over the six-minute walk. We did the North Star ambulatory timed up and go, and stair climbing. One of the most important things I think that we can offer in this trial that we'll be closely looking at are the cardiac MRIs. Cardiac MRIs are especially important in this study because of what Louise Rodino-Klapac showed you was our high delivery and high expression, which was way beyond our initial expectations in the myocardium. The results from the first trial are starting off with CK.
We have CK levels at baseline, which are pretty much what I mentioned, somewhere close to 20,000 all the way up to 35,000. These are the first four patients we've enrolled in this trial currently. CK is an important potential biomarker because it represents when the muscle membrane is very fragile, there's leakage of CK from muscle fibers. Obviously, then CK becomes the hallmark for muscle damage. Generally, in this group that we're studying, the four to seven-year-olds, the peak of CK is very high. It actually peaks in our overlapping group in the young and older cohort that we planned for this study at two to five.
Once the patients get to seven, this is no coincidence, because if you know the natural history of Duchenne very well, what you know is that there are two remarkable events that happen in the lifetime of a Duchenne boy. I think what you saw on those opening videos is the patients do improve functionally, but they improve at a rate of improvement that is far below normal. What you see is these milestones of improvement increase till age seven. At age seven, the brain can no longer compensate. The brain-induced milestones of improving walking, improving running, stair climbing, can no longer compensate for the muscle degeneration. It is very reproducible.
If you look at all the initial clinical trials that were done, including our initial steroid study in Duchenne dystrophy, we thought we were so smart we would start the studies at age seven because then there could be no one who refuted success, because at age seven, patients were supposed to decline. Even the eteplirsen study was targeted that way. Now we know, especially from this study, as you'll see, and our SMA trial where we clearly identified patients who were in the older cohort who didn't do nearly as well as the younger patients. We know that it's much smarter to start before there's any significant muscle loss. The seven-year-old, at age seven, the CK also, as a biomarker, begins to decline. Obviously in wheelchair-dependent patients, when there is marked loss, the CK drops dramatically.
In this trial, the CK, one of the most remarkable things, I think, when we first saw this, I was incredibly ecstatic, believing that we were really on the right path here. We've had an 87% decrease in serum CK in the patients that we've treated so far. Yesterday, my research nurse practitioner sent me the results of our fourth patient. This represents the first three patients. Our fourth patient's CK yesterday was almost back to normal at 540, it was really great to see that in preparation for this talk and any questions that might come up today. As a biomarker, we have won the first round. More importantly, I think, at least as far as many aspects of what we anticipate is dystrophin expression. How much dystrophin expression can we get?
When Dr. Braun showed you those Golden Retrievers this morning, where there was an anticipated expression of over 50%, the natural history of those laboratory retrievers changed dramatically, that's pretty much, I think, what we're experiencing here. We've had microdystrophin levels that you can see in the first three patients, these are biopsies that are done at 90 days. You can see subjects one, two, and three that have reached that time point have virtually no gene expression. In Duchenne dystrophy, gene expression for Duchenne is below 5% level. Now all of these patients, you can see on the next slide, have dystrophin expression over in your left far column, dystrophin expressions that are far over 70%. One of the things I'd like to point out to you, we are doing a natural history study with carrier detection study with PPMD.
The generosity of the PPMD in working with Pat Furlong and her expectations for what are possible in this disease. What we've seen is that most patients who have gene expression who are at over 50% in carriers have very little in the way of clinical manifestations, at least in skeletal muscle. Cardiac muscle is a different story. Even in the carrier state, of course, we can see significant abnormalities in the heart when the skeletal muscle is completely normal. That emphasizes the importance of what we're doing in this clinical trial. We see, again, % gene expression. Another aspect of that that's so important is what is the membrane really showing when you put in the gene? How much of the expression is showing at the muscle membrane?
There's this combination of intensity at the membrane that we can measure with BIOQUANT analysis and the % dystrophin positive fibers. In the first 3 patients we've had another, I would say, very successful preliminary trial in both of these areas. When we look at Western blots, you see here 4 boxes that are highlighted, 1 for subject 1 for subject 2, and 2 for subject 3. These are from needle biopsies that are taken from the gastroc pre-gene delivery. What you see there are virtually no gene expression with Western blot analysis. When we do the biopsies at 90 days, what we've seen by Western blot analysis is over 50%. This is the target that Dr. Serge Braun wanted to achieve, so we have satisfied him, and I hope we've satisfied others.
Sarepta wisely chose another method for confirmatory results and got results that were slightly lower, but not at all discrepant in terms of good gene expression. The other thing that's important, I think again, the way these talks were lined up was fortunate for me In the sense that Dr. Serge Braun said that 1 copy of a vector genome was what a cell needed to express, what we've seen in our trial now is over 1 copy. This means that when we put in the gene, this is really an unknown factor, how much gene could we get in and deliver to widespread areas of skeletal muscle? The widespread areas are told by the CK elevation, when we measure it directly in the muscle fibers, we see over 1 copy per nucleus, consistent with the microdystrophin levels that we're seeing.
If that is not enough, I can tell you that 1 of the most important things, again, the former talks told you about the importance of the DAPC, what you see here is restoration of the DAPC. You see alpha and beta sarcoglycan. 2 important components of the DAPC are completely restored. This means that despite the fact that this microdystrophin is basically less than 50% of the size of the normal dystrophin, it's really doing its job. It shows ligands at the myofilament level, ligands at the membrane level to other dystrophin associated glycoproteins, and dystrophin at the membrane at the basal lamina. If that's not enough, I'll show you really what makes my day, what makes my day are the kids that we have in this trial.
This is one of the boys that was two days post gene delivery, going up the stairs, cannot do reciprocal stairs. You saw that happening when you saw the pre-delivery. This really illustrates how important this is. Watch on the other side. This is 60 days post gene delivery, and this is beautifully illustrated here that he's got good proximal and good distal muscles that will allow him to push off with his lower limbs and to extend his muscles both at the glutei and at the hip flexors. We see another one of our patients post-treatment who easily negotiates the stairs at reciprocal stair climbing. This is one that, as a former soccer coach for my son, if we can achieve that, let's go back over that because it's such an inspiring one. Can we redo that video probably? Yeah, there we go.
You saw that one child running who, at my age, my running is more like the Duchenne child. This is one that is really truly inspiring. Good proximal and distal muscles, good leg extension, and ready to go out and knock it out of the park. This is 90 days post-treatment. I don't want to belabor too much, except that safety is something we saw. This is very reproducible from the gene delivery that we had in the SMA trial. In the first instance in our SMA trial, at doses of 2E to the 14 vector genomes per kilo, we had AST and ALT elevations in the 13 to 1,500 range. Frankly, I had never seen an SMA trial with elevations of liver enzymes that high.
First thing I did was go back to the FDA, and to our clinical reviewer, called her on the phone and said, "This is what we're seeing. This is the first child. What would you recommend?" What she recommended was that we suppress the elevated liver enzymes with steroids. Remember, this is SMA, and we don't treat SMA kids with steroids. We did that, and then we started another principle. Before we would deliver the gene, we started prophylactic steroids, prednisolone in the babies one day before gene delivery and maintained it for the first 30 days. In Duchenne, we have a little different circumstance, and we have patients who, four to seven, are naturally getting steroids. In this trial, one of the things I advocate very strongly, and this is highly controversial amongst different clinicians for DMD, I like to use weekend dosing.
That drug holiday maintains a long bone growth, and it prevents the cushingoid side effects. All of the kids in our trial are on weekend dosing. In this trial, what we did was we started daily dosing one day before and maintained it until. We've seen elevated GGT levels. GGT is the one way we have a window of opportunity to look at liver enzyme elevation, because AST, the aminotransaminases, as mentioned by the previous speakers, is elevated from muscle, not from liver. It does obscure any elevation from skeletal muscle. We use GGT, and actually, three of the patients have had elevated GGTs, so not surprising. It has not reached the mark that we set as an SAE. None of them have had elevations over three to five times normal.
That's where we are, actually there are no other significant clinical abnormalities except that we have seen boys who have had nausea during the trial, and we're frankly not sure whether that's from steroids and from reflux. Most of them have not been sick at all. Usually at times in the morning we'll have an episode of vomiting, then we'll be fine for the rest of the day. We've had very good safety results so far in this trial, despite these doses. I might remind you that 2E to the 14th vector genomes per kilo is the highest dosing level that have ever been used in any clinical trial. I'm sure we'll want to discuss that a little bit more. Where are we going with this trial currently?
We have been working with Sarepta, and it's been really my privilege to work with them for different clinical trials. This one, I'm very grateful for the opportunity to expand this in a way that we probably wouldn't have the opportunity unless we worked with industry. We have planned a placebo-controlled trial, and you can imagine from my comments why we need a placebo-controlled trial in this group. It's this very 4 to 7-year-olds that we're continuing to see. We see some milestone improvement no matter, even as part of the natural history of the disease. We have to ask, how much more improvement can we achieve with this? I think we've really demonstrated that with our video, since we don't see reciprocal stair climbing at this age. Nevertheless, we'll have skeptics out there.
When we first introduced prednisone, we had multiple skeptics, and since that time, we've had 6 double-blind, randomized controlled trials that have confirmed our results. I'm confident that we'll be able to continue this, but it will be reassuring, especially to the industry arm of this disease that is interested in this trial. We'll have 12 treated and 12 placebo for the first year, then all patients will be rolled over to be treated. The endpoints will naturally be safety, the demonstration of microdystrophin levels, and the other things that I mentioned, decreased CK, the 100-meter walk test, and so forth. That's where we're going with this. We hope to start that in a reasonable time. We'd have to, I guess, rely on Doug to give us some timeline for that. We're very happy to do that.
In summary then, we can say that our results, again, here, as they were in the SMA trial, I didn't get to show you any of the preclinical data there, but it was very predictive of what we'd see clinically. Here we're seeing consistent data with the preclinical results, widespread microdystrophin expression, upregulation of the dystrophin-associated protein complex, reduction in CK, good vector genome copies telling us we have delivery of the gene. The use of the MHCK7, I didn't emphasize this enough. We believe that as you see clinically, I mean, as we saw preclinically with 98% or so, not much less than that gene expression in the myocardium, we should be able to really correct the cardiac defect. Think of it this way, because the cardiac cells or the cardiomyocytes have one nucleus.
That picture that Louise showed you where there was transduction of one cell, where the gene goes into the cell, is delivered to the cell, then is transported to the nucleus, and expresses there for an indefinite period of time. If that is able to as well in our patients as it does preclinically, we will really have saved the heart. That is actually an easier task, and this is one of the concepts I learned in our SMA trial. Duchenne is a very challenging disease, and all muscle is very challenging because we have a fiber that is very long, and throughout that fiber we have multiple nuclei. We have to transduce all those nuclei over time, and with that one single injection. If we miss a section there, then we expose that domain of the muscle fiber to undergo necrosis.
What is saving us there, and I hope that we will be able to see that with Charlie Gersbach's trial, where he has been able to demonstrate. I said Gershon, that was good. Gersbach. Anyway, it was a compliment. He is able to show that it is possible to transduce Pax7 cells, which reflect the stem cells of muscle. If we can, and we are beginning to be able to do that, and I will let Louise comment on that in a question and answer session, but if we can translate or transduce the satellite cells, then that means that these domains of the muscle fiber that are not transduced initially, if they undergo necrosis, they will be selectively then over time, and this is what we see in the carrier state many times. We see fiber selectively increasing dystrophin over time because the stem cells are transduced.
In the heart, I hope I am keeping my train of thought here, because this is very exciting stuff. If we can transduce the heart in the first injection, all the nuclei in the heart at the 98% level, we will be able to save the heart. Really, what does that mean? It means that the question we get asked so often is, "Okay, so you treat the younger boys. What happens to the remaining patients that are eight, 10, 12 years old?" I believe we can save their myocardium, and if we can save their myocardium at a time when we can improve muscle function in their upper limbs, even if their lower limbs are not working well, we can save the life of that child, and we can continue to improve function. I think probably have said too much.
Maybe I still have a little time, but I am very grateful for this opportunity. This really, for me, fulfills a lifetime of work. Thank you very much. Are the boys coming? I have invited the patients that we have treated so far to come and visit us here at this session. Here comes my soccer player. My little other guy is here, and I want two things. Remember that these are Jerry's kids. Remember, please do not take photographs. This is really a private matter, but I am very grateful they have agreed to come. Ian, you brought them up here, or Francesca, how do you want to proceed with these wonderful boys? Thanks for coming. How are you? Great seeing you guys.
You too.
Thank you. All right. I've said enough, I'll turn it over.
Deep breath. Thank you. Thank you so much. I think as we continue R&D Day, you'll actually see the evolution of the story, where I think probably the most incredible thing is to actually see not just science and science applied to the clinic, but to actually see people, patients. Thank you so much. Now we're going to continue our story as we, again, focus on the core of dystrophin, I'm going to ask Dr. Charles Gersbach to come up and talk about his work looking at genome editing for Duchenne muscular dystrophy, with specifically a focus on dystrophin. He's going to walk you through the status of gene editing programs currently in development. He is the Rooney Family Associate Professor at Duke University, Department of Biomedical Engineering and Orthopedic Surgery.
