Foremost, welcome. Thank you for taking the time out of all of your day. For everyone in this room, I'm grateful, and we're grateful. I know you all have busy schedules, so we appreciate you spending some time with us to learn about what's happening at Wave. To those of you who are logged on to the webcast, we appreciate you taking the time out of your day as well, and we hope to have it to be a productive one. To make it productive, we'll quickly talk about some forward-looking statements. We will be making forward-looking statements during this presentation, so again, please refer to our SEC filings for current updates. With that said, I know many of you are here eagerly awaiting our clinical data for Duchenne Muscular Dystrophy, our Exon 51 suvodirsen program, as well as our HD programs. We are too.
It's important for you all to know that we remain on track for delivering that data this year. We anticipate delivering the suvodirsen biopsy data from our ongoing open label extension study in this quarter, and we anticipate delivering top-line data from our PRECISION-HD2 study by the end of the year. Top-line data, to remind everyone, means safety data, it means mutant protein reduction, and it means total HTT to assess wild type. While that's all about the clinical data, today is really about research. Research and discovery has been what's driven this company forward. It's what supported the initiation of suvodirsen and our two SNP programs, our PRECISION-HD1 and PRECISION-HD2 programs. It's also what powers everything we do. It's what's bringing the next wave of programs forward.
We'll talk about Exon 53C9 for ALS, our USH2A program in ophthalmology, it's also been about what's accelerated the platform. We're excited today to introduce what we've been doing around the world of RNA editing and our ADAR platform as well. The investment that we've made in people who are focused on advancing continued progression of the science, we're excited. We're excited also about an agenda that has two other features to it. While today is not about HD in the terms of our program, we are fortunate enough to have two experts in the world of HD biology, and they'll spend time speaking with all of you as it relates to the importance of preservation of wild type and what the role of the healthy protein is.
In addition, we're excited for Greg to go into some of the insights that really led to unlocking the potential of chiral chemistry and how we could apply that to the next generation of stereopure oligonucleotides. The approach we continue to take at Wave in treating these devastating diseases is one where we're focused intrinsically on knocking down and interfering with the genetic target. We make genetically defined drugs, and we've built over the course of time a genetic toolbox where we can focus on chirally controlled medicine. What does this mean? What this means is that the current state of art that's been developed over the last several decades is all about taking one tool and applying that tool to a mixture.
You'd use different opportunities to optimize that than if you have the resolution of what eventually becomes understanding and amplifying that signal around a single drug. If you look at the difference of the now versus the different resolutions we can see in this case of different screws, you realize that precision comes at a different cost. You wouldn't use the same tool on any of one of these mechanisms as you would others. What's exciting about Wave is the improvement we have of building additional tools to do those jobs. We call that our genetic medicines toolbox. That means that we can work in silencing, it means we can work in splicing, both programs now moving and generating clinical data. It means we can move into new areas and applications of those tools, like ADAR RNA editing, as you'll learn about today.
It means we've been able to learn new things with this toolbox. By having a chirally controlled drug, it means that we can now characterize our medicine before we go in the clinic, understand how it works, why it works, and apply that back in the setting. You'll learn how we're instituting that in our PRISM machine learning applications to accelerate the ability to develop drugs. What's also unique is our ability to see that we can do genetic medicines without the need for viruses and lipo nanoparticles for delivery. This is not a delivery problem, being able to deliver our nucleic acids across a broad range of tissues. While genetic medicine toolboxes are important, we are making medicines, and so what's key for us in each disease we focus on is what is the underlying mechanism? What is it that we're trying to do?
In most of these diseases, there's one of two things. Either the patient has this disease because they're missing a protein. In the case of Duchenne Muscular Dystrophy, the largest gene in the body is responsible for making the dystrophin protein. Our approach, and the tenacity with which our team approached it, was how can we functionally restore that protein? There's a variety of different ways of trying to amplify that protein. We took the approach of could we amplify the full functional protein? That's how we approach DMD. It's what's not just led to our program for suvodirsen for Exon 51, but we're now using that across the platform in Exon 53 and other exon-amenable deletions. Shifting gears, we've also attacked the function of how do we think about proteins that shouldn't be there?
Where there's a toxic protein, and how do we do that in a way where we can be very specific. We call that allele specificity. The goal here is in Huntington's disease, where you have a mutant protein and you have a healthy protein, and this healthy protein is important, evolutionarily conserved, as you'll hear about. The ability to selectively silence or remove the toxic insult while preserving wild type function, and to be able to do that while preserving potencyMaintaining durability and maintaining that specificity, we'll show you as not only we have our PRECISION-HD1 and 2 program, but as we show you data subsequently on our SNP3 targeting program, where we can show that both in vitro as well as with in vivo data.
Lastly, in terms of thinking about these diseases, is pushing the boundaries beyond just the central nervous system and the neuromuscular system, and thinking about diseases like inherited retinal diseases. There we'll show you our new data in USH2A, where we're utilizing what we've learned in splicing and applying it to the eye, in addition, a silencing context. At the end of the day, it's also about the translation of those diseases to patients and innovating in that translation to patients. We've done that on the clinical trial design, so we've worked collaboratively with the FDA as part of our DYSTANCE 51 study.
We are accepted into the CID program, a new program at FDA, which led to the first study where we're using an augmented placebo design to maintain a placebo cohort of real patients and virtual patients that enables us to run a placebo-controlled efficacy study in DMD. That study initiated in June and is core to our program as we advance not just our open label extension study with biopsy data, but to have that ongoing pivotal trial running. We've scaled our manufacturing and built the expertise required to be able to support a potential launch with material at that time so that we can get as many patients who want to be on therapy on therapy. Core to our strategy, beyond just the discovery chemistry, the development strategy, was making sure that our manufacturing strategy paralleled our development.
We continue to build our commercial infrastructure and to work on novel payer strategies to make sure that patients, if we are approved, have access to drug, and that's very important. While today a lot of that discussion was about development and about payer strategy, today is about research, and it's about taking it back to what we're doing in terms of advancing our portfolio and our program. The agenda today, I'll be followed by Dr. Greg Verdine. Greg will speak about the chirality and stereochemistry. We'll move to Chandra Vargeese. Chandra will be talking about that evolution and moving from the chemistry understanding of stereochemistry to how we've unlocked that with PRISM in terms of drug discovery efforts. We'll shift gears and focus on programs and biology, we'll talk about the biology of the healthy protein, both Dr. Cattaneo and Dr. Saudou.
Then move into our SNP3 program with an update on our in vitro and in vivo data in SNP3 targeting HD. Then transition to ophthalmology with new work that's been done by Michael Byrne from our ophthalmology group, we'll close at the end. To transition, I'd like to introduce Dr. Verdine. Dr. Verdine co-founded Wave. He was the visionary, and I always say, the creative visionary, who saw forth to imagine that chirality in every other area of chemistry should apply to nucleic acids as much as it does to other molecules. He is a passionate and accomplished inventor of novel approaches and drug classes to engage targets widely believed to be intractable. In fact, he coined the phrase "drugging the undruggable" to describe his life's mission.
Dr. Verdine currently serves as the CEO for FogPharma and LifeMine Therapeutics, which was born from his own new modality scientific work. As Irving Professor at Harvard University and Harvard Medical School, he invented stable peptides and also made seminal contributions to understanding fundamental mechanisms of DNA repair and epigenetic DNA methylation. As an entrepreneur, he founded multiple public biotech companies in addition to Wave, including Variagenics, Enanta, Eleven Bio, Tokai, Aileron, and Gloucester Pharmaceuticals, which was acquired by Celgene. These companies have succeeded in achieving FDA approval for three marketed drugs. He has served on the board of directors of Wave, Enanta, Warp Drive, FogPharma, and LifeMine Therapeutics. Dr. Verdine earned his PhD in chemistry from Columbia University and served as an NIH postdoctoral fellow in molecular biology at MIT and Harvard Medical School. I'd like to welcome Greg Verdine.
Thank you, Paul. I'm standing here thinking, having failed miserably to teach organic chemistry to undergraduates at Harvard for over 30 years, have I just ratcheted up or down the difficulty by now trying to teach organic chemistry to investors and analysts? You guys have to tell me where I'm at on that spectrum. It's really a great pleasure to be here. I want to start with the beginning, with the fundamentals and what's fundamental to what the company is doing and its mission. This is all around chirality, which is a fundamental property of molecules. It's as fundamental as their color or whether they're a liquid or a solid, or whether they dissolve in water or oil, and I want to tell you more about that.
To begin with, I want to take a specific example where this property exhibits itself and to help you understand why chirality, the property of handedness, matters in biology. First, I'm going to show you a molecule, which is known as carvone. This was first isolated from the leaves of spearmint. We're going to pass out a test strip of this, and you're all going to take a whiff of this and perceive, using the olfactory receptors in your nasal cavity, the scent of carvone isolated from spearmint. Just take a whiff of it, and the room is going to smell glorious after this. You might have gotten a little bit of when you're out there in the hallway. It wasn't the Four Seasons scent, it was our peppermint or our spearmint.
Carvone was then isolated separately from caraway seeds, and the version isolated from caraway seeds has the same structure in two dimensions. It has the same connectivity of atoms. It has the same weight. It has the same properties except for one property, and that is its interaction with biological molecules, specifically olfactory receptors. This is the version now that comes from caraway seeds. You'll notice that it doesn't smell anything like peppermint, and yet these two have exactly the same chemical structure. There's obviously something missing from the chemical structure that doesn't describe its full structure. In order to get to that last element, you have to now allow a three-dimensional world. This is a two-dimensional world. In a two-dimensional world, they're identical.
In a three-dimensional world, these actually have specific shapes, and the way that atoms in the molecule project from the structure, it's very much like hands. You have two hands. They look the same. They're actually mirror images of each other. They're not the same because you can't superimpose them. You can do the superimposition test, and you can see these are mirror images. Molecules also have this mirror image property that's called chirality. The way chemists like to describe this is using these kinds of wedges and dashes. All this wedge means is that this group of atoms is pointed out at you, and that group of that atom is pointed backward. They can occur in two different projections, one where this group is pointed outward at you and one where it's pointed backward.
This comes from having a core atom right here, this carbon atom that has one, two, three, four different things attached to it. Whenever you have this kind of a core atom that turns out to have tetrahedral geometry with four different substituents on it, that gives rise to chirality. Because the olfactory receptors in your nasal cavity are chiral, they interact differently with chiral objects. That is, they interact differently with carvone. One smell receptor interacts with R-carvone. That R stands for rectus or right. S stands for sinister or left. Left. This is sinister. This one is rectus. They're handed. What does this have to do with drugs? Well, as you've perceived when you smell these, you can tell the difference between these two molecules based on only their stereochemistry, because they interact differently with olfactory receptors.
That means all drugs that interact with proteins, chirality is important for their interaction. The most notorious example of this in the drug world is thalidomide. One of the forms of thalidomide is shown here. This molecule was prescribed for the treatment of morning sickness in pregnant women. Tragically between 1957 and 1962, this caused more than 10,000 birth defects, severe birth defects in children. It turns out that thalidomide is also a chiral molecule. If you look here, it has a core atom with one, two, three, four different substituents on it, so it has handedness. It exists as a mixture of what are called stereoisomers. These are mixtures of the two mirror images.