He's an investigator at the Duke Center for Genomic and Computational Biology, and is director of the Duke Center for Biomolecular and Tissue Engineering. His research interests are in the editing of the genome and epigenome in gene therapy, regenerative medicine, biomolecular and cellular engineering, synthetic biology, and genomics. He earned his PhD in biomedical engineering from Georgia Tech and Emory University before completing postdoctoral studies at the Scripps Research Institute on the West Coast. I'll say, came to Duke, where his work has been recognized through a number of awards, including the NIH Director's New Innovator Award, the National Science Foundation's Career Award, and the Outstanding New Investigator Award from the American Society of Gene & Cell Therapy. In 2017, he was inducted as a fellow of the American Institute for Medical and Biological Engineering. Please help me to welcome Dr. Gersbach to the stage.
Thank you. I'd like to thank the Sarepta team for the opportunity to come and talk to you today about our Sarepta-supported work in the area of gene editing for Duchenne that we're doing at Duke. Also thank them for the honor and the tremendous challenge of following what we just heard in the earlier speakers. Obviously, that work was focused on gene therapy and on the introduction of new genes into cells. What I'm going to talk about is gene editing. The summary of today's presentation is first to talk about an overview of gene editing, follow that up with how we're specifically applying it to Duchenne, then I'll walk you through the different models that we've explored this approach in.
Starting with patient cells, then moving in vivo in the mdx mouse model, followed by our most recent work in generating a novel humanized mouse for preclinical development, then I'll touch on the end, what Dr. Mendell was finished with, talking about the editing of satellite cells, the adult stem cell of skeletal muscle in vivo, and then summarize. As I mentioned, the talks we've heard about earlier today are about gene therapy and adding extra genes to cells that either compensate or replace the function of mutated genes. Those are, as was described earlier, are expressed typically from these AAV viral vectors.
With gene editing approaches, what we're really aiming to do is to go in and correct the mutation in the endogenous position in the chromosomal DNA where that gene exists naturally and see if we can restore the expression of a functional gene in that context. There's many unique advantages and also great challenges in using gene editing compared to gene therapy approaches. Nevertheless, I think I've been particularly impressed in working with Sarepta over the last year at their aggressive pursuit of multiple platforms for tackling this very difficult disease. I'm going to talk about this approach that we're using to use gene editing to correct endogenous dystrophin mutations.
Gene editing in general is based on the concept of introducing breaks into DNA and then taking advantage of the DNA repair mechanisms that the cell normally uses to repair those breaks to introduce target-specific sequence changes into the genome. It's important to recognize that these breaks that we're introducing into DNA are very common and naturally occur in our cells all the time. Every time a cell divides and copies its genome, new breaks get introduced. If you go out in the sun and expose the UV light, new breaks get introduced. Cells have evolved very good mechanisms at efficiently and precisely going in and repairing these breaks. What was realized in the mid-'90s was that if we could control exactly where this break was introduced, then we could either insert or exchange DNA at that site using a process called homology-directed repair or HDR.
We could go and shift by adding or removing a few base pairs, shift the DNA through a mechanism called non-homologous end joining or NHEJ, and that can be used to knock out splice sites or knock out genes or shift the reading frame of genes. What actually I'm going to focus on is the idea of making two of these breaks at adjacent regions in the chromosome and removing that segment of DNA very precisely. That early work, more than two decades ago now, described the concept that if you could create these breaks, then you could control those different repair outcomes. That started off the race to develop new technologies that would allow you to go and make those breaks at very specific sites.
We now have more than two decades of work of developing a variety of platform technologies that allow you to go in and precisely make those breaks with enzymes that are called nucleases due to their function of breaking nucleic acids or cutting DNA. That starts with a variety of protein-based platforms, including meganucleases, zinc-finger nucleases, and TALENs, and of course, most recently, the RNA-based platform, CRISPR-Cas9. CRISPR-Cas9 has really transformed the gene editing field, particularly because of how easy, fast, and robust the technology can be.
I won't go into too much detail over the basics of the technology or how CRISPR functions in its normal place in bacteria, but in its repurposed form for genome editing, it has two components, the Cas9 protein, the nuclease that is the blue blob shown in this cartoon, and the guide RNA, which is a short 100 nucleotide RNA that makes a complex with Cas9, as shown in this diagram. Then that complex goes and hunts for its target site in the genome, which it finds by complementary base pairing between 20 nucleotides of RNA sequence matching a sequence that it's programmed to target in the DNA. Once it finds that site, it forms a stable complex. Based on the precision of the targeting, there's then a conformational change to the Cas9 protein, which then leads to cutting of the DNA.
Now with the knowledge in hand of how gene editing can be used to introduce sequence changes into the genome, and robust technologies like CRISPR-Cas9 available to go in and make those targeted breaks, it's possible to envision a large number of different ways in which gene editing can be used to correct mutations that cause genetic disease. However, we believe that Duchenne is a particularly compelling application for gene editing technology. Of course, the reasons for that start with the current unmet clinical need. It also extends to the fact that, as we've heard this morning, there are a variety of delivery options available for getting nucleic acids or gene editing tools to skeletal muscle using platforms like AAV.
As we heard earlier, the multinucleated nature of muscle fibers, and the fact that each muscle fiber has hundreds of nuclei, we believe allows for multiple shots on goal, where even correction of or gene editing of a fraction of those nuclei would allow dystrophin production along the length of that, and protection of that fiber. Additionally, we'll talk about this later in the presentation, because there are adult resident stem cells in skeletal muscle that continuously participate in regeneration and repair, we think that if it's possible to edit those stem cells, that that would allow for a long-term sustained restoration of dystrophin expression and compensation for the disease. Also, there's many options available for editing dystrophin.
As we've shown in this cartoon, there's options for adding or removing DNA, there's options for ablating splice sites or shifting the reading frame, also options for removing whole exons. That really guides how we think about using gene editing to target the dystrophin gene. It's also motivated by this histogram that shows where along the length of the gene most of these mutations occur. Most of the mutations that lead to Duchenne are actually deletions to the middle part of the dystrophin gene. As shown here, those tend to pile up in that exon 45-55 region, has been highlighted throughout this morning, that actually that 45-55 region occurs in this rod domain that is actually expendable for the overall function of dystrophin.
Additionally, removal of just exons 45-55 is a strategy that could address about half of all of the Duchenne patients. This really motivates our strategy, which is to go in and remove exons, which was also inspired by the work of Sarepta and others over many years using oligonucleotide-mediated exon skipping at the RNA level and removing exons from the RNA. In this case, if we use gene editing, we can remove those exons from the DNA, and with a one-time treatment, permanently correct functional dystrophin expression from that cell and all of its daughter cells. Why are we focused on exon deletion versus the variety of other approaches that we could use for editing the dystrophin gene?
First of all, it does not require that homology-directed repair pathway, HDR, which we know is much less efficient in skeletal muscle cells, and in particularly in post-mitotic cells like muscle fibers and cardiomyocytes in the heart. Instead, it relies on this non-homologous end joining pathway, so a very constitutive mechanism of DNA repair. Also, because we're cutting in the intronic regions flanking the exon, the introns in the dystrophin gene are exceptionally large, many of them hundreds of thousands of base pairs. That gives us a lot of flexibility of design space to pick out the very best guide RNAs that are most specific and most efficient at editing the dystrophin gene, rather than restricting the design space for guide RNA engineering to a narrow range.
Finally, as I mentioned earlier, if we're thinking about exon excision, we can envision a single gene editing approach that removes exons 45-55 all at one time and gets about half of all of the patients with a single gene editing strategy. To give you some idea of how we're thinking about moving forward with this technology, one of the first things that we did was to work on restoring dystrophin expression in patient cells. In this case, we started with patient-derived skeletal myoblasts that the inherited mutation was deletion of exons 48-50. As a result, exon 51 is now out of frame and you don't get any of the correct dystrophin being produced.
Inspired by what had been demonstrated for exon 51 and other exon skipping strategies, we designed a CRISPR system to go in and remove exon 51 that would then restore functional dystrophin. That removal of exon 51, as we know, is applicable to about 13% of the dystrophin patients. With this type of approach, we have these patient cells in culture. We design a variety of guide RNAs that target in or around exon 51. Then we want to deliver these guide RNAs to the cells, in this case, by plasmid electroporation. In particular, we want to use pairs of guide RNAs that flank exon 51. It actually removes exon 51 from the genome. For example, in this cartoon, CRISPR 1 and 5, or CRISPR 2 and 5.
In fact, on the right, when after three days after treating these cells, we collect the genomic DNA and run PCR across the locus, we see that either with no guide RNA or only one of the guide RNAs, the only band that we get is that inherited delta 48-50 band. However, if we deliver 1 and 5 or 2 and 5 together, we get a smaller band. It occurs in about 10%-15% of the alleles. We can sequence that band and see, in fact, it's the perfect deletion between the two predicted guide RNA cut sites in the intronic regions. That shows that we can remove exon 51 from the DNA.
Obviously, we ultimately want to restore dystrophin expression. We can take those muscle cells in culture, differentiate them so they upregulate dystrophin expression. Then look at the RNA, and we see that, in fact, only when we deliver the two CRISPRs together do we get that smaller band that is missing exon 51 that we can confirm by sequencing is the exact predicted junction of exons 47 and 52. By Western blot, we also demonstrate that there is now restored expression of what we know is a partially functional dystrophin protein by these cells from this Duchenne patient. That was focusing on removal of exon 51 that we know can address 13% of patients.
I motivated the talk earlier by saying that what would be particularly exciting is a single gene editing strategy that could address a larger patient population, in particular, removing all of exons 45-55 to get to half of the Duchenne patients. This is an example of our results in that area where we took those same patient cells, but this time treated them with guide RNAs targeting introns 44 and 55 with the idea that it should remove this whole 336,000 base pair region from the genome. In fact, that's what we see, whether we use a human cell line or we use the actual patient myoblasts, we can confirm by PCR. In fact, again, it's the exact junction between the predicted cut sites in introns 44 and 55.
Again, looking at the mRNA level, when we treat with both of those CRISPR RNAs, we get a new mRNA species. It's the exact junction of exons 44 and 56, which then leads to restoration of dystrophin expression. All of that work was with patient cells in culture, to move in vivo, we then adapted the system to explore in the mdx mouse model of Duchenne. The mdx mouse model has been the workhorse for Duchenne research for decades now because it recapitulates at least some of the symptoms of the disease. In this case, it results from a point mutation to exon 23 of the mouse gene that then causes a premature stop codon and prevents expression of this full length dystrophin gene.
However, this exon 23 is very similar to human exon 51, and that if you remove it or skip it, that the rest of the gene is then in frame, and you can restore expression of a dystrophin protein. Now in this case, we redesigned our CRISPR system first so that we were using guide RNAs that were targeting around mouse exon 23, then using a new Cas9 that is small enough to fit into AAV vectors. In this case, we combined those guide RNAs and Cas9 onto an AAV vector. We inject it into the leg muscle of a mouse, and after eight weeks, we explored looking for dystrophin restoration. As has been shown in several other talks this morning, if you take a cross-section of wild-type muscle and stain for the dystrophin protein, you see that all of the fibers are lined with dystrophin.
In the mdx mouse, none of that dystrophin is present. If we take that same mouse and treat it with this AAV vector encoding the CRISPR system, now we see a significant amount of dystrophin restoration in many of these muscle fibers, about 70% after quantification. That, of course, is coming from the endogenous chromosomal position of the gene that we've now edited and restored expression from. We've suspected that about 70% restoration should lead to functional benefit, particularly because when we take, as shown on the left, a complete cross-section of the muscle, we see that the dystrophin restoration exists throughout the muscle tissue.
Moving from left to right, whether with our collaborators looking at force production by these muscles, first at twitch force, looking at generation of a contraction, tetanic force, looking at holding a muscle contraction, or looking at fatigue over repeated rounds of contraction. In all cases, these muscles got stronger with the AAV CRISPR treatment. That was some of the earlier work, a lot of that was focused on local administration to a single muscle. In all of our work, or most of our work since then, we've really been focused on systemic administration to adult mice with clinically relevant doses guided by Dr. Mendell's 2E14 vector genomes per kilogram dosing regimen.
What we've seen in those mice is not only dystrophin restoration throughout all of the muscle tissues, including the heart, but when we look at long-term time points, we see that that editing of the dystrophin gene is maintained in the heart and in the skeletal muscle tissues, even increases over time between eight weeks and one year. I also want to note that we've seen with all of the mice that we've treated, we've never seen any type of toxicity or adverse event with these mice, even taking them out well past one year post-treatment. That demonstrates in vivo editing and long-term restoration of dystrophin and functional benefit in these mice through this gene editing approach. It's using this CRISPR system that targets mouse exon 23.