One of those mirror image isomers, the R version, is responsible for the therapeutic effects, and the other isomer, which was originally present as a contaminant in the desired isomer, the S isomer, is the one that causes birth defects. After this thalidomide tragedy evolved, there was a movement across the industry to make all drugs stereochemically pure, and that is to avoid the unintentional downside of drugs that are fundamentally impure. When I began the journey that led to the founding of Wave Life Sciences, I realized shockingly, that there was one area of therapy in which synthetic molecules, namely oligonucleotide therapeutics, were being made as horrendous mixtures of stereoisomers, and it was the one area in which it was not true that one needed to get the stereochemical purity in order to take forward a molecule into human clinical testing.
It seemed to me that this field shouldn't be any different than the rest of drug discovery. Drugs should be stereochemically pure, period. What's going on in oligonucleotide therapeutics? I'm just showing you the fundamental building block here on the left of oligonucleotide therapeutics. It has two elements of nucleic acid with a phosphate connecting them, two nucleosides connected by a phosphate, and these are strung together in a repeating sequence where B changes from one unit to the other unit. I want to draw your attention to this phosphate. If you look at this phosphate, it turns out these two oxygen atoms are equivalent. There are only three substituents around this phosphate. It's not chiral. It turns out these are unstable.
The phosphate is unstable, and in order to stabilize it, what was done was to introduce a sulfur atom. The introduction of that sulfur atom breaks the symmetry in the molecule such that you now have one, two, three, four different substituents. You all know what happens when you put four different substituents around a chiral, a tetrahedral atom. You get stereochemistry. Every time you drop this sulfur atom into this molecule, it provides two different stereoisomers at each phosphate that is substituted. You need to put the sulfur in in order to stabilize the molecule to prevent it from being degraded, but that comes along with a cost. It creates a mixture of two stereoisomers. There's an Rp isomer and an Sp isomer. P just stands for around phosphate.
This is now moving into a schematic diagram from a structural diagram where these circles just represent these nucleosides, and the carets represent the phosphate. When there are two of them, this means there's a mixture. When you make a nucleic acid drug, an antisense oligonucleotide, these units are strung together with phosphate connecting them in between. The way this has been done through the entire history of the field is that every time one of these phosphates had this phosphorothioate substitution introduced, there was no way to control the geometry of introduction of that sulfur atom. Therefore, it generates a mixture of two stereoisomers each time you do the substitution. Two times two times two times two times two times two. You see how you get up to a horrendous mixture. If there are 15 of them, that's almost 33,000 stereoisomers.
These drugs are not just kind of casual mixtures. Literally, they're horrendous mixtures. What Wave is doing is to now take forward individual stereochemically pure molecules that are a single molecule, not a mixture of 32,000 or 500,000 or whatever thousand. What I want to do is just to provide a visual so you can see that these sulfur atoms are different. This is showing an oligonucleotide now, the fully blown structure with all atoms, where yellow are the sulfur atoms shown here. You can see they're all kind of pointed upward. Right? They're aiming in this direction, and that's the Rp isomer shown here. In the case of the S isomer, where these are all S, they're pointed out at the equator. They're more or less pointed out at you.
If you were an object and you were coming up to grab this DNA, you'd notice they look different. The pattern of sulfur atoms looks different from there. That creates the basis of what you just perceived with Spearmint versus Caraway, that biology perceives these isomers as being different. This is a case where they're all the same, they're all R and they're all S, but what Wave has developed the capability to do is to program individually each single phosphorus atom to control precisely, exactly the stereochemistry, whether it's R or S, and to learn, therefore, what makes the most drug in terms of its stereochemical program. If you just look here in terms of the total number of isomers, if you get to an oligonucleotide that has 19 phosphates, shown here, this is over 500,000 individual isomers.
As you go up to larger N, ADAR, these are adenosine deaminase targeting oligonucleotides higher. You're getting up to 30 billion, and then CRISPRs are on the orders of trillions of individual. Every single molecule is different. They're not the same, and biology perceives them as being different. Let me just show you. Why don't you just separate them? The simplest thing would be to say, take the 30,000 and just separate them. Of course, you'd imagine that you couldn't possibly separate one out of 30,000 components. First of all, you'd be throwing away 29,000 whatever and then keeping the one. It's physically impossible to separate them. They're physically impossible.
Just imagine that in a way, as if you had black paint in front of you, say, "Well, why don't you just take it apart into the individual colors that make up black paint?" Once they're together, you can't take them apart. What this shows is the clinically approved drug, mipomersen. This is kind of a Lewisian technique where you try to separate things, if they separate, you see individual peaks that correspond to individual components. Here you can see there's no feature at all. There's no basis for separating these molecules physically. Separation is out, just as with paint.
What you could do, as with paint, is make the individual colors individually. That's exactly what we, in the early days of Wave, we developed the capability to, for the first time, to synthesize oligonucleotides with individual stereochemically programmed composition at each individual phosphate to have an addressable system where the phosphate chirality could be programmed, and then to ask the question, how do we optimize this for properties? Here you can see in this eluting technology, if you now have an individual composition of stereoisomers, this is from mipomersen, this clinically approved molecule. You see that one of them elutes is here, another one's elutes is later, another one's elutes is later, and these obviously have individual features and peaks, which means they're separable, purifiable, put into a bottle as a single pure entity. This is transformative for this field.
It has never existed, the capability to isolate individual molecules. Imagine if you put this into a patient and you say, "I want to track how mipomersen is behaving." Into the patient, you're putting 500,000 molecules, each of which has different cell penetration, different metabolism, different plasma protein binding, different interaction inside of the cell with the target, and so on. Every aspect, stereochemistry affects every aspect of the molecule. Over here, these are trackable, they're optimizable, and so on. I make the argument that this is a different field altogether once you impose stereochemical purity from everything that existed previously, where one was dealing with untrackable, unoptimizable mixtures of drugs that are beyond imagination. Okay. Very quickly, how do these oligonucleotide therapeutics work? First of all, they target RNA, so these are new modality drugs. They don't target proteins, they target the RNA that encodes proteins.
This is the drug now, and it combines together using the Watson-Crick pairing to form a complex between these two. Once these two come together, the drug plus the target, they recruit an abundant conserved cellular protein called RNase H. The RNase H will only bind after the drug combines with the target. RNase H then does a cut on the red strand. It literally chops it into two pieces. Now, in the past, first of all, where these are not substituted with sulfur, they're unstable, as I mentioned. RNase H couldn't recognize any feature of this molecule. Everything kind of looks the same. It would slide along, and it would not make a kind of defined complex. That's the same thing with stereorandom oligonucleotides. If you have half a million different isomers, RNase H interacts differently with every single one of those.
You have this ensemble effect where you can't really understand what's going on at the level of each individual drug, because there are so many, there's no way to deconvolve them. What I'm showing over here is work that was recently done at Wave Life Sciences that represents a real move forward for this field. This is an X-ray crystal structure, which is a molecular image where you can define individual atoms here in the protein and here in the target of therapeutic action, where RNase H, by virtue of stereochemical programming, binds at a single spot on this target, and it then directs the catalytic power, the cleavage power, to a single spot on this. This represents harnessing RNase H in a way to focus its catalytic and cleavage power. What this means from a practical standpoint, actually, it means a lot of things.
As you'll see, it greatly improves the catalytic power of the enzyme. What it also does is, instead of giving random cleavage all over the place, it directs the enzyme to focus on an individual predetermined spot that is programmable on the RNA, and that allows you to discriminate between the two alleles, where a patient has, for example, an A at one site and a C at the other site, and it allows you to take out one of those alleles selectively. You'll be seeing more of that. This is a remarkable achievement because what we can do now is look at the interface. This is now in blue is the antisense oligonucleotide. In red is the target where it's going to get cleaved right over there, so it binds there, cleaves right at the scissile phosphate. There is a precise interaction of each phosphate.
You can see this phosphate has the sulfur pointed out at you, this phosphorothioate, that's one stereochemistry. This one has the sulfur pointed back. That's the other stereochemistry, S. This code, SSR, causes the enzyme to position itself to dock and to cleave specifically at that site. There has never before been the ability to look at any antisense oligonucleotide at the level of atomic precision. This is the first time it's ever been seen, and what it means is that now one can apply principles of rational drug design for the first time to the creation of oligonucleotide therapeutics.
For that reason, I would argue that this represents a rebirth of this field in a completely different guise. I'm coming down to the end of my talk, I just want to point out, now, when you have this ensemble of things, there are some of them that are not very effective and some that are effective. I mentioned to you early on that when we put sulfur atoms into these oligonucleotides to make them stable, early on at Wave, we discovered that that's actually only partially true. That sulfur atom only stabilizes the S isomer. It doesn't stabilize at all the R isomer. What that means is when you're dosing a patient, every time it has an R configuration in it, that's a site of metabolic liability. If you have a lot of them, those drugs are arriving in cells dead on arrival.
What Wave can now do is judiciously put only the right number of Rs into an otherwise S oligonucleotide, again, allowing optimization. This just shows the differences in rates of cleavage of a stereopure oligonucleotide versus a stereorandom. Interestingly, even though there are fewer cut sites, the enzyme is faster. The enzyme prefers a stereochemically pure. What's not shown here is that the overall extent of cleavage is also dramatically increased. Each molecule is giving more turnover of each target RNA. If you look at also, now this is looking in neuronal cells and now administration of oligonucleotides, you can see there's roughly 24-fold better potency here. Now this is looking at intraocular injection.
The thing is, when you have all these R configurated stereophosphorothioates, they create instability in the molecule, when you concentrate S configuration, you increase the stability of the molecule biologically in a very rational way. What you can see is by intraocular injection, there's a roughly 50-fold, this is now looking one week after intraocular injection, there's a 50-fold increase in potency. There are other properties that are not shown here, for example, like TLR activation and cellular penetration, et cetera. Stereochemistry is a complete game changer for being able to rationally discover drugs. I'm going to stop there. I'll be happy to take questions at the very end and to introduce to the podium Chandra Vargeese, who's our SVP and Head of Drug Discovery and a legend in the world of oligonucleotide therapeutics.
I want to also just say that I didn't want you to get spearmint and caraway all over your hands and then have to leave and explain for it. You'll get one of these that you can take home with you to experiment with the kids. Just be careful when you open it up because it's glass. Okay? There'll be a stereochemical takeaway on the way out the door for all of you guys. Thank you very much.
Thank you very much, Greg. That was such an inspiring talk on the stereochemistry. This actually helps me to introduce our platform. Like Greg pointed out, chirality is important in biology. Really what we have uncovered at Wave is the use of chirality or use of stereochemistry to design our oligonucleotides. What we call our platform is PRISM. I want to explain, walk you through some of the concepts of PRISM, then review some of the advantages of using this across multiple modalities. The first, I want to provide a brief review on the PRISM platform, how we came up with the technology and using stereochemistry as the fundamental basis and chirality as the fundamental basis for designing oligonucleotides, which to Greg's point, it was ignored for the last 30 years. PRISM.
Here, what we're talking about is when we start designing or when we start thinking about generating oligonucleotides, we look at target indications, we start generating the target that we want to transcript, that we want to attack, put the product profile in place. Once we have that, we now have an option to use any of these modalities starting from silencing or splicing or editing. You can use any of these modalities to make a therapeutic. We come to the middle point where we call our platform as PRISM, which takes into account not the two-dimensional interactions of Watson-Crick base pair. It actually incorporates the stereochemistry so that we now have, to Greg's point, a three-dimensional interaction.
We take into consideration the sequence and the chemistry, which you all know in oligonucleotides, there are two biomodifications. We could use all different chemistries. That's not enough. We have to introduce the chirality, that is the stereochemistry. With all these three things in place, now we have a platform that actually can construct specific stereopure molecules. You can control stereochemistry at every position wherever we incorporate a heteroatom in the phosphorus. Phosphorothioate is an example, but there are also other heteroatoms like methylenos, which is exactly the same. It's a mixture of several diastereomers. For us, we can control, we can use our technology to control each of these parameters.
At Wave, the scientists are now developing stereopure molecules using this technology and trying to understand about different parameters of the drug very specifically. In order to design, we also need to have assays in place to characterize each one of these isomers. What we have introduced internally is developing cell lines that can take up the oligos genetically. One thing that is forgotten in the industry is that we use transfection agents or electroporations to introduce oligos into the cells, because oligonucleotides are heavily negatively charged, so they do not enter the cells, but we force them to enter the cells using transfection agents and electroporations.
What we decided to do is to mimic closely and to actually eliminate all the false positives, we need to have all of the nucleotides that enter the cells by itself, by genetically or free uptake, just like small molecules. That means we need to develop in vitro systems that can take it up readily. Once we have the systems in place, we have a number of assays to look at the potency of the molecule, the duration of activity of the molecule and the tox profile. We are developing therapeutics which has got all the potentials, which contains the potency, stability, immunogenicity, and also having very good safety profile. We can characterize the molecule precisely with these individual constructs.
Once we have them and we show genetically that we meet all the aspects of in vitro potencies, we can then switch over to animal models if they're available. In some cases, we may not find animal models, especially in rare diseases. In that case, we can actually move on to looking at the PK properties in animals and then use that to drive our clinical potency calculations. We could actually do a validate in an animal model and move on to generating clinical candidates. What we have also learned over time is we have created a lot of data set, and we have understood several parameters, and we have understood the interactions between sequence and chemistry and stereochemistry, and we have developed a knowledge base. Using this knowledge base, now we can create sequences that are also useful for finding additional target sequences.
Traditionally, when we get a target, we also design oligonucleotides to see which portion of the transcript is accessible and which portion of the transcripts provides a good oligonucleotide. In order to do that, we create oligonucleotide in stereo random formats and do a screening across the transcript purely to select the sites. If we do that, what we identify is that our hit rate is only 10% to 15%, meaning if you generate 100 oligos, you only can have 10 or 15 oligos that can show potency, a 50% or 70% reduction in target modulation. This hit rate is actually fairly low. If we incorporate all the knowledge base screening using our stereo pure format, what we observe is that the hit rates actually dramatically increases.
With the knowledge base that we have developed over time with multiple sequences, right now, our screening hit rate for identifying the target sequence is over 50%, which is almost fivefold better than what we were with the stereo random compounds. Clearly shows that with the stereo random formats, we are not able to access all different sites. To Greg's point, we cannot optimize a stereo random molecule, and we cannot get target sequences that are amenable for biological activities. Now, because of the high hit rate, this has exposed us to different target sites, and we now have the option to actually investigate many more target sites than we had before. This is still advancing, and we actually have a knowledge base that we are developing, and we are incorporating every day to improve our screening technologies for purely identifying target space.
The next topic I want to talk about is how we have used our technology to optimize, to rationally design our drugs, and to actually connect the in vitro potency with the in vivo potency. In order to do that, I want to walk you through a surrogate sequence. Our targets, predominantly what we are targeting resides in the nucleus, primarily pre-mRNA and intronic sequences are present in the nucleus. What we decided to do is to use a target that is present in the nucleus. We have been using MALAT1 as a surrogate target. This is a long non-coding RNA which resides in the nucleus, and it's ubiquitously expressed. We should be able to do our SAR development and understand about the broad distribution and target engagements and nuclear uptake by using ASOs that are directed to a MALAT1 target.
The first question that we ask is: What are the implications of using stereopure MALAT1 oligos? What we find here is with a single IV injection at 25 mg/kg, which is equivalent to roughly 1.6 mg/kg, what we see is a very broad distribution of person generated MALAT1 oligos. We do have two constructs that are shown here, and what we see here is a broad distribution and target knockdown in multiple tissues in different cell types, clearly showing that we are able to target the nucleus. This provides evidences for intracellular delivery into the nucleus. Secondly, the target knockdown that we see is also substantial, and it persists for up to eight weeks.
This type of data is unprecedented in the case of oligonucleotides, especially when we give a systemic delivery and looking at not liver, not kidney, looking at multiple target tissues, that we are getting continuous knockdown. With this in mind, what we have shown here is some of the muscle tissues, because we are very much interested in neuromuscular and DMD, we wanted to see how these oligos are taken up and how they're effective, and we wanted to look at the longevity of the duration of action of these molecules. Clearly what we see here is we can develop drugs that target multiple tissues. Similar point is true when we go into CNS space. Since we have a number of neurology programs, we also want to understand, after an IV injection, how are these oligos distributing?
What we clearly see here is the MALAT1 knockdown is actually present throughout the brain in multiple regions, including cortex, striatum, cerebellum, hippocampus, and brainstem, everywhere. In spinal cord, we see very good knockdown, and we see sustainable knockdown and persistent knockdown up to 10 weeks. We see greater than 80% knockdown, clearly showing that our oligonucleotides are distributing to multiple cell types, neurons, astrocytes, microglia, and so on. Again, to the point that our stereopure oligonucleotides generated by PRISM is capable of entering into multiple cell types, which is very important for CNS programs. Now that we have seen proof of concept using MALAT1, we wanted to switch over to other programs, including C9orf72 for ALS and FTD. In this case, what you're looking at is a hexanucleotide repeat, and the transcript it's a genetic cause for ALS and FTD.
In this particular case, we want to knock down the hexanucleotide repeat, which produces toxic dipeptides that causes progression of diseases. Now how do we selectively target the hexanucleotide repeat-containing transcript while we maintain still the normal protein? Just like Huntington's, which you'll hear later today, the C9 protein is very much important for the neuronal strength. Here I'm showing a lead compound which we targeted specifically to knock down the repeat-containing transcript. We see a substantial knockdown with a single dose of oligos. What we also see here is that we are still maintaining the C9orf72 protein that is required. It's a different kind of selectivity where we are only targeting a specific transcript. We are doing currently several in vivo studies, and we expect to be in clinical development by second half of 2020.
The next question here is an interesting one, which Greg touched upon regarding the toxicity. We always want the drug to be potent, durable, but we also want to make sure that the drugs also have a good tox profile. When you're making a compound with hundreds and thousands of stereoisomers, it's impossible for us to understand about the toxicity of each one of the isomer. You get a whole reading, but it's not easy to characterize. When we see tox, we wonder, what is causing the toxicity or is it a class effect? The answer is that that's not very simple and it's not true.
Here, what I wanted to show you is two molecules which have got exactly the same composition, exactly the same chemical modification, it's the same sequence. In vitro it has the same potency, in vivo, it has the same potency. It's conjugated to GalNAc, so it gets delivered to the liver very effectively. By all means, the molecule is behaving very similarly. However, you can see one difference in the stereochemistry at one position where you have a right-hand and a left-hand switch. This particular switch, although it appears to be extremely subtle, shows dramatic changes in the tox profile in hepatocytes. We see substantial elevation of ALT and AST with one isomer, the other isomer is quiet. With just two isomers and with a small change, we can see drastic difference in toxicity.
This is very clear because we are able to make stereopure molecules. When you do a stereorandom molecule, and if that was our lead, we would not be able to tell which one caused toxicity and which one is silent. This is the power of using chirality and trying to understand the drug better so that when we start, we start with a single molecule all the way through, and the toxicities and other effects that we see continues throughout. This is the power of Wave's technology. Switching to exon-skipping oligos. What we always saw in our oligos is that they were taken up so readily by cells, by passive uptake, by genomic delivery or free uptake. We were wondering why these oligos get in. We did some experiments to understand how these oligos distribute.
On the left-hand side, you can see the myoblasts when they were treated with our stereopure exon-skipping oligos, what we saw was a dramatic uptake in the nucleus, which is shown in blue, and the red dots there are the oligonucleotides. We counted the number of foci and plotted this against to understand about how many molecules are present in the nucleus. You can see with the stereorandom oligos, we see minimal amount of molecules in the nucleus. With stereopure oligos, we see substantial copies in the nucleus, which clearly might show that why our oligos keep much better or why our oligos are more potent. We also see the same phenomena when we move into in vivo in mice. We injected a single dose and looked at the tissues.
Here, we are looking at muscle tissues, and what we see here is a very good distribution of oligos in the muscle tissues. What you can clearly see, observe is the nucleus, the myonucleus or the central nuclei, has a lot of oligos, which is where you want an exon skipping oligo to be present in order for the molecules to work. Clearly stating that stereopure oligos have a completely different intracellular penetrating properties. This could be the reason why we see very good skipping efficiency with the Exon 51 oligo, with our shorter molecule. It is targeting Exon 51, and the molecule that targets Exon 53 also shows substantial amount of dystrophin production in vitro. This is using free uptake, and these two molecules have produced enormous amount of dystrophin, truncated dystrophins.
You can see that from Paul's talk, it was very clear that we are going to get readouts by the end of this year for clinical DMD for dystrophin production, and we are pretty excited internally to see what the data is going to look like. The next data point that we would get is from Exon 53 oligos. We are going to be entering into the clinic, and we expect top line dystrophin data by the second half of 2020. Now I'm going to switch over to another new modality, which is ADAR-mediated RNA editing modality, which is actually a pretty exciting field. Now, this is emerging to be another oligonucleotide modality that is very versatile. Besides silencing and mRNA splicing, there are other targets that we cannot target with regular ASO or skipping oligos.
With ADAR, we could actually expand our target profile, and we can get into new targets. What ADAR can do here is a piece of oligonucleotide, now you can design it to the mRNA target, where what we can do with the help of ADAR is convert an A in an mRNA to an I. This I is equivalent to a G. Essentially what we are doing with the help of oligonucleotide and ADAR is converting an A to a G, and this can be done to improve certain target profiles. Some of the missense mutations and nonsense mutations are not amenable for splicing. You cannot use a splicing technology, but you can use ADAR to restore the protein expression for these mutations. On the other hand, we can also cause mutations to alter the protein function or to increase the protein expressions.