The mdx mouse has been valuable for treating the symptoms of the disease for decades because it does replicate some of those symptoms. Since we're actually treating the specific sequence mutations in the gene, any type of preclinical candidate would have to be targeting human gene sequence. Targeting mouse exon 23 doesn't do us much good. For that reason, we've now moved forward for preclinical development and created our own novel humanized mouse model of Duchenne. For that, we started with a mouse generated at the Leiden University Medical Center, where they inserted the whole full length human DMD gene onto the mdx mouse background. Because it's on the mdx background, it doesn't produce any of the mouse dystrophin, but it has the full length human gene inserted on chromosome 5, including all of the human exons, introns, and promoter structure.
We acquired that mouse. However, it expresses the full length human gene, it doesn't do us much good in terms of being able to treat the disease or look for restoration of dystrophin expression. For that reason, we used the gene editing to go into the germline of these mice and create a mutation that mimics the human disease. What we started with is actually removal of exon 52 from the human gene. The reason why we did that is because that now gives us a model that can be treated by either removal of exon 51, removal of exon 53, or removal of the full exons 45 through 55 region that we discussed earlier.
The first place that we explored testing this model was to re-engineer our CRISPR system to flank human exon 51, again, with the smaller Cas9 that is compatible with the packaging limits of AAV. First, we wanted to confirm that this mouse we've created the model that we're interested in. We confirmed by immunohistochemistry and Western blot that both the heart and skeletal muscle tissues are no longer making that human dystrophin protein that the parental mouse was expressing, and also that these mice have a dystrophic phenotype, including decreased overall activity and decreased grip strength. This demonstrates that this is a viable model for developing human-targeted genetic medicines and testing them in this small animal model.
In fact, when we take that new CRISPR system targeting the human gene in the AAV vector and deliver it systemically into this mouse, either as an adult or a neonate, we see very efficient editing of the heart, efficient editing of the skeletal muscle, and we see restoration of dystrophin expression in these tissues as well. All of that work kind of was focused on editing of the whole muscle tissue, primarily looking at muscle fibers and restoration of dystrophin expression of those fibers. A longstanding question in the field has been, how long will this dystrophin restoration continue for, and what is the sustainability over repeated rounds of regeneration in this muscle?
In particular, because we know that skeletal muscle is continuously regenerated by what are called satellite cells that are basically the adult stem cell or progenitor cell of skeletal muscle. The satellite cells exist on the outside of muscle fibers, and when there's either damage or repair to muscle fibers, these satellite cells asymmetrically divide, where one of the daughter cells stays as a satellite cell, the other one fuses into the muscle fiber and becomes a myonuclei and participates in muscle regeneration. Obviously, correcting the dystrophin gene in these progenitor cells and having them continue to divide and proliferate and regenerate this muscle tissue over the lifetime of the patients would be highly desirable.
A longstanding question in the field has been whether or not AAV transduces these satellite cells in vivo, and then obviously whether CRISPR can edit these satellite cells in vivo, and then finally, would satellite cell editing facilitate long-term dystrophin restoration? We've begun to pursue some of these questions building on other work in the field from Amy Wagers' group and others, where we used a mouse that has GFP inserted into exon 51 of PAX7. PAX7 is a transcription factor that is specific to satellite cells and is responsible for coordinating the gene expression that maintains satellite cell fate. In this mouse, all of the satellite cells should then be labeled green from expression of this green fluorescent protein from the PAX7 locus.
In fact, if we take all of the mononuclear cells from the skeletal muscle and analyze them by flow cytometry, as shown here, we see about 5% of those cells are GFP positive, which is consistent with earlier estimates of the frequency of satellite cells in skeletal muscle. We take that mouse and we treat it with the CRISPR system targeting mouse exon 23 of the dystrophin gene and ask the question, in those green satellite cells, do we see gene editing? In fact, if we sort out those cells using flow cytometry, that is exactly what we do see, that the satellite cells specifically have a population where exon 23 has been removed, whereas the other mononuclear cells in the skeletal muscle, which probably consist of blood cells and other cell types, we do not see that editing after AAV delivery to these muscles.
This is exciting in that we might be not just editing the muscle fibers, but also editing the stem cells in vivo and restoring dystrophin expression. To further explore this phenomenon in high throughput, we're actually using a mouse where following AAV delivery of a Cre recombinase enzyme, it will remove a stop cassette inserted in this mouse genome upstream of tdTomato, which is a red fluorescent protein, remove that stop cassette and turn those cells red. The question is, of the green cells that we know are the satellite cells, after AAV delivery, how many of them turn red? That's the question we asked across a panel of different AAV serotypes.
We see either after local delivery, we can find several that give us very high genome editing by Cre recombinase in these cells, approaching 60% from multiple different serotypes by local delivery, and hovering between 20%-40% across many different skeletal muscle tissues following systemic delivery to adult mice. We think that this is a particularly exciting and unique application of gene editing, is to correct the endogenous progenitor cells. We can use immunofluorescence staining to go in and confirm that these edited cells that are now expressing this tdTomato red fluorescent protein are in fact also the Pax7-positive satellite cells in the proper location in the skeletal muscle adjacent to the muscle fibers and underneath the basal lamina. To summarize, I think we've shown that genome editing can be used as an effective approach to restore dystrophin expression.
We believe that exon excision provides the greatest flexibility in designing safe and efficient editing strategy that can address the largest group of Duchenne patients. AAV is an effective delivery for these systems, and with that approach, we see dystrophin restoration out for over a year, increasing over time with no obvious adverse events. For exploring preclinical development of human-targeted genetic medicines, including CRISPR, we've created this new humanized mouse model of Duchenne and validated that that works for evaluating these tools, and we're particularly excited about the potential for editing of muscle stem cells. The ongoing efforts involve optimizing our design, including efficiency of editing, and scaling this up for large animal studies in collaboration with Sarepta, and doing a rigorous evaluation of the safety and immunogenicity of the different components that we're delivering.
I also want to mention that I think it's important to recognize that all of the lessons that we're learning and technology that we're developing for gene editing in the context of Duchenne would be likely equally relevant to exploring gene editing for other inherited neuromuscular genetic diseases. With that, I want to thank the recognized, the lab members and collaborators who are responsible for this work, particularly David Ousterout, Christopher Nelson, and Jacqueline Robinson-Hamm, and Jennifer Quan, who generated the results that I showed today, and also those who provided support, and particularly most recently, Sarepta Therapeutics, that's really catalyzed the development of this program. Thank you for your time.
Thanks very much. That was great. Thanks very much. We actually realized that we started you very early this morning, and it's a long day, and we've actually given you a lot of information. What we decided to do was humbly submit that we take a break now, lunch break now for 30 minutes, returning at 11:20. Lunch is actually outside, believe it or not, so you get to attend to 11 lunch. Not something I ever anticipated experiencing when I grew up, where I grew up. We're very open to join us for lunch, and then we return 11:20, where we will continue the walk along dystrophin and into the sarcoglycan complex and beyond. Thanks very much. Okay. I'd love to say good afternoon, but it's not good afternoon yet. Welcome back to the remainder of R&D Day.
What I'd like to do is invite Dr. Rodino-Klapac back up to the podium to continue our walk beyond dystrophin and over to some of the components of the dystrophin associated glycoprotein complex that we've talked about, and actually go a little beyond that. What Dr. Rodino-Klapac will talk about is gene therapy approaches to the treatment of limb girdle muscular dystrophy. Help me welcome her to the podium again. Where's the clicker? There, perfect.
All right, thank you. Now I have the unique opportunity to follow up on our microdystrophin program and really walk you through how what we've shown with microdystrophin has direct application to a whole another class of diseases called the limb girdle muscular dystrophies. I hope that you'll be excited about the platform approach that we're taking to being able to accelerate multiple programs forward and help another class of diseases with really no current treatment options, and a significant unmet need. In today's presentation, I'll walk you through a general introduction of limb girdle muscular dystrophies and really hone in on the shared therapeutic design principles that we've employed amongst LGMDs and DMD. What you don't know is that we've been working on LGMD in parallel to DMD for over a decade now.
We've been learning from one program to another, passing along knowledge from one program to the other, and this has allowed us to really accelerate all of our programs. I'll touch on the fact that there's some history involved in our limb girdle programs that goes back to 15 years ago, and that we took a very stepwise approach to our program, starting with small intramuscular proof of principle studies before moving to vascular delivery. I'll again touch on the rational design of each of these programs using the same AAV serotypes and muscle specific promoters, and really show you some robust preclinical data in many of our LGMD programs that have allowed us to gain approval directly to a phase I systemic study for LGMD. What are LGMDs? Limb girdle muscular dystrophies are a group of approximately 20 different disorders.
As the name implies, limb girdle, the weakness in limb girdles often begins in the shoulders and the hip muscles, but it eventually progresses distally and affects all muscles. What's unique about the LGMDs, or the Type 2 LGMDs, is these are autosomal recessive, so they affect both males and females equally. They affect all skeletal muscle, and also cardiac muscle in many of the subtypes. We see many similarities to Duchenne. We see elevated CK. We often see symptoms develop in many of the subtypes before the age of 10. In the case of the sarcoglycans, before the age of five. We see loss of ambulation in the early teens, and death from cardiac and pulmonary complications in many of the forms. There's so many parallels between Duchenne and many of the LGMDs, and so we've been able to apply knowledge across our programs.
When we look at the LGMDs collectively, the prevalence is about 1 in 14,000, so we're looking at a similar range of Duchenne. The MyoNexus programs are targeting up to 70% of these patients collectively. The LGMDs, and I mentioned there was about 20 different forms, are caused by defects in proteins in the dystrophin-associated protein c omplex. Each of these LGMD subtypes is caused by a defect in a unique gene. The MyoNexus programs are really focusing on two different pathogenesis. One, in the sarcoglycan complex. This we've talked a lot about is a component of the dystrophin-associated protein c omplex. The sarcoglycans are embedded in the membrane, and they're more of a functional connection between dystrophin and the extracellular matrix. The other class of proteins we're working on are involved in a process called membrane repair.
Through normal daily activity, you get little micro tears in your muscle. Proteins like dysferlin and annexin A5, which we'll talk about, come in to be able to repair those micro tears, so that you can function normally. When these proteins are absent, you're not able to repair those little micro tears, and eventually you get chronic muscle weakness and wasting over time. To describe two classes of molecules, some that are embedded in the membrane, some that are involved in membrane repair. The primary defect may be different, but the end result is the same. All limb girdle muscular dystrophies, you have loss of muscle over time, infiltration of the muscle by fat and fibrosis. Our lead program is focused on gene beta-sarcoglycan for the LGMD2E subtype.
I wanted to spend some time on beta-sarcoglycan because it's the core component of the sarcoglycan complex. In the formation of that complex, beta-sarcoglycan forms at the core, and the other sarcoglycan proteins bind around it. The beta-sarcoglycan protein is shown in purple, the other sarcoglycans in red. This binds to the dystroglycan, which is shown in dark blue, as well as dystrophin. It's critical for forming the dystrophin-associated protein complex. We also see that when we lose beta-sarcoglycan, we lose dystrophin. It essentially ends up being a dystrophinopathy where you have a loss of dystrophin, and ends up having very similar phenotypes to DMD. For the MyoNexus programs, we really focused on targeting the most severe and common forms of LGMD to start. We have 3 clinical stage programs.
LGMD2E is our first program, which will start an IV trial later this year. We'll talk a lot more about that. alpha-sarcoglycan is finishing up in a limb perfusion study, and our dysferlin program for LGMD2B is in an intramuscular proof of concept study. We have two preclinical studies, and those are focused on LGMD2C, gamma sarcoglycan, and ANO5. As we go through these names will become more apparent and familiar. The LGMD as a whole, as I mentioned, represent a very large unmet need. There's currently no treatments for these. There are several tens of thousands of patients across the world that have no treatment, and we're targeting a large percentage of this population. Spending a little bit of time on the shared design principles. We're having very good success in DMD using the Rh74 AAV serotype, as well as the MHCK7 promoter.
We've been able to use these along the way. We've identified Rh74 10 years ago, and been using it in our limb girdle programs for quite some time. As you can see, we're using a modular design. We can decrease the time. We've been able to get to approval of the clinical trial much faster because we can essentially refer to the other programs. Not to make light of it, but basically we can take the same genetic cassette, remove the gene and replace a different gene. It's a very platform approach to therapy. We chose Rh74 for LGMD in addition to DMD for the same reason. It has widespread distribution across muscle, as well as cardiac tissue.
We've chosen to use the MHCK7 promoter in cases where there's associated cardiomyopathy, but there are some forms of limb girdle that do not have cardiac involvement, such as LGMD2D, and for that we're able to use different cardiac or skeletal muscle specific promoters that don't express as highly in the heart. In focusing on our lead program, beta-sarcoglycan, we have robust preclinical data that really led to the approval of the upcoming clinical trial. What's unique about the sarcoglycan proteins is that they're much smaller than dystrophin. We don't have the same limitation as far as fitting into the AAV vector. The cDNA for beta-sarcoglycan, for example, is less than one kilobase. When we add up the promoter and the rest of the cassette, we're at less than 2 kb. This allows us to fit into an AAV vector that's called a self-complementary vector.