ADAR is so versatile, and unlike CRISPR, where we are editing the genome, here we are editing the RNA. We will not cause any damage to the genomic DNA, and this is not inherited. It's also reversible. This is going to be a versatile tool for us to use in the future. Now we also approach targeting ADAR in the same way as RNase H mechanisms. What Greg pointed out is understanding about the stereochemical interactions. When we look at this crystal structure of ADAR, you can see ADAR enzyme binds to the double stranded RNA, and it makes contacts at several positions. It makes contacts at the backbone, and it also makes contacts with the sugar and also with the nuclear base.
That means we can work around ADAR using our PRISM technology to optimize for backbone interactions and sugar modification and nuclear base modifications. The discovery team, the scientists started looking at it more intensely, and we wanted to use our PRISM platform to actually incorporate all the changes so that we can have an ADAR editing oligos. Now we generated about 1,000 oligos or so in the last year to develop an ADAR. In order to do that, we need to understand about two-prime modifications. We incorporated that. We incorporated backbone stereochemistry and backbone modifications, and we looked at various sizes and structures and modified nuclear bases. Now the team has developed an ADAR so that you can generate a single stereopure oligonucleotide to cause editing using an endogenous ADAR.
We believe that this RNA editing platform is much superior than the existing technologies in a way that we have developed a fully modified or chemically modified stereopure oligonucleotides that can be taken freely without any involvement of an AAV or a lipid nanoparticle. These oligos, as I showed before, they enter cells very easily. What we also found is these kind of chemical modified sequences can use endogenous ADAR to edit, and we do not require any exogenous protein like Cas13 or chimeric ADAR. With all this advantage, we have shown in vitro high level of RNA editing. In this case, what you're looking at is editing at UAG sites in an actin mRNA. It's an endogenous mRNA. It is present in all the human primary cells. What we are looking at is a GalNAc conjugated ADAR editing ASOs or ADAR editing oligonucleotide.
We see here in human primary hepatocytes a dose-dependent increase in editing efficiency. The ED50 or the EC50 of this molecule is 100 nanomolar, which clearly shows that without the use of any exogenous ADAR, we can have an efficient RNA editing. Now we also took the same molecule and took away the GalNAc and put it in genomic delivery in different cell types. Here again, what we see, the stereopure oligos gives you editing in hepatocytes and also in bronchial epithelial cells. We see editing. However, the same oligo, which is stereorandom, gives some amount of editing in hepatocytes, but completely absent in other cell types. Clearly showing that we have a technology, RNA editing technology, that we can make use of using endogenous ADAR for editing without the use of exogenous ADAR or delivery systems.
We expect to have in vivo data for this modality sometime in 2020. We are pretty excited about the progress the team has made in RNA editing. Now I'm giving back to Paul.
Well, as you can see, we're pretty excited about the chemistry platform. As we said, one of the things that we've done and focused on early is building a dominance in understanding the interactions between RNA and protein. As you heard from Greg, the crystal structure unlocked a lot for us in understanding this code, because we often got this question of, well, do you have to make a half a million different drugs every time you want to reduce it back to practice? I think one of the things that we've learned as we've gotten better at the interactions with enzymes is really understanding that interplay between where do you put your modifications and how does that translate ultimately to stability, to potency. As Chandra alluded to, you take a sulfur from this one position, and you turn it slightly, and you can radically change safety.
This idea that we've built fundamentally and foundationally, that rational drug design shouldn't elude the nucleic acid space. With rational drug design, it means the same drug that we're testing in vitro is the same drug that we're putting into our animal models and assessing, is the same drug that's going into toxicology studies, and ultimately becomes the same drug that would eventually translate to patients. That level of characterization is how we can translate a lot of the chemistry that you learned about across the platform and be able to ultimately apply it into how we make therapeutics. As we talked about too, increasing the toolbox and the array of tools, meaning single-strand RNAi, antisense, splice correction, and now with RNA editing and ADAR, lets us increase the number of targets that we're amenable to.
Now we're also staying resolutely focused right now in the areas of biology that we're pursuing. Bringing that convergence between a chemistry platform and a biology platform, where do we apply it? What we're focusing on the portfolio, obviously spent a lot of time in other meetings and places talking about our suvodirsen program for Exon 51. We're able to apply that learning continually, as we said, as we emerge into new exons and new muscular diseases. As Chandra showed, being able to get into additional muscle cells.
The MALAT1 study where we could show at a human equivalent dose of 1.6 mg per kg, durable distribution, so it was eight-week distribution and knockdown, tells us that we're getting to the right compartment of the cells to be able to do this, not just as a single dose, but get that durability within genetic therapies, prolong that exposure time, and be able to redose. Oftentimes we talk about genetic medicines as redosing is sometimes detrimental. In a lot of ways, we're excited about redosing. In fact, the more we hear about gene therapy programs in areas where there's turnover, like muscle, redosing is a problem elsewhere. For us, that's not a problem. We see that as a huge benefit. Every time we redose drug in intervals, we see more dystrophin protein get made across more cell types. We actually see that as a distinct advantage.
We talk about more, and I'll use this as a transition point next in terms of Huntington's disease, but you'll learn more about SNP3 and being able to take what we can learn to Chandra's point around sequence space. What sometimes gets unappreciated is that there's target sequence space that others would say is nontractable and thereby limit what they can do. I think the advantage of what we've built across our tool platform, so being able now to open up the power of that 55%, which I know goes kind of unnoticed to folks, is the ability to unlock sequence space that would otherwise people would say is a non-active sequence space. It opens up our ability to find new and potent sequences.
That's important as we talk about our C9 program, because there, unlike SNP targeting in Huntington's, we're using the sequence space to be able to do allele specificity. What that doesn't require us to do is reduce the number of patients based on a SNP. It means that all patients with C9 positive ALS or FTD would be amenable for our therapy. Opening up and broadening those definitions is because we're getting access to more and bigger sequence space. We'll talk soon about our excitement about the emerging areas of ophthalmology, as you saw a little bit of the in vivo data that's getting us to that point. The key here is broad therapeutic space, continuing to move programs forward in the clinic, continuing to increase a robust portfolio. We now have over 17 programs across discovery and development.
Remember, we do have a collaboration with our great partners at Takeda on the discovery side, increasing the ability and our engine across drug discovery. The next transition in really thinking about the portfolio discussion is now to talk about where are we going. I'd like to introduce two amazing KOLs who've really been instrumental in the field of Huntington's disease. We oftentimes hear from clinicians who are in the space. We often get questions about, well, what about this protein? What does this protein do? Why is it important to selectively silence it? We're very fortunate to have two KOLs who are going to talk to us today about huntingtin as the protein, both the mutant and the wild type. It's my pleasure to introduce Dr. Elena Cattaneo.
Dr. Cattaneo is a full professor of pharmacology and director of the Laboratory of Stem Cell Biology and Pharmacology of Neurodegenerative Diseases at the Department of Biosciences of the University of Milan. She's also director of UniStem, the Center for Stem Cell Research of the University of Milan. Dr. Cattaneo was born in Milan, where she earned a PhD in biotechnology applied pharmacology. She spent a few years at MIT as a postdoc under the supervision of Professor Ronald McKay. At MIT, she studied neural stem cell differentiation and its association with neurodegenerative conditions. She also spent time at Lund University and then returned to Italy to become a researcher at the University of Milan, where she was appointed full professor in 2003. Today, the main research focus of her lab is the molecular pathophysiology of Huntington's disease.
This important scientific work and Dr. Cattaneo's social merit led the President of the Italian Republic, Giorgio Napolitano, to appoint her as Senator for life in August of 2013. Today, we're also joined by Dr. Frédéric Saudou. He will follow Dr. Cattaneo. Dr. Saudou served as a hospital Professor at the Université Grenoble Alpes and CHU, Director of Grenoble Institute of Neuroscience. He is also the group leader of the team Intracellular Dynamics and Neurodegeneration, and Director of the Grenoble Excellence in Neurodegeneration. He undertook his thesis at the University of Strasbourg with Professor René Hen on serotonin receptors and completed his first postdoctoral fellowship in Strasbourg with Professor Jean-Louis Mandel in human genetics. A second postdoc at Harvard Medical School with Professor Michael Greenberg on neuronal signaling.
He then moved back to France to lead the research team at Institut Curie and became the director of the department in 2010. Today, his research team focuses on understanding huntingtin protein function and dysfunction in intracellular trafficking. In 2014, he also received the Richard Lounsbery Prize for Medicine and Biology from the French and U.S. National Academies of Science. With that, Dr. Cattaneo.
Many thanks for the opportunity. As you see from my title, I would like to lead you through the billion-year-long history of the healthy allele, as we want to know why it came to us, I actually want to know why we carry the gene in our genome, given that it can be so dangerous. I will deal with this. First of all, I would like to spend the first four slides on just introducing the disease. Of course, this is the gene causing Huntington's disease. It was cloned in 1993, and it is a big gene, 67 exons. We know that in exon one we have this CAG repetition. We also know that the normal population has a number of CAG repeats below 35. While when the CAG gets above 36, of course, this is a disease range.
We also know that the longest is the CAG, the earliest are the symptoms. The CAG is translated into glutamine. The protein, whose name is huntingtin, is fairly large protein, 340 kilodalton protein, again, carrying this elongated polyQ in the pathological range. As a consequence of that mutation, we have neurodegeneration in the brain. I just want to highlight that neurodegeneration not only hits the striatal neurons but also there is cortical atrophy and cortical degeneration. At the end stage, of course, there is a general atrophy in the brain. The gene, the protein is broadly expressed in all cells of the body. I would say it is highly expressed in the nervous system and within the brain.
We have to keep in mind it is even more expressed in the cerebral cortex or in the cortical neurons, actually, that project to the striatum. This very large protein is also the subcellular localization is quite complex because you can find the huntingtin, of course, in the cytoplasm bound to microtubules with a role in intracellular trafficking, exocytosis and endocytosis. It is also associated with many other organelles, including the mitochondria, the endoplasmic reticulum, and the lysosomes, synaptic vesicles, the Golgi complex, and the nucleus. It is also found within neurites and at the level of the synapses where it is found associated with proteins that are very important for synaptic function and vesicular structure. Having said that, these are the three points that I want to cover with you today.
Yes, huntingtin is expressed everywhere, but I would like to give you my view of, let's say, the three, four key experiments and Frédéric Saudou will add a few more. Really highlighting how exactly mutant huntingtin becomes toxic to neurons. I would like to start with four pieces of data. The very first one is this one, and this is an older study, but I would say this is a seminal paper really showing that, okay, if you block mutant huntingtin expression. These are animals that express mutant huntingtin starting from postnatal day 0. Okay. Expression of mutant huntingtin is maintained until week 18. At that time, you administer doxycycline. This is an inducible model, and you turn off mutant huntingtin, and then they've been waiting for another 16 weeks.
Basically, the message is that there is a reversal of many or basically all phenotypes. I think this is really a key message for all of us, certainly for me, because it says that turning off mutant huntingtin is sufficient to reverse the disease in mice. Okay. This is the first piece of data. The second piece of data is, while we would like to know, how exactly mutant huntingtin exerts its toxicity now that we know that we can reverse toxicity. This is a very nice piece of data coming from two groups and two papers, 2009 and 2010. Basically, in this paper, they are focusing on the cortical striatal synapse. Okay.
The striatal neurons die in Huntington's disease, but the cortex, I mean, the afferents from cortex, of course, are very important for the normal functionality of the striatal neuron and as you might guess, also for the dysfunction of the striatal neurons in Huntington's disease. One first evidence of this is from this paper. Here, basically, they show that, yes, at the level of the post-synapse for those striatal neurons, we know that they carry this important NMDA receptor, and we know that the synaptic NMDA receptor are very important for the functionality of the circuitry. In this paper, they deal with the extra-synaptic NMDA receptor because we learned that the extra-synaptic NMDA receptor exerts toxicity. An exacerbation of the activity of this extra-synaptic receptor can be toxic to those striatal neurons.
The paper suggests that the balance between synaptic and extra-synaptic NMDA activity is critical in determining neuronal survival in Huntington's disease. They prove that in a very nice manner by administering to cells in vitro or to mice in vivo, memantine. Okay. This is an NMDA receptor blocker, and we know that a low dose of memantine can block specifically the extra-synaptic receptor. By blocking this extra-synaptic receptor, of course, you rescue the pro-survival pathway, and you reverse the disease. The key message is that there is something happening at the cortical striatal synapse, which is very important. I have to look at this compartment very closely. There is a second paper that goes in the same direction by using a completely different paradigm. This is William Yang's study. Here, basically what they did. This is another in vivo mouse study.
Here the full-length mutant huntingtin was genetically reduced in striatum with these Cre mice, a recombination that targets specifically the striatum. They were able to reduce mutant huntingtin only in striatum, or with this other version, they were able to reduce mutant huntingtin only in cortex. With this final mice, they were able to reduce mutant huntingtin in cortex and striatum. They were able really to dissect out what is the role of the two compartments. Is there a role for the cortical afferents? The result to me are really striking because basically they show that if you reduce mutant huntingtin in striatum, you are looking at the cell-autonomous effect of mutant huntingtin, you are able indeed to restore some activities of the post-synaptic compartment because you are restoring in these mice when you remove mutant huntingtin, the activity of these molecules. Okay?
There is cell-autonomous toxicity of mutant huntingtin in striatum. When they look at reduction of mutant huntingtin in cortex with this other mouse line, basically they were able to show that they could restore some cortical function. Quite remarkably, removal of mutant huntingtin in cortex was beneficial to the striatal neurons because they were able also to restore parameters associated with the post-synaptic density. The message is there are also non-cell autonomous toxicity of mutant huntingtin in striatum. In the final portion of the paper, they were able to show that in the mice in which mutant huntingtin was repressed both in cortex and striatum, there was basically total recovery of several functional parameters.
The key message is, to me, if we want to cure HD, not only we have to save the striatal neurons, but we also have to work on the cortical neurons.The fourth piece of data that I want to show you is that we have been discussing neuronal toxicity, right? Striatal and cortical toxicity due to mutant huntingtin. Huntingtin is not only expressed in neurons, it is also expressed in astrocytes. What is mutant huntingtin doing in astrocytes? For example, here we were able to show that mutant huntingtin in astrocytes suppresses the production of cholesterol, turns down the transcription of many genes implicated in the cholesterol biosynthetic pathway. Not having cholesterol or having a reduced activity of this cholesterol synthesis pathway is very problematic because the cholesterol that we have in brain is synthesized locally. It doesn't come from periphery.
There is a local synthesis of cholesterol in the brain, in the astrocytes. The astrocytes are the producer of cholesterol in the brain. Of course, this cholesterol, as you may imagine, is very important for neuronal function. Once again, for the cortical striatal synapse where because, of course, the cholesterol, and also many metabolites and catabolites participate in many different function in the brain. This story is so strong that there are two therapeutical strategies actually going on, one in my lab and one in a French lab where we are trying to restore cholesterol biosynthesis and cholesterol level in HD. This is to tell you that this cortical striatal synapse with the other cellular elements are, I think, critical in HD. What is the first conclusion? Mutant huntingtin toxicity causes nuclear cytoplasmic and mitochondrial pathology that affects neurons and astrocytes.
It acts in a cell-autonomous and non-cell autonomous manner. Toxicity can be reversed by turning off mutant huntingtin. In a disease-modifying therapy, we have to cure not only the striatum but also cortex, the cortical neuron. Second part of the talk, what is the evidence that wild-type huntingtin is important? We have also been working on that. We are cell biologists, and when I started on HD, I started with the idea of really learning what is the normal function of wild-type huntingtin. I want to review this data, okay? What is the evidence that wild-type huntingtin is important? I would say there are two type of evidence. One evidence I call nature's evidence, which is evolution of the gene, genetics, and some human studies. Then the second one is the experimental evidence, mainly in mice.
Okay, what is nature's evidence in favor of wild-type huntingtin being so important? Well, this is a protein. This is the huntingtin protein, and you see up here the polyQ, okay, that we want to address. This is a human protein. It is a 340 kilodalton protein, 67 exons. Now, if we turn our eyes to 1 billion year ago, this is how the protein is in Dictyostelium discoideum. There is no Q. Actually, Dicty is the first specie to carry the huntingtin gene, and Dicty is the first pluricellular organism that has appeared on Earth. Before Dicty, you have yeast. Yeast has no huntingtin. Dicty has huntingtin, but with no Q, with no CAG repeats. This is why we are used to say that when the gene was born, it was born innocent with no CAG repeats.
Then, of course, we were curious to know what happens exactly from Dicty to human because we know that the CAG is in our gene. To make a very long story short, and of course, this is something the lab is really fascinated by this story, and we are doing a lot of work. To make a long story short, this is a story of the gene, okay? The gene was born with no CAG repeats, and then you have the two branches of evolution. You look at the protostomes, the insects. The gene is there, but there are no CAGs, so you cannot find a single specie with two CAG repeats.
Instead, if you look at the deuterostome branch, so to our branch, we discover that the sea urchin is the first specie to carry a CAG repetition in the gene, and we found two CAG repeats. By the way, the CAG repeats in sea urchin is found exactly in the same position where the CAG repeat is in our gene. The sea urchin, of course, is a fantastic specie also because it is the first specie to carry a very primitive ring of a nervous system. Dicty has no CAG repeats and no nervous system. Okay? Sea urchin has a very primitive nervous system, and it has two CAG repeats.
If you look at the next step at species with a progressively more evolved nervous system, you see that not only the CAG has been maintained throughout evolution, but the CAG repeat has increased in length. Okay? As you go from zebrafish to mice, to other species. The other amazing thing, and actually this is the protein version of the story, and in genetics, this kind of repeat that is elongated in more evolved species are called dynamic mutation. We know that dynamic mutation represents critical point in evolution. Basically, a dynamic mutation is, I would say, is an heritable element associated with the phenotype, whose expression is a function of the number of copies of the mutation.
The suggestion here is that not only huntingtin per se is being conserved during evolution, but that during evolution, this CAG repeat track has elongated probably for a good reason. Probably, to finely tune some function of huntingtin that we believe have to do with the nervous system formation and function and activity. To prove that, we did an experiment in vitro. Now we have more ongoing. We are stem cell biology. Actually, we use stem cells to address questions related to normal and mutant huntingtin. Starting from stem cells in vitro, we can create these beautiful structures that look like a neural tube, like a cross-section of a neural tube. We call them neural rosettes. Here you have the lumen. These are the neural cysts. This structure really mimic aspects of brain formation and maturation.
We can use this structure and this assays to address questions related to huntingtin and CAG function, but also related to any huntingtin modification. Here is the experiment that we did. We took this cell culture system, and basically we remove the normal huntingtin, and you will see later on in the presentation that in the absence of huntingtin, you cannot form these rosettes. Okay? These rosettes form only if you have the two copies of the wild type huntingtin gene, suggesting that huntingtin is important for neural rosette formation. The experiment testing this evolutionary paradigm is we then express in the minus cells, huntingtin, heterologous huntingtin from different species. Okay? From Dicty, from sea urchin, from Ciona, from zebrafish, and so on and so forth. Here is the result.
The result shows that in the normal condition, when you have the two copies of wild type huntingtin, you have plenty of rosettes, and they're really beautiful. If you remove wild type huntingtin, black column, basically the rosettes are gone. You have the cells, they don't talk to each other. I will come back to this in a second. The cool thing is that if in these minus cells, you express huntingtin from Dicty, from Drosophila or Dicty or Strongylocentrotus, which is the sea urchin, Ciona and amphioxus, and so on and so forth, you see that as you go in, heterologous huntingtin that carry more CAG repeats. Basically, you are restoring rosette formation. This is, I think, the first experiment suggesting that the CAG repeat in huntingtin is not neutral, but is functionally relevant for the activity of the protein.
On the other hand, it's nice to see that if we do another type of experiment, instead of looking at the neural rosette, we look at the apoptosis. We know that once again in the plus cells, so the normal wild type cells, there is a certain degree of cell death if we challenge a cell. If we remove huntingtin, so the black column is in the absence of huntingtin, there is more cell death. What happens if we express in these cells, once again, the same heterologous huntingtin as done here? Nothing happens. Suggesting that the CAG elongation probably instructs some neural function, imposes some neural function to the protein, and there is no influence on other activity like the anti-apoptotic activity of huntingtin. This was from the evolutionary side. I think the message is quite relevant.
I would say, this is another piece of data from Michael Hayden, in which now he looks at the human allele, the healthy allele, okay? Basically what this graph shows is that the healthy alleles show an abnormal distribution. There is one in 17 individuals, so probably three of us in this room, carry an intermediate allele. This is a normal range, again, within the normal range, but very high. Between 27 and 35. One in 17 is a lot. As if evolution keeps pushing toward probably, I don't know, adding more and more CAG repeats in our gene because probably it is functionally important.
If we would do an MRI scan of all of us in this room, each of us with a different CAG repeat in the gene, by the way, of course, it is polymorphic, this is another question, why is it polymorphic in human? Why the CAG in our gene isn't just a fixed number, 17? Mice have 7. Okay? We look-To many mouse strain, not only from Charles River but also wild mice. They have a fixed number of CGG, so why are we polymorphic?
If we would do an MRI scan of our brain in this room, we would discover that those of us that have more CGG repeats in the normal range also have more gray matter in the globus pallidus, okay, which is involved in Huntington's disease, suggesting that maybe there is some functional correlation also in human, between the more CGG repeats in the normal range and some parameters related to brain function. I don't know whether more gray matter means more intelligence, there are groups that are working on that. I will skip this slide because the key message of this second piece of data is that while I would say that for a billion years, nature has not eliminated huntingtin but implemented its function by lengthening its CGG repeat. I think we don't want to reduce wild type huntingtin as well.