If you remember back to the first presentation, we talked about the fact that that was a single-stranded vector that had to be converted to double strand. What self-complementary allows is that it's been engineered so that when it goes into the cell, it automatically converts to double stranded, and this allows for more rapid and robust expression early on in the cases where you have a small gene like this. Using this vector, we've achieved up to 99% of muscle fibers expressing following our studies, and I'll go through those in very much detail, with no associated safety issues. This trial will begin in the third quarter 2018 with early data in 2019. This program has been granted orphan drug as well as rare pediatric disease designation.
These are results following a single IV injection in the beta-sarcoglycan knockout mouse model for the disease, which is a very good model that recapitulates it, as we see in patients. This is gene expression following six months of treatment. What you'll see on the left two panels, we're showing gene expression in the diaphragm and the heart here. For first you see beta-sarcoglycan is expressed at the membrane similar to what we see with dystrophin. In the knockout mouse, you see a loss of expression. These are just two examples of treated animals. On the right two panels in both the diaphragm and the heart, you see very robust expression that exceeds 95% of all muscle fibers.
We looked at all skeletal muscles in surveying those, dramatically what we see is greater than 95% of muscle fibers in every single muscle are transduced following six months of treatment. Very robust levels of gene expression using this vector. Importantly, as you've all become experts in the DAPC, not only are we restoring beta-sarcoglycan, which we're showing on the right, which is lost in these mice. Dystrophin is also greatly reduced, and we see complete restoration of dystrophin following treatments of the membrane. This beta-sarcoglycan is expressing and it's functional. It's restoring the DAPC complex. How does this expression translate into clinical benefit and function? One of the first things that we looked at was the overall health of the animal. These animals have scoliosis akin to what we see in patients.
We're able to take X-rays of the animals, and in the knockout mouse, you can see that there's a significant curvature of the spine that's shown in the upper right-hand panel. Following treatment, this curvature is reduced, and we can quantify that with what's called a kyphotic index. On the right, this is a graph quantifying that. You can see the wild-type mouse is in blue, the knockout mouse in tan, and following treatment, we're seeing restoration or similar to wild-type levels following treatment. To further look at function, we also wanted to look at the diaphragm muscle, which was severely affected in these patients as well. For this, we take out muscle strips of the diaphragm. We subject them to force measurements, here again, we're seeing complete restoration of force in the diaphragm following treatment.
We also use a fatigue model where we continuously stimulate the diaphragm and look at resulting force. Here also we see significant benefit from fatigue in the diaphragm, lending to the fact that we'll have significant benefit on respiration as well in patients. CK. Again, we're all experts in CK now. CK is leaked into the serum, in the beta-sarcoglycan knockout mouse. We see very high levels in this model, and following treatment, we see a drastic reduction following 6 months of treatment. Also looking at the histology of the muscle. How much fibrosis and fat infiltration. This model particularly has a robust inflammation and fibrosis within the muscle. We're looking at the top panel here. This is skeletal muscle. We see wild-type on the left panel. In the middle is the beta-sarcoglycan knockout mouse. You see a lot of fibrosis indicated by pink.
Following treatment, this is very much reduced. We're showing this in both the gastrocnemius, which is skeletal muscle, and the diaphragm. The two graphs on the right are quantifying this. You see high levels in the tan bars in the knockout mouse, which is significantly reduced following treatment. If that's not enough to quote Dr. Mendell, we also looked at durability of the effect in the beta-sarcoglycan knockout mouse out to 27 months, which is the lifespan of the mouse. This is one of the questions that we're really interested in, is how long this will express. We looked at the same dose, 5 x 10^13 vector genes per kilogram, and then looked at the animal at 27 months. What we can note is that we saw no loss of gene expression over the time.
If you really want to split hairs, we actually see a few percentage of points above what we saw at 6 months. No loss of expression over that time in both skeletal muscle and heart. It really begs the question that we should have a very durable response following treatment. Now that I've shown you all of that preclinical data, this really lended to approval of a phase I trial directly to systemic delivery for this LGMD subtype. The first phase I trial will consist of nine patients, and will be a randomized, double-blind, placebo-controlled trial consisting of two different cohorts. In the first cohort, we will dose at 5 x 10^13 vector genes per kilogram, as we did on our preclinical studies. What we built in is the possibility for dose escalation if needed.
At 60 days, we'll be doing a muscle biopsy. If 50% or greater of the muscle fibers are expressing, we'll keep the dose the same. If it's less than 50%, we have the opportunity to raise the dose. Based on the preclinical data that I showed you, we don't anticipate having to raise the dose, but that's built in as a possibility. Primary objectives are safety and demonstration of gene expression with functional outcome measurements similar to what we see in Duchenne, consisting of the 100-meter on the North Star, as well as manual muscle testing. Moving on to the second program, which is alpha-sarcoglycan deficiency or LGMD2D. This is really the program that we have the most historical data. Dr. Mendell originally did a small trial in 1999, we began a trial in 2006 for proof of concept IM injection.
Just thinking back to over a decade ago, we were in a much different place with gene therapy. We didn't have the manufacturing capabilities that we have now, and safety wasn't established. Trials were designed differently. It was really to design it to look for safety, demonstration of gene expression. This first trial that was done in 2006 was the first time that we employed the use of a muscle specific promoter, and this was the tMCK promoter, and I'll show you data from that. We then progressed to an isolated limb infusion study, which we'll present the data. Now as we are moving all of our programs to systemic delivery, we've performed bridging studies to enable that within the next year.
In this first LGMD2D trial, as I mentioned about 10 years ago now, was a placebo-controlled trial in that each patient was dosed in two different muscles. This used the extensor digitorum brevis muscle. This is a vestigial muscle at the top of the foot that can be completely removed with no harm to the patient, so we can do full biopsies following treatment. One of the muscle biopsies received saline, one of the muscle biopsies received the gene, and then we did biopsies at 6 weeks, 12 weeks, or 24 weeks. What we're showing here is two patients from that trial that were dosed and did biopsies at 6 months following treatment. I want to point out a few things. On the right two panels, we're showing the saline treated side.
This is the baseline levels of expression, and on the left-hand side is following treatment. What you note is that there's varying degrees of baseline expression in these two patients. If you remember, this is a mutant protein at the membrane. What you can appreciate following treatment in the first top patient, for example, is that we see a dramatic improvement in the muscle fiber size following treatment. Just because we saw expression previously, it wasn't functional, but we were restoring the full length functional protein in this case, which was also restoring health of the muscle fibers. You can note the robust gene expression in the bottom patient as well, also indicated by Western blot on the right.
This trial really set the stage that, yes, we can deliver the alpha-sarcoglycan gene safely, and we can see robust expression that didn't diminish from 6 weeks to 24 weeks at that time. Next we move to a vascular delivery model, and at this point, no one had done a vascular delivery trial, and we were really trying to do proof of concept that we can deliver AAV through the vasculature, express the gene safely. At this point, we transitioned to AAVrh74 for this trial. Dr. Flanagan did a nice job of explaining the isolated limb infusion procedure. I'll just touch on it quickly. This is where we insert two catheters into the femoral artery and vein. We blow up the balloons to restrict blood flow.
There's also a tourniquet placed, the virus is delivered over 10 minutes, allowed to circulate, then we remove the balloons, and the virus is left to go into the circulation. This was a phase I trial, really focused primarily on safety. This is the first vascular delivery trial with some secondary outcomes looking at function as well. The first patient in this study, 6 patients have been dosed. First patient was a single limb, non-ambulatory patient. This was really focused on safety, the FDA was critical about dosing a patient that was non-ambulatory as well as in a single limb. Although the patients were ambulatory and dosed in both limbs at either 1 times 10 to the 12 vector genomes per kilogram or 3 times 10 to the 12 vector genomes per kilogram. Just a comment on predicted dose.
If you compare this to the systemic delivery doses that we're using, we're anywhere from 2%-10% of what we would expect to be effective systemically following gene transfer. All of these biopsies were done at 6 months. There was 1 patient, due to a fall, that could not have post muscle biopsies. As you can see, we had robust vector genome copy levels following treatment in all patients that we looked at. This then correlated to gene expression in all patients. We've quantified gene expression. We're looking here at the right. On the left panels is pre-treatment biopsies, followed by post-treatment biopsies, as well as Western blot. When we quantified gene expression by Western blot, we saw it ranges anywhere from 14%-25% of normal at this low dose.
This is really proof of concept that we could express the gene through the vasculature safely before proceeding to intravascular systemic delivery. We've now performed a very detailed study in the alpha-sarcoglycan knockout mouse model for the disease or the LGMD2D, looking at 3 different dosing levels ranging from 5 times 10 to the 13th up to 2E to the 14th, looking at a 3-month endpoint. This study was designed to look at both safety and efficacy, like we did with LGMD2E. Here we're showing widespread gene expression by immunofluorescence. This is 3 months following treatment across all muscle groups. This has been quantified on the right, what you'll notice at all 3 dosing levels, we see very high levels of gene expression across skeletal muscle as well as the diaphragm. How does this gene expression translate to function?
We are looking at creatine kinase levels in this animal model as well. We see very high levels of CK in this animal model, at all 3 dosing levels, we saw a significant reduction in CK following treatment. We're also able to look at the animal clinically. We can quantify how they ambulate, how they rear on their hind limbs vertically, also saw a dose-dependent improvement in all of these functional measures as well. Looking at force measurements specific in muscles in both the skeletal muscle, which is a tibialis anterior muscle, the diaphragm. In this mouse model in the light purple, you see we have a deficiency in force in both the skeletal muscle and diaphragm. At all 3 dosing levels, we see significant improvement in these measurements. We also see protection from eccentric contraction, as you'll note from the previous studies.
In the diaphragm, significantly, we have very good protection from damage. This data, collectively with the broad range of safety data we have from both animals and the clinical trial, we are now amending the IND to include, again, a randomized placebo-controlled study that will enable us to look at gene expression as well as safety. This trial also has a dose escalation built in with the possibility for raising the dose if the gene expression is less than 50%. The goal here is to make sure that we're dosing patients effectively with the highest dose possible to protect their muscle for the longest amount of time. We want to make sure that we're building in the possibility to go up as needed to the levels that we're using with microdystrophin.
Primary safety, demonstration of gene expression, and the same functional outcome measures that we've seen with LGMD2E trial, 100 meter walk, North Star, et cetera. We're definitely seeing a pattern here, right? This is a platform. We're seeing very similar studies that we're able to accelerate to the others. LGMD2C, or gamma sarcoglycan, we just started working earlier this year on obtaining the mouse model and perform these dose escalation studies. We're just starting to get the data in from this study. At three months following treatment, at 5 times 10 to the 13th vector genomes per kilogram, we're seeing very high levels of gene expression across all muscle groups. As you can see here, 88% of muscle fibers are above.
We have an early look at function in these animals, looking at specific force in the TA. We also see that function is restored in this animal model as well. All this data collectively bodes very well for how we would expect to see significant benefit in the sarcoglycan LGMD subtypes. This is just the formal study design for the LGMD2C trial. The preliminary data is what I showed you in the previous slide. All right. In the last part of the talk, I'm going to focus briefly on two different programs that are really focused on more of the repair phenotype. Both of these subtypes have a little bit of later onset in the teens, but they represent a very significant population of LGMDs without any treatment.
The MYO-201 program, or LGMD2B, which is dysferlin deficiency, as you remember, I talked about when it's deficient, muscle fibers cannot repair themselves. This program is quite unique, and I'd like to, in future presentations, delve into a little bit more detail. We've taken a unique approach to use a dual vector strategy to deliver the entire full-length dysferlin gene with really very promising results, both in an intramuscular safety trial, as well as preclinical studies. We've already done a GLP toxicology study for intravascular delivery. Again, we're amending our IND to move to vascular delivery for this LGMD subtype with proof data out to 15 months, showing protein restoration, as well as functional restoration and MRI. This study will be amended this year, enabling a rapid regression to an intravascular IV systemic trial. Our Annexin-5 program is our earliest program in the preclinical stage.
ANXA5 deficiency, again, is involved in membrane repair process, as well as many membrane dynamic processes within the cell. When we started this program, there was no animal model available, so we generated a knockout mouse, demonstrated that it had a phenotype that recapitulated the disease quite well, and then made our AAV vector and showed restoration of both protein expression, which you're seeing on the right. Then we were also able to show that we could restore the membrane repair capability of the muscle. Just to take a minute to walk you through this image. On the bottom left-hand image, we can take a single muscle fiber and damage it with a laser, and then quantify the amount of dye that comes into the cell.
In a knockout mouse model, you have much more dye infiltrating the cell versus a wild type fiber. Following treatment, we see less dye coming into the cell, and then we can quantify that. This is another way of looking at a functional repair within the cell. This middle graph here in the bottom, this is just looking at the infiltration of dye over time. It's time on the x-axis and the amount of dye on the y-axis. In blue, we're showing a wild type fiber. On the tan, we're showing the knockout mouse, and we're showing an improvement following treatment. This, we've shown proof of principle in the preclinical stage, and now we're doing the formal dose ranging and toxicology studies to support a clinical trial. Collectively, all of these programs are moving very rapidly to the clinic.
We've shown proof of principle in many of these, and very excited to see the LGMD2E trial, which has so many parallels to what we're seeing with microdystrophin and Duchenne. The LGMDs represent a very significant unmet need. There are no treatments out there, and the programs that we've developed have a very good chance of helping this population significantly. The programs that we've targeted thus far as part of MyoNexus represent about 70% of the LGMD population, and there's many shared design principles that have enabled us to get to the point where we are. Again, the phase I trial will begin later this year, and we'll look forward to those results early in next year for the early biopsy data. That's it. Thank you.
Thank you very much. That kind of completes our molecular walk out along the dystrophin to the sarcolemma. Now what I'd like to do is move from the genome and the sarcolemma back to the transcriptome, if I can use that term, and dystrophin. Doctors Gunnar Hanson and Marco Pacini are going to conclude the prepared part of today's R&D Day by discussing the RNA-targeted therapies in our pipeline, and to talk a lot about the next generation RNA-targeted therapy program known as PPMO. Dr. Hanson, let me start with him, has been with Sarepta Therapeutics for the past 12 years. He currently serves as Senior Director of Research Chemistry and has played a key role in the formation and advancement of our follow-on RNA chemistry platforms and resulting programs.
He earned his PhD in Organic Chemistry from MIT and completed his postdoctoral fellow at Stanford before coming into industry. Dr. Pacini joined Sarepta Therapeutics 4 years ago after a distinguished career at Genzyme, which spanned more than a decade. He currently serves as the Senior Director of Biology. He earned his PhD in Molecular Biology from SUNY at Stony Brook, and completed his postdoctoral fellowship at University of Pennsylvania. What they're going to talk about now is, Dr. Hanson is going to talk about the chemistry, and Dr. Pacini about the biology of our PPMO and show how that complements and is a critical part of our portfolio. I'll call on Dr. Hanson to join us now. Thanks very much. There you go.
Thank you. Okay. Good afternoon. Thank you for not running for the exits. We're going to do a little bit of a chemistry presentation. Before we get started, I just wanted to recap what Doug had said, and that is we are, at Sarepta, all about patient focus. The RNA therapeutics platform. The transformative platform that I'll be discussing today is PPMO. PPMO stands for peptide conjugative phosphorodiamidate morpholino oligomers. That's a huge mouthful. Even after 12 years, I can't even say it right. PPMO for short. The base structure, of course, is PMO, which is our antisense oligonucleotide. What we're trying to do with PMO is treat rare disease. In order to do that, we need to basically focus on not only the RNA target and its identity, but delivery.
Delivery is everything, and this platform has the capability of really transforming RNA therapeutics. Before we get into the chemistry part, I just wanted to tell you that I will be discussing a lot of ideational thinking here. This is a relatively data-free zone. Then Dr. Pacini will discuss in detail the pharmacology of what happens with PPMO. The first thing that you have to do is clearly get PMO inside the cell, and this is how PMO works inside the cell. Brought in from the outside, PMO hybridizes to an RNA target, and it does it in, of course, a sequence-specific fashion, and therefore, we derive our power from that sequence-specific approach. Once it gets in, there's hybridization that goes on inside the nucleus to the target RNA. We thereby, at Sarepta, redefine intron-exon junctions.
Therefore, we can do all kinds of things like exon skipping, exon inclusion. We can treat DMD, we can treat SMA. All these kinds of genetic diseases are basically under our control. What's different about this kind of genetic approach from the approach that Louise and Jerry have been talking about, as well as our other speakers, is that we don't use a viral vector, that's one thing, and we can redose. We do develop a kind of an orthogonal approach. We have the ability to redose and recontrol our therapies. The first thing we have to do with any precision genetic drug is get it in the cell. It turns out this red thing called the cell membrane isn't so cooperative. It basically resists entry of anything that's over 1,500 Daltons, anything that's polar, anything that basically just isn't a lipid.
We don't have the capability of going directly through the cell membrane that we would have with small molecule drugs. Of course, that's okay because we're going to solve the problem, and my invention of PPMO does actually solve the problem and gets the antisense oligo into the nucleus. Just to back up for a second on why I think all of this is interesting, before I got to Sarepta, I was in big pharma working on small molecules. What I wanted to do was something a little bit different, and that is small molecules in general inhibit proteins. With the Sarepta PPMO technology, we have the power to actually make new proteins. We can reshape proteins. We can change the nature of proteins. We're not just inhibiting proteins, we're actually creating new functions inside the cell.
What is the form of peptide conjugate PMO? Well, it takes the form of the addition of a cell-penetrating peptide to the PMO, thereby unlocks the capability and potential of PMO. I have really one message that I want to deliver, if you get this, it's really the whole thing, that is what we are trying to do is to take an uncharged PMO, we're going to join it to a positively charged peptide. That really is the whole thing, because when you do that, you have the capability of entering the cell by endocytosis and causing all these great therapeutic advantages. That actually is pretty profound because it's something no one else can do. You have to have an uncharged antisense agent in order to use a cationic or positively charged cell-penetrating peptide.
If you try this, I don't recommend you try it at home, if you try this with, for example, a phosphorothioate, what you're going to do is get the negative charges of the phosphorothioate collapsing onto the peptide that's positively charged, that will create aggregation, that will create a loss of solubility in water, basically you'll have floating on top of your Eppendorf solution brick dust, that isn't going to be of any use to anyone. The PMO is so unique in that its great attribute is that it's capable of being joined to a cell-penetrating peptide of the positive type of cell-penetrating peptide. That really is the whole message. I can probably sit down right here, I'll continue a little while longer because I want to tell you that we really are investing a lot in these cell-penetrating peptide conjugates.
We have SRP-5051 in the clinic right now, we're putting four others into the clinic. We're making a larger investment in this than just about anything, for good reason. Let's continue. I want to show you the architecture of PPMO. Let's build a PPMO together, I think this will illustrate where we're coming with this, it'll teach you a little bit of chemistry. Not a lot. You can pretty much look at this modularly, maybe I hope when we're done to convince you that you too could have gotten an A in high school or college chemistry if you'd had a decent teacher. The first thing is PMO is made. How do we make that? Well, in the beginning, there were some primordial subunits that our founders, Dwight Weller and James Summerton, put together.
One of them was a morpholino scaffold that contained a base. You can think of this as a wire frame containing the base. You can think of it as chicken wire containing the base. Doesn't really matter. The point is it's a scaffold. Coupled to that morpholino ring, our founders introduced this other very interesting unit, the phosphorodiamidate. There is a lot of significance to this because this unit is the uncharged unit that enables the coupling of a cationic or positively charged cell-penetrating peptide. In a sense, ideationally, the founders put these things together by docking these two groups at the nitrogen atoms you see there. They were intersected and then joined. Here are the two basic primordial units put together. What happens when you do that? You make the PMO core structure.
This is the train that got started in the mid-'80s that formed Sarepta, and that's really brought us to this position. This is the constituent of eteplirsen and casimersen. This is really important stuff. What's in the blue and the red is really the core unit of PMO. Once we've assembled a core unit, we of course want to add more nucleobases so we can get great sequence-specific Watson-Crick interactions with our target RNA. That is easily done through solid phase synthesis, and here's an example of just adding some other additional nucleobases. Once all that's done, everything will work great as long as we can get this inside a cell. To do that, we're going to add the peptide. We add the peptide finally, the CPP, which stands for cell-penetrating peptide. There it is.
We add it to the morpholino preassembled piece, and that's called the PPMO. Now, I want to point out that where I've circled in red and where I've circled in green, again, coming back to my theme, those two units are compatible because they're compatible charge-wise. Without that compatibility, nothing good is really going to happen. Let's go on here. The PPMO is composed of a PMO and a CPP, and it gives rise to a lot of really, really great properties, exon skipping, exon inclusion. We can target muscle. We can do something nobody else does. In the antisense field, targeting muscle is really tough, and Sarepta's got a total lock on that. There's also the capability of binding to targets that are in the CNS, so we can do that. We have great stability to nucleases.
All these things are very interesting properties, but really what I want to do is get back to the core structure and why it's so important. Let's move on here. This is a representation of that PMO, that all-important PMO core. As you can see, the parallel units that are forming here, these stacking units, those are the nucleobases, and this is a 3D picture. That big, bright red sphere is the oxygen attached to the phosphorus. If you go to the right and you take a look at this in our chemistry lesson, you can see this unit that's flashing there. Now, this is non-trivial. This is so important. We all know, and there's so many products out there, that hydration is important. We drink water, we hydrate our skin. We've got all kinds of cosmetics based on hydration.
What I'm trying to tell you is that unit in red is the reason why it all works, because that is a highly water-attracting unit. It basically allows the PMO to hydrate and essentially humidify the entire PMO structure. Now, that's extremely important because the PMO is a long polymer, and just having a cell-penetrating peptide on the end is not going to allow the molecule to relax in solution and get ready for the antisense interaction in the cell. That unit in red is so important because it actually attracts water, creates great water solubility, and allows this whole chemistry to take place. Let's go into a commercial product. I want to describe what SRP-5051 is, and it is a PMO containing an exon 51 targeted sequence coupled to a cell-penetrating peptide.
This is what we have in the clinic, and it's a sequence that works very well for us, and it is using the cell-penetrating peptide that I developed. This is our first commercial product, and I think it was the December 2015 we decided to go forward with this and use this. One of the members of the board of directors, Hans Wigzell and I, discussed this, and we hatched an idea of moving this forward into the clinic. This is, right now, our first entry in the clinic. I wanted to just go, before I conclude, with how did I come up with this cell-penetrating peptide in the first place.
I spent 10 years studying the cell-penetrating peptide and macromolecular delivery field, and what always struck me is the importance of tailoring your delivery agent to the cargo that you're trying to actually get in the cell. Most researchers don't do a good job of this. They tend to either look at the peptide alone, or they don't look at cargo or enough cargo. I really started this whole thing off with the idea we had to tailor-make the cell-penetrating peptide specifically for working with PMO and in concert with PMO. I got this idea in one of these great fiery Pacific Northwest afternoons where the sun just comes down under that cloud deck momentarily.
It was bathing the office with light, and I had finished this analysis, and I was basically describing it to somebody, and that was that I wanted to take what 10 years of research had yielded before, which was a compound called a B peptide, which was extremely interesting. It was highly potent, but it had the problem that it was toxic. I wanted to start with this as a model and basically use a lot of symbolic logic and molecular algebra to essentially do a subtractive synthesis to yield something that would be non-toxic yet highly effective. Out of that analysis came four cell-penetrating peptides. There were four candidates here, not thousands, but a very directed, rational thinking. I was telling my boss at the time that, "Boy, this is really great.
I'm sure this is great, and I think it'll work really well." I probably should never have said anything like that because chemistry and biology have a way of changing things and making your predictions not to work out so well. In the end, I set the chemistry team to making the PPMOs out of this using a sequence that we use in our preclinical model. We arrived at four PPMOs and then went ahead and tested them biologically, because that's really the only way we can find out if anything we're thinking about makes sense. Out of that, one of them was selected as the optimized PPMO, and it's that one that we're using now in SRP-5051. This is one of the few pieces of data that I have for you, and Marco will show you really all the advanced stuff.
What came out of that exercise was testing of the four PPMOs individually, one of them, the one in red, gave the best activity. They were all similar in activity, the one in red gave the best activity, and ultimately in follow-up studies with the toxicology, that turned out to be the safest. It was really the sweet spot in this whole looking at the four individual candidates. We took that compound in red and did a duration of action study where we dosed at two doses and followed it out for 85 days. I think as you can see here, this thing has got legs. It's got huge activity. For example, even at the low dose, at 40 mg/kg, you get 40% skipping in the quads at 40 days. This is actually extremely long-lived.
It gave rise to the idea that our PPMOs could be dosed once a month. I think this was really encouraging, and we have follow-up studies as Marco will show. In my summary slide here, I hope you've learned a little bit about chemical architecture and how that can bear on these molecules. I hope you've taken away the lesson that the core structure is just as important as the cell-penetrating peptide part, and that the major point here is I think that these PPMOs not only unlock the capability of PMO, but they unlock the capability and potential of antisense oligos in general. This is really a general approach that we can apply to multiple rare diseases, SMA, DM1, DMD, and I think really go to town on it.
Right now I'd like to turn over the podium to Marco to talk about all of the advanced pharmacology of this. Okay, thank you.
Okay, well, thank you very much. It's a pleasure to be the final speaker today. My name is Marco Pacini, I'm Senior Director of Biology. One of my primary responsibilities for the company is to help advance our preclinical pipeline. As Gunnar Hanson just mentioned, he described the chemistry regarding the cell-penetrating peptides and PMOs. The mission of my presentation is to give you a high-level overview of our PPMO efficacy and pharmacology data in preclinical models. PMOs are designed to bind to target RNA in cells. In regards to DMD, the dystrophin RNA is mutated and out of frame. As a result, the cell cannot read the message properly and no dystrophin protein is made.