What is the experimental evidence? Experimental evidence means what happens if you increase wild type huntingtin level on a normal background? What happens if you reduce wild type huntingtin level? What happens if you do this modification of wild type huntingtin level on an HD background? I will review this data. While we know that wild type huntingtin is important during development and also depending on the dose, the dose of wild type huntingtin is very important, and Frédéric Saudou, I think, will talk more about this. I want to focus on whether there is evidence that wild type huntingtin continues to be required throughout life to support neuronal function. I would like to review what happens if we reduce wild type huntingtin in an HD background. I think I have five piece of data that I want to show you.
This is a very old, but I would say quite a nice experiment. These are YAC18 mice. Okay? These are mice that overexpress wild-type huntingtin. The mice have the two normal alleles and then on top of that, there is human huntingtin. This group, what they've done, they have induced an ischemic lesion by middle cerebral artery occlusion. As you see here, the mice, the YAC18 mice overexpressing wild-type huntingtin show a reduction of lesion volume, suggesting that maybe wild-type huntingtin is neuroprotective, at least in this assay. There is a second assay that was done by another group, they used, again, the YAC18 mice. First, they've done this experiment in vitro, they prepared neuronal primary cultures, striatal cultures, in black here, these are the YAC18 primary cultures which were exposed to the excitotoxic, this toxin, NMDA.
Basically, they were able to show that these YAC18 neurons in vitro were resistant to cell death. Okay? Again, neuroprotection. They did this other experiment in vivo, again, the YAC18 mice, which were injected with quinolinic acid, which is a lesion model for Huntington's disease. As you see, the YAC18 show a reduction in lesion volume and also an increase in neuronal number. This effect is huntingtin dosage-dependent because they were able to show in other version of the YAC18 mice that depending on the dose, the more huntingtin you have, the more protection you have in your mice. This was the second piece of data. There is a third piece of data which is very interesting. More recent paper from Ioannis Dragatsis.
Basically, here they conditionally inactivate the wild-type huntingtin gene in this Cre-estrogen receptor transgenic line. They use tamoxifen to induce the conditional removal of wild-type huntingtin at three, six, and nine months. In this case, this is the experiment deals with the long-term global, okay? It is not brain-specific. They are removing huntingtin from the entire organism. They were able to show very strong data about, so you remove huntingtin and there is a reduced longevity. Not only that, there are motor and behavioral deficits which are quite strong. In addition, they were able to show that there is brain atrophy in the absence of huntingtin, calcification, and iron depletion. Removing huntingtin in the adult brain causes all these problems.
The other important message about this paper is that regardless of the time of huntingtin elimination, okay, if you eliminate at three, six, or nine months, basically you go through the same step and all mice develop all these abnormalities. I want to show you, so all of this was the removal of huntingtin or what happens if you change huntingtin level in the adult mice on a normal background. Now two pieces of data that I want to review with you, showing that basically now here we are on an HD background. We remove wild type huntingtin. This was published some years ago. If you cross, again, the YAC128 mice, so these are the HD mice. They have a very clear phenotype.
If you cross them with the huntingtin knockout mice to generate mice that have no endogenous mouse huntingtin, you reduce the level of endogenous wild type huntingtin, basically, you have a worsening of some phenotypes. The opposite is also true. If you overexpress wild type huntingtin on an HD background, basically you have an improvement of, for example, striatal neuropathology. I would say that there is evidence that in the adult brain, wild type huntingtin continues to be necessary and is neuroprotective, while mutant huntingtin is unable to support, for example, BDNF production. I will come to the BDNF in a second. This is my final part. I would say yes, wild type huntingtin is important. Can we demonstrate that Huntington's disease is possibly also due to the loss of wild type huntingtin function?
If the answer is yes, we have to be careful at not exacerbating this pathology. What is the evidence? I will show you three data. Again, this was an older study, and here these were cells in vitro. We were showing that if we overexpress wild type huntingtin, so these are the overexpressing cells, the cells become resistant to any toxic stimuli. Okay. This was one of the first data that we had, that led us to the idea that huntingtin could be neuroprotective. Instead, if you overexpress mutant huntingtin, there is increased cell death, so wild type huntingtin is anti-apoptotic, while in the same assay, mutant huntingtin increases apoptosis. If you look at the pathways, they really go the other way around. The absence of huntingtin, or in the presence of wild type or mutant, really suggesting a loss of function.
Probably a stronger evidence of that comes from the BDNF story, and Frédéric will also add more. The BDNF, brain-derived neurotrophic factor, is very important for the survival and the function of the striatal neurons. The BDNF is not produced within striatum. 90% of the BDNF that you find in striatum comes from cortex. Okay? The BDNF is made here, and then it travels to the striatal neurons through the cortical striatal afferents. Well, we discover that if you look at cells overexpressing wild type huntingtin, there is more BDNF. Instead, mutant huntingtin is unable to support BDNF production. This is a knock-in series of cell lines with two copies of wild type, one copy or zero copy of wild type. As you go from two to one to zero of wild type huntingtin, you see that BDNF production is progressively reduced. These are mice. Okay?
These are HD mice. Once again, you take the cortex from the YAC18 mice that overexpress the wild-type huntingtin, you have a lot of BDNF. Instead, you take the cortex from the mutant, you see that you have much less BDNF. The idea is that, yes, wild-type huntingtin in cortex stimulates the production of BDNF. One further demonstration of that comes from this experiment in which we measure BDNF, not only protein but also messenger RNA because we discover that the function of wild-type huntingtin on BDNF is through facilitation, is through increased transcription. Basically, we took a cortex from these conditional knockout mice. These are mice in which huntingtin has been removed in cortex.
As you go from the normal mice to the heterozygous conditional knockout or homozygous conditional knockout, and you look at BDNF messenger RNA, you see that there is less transcription in the absence of huntingtin. We actually know since the BDNF is composed of different exons which are transcribed in a stimulus and time-dependent manner because they have different promoters. We found that the target of wild type huntingtin on the BDNF gene is BDNF exon 2. The story is, I would say, quite strong. Frédéric Saudou lab has added to the wild type huntingtin function on the BDNF gene transcription, the evidence that wild type huntingtin also stimulates the transport of BDNF from cortex to striatum. This is just a list of several papers that show this data.
Just some recent data show that also in neurons, human neurons from stem cells, either iPS or human ES lines carrying different CAG repeats, you see that as you have the mutation, there is less BDNF. In this graph, you also show that not only the mutation reduces BDNF gene transcription, but you have the same phenotype once again in the wild-type huntingtin knockout cells. The BDNF story has continued over the years and to show that you can rescue HD phenotype by crossing, for example, mice with overexpressing BDNF or another beautiful data from another group shows that if you remove BDNF from cortex, if you do a conditional deletion of BDNF in cortex, these are normal mice. Of course, you have phenotypes and clinical symptoms in mice that mimic what you see in HD mice.
You look at the striatum, and you look at the gene expression in striatum of these mice in which you have removed the BDNF, and basically, the gene expression patterns overlap with what you see in HD mice or human HD brain, really saying that the BDNF and cortex is really important. The story is even bigger because not only BDNF is regulated by the wild type, but we found a mechanism that regulates through wild-type huntingtin, that regulates the BDNF and also many other neuronal genes. Wild-type huntingtin stimulates the transcription of the BDNF gene, the transport of the protein, but wild-type huntingtin also stimulates the transcription of many other neuronal genes and mutant huntingtin is unable to do so. To close, I would like to show you the last piece of evidence of a loss of wild-type huntingtin function in Huntington's disease.
I mentioned this paper, okay? The beautiful rosette that we get from just regular stem cells and the fact that in the absence of huntingtin, basically, you had the cells in vitro, but they are unable to talk to each other. We discover that they don't form the rosette, in the absence of wild-type huntingtin because in the absence of wild-type huntingtin, you have no rosette because there is an hyperactivity of this important metalloprotease, ADAM10. ADAM10 is up in the absence of huntingtin, and when ADAM10 is up, it cleaves several targets, downstream targets, including N-cadherin, which is a cell adhesion molecule. Basically, if you cleave N-cadherin, the cell-cell contact are gone, and this is why you have no rosette. Basically, the function in this assay of wild-type huntingtin is to prevent ADAM10 proteolytic activity. Okay? This was cells in vitro.
Is this relevant for HD? We look in vivo and this is, again, wild-type huntingtin. Again, these are the conditional cortical knockout mice. You remove huntingtin from cortex. You remember, this beautiful cortical striatal synapse, we found the same phenotype. In the absence of wild-type huntingtin in cortex, now we are in mice, there is increased ADAM10 and proteolytic cleavage. I didn't tell you that, of course, the story is important because ADAM10 is critically involved exactly at the level of to regulate the functionality of this excitatory cortical striatal synapse. Therefore, ADAM10 activity is under wild-type huntingtin function. You remove wild type ADAM10 is hyperactive, N-cadherin is cleaved, at the level of this synapse, you have really a lot of problems.
The very final piece of data, this is what happens in the absence of huntingtin. We look at the HD brain, we found that the HD brain phenocopies what we see in the absence of wild-type huntingtin. Once again, in the HD brain, two mouse models, human postmortem material, we always find hyperactive ADAM10, cleavage of N-cadherin, and the data were so relevant that we were able to rescue the ADAM10 hyperactivity either genetically, and we rescue the phenotype in the HD mice either genetically or pharmacologically with a compound that inhibits ADAM10. To close, I would say that in addition to being neuroprotective, there are definitely phenotypes in Huntington disease which are due to loss of wild-type huntingtin function.
My conclusion is that we definitely need to preserve wild-type huntingtin function in the HD brain, especially at the level of the cortical striatal synapse. These are the several people in my lab, and I want to thank you for your attention.
Good afternoon, everyone. I will actually follow up on Elena. Actually, my lab has been working for many years on the function and trying to understand how the function and more specifically, the dysfunction of huntingtin protein could actually participate to disease pathogenic mechanism. I will briefly the outline of the talk. I will come back to a few points, kind of key facts about Huntington's disease and huntingtin protein, and then I will give you two example of key cellular functions that I think are important to describe the disease, and then I will move to the concluding remarks. Basically what I would like to stress out is that, as Elena said, is that we have a gene with an expanded CAG that become abnormally expanded in a disease situation.
Basically, one of the mechanism by which mutant huntingtin could lead to a death of the neurons is the gain of new toxic function for the protein. As you know, and as Elena said, the disease is dominant, which means one allele is wild type and one allele is mutated with the abnormal CAG expansion. What we have at the level of the protein, we have two proteins, one that is wild type, one that is mutated, and basically they are all expressed at the same level. This will lead to the disease because the mutant form is actually dominant.
In agreement with the gain of a new toxic function for the protein is the fact that the huntingtin knockout is embryonic lethal, which is very different from what happened in the patient because as we said several times, Elena mentioned that, most of the patients developed the disease around the age of 40 years, 40 to 50 years. Which is very different from a complete loss of the protein function, which leads to actually embryonic lethality at 7.5 of development, so really like mice develop around the age of 18 days. Now, in addition to this gain of a new toxic function, we have also the possibility, there are several arguments, genetic arguments, or functional arguments pointing out the fact that loss of normal function or alteration of the normal function could actually participate to the disease mechanism.