What we do is we design our PMO to bind to the dystrophin RNA, and in doing so, a process called exon skipping undergoes, and that puts the dystrophin RNA back in frame, and now the cells can properly read it into functional protein. Our thesis is actually very simple. If we can drive more of that PMO into cells using a cell-penetrating peptide, we can deliver that PMO more effectively into muscle using a cell-penetrating peptide, then that should result in higher levels of exon skipping, higher levels of protein production, and ultimately potential transformative improvement in muscle function. We can perform these proof of concept analysis in preclinical models, and we can do it using PPMO compounds that have our proprietary lead cell-penetrating peptide covalently linked to our PMO sequences. We can test this in different models.
On the most commonly used model, you heard about the MDX mouse model for DMD earlier today. That's a very nice model. It recapitulates many of the features of the human disease, and we can really investigate the therapeutic potential of these compounds. More exciting is that we can take the same lead penetrating peptide and put it onto our human sequences, such as eteplirsen and golodirsen, and treat non-human primates. These are large preclinical models whose size and anatomy resembles more of the human, and we can actually learn about the potential of widespread global delivery of our human compounds in these models. This just gets right to the point of delivery. This is an MDX mouse experiment here.
On the left you can see, and you've seen this before with our other talks, you have a normal healthy muscle, and all those rings you see there, those green rings, are individual muscle cells that are all expressing dystrophin. That's what a healthy muscle looks like. On the middle panel is a dystrophic muscle from the MDX model, and here you can see it's very dark because the majority of the muscle cells aren't expressing dystrophin. The only reason why I like this panel is because you do see occasionally what we call revertant fibers in the MDX model, and the significance of that is that even in humans, in patients, you have revertant fibers.
These are cells that are able to spontaneously figure out how to make dystrophin on their own, a very, very low % population of these cells in both our MDX model and humans. The point is, here's another example of the MDX model recapitulating a feature of the human disease. The take-home message is really the last panel, on the right-hand side. You can clearly see that a single dose of PPMO is able to widely deliver the oligos and produce widespread expression of the dystrophin protein throughout the myofibers, throughout the muscle. Our next question is: How long does this ability last? How is the duration of effect? We did another study in which we did a single dose of our PPMO.
Again, this contains our lead cell-penetrating peptide. We treated the MDX model, and we looked at 7, 30, 60, and 90 days later to get an idea about the duration of effect. On the top row, you can see, is our Western blots. This is a method that we use to measure the levels of dystrophin and the method that's being used currently in our clinical trials in our patient biopsies. What I'm showing you is four blots, and there's two treated subjects in each blot. You can see very nice dystrophin bands being restored, and we can measure the levels of dystrophin by comparing the intensity of those bands to the standard curve, and that generates the graphs on the bottom. Those graphs require 36 Western blots to generate those graphs.
What you can see is that in the first two months, between seven and 60 days, the levels of dystrophin are relatively similar within a given muscle group. Even at 90 days, we can see expression. You can look at the top right-hand picture of the quadriceps at 90 days. You can clearly see expression there. Even in the heart, although it's less than the other structures, the heart we're generating about 1% to 2% dystrophin within the first two months, and even at 90 days, you can detect some levels of dystrophin, and it's below our lowest limit of detection. The next question is, well, what this data's showing is that we can achieve, from a single dose, high levels of expression. The question is, can we sustain those levels of expression with repeat dosing?
Our next experiment, our next study, was to do just that. What we did here was ask the question: If we repeat dose, can we sustain the levels of dystrophin and prevent the waning of expression over time? What we have here is group one was treated with our PPMO. As a reference point, we analyzed group one at 30 days to get you a reference point. Then in group two, we actually did three monthly injections at month zero, month one, and month two. Then we analyzed the tissues at month three. You can see, not only did we sustain the levels of dystrophin, but we actually statistically significantly improved the levels of dystrophin compared to our one-month reference point.
That's important because this is going to be a repeat administration therapy in the clinic, and our preclinical models support that repeat dosing is going to increase the levels of dystrophin, not just sustain, but increase those levels. If you just compare it to our single-dose study that I just showed you before, and look at 90 days, again, a single dose, and you look at 90 days, the expression eventually goes down, and the group that received the repeat dosing was significantly higher than that that received the single dose at three months. We can also look at the benefit of repeat dosing histologically. If we compare those two points to each other, you can see a very nice phenomenon. In the first panel is your, again, your control dystrophic tissue, and on the right panel is your normal healthy tissue.
If you look at panel number 2, our MDX PPMO single-dose panel, you see, remarkably, I'm still very impressed that at 90 days after a single dose, you see myofibers still positive for dystrophin, which is predicted based on the Western blot. You also start seeing areas of darkness. You start seeing some cells lose the dystrophin expression. With repeat dosing, not only are we increasing the levels of dystrophin, but we're maintaining a uniform expression pattern of dystrophin across the muscle. All the myofibers are continuing to participate in dystrophin production. Up to this point, what we've done so far is talk about dystrophin expression, and that's great. When we talked about our thesis in the beginning, we mentioned that PPMO is going to hopefully increase the levels of exon skipping and increase the levels of dystrophin.
I'll show you some exon skipping in a second. That's the first part. The second part is that these high levels of dystrophin we're generating should result in functional improvement of muscle. We went ahead and we did a dose response study, and the goal of this study is to demonstrate, can we reverse the hallmark pathologies of DMD? Can we reduce cell inflammation? Can we reduce the hallmark features of fibrosis? Moreover, can we improve muscle function? We did a dose response, as shown here, and we chose doses from 10 to 80 mgs per kg in the mouse. Those are the mouse doses. If you can see, there's another column there that says HED. That stands for the human equivalent dose.
I want to bring this up because I don't want the audience to go away thinking that the doses we use in our preclinical mouse models of the doses that necessarily are going to be used in the human, there's a convention calculation based on body surface area and body weight called allometric scaling. Allometric scaling is a widely used calculation to compare doses across different species and extrapolate what the doses may be. Between mice and humans, the allometric scaling is 12.3x. This is actually used widely. In fact, it's even used in the FDA guidelines to help investigators determine the first dose in man based in these single ascending dose studies. Based on those, we're in a range of somewhere between one and six mgs per kg. This shows you our dose response.
We treated just one time in this case, we're going to look over 30 days and show dose response, show therapeutic efficacy. You can see the dose response in action here at the level of histology. The difference here is that we actually counter-stained, immunostained, the tissues with a red laminin stain, you can see where all the cells are rather than just looking at something that's dark and black. If you look at the quadriceps at 10 mgs per kg, you're showing the increasing levels of dystrophin in individual myofibers. Then by 40 and 80 mgs per kg, you're pretty much similar to a healthy tissue. I also want to impress on you the impact on heart expression. Heart, again, is a very important tissue that we want to go after.
You can see that at 10 mgs per kg and 20 mgs per kg, you start seeing some cells appear in the heart. By 40 mgs per kg, we have a lot of expression in the heart in individual cardiomyocytes, also 80 mgs per kg looking similar to wild-type animals. The key is, what does this all mean in regards to therapeutic efficacy? This is quantifying the data we just saw before at the level of Western blots and qPCR. We can look at the levels of dystrophin protein, we can look at the levels of exon skipping. Again, everything is generated in a dose-response manner, we like to see that in biology. We like seeing, when we do a dose response, the resulting efficacy data coming back in a dose-response manner.
What does this all mean in terms of therapeutic efficacy? What we did here was look at the hallmark pathologies of DMD. We heard earlier from Jerry regarding CK levels. Other biomarkers that we can look after are inflammation, fibrosis. The inflammation is on the left column there, both in the quadriceps and the diaphragm. Again, with increasing doses, you're seeing increasing impact on reversing the inflammatory response in DMD. Again, a hallmark feature of DMD is inflammation. We're reducing that. The same thing with the fibrosis. As you increase the doses, you show an impact and a reduction in fibrotic tissue. That's very impressive. That's what we want to see. What's really impressive to me is especially the function improvement. We saw some of this function improvement data with our previous talk, with Louise especially.
Here in the DMD mouse model, in the MDX mouse model, when you treat at 10 mgs per kg in the grip strength, which is just a static test of muscle strength, you have a measurable improvement in grip strength. It's not significant, but there's something there, something that we can pick up. When you get to 20 mgs per kg, you have a significant improvement in grip strength compared to our MDX controls. By 40 and 80, we normalize grip strength. It's indistinguishable to healthy, normal wild-type mice. The second test is Rotarod. This is a little bit more of a complex test. This is a locomotion test. It's a test of ambulation. It involves muscle strength, coordination, endurance, it's a much higher hurdle to try to achieve this test. At 10 mgs per kg, we're not so surprised that it didn't necessarily work.
By 20 mg/kg, significant improvement. At 40 and 80 mg/kg, normalization of muscle function, endurance, and coordination. We can take all this data and put it together and come up with themes of what levels of dystrophin in our preclinical models results in function recovery, and what degree of function recovery do we achieve. For the test that I showed you, the biceps and the quadriceps are the major players. Just focusing on those two muscles, if we inject our placebo control, saline, we have no expression of dystrophin, obviously no improvement in muscle function. As low as 0.3 mg/kg, again, the PPMO at the 10 mg/kg dose, we had this measurable improvement in grip strength. It's not significant. It didn't impact the Rotarod. We cannot ignore that low levels of dystrophin provide some therapeutic relief.
These cells are starving for dystrophin, and low levels have an impact on muscle function. If you can get to 10%, as shown there in the 20 mg/kg column, then we're getting significant improvements in muscle function in our preclinical models. If you can eclipse 20%, then we have normalization of muscle function. The take home message here is that we don't need to get to 100%, but if we can eclipse a 20% margin, our preclinical models predicts a highly successful outcome. We can also do the same exercise using exon skipping. In this exercise here, the same theme. I'm not going to go through every point here, again, at low levels, at 0.6%, you get some measurable effect on grip strength. Greater than 10%, you get significant improvements.
If you can hit 50% exon skipping, we have normalization of muscle function. What I like about this slide is it sets up the last two slides I'm going to show you today. The cell-penetrating peptide, our lead proprietary cell-penetrating peptide that was used in the MDX preclinical model, that same peptide is going to be placed on the human sequences now and tested in non-human primates. As you remember, non-human primates there, and Louise mentioned this earlier, there's no DMD non-human primate. These are normal preclinical models. Our main outcome measurement's going to be exon skipping, and we have this result here in our MDX mouse to give you a guide what levels of exon skipping provided what effect in terms of function recovery. This is our SRP-5051 compound.
This is our exon 51 PMO skipper, eteplirsen, that's been conjugated with this lead cell-penetrating peptide that was used in the mouse studies. When we treat our large preclinical model, we see something very impressive. We see widespread global delivery of our PMO to the major muscle tissues that are affected in DMD. All structures shown here are highly affected in DMD, and we're able to target those muscles and achieve high levels of exon skipping. In fact, if you go back to our preclinical model of the previous slide, we are achieving levels that correlated with significant improvements on the muscle function, in some cases, normalization of improvement. Also, the data shown in the dose response manner, that's just the way we want to see. The next data set is brand-new data, and we're very excited about this data.
The activity we see with SRP-5053 is really remarkable. Here, we have the same cell-penetrating peptide. We put it on the golodirsen sequence to generate SRP-5053. We treat non-human primates, and we look at where are we seeing exon skipping, and clearly, what you see is an amazing widespread, robust effect. Again, all the major muscle groups that are affected in DMD are shown here, and they show high levels of exon skipping. This data is brand new. This is the first time we're showing this data. We generated this data about three to four weeks ago. It's really a pleasure to share this type of data set with the audience. You can also look at the heart. Let's not forget the impact of the heart that we were trying to achieve here.
I know Jerry and Louise were talking a lot about the heart earlier today, and with our SRP-5051, but especially SRP-5053. Even with the low dose, we're getting high levels of exon skipping. Again, the levels are according to our first preclinical model, are above the levels needed for significant improvements. The only other point I want to make is, although we're really, in some ways, just extremely excited, and I don't want you to say blown away, but we're really excited about this. Actually, we were blown away. The thing is that it's somewhat not unexpected. The reason why I say that is because if you look at our clinical trials for golodirsen and eteplirsen, golodirsen does appear to have higher activity in our clinical trials. That higher activity is just also being recapitulated here with our PPMO versions of these compounds.
We celebrate the consistency of our data. Although, again, SRP-5053 was really an impressive active compound, it's not that unsurprising that it performed this well. This is my last slide. In conclusion, we're using the same proprietary cell-penetrating peptide in two preclinical models. We're able to achieve our ultimate goal, which is widespread delivery, widespread protein production, and an improvement in muscle function. The preclinical data was so robust that it clearly supports the clinical development of this program. As Gunnar mentioned earlier, the 5051 is in clinical development. We have already treated several patients, and they're in phase I. The SRP-5053 compound, we're going to file an IND at the end of this year, and we're going to move on to the other follow-on PPMO compounds for IND filings in 2019.
In conclusion, we're really excited and enthusiastic by the possibility and potential of our PPMO compounds to transform the lives of DMD children. Thank you very much.
Thanks very much, Marco Pacini. That was great. Thank you very much. That was a very nice demonstration of the evolving platform and the CPP technology that is contributing to PPMO and its potential, both to meet along with our gene therapy platform, the various diverse needs of people affected by both Duchenne and a broader set of muscular dystrophies, but even actually look at potential synergies. Now, the R&D Day continues. Don't move yet. The formal didactic session, if you will, is complete, but we're now going to move into a Q&A session, and I'm going to invite all the speakers for today to come up and take a seat here, and Doug is going to stand where I'm standing and compare the Q&A session. Thank you very much. If the other speakers can come up and join me.
Thank you.
Thank you very much.
Welcome aboard again.
Thank you all for hanging in there.
It's a kidney room.
Kidney room.
What's that?
Turn talking here on.
Okay.
Your microphones.
Oh, that explains why no one can hear me. All right. We're going to do about a 30-minute Q&A, but just so you know, we won't be disappearing. We will be in the community after this, so this won't be the last time to ask us questions. I'm sorry. First question was for whom?
Thanks very much. Matthew Harrison, Morgan Stanley. I guess two from me. First, just given the AveXis experience and what you've seen with other gene therapies, can you talk a little bit about what size of a data set you need to deliver to the FDA to have discussions around pivotal data sets and what sort of discussions you think you need to have? Then maybe a specific follow-up for Dr. Mendell. Have you seen any complement activation in patients? I know that was one of the risks that sort of Solid had highlighted. Thanks.
I'll answer the first question, then I'll send the second to Dr. Mendell. Just the short answer on when we engage with the FDA, it's now. We are going to seek a meeting with the FDA certainly before the end of this year to discuss with them the pathway to an approval. That will require us to talk both the development plan and get their buy-in for our bridging strategy for manufacturing as well. It won't come as a surprise to you that it's our goal and hope that what we call currently Cohort C, which has already cleared IND, and we can begin executing on it immediately, could form the basis for our approval, our registration trial, which is a one-year trial, 24 patients. 1 to 1 with a placebo, and then a crossover at the end so everyone gets gene therapy.
It would be a mistake for us to claim that we know that that's the pathway. That's going to require discussions with the FDA, and we'll certainly update people once we know what the FDA's perspective on that would be. With that, to Dr. Mendell on complement.
The simple answer is at 90 days, we don't see that. The more complex answer is if we did serial sectioning from very early on, we might. When we have degradation of the capsid, which probably starts taking place sometime after two weeks or so. I think it's possible that we could see a localized inflammatory response with some complement activation. Obviously, it doesn't impair long-term gene expression.
Next question.
Hi there. Lisa Bago from JMP. Just a first question for Doug. This is obviously very exciting data with gene therapy seeming to have a very meaningful impact already. How do you see the entire portfolio, just thinking about DMD playing out? You have PPMO coming forward. You're working on gene editing. You have the gene therapy that obviously looks exciting. Talk to us about portfolio management and how it all works together.
There's two ways to look at that. First of all, one way is that we're going to pursue everything that has the potential of transforming the lives of these children. That we're taking an agnostic approach to genetic medicine. That's why we have the RNA gene therapy and gene editing. There's a second way to look at that as well, which is it is very likely the case, presuming that what we're seeing in gene therapy holds out, that there is actually a place, a complement in place for RNA technology and gene therapy. Our goal isn't to pick a therapy and get behind it. Our goal is to transform the lives of these boys into men, into hopefully elderly people.
With that said, there is already some literature and publication on the opportunity for an RNA-based technology, PMO specifically in the case of this study, to benefit gene therapy even in advance of getting the gene therapy. There's a paper out, I think Dr. Voigt was the author of the paper, in which he found that pre-treating with a PMO or hopefully in the future, a PPMO, would actually increase essentially the amount of expression by decreasing the leakiness of the muscles. One could envision the thesis that to give the maximum benefit to these children, that a combination of truncated dystrophin and microdystrophin going forward would be synergistic and even potentially increase the durability of gene therapy. We actually see these as complementary.
Just a question, I guess, for the panel. The last question is from me. I just wanted to understand how you think about the durability of response with some of these early responses we've seen that obviously look really great. In terms of we know we have these satellite cells. How important is that to the story, or is it really just about these progeny cells, and how long do you think that will last in there, given that maybe the cells do divide if they're damaged, maybe stop dividing if they're not damaged? I don't know. I'd just be curious about how you think about that longer term. Thanks.
Well, I can take a shot at it. Right now, we're seeing persistence of long-term expression. Take the SMA trial, we have persistence over 4 years with continued improvement. We have a different model there, and I tried to point that out when I talked about we have contrasting issues. We have 1 nucleus and transduce 1 nucleus and get that nucleus to set up a circular DNA and express from outside the genome. There's really no threat to integration. We've got a safe paradigm, so to speak. The long-term expression does depend on what Charlie Gersbach said. No. What Charlie said, and that is how much satellite cell transduction we have. I don't know whether Louise wants to comment on that, because I think that's critical.
Just a few comment. Based on our preclinical data, we're not seeing loss of expression for as long as we can look in the mouse model. We have primate studies out longer as well, we continue to look at that. As far as durability, we're not seeing a lot. It's very early days, in the biopsies, we're also noticing that we are getting a fair amount of transduction of the satellite cells, and a lot more work has to be done to quantify that. We're particularly intrigued about the level of transduction that we're getting in the satellite cells. That bodes well to the long-term durability beyond what we thought was possible.
We're particularly intrigued about the durability of response, and hopeful based on not only our own preclinical studies, but the literature in the field of up to 15 years in non-human primate models for other diseases. I think the durability is going to be key, and I think we have a good possibility of a long-term response.
Dr. Braun?
Yes. There are data in non-human primates, really. Some has been published, but the longer follow-up we know from our colleagues in Nantes, in something like 15 years through an administration in the field expression, the different functioning of people, which is in use so they can follow the evolution. What we also have to remind you is that if we are treating very young boys, they will grow up, their muscles will grow up. We better be very efficient with the first shot to take this phenomenon into account. Be very efficient upfront.
Should also say, as encouraging as the durability looks like it is in animal models, that Sarepta and Louise group are going to focus on the concept of redosing, which of course at this point is only theoretical. Assuming that we have a successful gene therapy program in the future, I think the next big breakthrough, probably in race with gene editing, is the concept of redosing, topping up, that sort of thing. That will be work that we'll do. Next question.
Hi. Brian Abrahams, RBC. I guess first for Dr. Mendell. Can you talk about the patient population that was enrolled in the study so far in terms of exon 18 to 58 patients? Do those sorts of patients tend to be similar to the broader DMD population? Can you tell us about any increases or initiation in steroids at the start of the study, and the degree to which those may have potentially impacted CK levels and functional changes?
First of all, the 18 to 58 are not a selective population for a milder course. The more 40, 45 to 55 of them are a milder course. I think we don't have any evidence that these mutations would have predicted anything other than what we see in fairly large sample natural history data. I don't think that is really a factor in this trial. I think I was very concerned about what you're bringing up about CK. What we saw is that there was no effect after the first 30 days. I was afraid that we were going to be seeing a surge in improvement, a lowering CK, and then after we took the steroid daily dosing away, that we would see a decline and an increase in CK, but it just hasn't happened.
Over time, we've seen continued improvement, and we've seen a further drop in CK. As I said yesterday, I had one that came in. I was following their chemistries through my research practitioning nurse, and she sent me the results. When I saw 540, first thing I did was call Luis. No, I mean, I was blown away by that. It's really practically back to normal.
That's really helpful. Maybe just a follow-up question for the company. As we think about the timelines, rate limiting steps, and the regulatory path forward here, do you have any sense as to the FDA and EMA's views of microdystrophin as a surrogate akin to dystrophin? How should we think about the timeline for cohort C to be enrolled? Are you going to be completing the six patients in the current cohort or moving straight to that study? Is there any manufacturing scale-up that would still be a rate-limiting step, even following conduct of that study? How should we put all that together in terms of timeline?
By the way, just so we're clear, the first boy, I believe, was exon 51. These boys are sort of the quintessential aggressively declining DMD kids. I think one of the other kids is exon 45. These are severe type DMD. The short answer, again, I really don't want to get over our skis on this. We're going to sit with the FDA and talk through with the FDA their concept. I can't say in advance precisely what the FDA's perspective is on microdystrophin as a surrogate endpoint, nor whether a surrogate endpoint or a combination of a surrogate endpoint and a biomarker like CK, or a surrogate endpoint and a functional endpoint is going to be what we're going to ultimately go after. What I can say is we're going to execute two things at the same time. We have cohort D.
We have three boys that have been, or four boys actually, that have already been dosed. Three we've seen biopsies on. The fourth biopsy hasn't been done yet. We'll complete those six Okay, those boys. We have the ability to move on cohort C now. We'll begin to execute on cohort C essentially, as soon as reasonably possible after today. We actually also have the ability to go faster than we were going with cohort B. Cohort B required us to go essentially one month between, a little bit more than a month essentially, with evaluation between each dose, so we can go faster. We can move very quickly on the study.
With the manufacturing, I can tell you, as we laid out the timelines, essentially using the most aggressive concepts that we can think of, more than I'm willing to sort of get out and admit to right now. That we are in a place where, assuming the FDA agrees with us on our bridging strategy and the like, and taking into account our relationship with Brammer Bio and our concepts of a hybrid gene therapy manufacturing model, that we'll be prepared to launch and completely serve physicians and patients that need our therapy, if we're fortunate and the gene therapy program works out, and we're able to get to the market fast. Just understand our view on all of this, why are we trying to be so aggressive?
When you see these videos and you see these CK level drops, and you see this expression, it isn't just an opportunity. It places a moral obligation on us to move as quickly as possible.
Right.
Hi, Christopher Marai from Nomura. I was wondering first, if we just talk about the study and the patients enrolled, please remind us if the patients currently are on background levels of steroids. Two, with respect to the data, I guess patient 2 had a slightly lower level of dystrophin production. I was just wondering if you could comment on, from your animal models, the level of variability we expect on a patient-to-patient basis, just so that when we see the next data, we're not totally surprised. Thank you.
The animal models?
Are they on baseline steroids?
A what?
Are they on baseline steroids?
Yeah. Well, we did change, as I mentioned. The patients all had to be on steroids, in this trial, for six months. No, for three months. The reason for the three-month time point was, you've probably been either too young or too long ago. When we reported the steroids in 1989, we had three cohorts. There were over 100 patients in that study. Actually, it was the largest clinical gene therapy trial ever done in Duchenne, and probably still is. What we found at that time was pretty remarkable and very consistent. There would be an increase in function for three months, and then there would be a plateau and a steady decline. What we did was change the rate of decline in those patients. Virtually every clinical trial now maintains boys on either three to six months.
It depends on who's calling the shots on that one. We wrestled over this. It was not an easy decision. No, it seems like every decision we make in these clinical trials is monumental. We decided that we would go ahead and do daily dosing, except for the weekend. We would keep the boys on the weekend dosing, Monday through Friday, we would put them on one milligram per kilo. That's basically what we've done and then tapered it off.
In terms of expression, I think, we focus on preclinically, and really what's most important is the distribution of expression. If you looked across the three patients, the % fibers was extremely consistent among the three. I think the one patient you said there was maybe slight variability in the intensity of the fibers, but I think that's within the range of what we'd expect. I think what I was struck by was the very consistent number of % positive fibers across every patient. We're extremely excited about that.
Okay. One quick follow-up for the company. Since you're getting great expression in the heart and the diaphragm, at least preclinically, is there any expectation or desire to look at treating older patients, later stage patients?
Yes
in a separate trial? Would you look at this new registration trial for, obviously a broad label that could treat those patients as well? Thank you.
The short answer is absolutely. We've got this cohort C, which we're maintaining the same age range. It's our goal on the path to approval to treat younger children as well as, in a safe way, to treat older children as well, so that we don't leave anyone behind as we track forward. As Dr. Mendell mentioned, with the ability to overexpress in the heart, and actually getting more expression even in the heart than we are in the skeletal muscle that we were able to see through biopsies today. It would be important to us to get to all those patients. We're going to look at that, and then we're also going to look at the other.
Even though they're rare, we're going to look at the other exons as well and see if we can find a way to actually treat the kids that are in the lower exon ranges as well as the ones that have the.
Just one other comment. I may be talking too much about the heart, but one of the initial charges when I first got into this gene therapy world was, "You guys are going to improve skeletal muscle, and you're going to put such stress on the heart that you don't have a chance of prolonging survival." Now, I think clearly we have overcome that anxiety about that. That's a big mark for me personally. Ritu Baral, Ritu Baral.