The first one is that if you remove huntingtin protein just after birth, actually the mice actually are going to develop progressively neurodegeneration. Indicating that if you lose the function of the protein, you will actually develop neurodegeneration. The second thing that points out the fact that the level of the wild type is very important, is the fact that mice that have only one copy of the wild type and not the second copy of the wild type, so only 50% of the wild type protein. Actually, those mice show cognitive defects and neurodegeneration. One thing that is very important is that now if you compare to the mutant situation, when you have the HTT gene, actually, you have already only one 50% of the wild type because the other 50% is mutant. Okay?
Now, there are two other arguments I would like to point out to complement what Elena said, is that there are some evidence in human, actually, patients that the level of wild type and mutant could regulate disease progression. The second thing I would like to mention also is that we and other labs have shown that the huntingtin has several central function in the body. The first one is the work from where they identify a SNP called 13-10. Basically, this is a G2A mutation. Basically, when the SNP is as the A, actually there is a decreased expression of the huntingtin protein. What is very interesting is that, in this case, if the A is on the HD allele, actually the HD gene, HD protein is less expressed than the wild type protein.
Here you have less HD compared to wild type, and then the age at onset HD allele, actually, they develop the disease later. In contrast, if now you look at the population where the A SNP is on the wild type haplotype, actually, you see that they develop the disease earlier. Basically, when you have a higher amount of HD protein, the mutant protein compared to the wild type protein, then the disease starts earlier. I think it's a good indication that actually subtle changes in the level of the wild type and the mutant can actually modify the disease progression. Now let's move to the second part, which is trying to understand more about the function of the protein. This is huntingtin protein. As Elena said, this is a very large protein.
In fact, this protein, what I would like to stress out on my view is the fact that this protein contains this region that's called heat repeats or has its antiparallel alpha helices, which are very important for protein-protein interactions. There are several of these domains, these heat domains. There are at least 7 domains which are going to make intramolecular interactions. Some part of the protein can interact with other part of the protein. In addition to that, actually, this huntingtin protein binds to many different other proteins. In fact, we identify, or we and others have identified more than 400 different protein interacting with huntingtin.
You can imagine that actually you have this single protein here that in fact is able to interact with many different interactors, forming many different protein complexes that are going to have several different function in the cells. Actually, Elena pointed out already several of these functions. This is also illustrated here by cryo-electron microscopy showing you here that at least huntingtin, by interacting with different proteins, forming different complex, can adopt several conformations. There were more like 100 different conformations that could be formed by huntingtin. Let's go now from these several functions. I'm not going to describe the 400 different interactors or nor the different functions that huntingtin could have. Actually, I would like to point out one single complex that huntingtin is interacting with, and that is the huntingtin and dynein, dynactin, and kinesin complex. This complex actually is extremely important.
It's called the molecular motors. Those molecular motors, that's basically motors that are able to move along a structure, in particular, along the microtubule, which are the cytoskeleton of the neuron or the cell. One of the function that is shared by this dynein, huntingtin, dynein, and actin complex is actually the capacity of this complex to transport vesicle along microtubule, to transport protein complex, for example, to the base of the cilia or actually control cell division by controlling actually the way the cell is dividing and making that cell is going to differentiate or divide. I'm not going to describe again. I will focus just on two of these functions that are elicited by huntingtin controlling this complex. As I told you, huntingtin is interacting with the dynein-dynactin complex, and in fact, here it plays a facilitating role.
huntingtin acts as like a turbo for vesicular transport, so moving the vesicle along the microtubule. If you have huntingtin, you go very fast. If you remove huntingtin, you decrease the speed of the vesicle. Now, if you add the mutation, the mutation that caused the disease actually leads to a change of conformation and detaches the motors from the microtubule. It's like the train is getting off the rails, basically. The result of that is that the transport is slowing down as well. Okay? You have a reduction in the transport of this vesicle. What's the impact of these changes? Now we have to go back to this cortico-striatal projection Elena mentioned. Elena mentioned that huntingtin has a role in the transcription of BDNF.
Here, what we showed is that in addition to that, huntingtin is transporting this vesicle that contains BDNF and provide BDNF to the cortico-striatal synapse. Again, Elena told you about the importance of this synapse. This is extremely important because striatal neurons are unable to produce BDNF. They strictly depend on the BDNF that is provided by the cortico-striatal projecting neurons. Basically what happen is here we have in a disease less transport of BDNF, then less activation of the TrkB receptor that signal within the striatal neurons to provide survival. They activate specific pathway, ERK, that provide survival of the striatal neurons. Basically what happen here, you have a dysregulation of this BDNF TrkB signaling that in turn will lead to the degeneration of the cortical neurons.
To address more specifically this circuit, because it's very difficult to address that in vivo, we were very much interested to reconstitute this cortico-striatal circuit that is observed in brain on a chip. Actually, we used these microfluidic chips that are as big as this. Basically in which we can actually reproduce, put cortical neurons in one of the chamber and the striatal neurons on the other chambers, and they are separated by three by three micrometer channels, if you want to have a look. Basically, they will form synapses there. Exactly reconstituting the circuits that Elena mentioned and that I just mentioned previously. Using this, using neurons from HD mice, we could show that actually the alteration of all the different events, from transport of BDNF to the synapse formation to the postsynaptic events occurring in the striatal neurons.
Basically, this is an example. If we go specifically in the cortical neurons, now we want to look at what happen at the trafficking. Here what we do is that we express using lentiviruses BDNF fused to mCherry, so that the vesicles are fluorescent. The BDNF, the brain-derived neurotrophic factor, is fluorescent. We use a confocal, a spinning confocal video microscopy to follow up the trafficking of the vesicle inside the axon. Each channel is 3 by 3 micron, and basically inside you have an axon, sometime branches, and inside, the little dots you see moving are corresponding to BDNF vesicle that are transporting along the microtubules. You can see them going from the cell body to the synapse, some of them go back to the synapse. We can quantify this movement of the vesicles.
Basically here you can see this is a kymograph from wild type and the HD, which is a distance of a time representation. All these little tracks there correspond to the movement of single vesicle inside an axon. We can quantify. You can clearly see that there is much less trafficking in the mutant situation compared to the wild type. If we go more into the details, we see that the velocity, the speed of the vesicle, is reduced, both for the one that goes from the cell body to the synapse and the one also that go back. If we look at the number of vesicles, we see much less vesicle moving in both directions.
Now if we do a calculation, which basically calculates the flow of the vesicle going from the cell body to the synapse, we can see that very early on, actually, there is a decrease in the flow of BDNF reaching the synapse. Basically, in disease situation, we have this alteration of BDNF and less BDNF reaching the synapse. One thing that is very cool with these little chips, actually, is that you can play with the Because they are physically isolated, you can put different neurons with different genetic status into this circuit. Here, what we compare first was the wild type circuit, cortex and striatum wild type compared to HD circuit.
Now what we can do is that we can put cortical neurons that are wild-type, that are connecting to HD striatum neurons, or conversely, we do cortical HD network connecting to wild-type neurons. Basically what we found, to make it brief, is that we found that when the cortical neurons are from HD genotype, they are able to induce the dysfunction of the striatal neurons, even though they are from wild-type genotype. Conversely, if the cortical neurons are from wild-type, they can make striatal neurons working correctly. Indicating that the presynaptic compartment actually determine the synaptic integrity of the network. I will come back to that point, but this means actually that the cortex has to be considered for therapies, and I'll come back to that point later.
That's why we came back to the trafficking defect that we observe in disease when we look at the BDNF trafficking. We use neural stem cells from human embryonic stem cells. You can compare here the trafficking of BDNF here, the white graph, the white bar graph corresponding to the trafficking of the BDNF vesicle in the anterograde direction or retrograde. These are two independent cortical progenitors from HD patients, where you can see the reduction in the trafficking of BDNF. Now we selected a line that is called VUB05, that is actually heterozygote for a SNP at exon 50. This was done in collaboration with Anselme Perrier and Nicole Déglon, and where they could see that the wild type allele has actually the SNP C, and while the mutant allele has the SNP T.
Now if you design SH that is specific for the 50T, you can selectively silence the mutant allele. Conversely, you can use SH50c that silence the wild type allele. Then if you go back now to your cells, looking at BDNF trafficking, this is again the mutant situation. Now, if we silence the mutant allele, you can see that you increase the trafficking, both in the anterograde and the retrograde direction. While if you silence the mutant allele, you're unable to change the trafficking. Really, making kind of a proof of concept that basically if you silence specifically the mutant allele, you can alleviate, I would say, the toxic effect or the dominant negative effect of the mutant over the wild type allele.
As a conclusion of the first part, I would like to emphasize the fact that defects in axonal transports are a potentially clearly component of the HD pathogenesis, that we can modify the wild type and the level of the wild type or the mutant, and this has a strong effect on the trafficking. This trafficking is important to maintain the cortico-striatal circuitry. As I mentioned a bit earlier, is that we know that the striatum is affected, and Elena mentioned that. We know also that the cortex has to be considered for therapies. Now I would like to move to the second step, to the second part of the talk, which is the other example I would like to share with you about the potential role of huntingtin, just because it modulates the dynein and actin complex, which is ciliogenesis.
In fact, every cells in our body have a cilia, which is kind of an antenna. Some cells are multiciliated, and I will come back to that point. Most of the cells in our body has a one single cilia, which is important for sensing the environment, nutrients, and everything. There are specific proteins that are transported to the base of the cilia. One of the proteins called PCM-1 or pericentriolar material 1, and this PCM is actually transported by huntingtin and the molecular motors. To be brief, actually, what we could show is that if we remove huntingtin in a given cell, we lose the capacity of huntingtin to bring these PCM proteins to the base of the cilia, and this leads to the disappearance of the cilia. No cilia are formed into these cells.
In contrast, what is interesting is that if now we have the mutant huntingtin in these cells, basically what happens is that the protein is transported but unable to go back in the other direction. Make this kind of balance, bringing material and removing some materials. Basically, there is accumulation of the mutant huntingtin there, and this make longer cilia. You would think more is better, maybe, but not sure. We check that. The first thing is that if we look at absence of huntingtin leads to the loss of cilia in the brain here, in the ventricle of the brain. You know that we have ventricles where there is cerebral spinal fluid that is circulated inside our ventricles, inside our brain. You can see that if you have no cilia, basically, you have mice that develop hydrocephalus.
Now, what happen in disease? In disease, this is the contrary. Actually, we have longer cilia and we have accumulation of PCM, accumulation of these cilia there. This is shown here on the ventricle of the brain, where you see these tufts. Those cells are actually multiciliated. They are several. It's kind of like tulips, like a bouquet of tulips. I don't know if you say, yeah, I guess. They are moving like this, and they make the flow circulating in the brain. I will come back to that point. You can see here that in disease, actually, they are longer. This is also true in humans. This is a section from Huntington's disease patient individuals there. To come back, what's the role of those cilia? In fact, inside our brain we have these ventricles.