I think it's my turn first, Patel. James Carney from Baird. Ian, who has known me for a long time, knows I'm extremely conservative in my use of the word congratulations. This gene therapy data is really inspiring. Congratulations across the board, especially you, Dr. Mendell. Really phenomenal data. I guess just digging in a little bit on the Western blot, when I look at the slide, it seems like there's four samples run, one on each of subjects 1 and 2, and then two on subject 3. Is that the totality of the samples that were run in Western blot? How is the mean calculated? Is it from across all four? Maybe if you could just detail what's the difference between block 1 and block 2.
Is that literally using a different agent for the blocking step, or is it a different block of the sample?
Yeah. For each patient, there's two pre-blocks and two post-blocks. The first two patients have to be the same block. We saw consistency among the blocks. The first blot was patient one and two, then the second blot happened to show both blocks, both post blocks of patient three. We take the mean of the blocks, then the mean expression that we're showing, or the mean value of the three patients.
The reason there's two blocks for one child and one block each for one and two is we got patients one and two done. Remember, we had committed to doing two patients, and then we were able to actually administratively get the third patient in. You get to see third patient's both blocks.
Thanks. That's all I had.
Yes, sir.
Ritu Baral, Cowen. First question is on what we can expect from the functional measures, a question for Dr. Flanagan and Mendell. I'll have a follow-up on gauging durability. As we look forward to upcoming functional data, how should we look at that given the age of these patients, age 4 to 7? What are the most important timed function tests, North Star? How should we interpret the data that comes out?
Well, we have control data. It's not a naive approach to do this, in that we have control data from our clinic. Every patient who comes in our clinic, and at least 90% are on steroids, we do the exact same testing. We have data, and the comparative controls that we have, they're based on age, height, and weight. I didn't give you the numbers, but every single patient has improved in the North Star, in the 100 meter, and in the stair climbing. It's variable. They're better than the predicted Duchenne controls. We don't want to overemphasize more than we can. These are early days, and we'll have to see. Do I believe it will continue? Absolutely. Believing and seeing are not exactly the same thing.
That's one of the reasons why I think although there are pros and cons about doing a controlled trial, but there is great wisdom in that, and I just said how great we were doing a controlled trial in 1989. I talk out of both sides of my mouth. It's painful to do a controlled trial, for sure. I think if we target this very young population, we've lost very little. The patients in this trial are now 4 to 6. I'd like to focus on The FDA. None of our colleagues who are set for target practice want you to be selective about the patients you take. Maybe we should just frame it. We'll do 4 to 6-year-olds, so we have plenty of room to improve. I believe, I don't know the age of the first Pfizer patient.
I think was around eight or so, wasn't it? Something like that, and the data looked pretty good. I do think we'll have room to improve the patients, but I would like to do a population of placebo patients that have a chance once we roll over to be able to improve. As Doug said, we're really in this business to help boys with Duchenne dystrophy. I guarantee you that's my goal, and I want to make sure that in the planning phase, that we think this through carefully about which patients we're going to be treating, and we want every patient to have the opportunity to improve. That's really important. I cried when those kids came in here today, and I'll do it again, but I want to make sure that all these boys have an equal opportunity to improve.
The follow-up on gauging durability, especially in cohort B. How long do you believe you need to follow these patients up, both on functional, is there a role for a repeat biopsy, which I don't think is in the protocol? Same thing for cohort C. How long should cohort C go on for to prove to you this is a durable effect?
Well, other people can weigh in. I maybe pass it along. I have my opinion about that.
I'll just say that the trial right now is set to look at three years in completion, but we'll be looking at these outcome measures consistently along the way. Adit?
Just to add to that, of course. The FDA mandates that we do, we've just been to the FDA about the SMA trial. They want long-term follow-up, and they're willing to take long-term visits after the end of the IND for at least five years, and then at least telephone visits for up to 15 years. They want long-term data, and they expect it. That's where the importance of partnering with a company like Sarepta is very important because, obviously, Pat supported this trial from day one. She doesn't want to invest the rest of all of PPMD's money in long-term 15-year follow-ups, because she wants to open up avenues for new treatment and new opportunities. That's what we want. We need partners to help us get the entire picture for the long run, and that's one of the advantages of partnering with industry.
Sorry, just one quick follow-up. Any role for repeat biopsies?
We can say in the GALGT2 trial, I'll just mention that we have biopsies actually at six and 12 months as well that are coming up in the current design of the isolated limb infusion study, and plans to use a similar protocol to test that in the systemic trial that's being designed.
Yeah.
Juno, I'm from Barclays. I have two questions for Dr. Mendell and also Dr. Rodino-Klapac. Dr. Mendell, in your trial, you enroll patient, exon 18 to 58 inclusive, and there's some other gene therapies that go after the entire population. Do you have any concerns there? You also comment, I'm wondering if you can share some thoughts on the approach go beyond the current patient population.
The transgenic data.
Some other trials are including mutations beyond 18 to 58.
Oh.
Do you have any concerns about that?
Yeah, I think that is, again, something we thought long and hard about, but I think you should appreciate that for the first trial, we wanted a homogeneous population. We wanted reasonable assurity that we weren't shooting ourselves in the foot by doing a population with potential antibody production since we had already seen that before. Having said that, we don't know the significance of the antibody that's produced. We were kind of always taught in this gene therapy world that satellite cells would be tolerizing, and we never had to worry about antibody. I don't know how many times I heard that story from the so-called experts. Dystrophin, you don't have to worry about producing a novel protein for antibody production. Well, one thing we did was prove that was wrong. Now, the other side of that question is what do they mean? We don't know.
Personally, I believe that it will not impair anything as we move forward. I think after this population, as Doug said, we want to extend it to further and further groups. We'll broaden the population. We'll broaden it in terms of those with, especially mutations, and I think we can also do the same with preexisting antibody to AAV. There's a threshold for that, for sure. I mentioned that we saw the threshold in our LGMD study. It was absolute at 1 to 3,200, and we had patients at about 1 to 800 AAV antibody who did carry that level of antibody to AAV, but expressed the gene well. We'll ratchet that down gradually, and I think we'll be able to know the exact threshold. I think that we could in 1 to 50 is a very conservative approach.
I think we could easily go up to 1 to 400 or 1 to 800 and not have any problem. We would probably be inclusive. We haven't seen a single patient that we've screened in this trial, or in the previous one that had antibodies over 1 to 800. The highest we're seeing some at about 1 to 200, 1 to 400, and I don't think that'll be a problem. We're being very conservative right now because we want homogeneity, and we want the best possible results for this first trial.
So-
I just simply note that even with respect to the conservative approach that Dr. Mendell and Louise Rodino-Klapac are taking, that the screen-out rate is less than 15%.
Less than that.
Significantly less, I guess.
Thank you. My second question is, how important is nNOS binding site for the clinical benefit? I think that Dr. Rodino-Klapac, you show one patient, Becker's patient-
Right
does not have that binding sites, right? Was that patient exercise intolerance?
Right. Our construct does not contain the nNOS. Over the years, we've compared to three or four different constructs, with and without the nNOS binding. In our head-to-head studies, we did not see a significant benefit of that construct over our own as far as nNOS. In our hands, we did not see any benefit to including it. Really you have to give and take, by including that, you're oftentimes limiting spectrin repeats one, two, and three. Two and three, which are extremely critical for force production. We found that the benefit including those surmounts at anything by adding the nNOS binding site.
How about one last question?
Yeah. Hi, Hartaj Singh with Oppenheimer. Thank you very much for a very comprehensive presentation. Really appreciate it. Just a quick question on your construct, which you've been working on for a very long period of time. What's the clinical manufacturing to the Brammer Bio? What's the tech transfer, the steps that you got to go through, Doug? About how long do you see that sort of happening? Then the other question is just on the AAV antibodies. I know in other disease where AAV vectors have been used, some antibodies have shown up, and I assume the regulators have dealt with that before. Just any thoughts with that? Appreciate the other questions.
Someone else answered the second part of that. The first part, what we've disclosed so far is simply that we have a relationship with Brammer Bio, that we have this hybrid model that will get us to a place where even under the most aggressive timelines, we'll be prepared to fully serve the physician and patient community. As we are tech transferring away from Nationwide to our new process, that without getting into too much detail right now, because I'll get way over my skis and misstate things, that we're making the least number of changes necessary consistent with the goal that we need to really ramp up, and we need the ability to scale up.
One of the things I said earlier was, and it's true, at launch, with our Brammer Bio relationship alone, we'll have more manufacturing capacity for gene therapy than any biopharmaceutical company has as we sit here today, and that's because that's what you need. This is neuromuscular. It's a significant amount of vector that's required to treat these boys. We're being very thoughtful about it, taking the least number of steps. We can probably provide a more thoughtful update once we talk through the FDA about our bridging strategy and ensure that we're all on the same page about that.
Regarding the antibodies, I just want to understand your question about seeing them pre-existing. Is that what you're meaning?
Yes, pre-existing. I know in other disease areas, there's been some antibodies to AAV vector. I assume the regulators have actually seen that in other diseases, and are used to sort of dealing with it. Just any thoughts of how that applies going forward? Would you just automatically not have those patients in clinical trials? Is there some kind of regulatory protocol you'll put in place for dealing with steroids, which is what's done in other rare diseases? Just any thoughts there. Thanks.
What we're doing right now is screening for them. We do have a low rate of antibodies in this population. We do have proof of concept data in primates showing that we can use something called plasmapheresis to remove these antibodies, and this is yet another population that we intend to go after to broaden our scope, to be able to remove the antibodies. The following treatment, we showed this is highly effective in primates. This is the steps that we'll take to be able to treat even patients with low level pre-existing antibodies.
Great. We got one more question here. One more question.
Thanks, Doug. I appreciate it. Salveen Richter from Goldman Sachs. You guys, when you gave the microdystrophin data, you gave two sets. You gave the Sarepta measure and the MHC measure.
Yeah.
Can you explain the difference between the two, which one will hold more importance in terms of functional and clinical translation?
Well, I think actually the interesting thing is, one of the things I said at the Goldman conference was that if you actually look at the literature, the best way to try to correlate, other than just empirically in the study itself, to try to correlate expression levels and function, is actually to look at the immunohistochemistry and look at dystrophin positive fibers. All the literature speaks to that. All of the presenters today spoke to that. That's really the right way to do it. On the Western blot, the reason we have two numbers is it's the same blots, the same images, just two different mathematical algorithms that were used, Nationwide versus Sarepta's. Sarepta's does not take into account the fact that in the blot, there is fibrotic tissue and fat. These kids have not only muscle, but also fibrotic tissue and fat.
Nationwide's approach uses a reference protein to take that into account. That's the delta. Obviously, Western blot's a regulatory requirement. I think one of the things we wanted to do, in addition to giving you the numbers, is just show you the images so that everyone can see that there is extraordinary expression, not only in the immunohistochemistry, but also in Western blot. You want to know one of the challenges for us on Western blot? The challenge is we're making so much darn protein, if you want to know the truth. In our prior Western blots, our standard curve went all the way up to 4%. We had a bit of an issue on this one. We've had to come up with a new standard curve to come up to 80%.
Our biggest challenge is dealing with success, if you want to really kind of understand it more specifically. With that, I want to first and foremost thank everyone, both on the web and here in the room, for sticking with us today for our inaugural R&D Day. I want to thank all of our presenters. Can everyone give our presenters a round of applause? Let me just say just two or three comments. Two comments. This is a day you would envision for those of us at Sarepta where we could be arrogant and thump our chest, but it is not that day for us. We feel quite the opposite, for two reasons.
One, the humility that comes from knowing that the great opportunity that we have in front of us relates to the extraordinary work that was done by our partners who have come up with such extraordinary therapies, or at least therapeutic options. Also to recognize that all this opportunity is nothing but obligation. That it's all about execution. We have to take all of these grand things, and through great work and single-minded focus, ensure that we're actually serving this community. Talking about the community finally. One of the first things Gilmore said, one of the things he said in the interview process, is we've got to listen very carefully to the patient community, and that's very true. How does one listen to the patient community? These families are going through very difficult times.
There are these individuals that develop organizations, these patient advocacy organizations, that in the face of extraordinary crises, familial crises, respond by not just helping their own families, but helping those around them. I want to speak really at a very personal level to PPMD and to Pat Furlong, who from some time ago began to invest in, for instance, the Nationwide Children's Hospital microdystrophin gene therapy program on behalf of kids with DMD. For those of you who don't know, if you got to know these patient advocacy groups like PPMD, which is the gold standard, you would be surprised. The level of commitment is unbelievable, and that won't surprise you. The level of passion is amazing, and that won't surprise you.
The level of sophistication, expertise, scientific knowledge, the things that they're doing go so far beyond what you envision a patient advocate would be doing if you were, as I was before I came to Sarepta, ignorant about the kind of work they're doing. They're driving so much of what's happening here. They're leading us in many ways. I want to say on behalf of all of us, thank you so much, Pat. Thank you all. Have a wonderful day. Thanks a lot, everybody.