Here, this is mice, but basically there are several of these cilia tufts here that are very important to make the CSF circulating in the brain that come from the back of the brain and circulate inside there and is renewed. In fact, the CSF is renewed five to six times a day and it's very important for clearing catabolites in the brain, but also to make newborn neurons to migrate and then innervate the olfactory bulb. What we found is that this is a movie of the particles that are moving inside the brain of a section here of a wild-type mice. You can see that basically there is a very homogeneous movement of the particle inside the brain. Which reflects the movement of the CSF in the brain.
If now we move back to the mutant condition, you can see that this is much less organized with some vesicles going into that direction and some other ones going there. If we go to the next slide, what you can see is time projection. You can see here, this is very constant. Here, this is a camograph showing you that all the particles are moving exactly with the same kinetics, while in the mutant situation, this is completely random directionality with very inorganized movement of the particles. Indicating that the CSF flow is altered in disease situation. This is my last two slides.
Just to conclude on this part, I hope I convinced you that for this other function, there is another function of huntingtin in ciliogenesis, and that has important physiological consequences by changing the circulation of the CSF in the brain and with clear consequences for brain homeostasis. Basically, this function is altered in HD situation. I would like to conclude now, and I hope I convinced you that we have here one protein that is quite complex with many interactors, many function, many physiological function in the cells, in the organism. Actually all these so far, all the functions that have been described for huntingtin, for wild-type huntingtin, has been found altered in disease.
We believe this is very important to understand the wild-type function because this gives clues about the HD pathogenesis, sorry, and identify new therapeutic targets and also one of the main consequences, and we already discussed that with Elena, that restoring or preserving wild-type HD function is essential. I would like to raise this question. Actually, I think one mechanism by which the mutant huntingtin is acting is probably by acting as a dominant negative on the wild-type function of the protein. I'd like to thank the people involved in this work. I would like also to mention collaboration with the lab of Sandrine Humbert, who's also been very much working on the function of the wild-type protein and assessing also other function that I didn't have the time to discuss with you today. I thank you very much for your attention.
Thank you very much, Fred. We have heard a wonderful talk about the importance of wild-type huntingtins, then taking away the mutant huntingtins. What we are now going to talk about is our program, our continuing to develop our allele-specific program for knocking down the mutant huntingtins with another SNP. We currently have three SNPs. We are targeting three SNPs. We have included SNP3. We do have SNP1 and 2 in the clinical trials with PRECISION-HD. This is the first time you'll be hearing about SNP3 for taking away the mutant huntingtins. We are using allele-specific RNA-based mediated silencing of mutant huntingtins transcript. Here is the population genetics. With the SNP1 and 2, each one of them can cover 50% of the population, and the SNP3 can cover approximately 40% of the huntingtin population.
The patients can have two or more of these SNPs associated with the CAG repeats. By taking all the three SNPs together, we believe that we can cover about 80% of the huntingtin population. By committing ourselves to developing another SNP, we are actually expanding on our Huntington's disease program, and we are also committed to serving the patient community. Because we are doing allele-specific targeting, we do have a slightly complex problem. We need to make sure that we can actually address each one of the questions for allele-specific targeting. One is trying to understand about the potency of the molecule in certain cell types. Secondly, measuring the allele selectivity both by biochemical assays and also looking out the patient-derived neurons.
Third, in the case of SNP3, we do have a back HD model where we can actually do the target engagement and durability in vivo. Let's actually start talking about each one of these steps carefully. Here what we are looking at is a homozygous isogenic neuron. This particular cell line only has SNP3. It does not have the wild-type allele, so you cannot measure allele selectivity in these cell lines. However, we can actually compare the potency of our allele selective molecule, which is generated by PRISM, against a clinical stage, Roche's pan silencer RG6042. In this particular cell line, the two molecules or the two potential leads for SNP3 are more potent than Roche's clinical compound 6042. This is an analog that we made internally. In the next slide, I want to show the allele-specific nature of the molecule.
This is a biochemical assay, and these two are two different compounds for the same SNP. What we have in this assay is the siRNA, which one is wild type and mutant, and using our oligos, what we can silence preferentially is a mutant copy for both molecules, leaving the wild type pretty much intact. Clearly demonstrating that we have allele selectivity in this biochemical assay. Next, we also developed a cell line. In this case, this is a patient-derived neuron, which has got both wild type and mutant, which is shown in this PBS group. What we have here clearly is the two compounds, which are labeled as SNP3 compound one and two, and we are measuring allele-specific target knockdown at two different concentrations here.
With our SNP3 compounds, compound one and two, what we can see is a clear allele-specific differentiation knockdown of the mutant while leaving the wild type pretty much untouched. The same thing is true even at a higher concentration, we do see exactly a knockdown of the mutant while leaving the wild type intact. On the other hand, if you take a look at the pan silencing RG6042, you can see a very nice potent reduction of both wild type and mutant at the low concentration. At the highest concentration, we see exactly the same effect. Clearly showing that the molecule is potent in silencing both mutant and wild type, whereas our molecules maintain allele selectivity. Now, we can look at the potency of these molecules in a BACHD transgenic mice.
There are caveats in this model, and I want to go through this model a little bit more carefully. What we have is, this model is homozygous for SNP3. That means you cannot measure allele selectivity in this model. However, we can measure potency and duration of activity in this model. Secondly, the transgene here, this is a human mutant Huntington's transgene, and the transgene is overexpressed. We have multiple copies of the transgene. However, not all copies have got the SNP3 in it. That means we cannot silence any of the mutant Huntingtons that does not have SNP3 in this transcript. This is a high bar. This mouse model is a high bar for us, and we only have this model for us to assess our potency of the molecule.
First, we did oligo distribution study, and we injected all the three molecules, the pan silencing RG6042, along with our two SNP3 compounds. What we clearly see is distribution in both cortex and striatum. We see our oligos, both our first SNP compounds show oligos, and the pan compound also shows distribution to cortex and striatum. However, we see more of our molecule present in cortex and striatum compared to the pan targeting molecule from Roche. The next is looking at the target knockdown of SNP3 in this model. What you can see here is all the three molecules were injected at the same quantity, and the one that is written in black is pan RG6042. That molecule plus a SNP3 molecule, within two and four weeks time point, give exactly the same knockdown in cortex and in striatum.
However, when you go into 8 weeks time point, the pan targeting molecule loses its potency and the transcript starts coming back. At 12 weeks time point, the pan targeting molecule again loses its potency. With both our molecules at the 8-week time point, we still maintain substantial amount of reduction of mutant huntingtin, clearly showing that the SNP targeting molecules are more potent than the pan silencing, and the duration also exists up to 12 weeks in this model. Again, keep in mind, all the huntingtin transcript, it's a highly expressed model, and all the transcripts do not have SNP3. There are a few transcripts that do not have the transcript. We are at a disadvantage of getting a lower percentage and knockdown of the huntingtin. This model is a difficult model.
Nevertheless, it clearly shows that our molecule is more potent than the pan-targeting molecule, and the duration of activity is also much higher than the pan-targeting molecules. In summary, what we have shown here with our SNP-targeting approach is that in a homozygous cell line, our SNP-targeting molecule actually is more potent than the pan-targeting analog, Roche's analog. We also see very good allele selectivity with our SNP-targeting molecule. We do see duration of activity in back transgenic mice, and we also see target knockdown up to 12 each. We get very good distribution in both striatum and cortex. We have a plan of doing our IND-enabling tox studies by 2020. With this, I would like to introduce Mike Byrne, who's the director of biology, and he's in charge of the ophthalmology program, and he's going to walk us through USH2A.
All right, everyone. Thank you. Understand we're tight for time. I'm excited to be here as I really want to give you some insight on what we've been doing in the ophthalmology space. Ophthalmology is an interesting space for oligonucleotides because of the ability for them to be delivered by intravitreal injection. An intravitreal injection, as you see here, is an injection where the needle penetrates essentially the eye, and you deliver into the back of the eye. You inject into this space called the vitreous. The advantage of this is that allows you to have broad distribution across all the layers of the retina. Think about this as a bowl. The retina essentially is the bottom of the bowl. You're injecting into the milk of your cereal, and it spreads across the entire bottom of your bowl.
This is a little bit different than gene therapy approaches, which have to be delivered by subretinal injection. Subretinal injection is essentially, if we use E here in this example, it's formed by a bleb. They have to make a retinal detachment and inject into that detachment. The distribution of an AAV vector is limited to where that bleb is created. The oligos that we inject intravitreally spread across the entire retina. This is really important for certain inherited retinal diseases that focus on peripheral rods. Rods and cones are the photoreceptors that we want to target. When you do an IVT injection, you get to distribute to all of those. What I'm going to walk you through is some MALAT1 data. Greg gave you an example of some early data, one-week time point.
I'm going to show you some more data there, and I'll talk about our lead program in USH2A. What you're looking at here is an extension of what Greg showed very early today. He showed a potency shift at one week after an intravitreal injection in mouse. I want you to pay attention to the x-axis. This is weeks. Now you're looking at three, five, and nine months after a single intravitreal injection. At nine months, using our PRISM oligos, we maintained 50% knockdown in MALAT1 after a single injection. Very importantly to understand is that the back of the eye, the cells of the retina are terminally differentiated. Once you get that delivery, the potency of this stereopure molecule extends for a long time. Here you're looking at the PK, so minimal exposure is detected at nine months. Here we're into the nanogram per gram of tissue.
The minimal exposure is fine because we still maintain nine-month duration. That's a mouse eye. Mouse eye is about half the size of your pinky fingernail. We want to go to a large animal and understand what happens when you do that. Here you're looking at non-human primate data. In this case, we've taken a non-human primate. We've done a single intravitreal injection, again, to look at MALAT1 knockdown, and we've dissected out the retina itself. Just the retina, nothing else. When we do that, you'll see that greater 90% knockdown is maintained for four months. This is with one of our compounds from PRISM. We wanted to understand, well, what does that mean?
Our oligo has an RNA species to it, we can actually look using an assay called ViewRNA, which is typically used to look in tissues for the RNA of a gene to identify a gene. We can specifically target that to our exact compound sequence. What you're looking at here on the right is a cross-section of the retina, I've highlighted the photoreceptors. The blue are the cell nuclei. The compound is in red. The drug is delivered across the retina, at four months it's still detectable in all layers. This matches up well with the qPCR data showing that we have knocked out of the target. What's important to point out is that both of those situations, nine months in the mouse, four months in non-human primate, that lends itself for one to two, maybe three doses per year.
Anti-VEGF therapies, depending on the indication, are dosed eight to nine times a year. We're talking about one to two times a year dosing here. Using that information on durability, understanding what we've done in the Huntington space and our DMD space, we started to look through inherited retinal diseases through the lens of our knowledge base. We identified Usher syndrome type 2, the progressive vision loss disorder. It's autosomal recessive. There is no cure. There's no approved disease-modifying therapy. It's a humongous gene. It's 72 exons. These kids present first with hearing loss, and then they have progressive, through adolescence and through about the fourth or fifth decade of life, they have vision loss. The problem in Usher is that the Usher protein identified here in the rod and cone cells is used to stabilize this last region here, this outer segment.
When the protein's not there, the outer segment's not intact, the rods and cones die. The rods usually die first. The rods are in the periphery. This is why an oligo-based IVT injection is critical. We want to hit those cells in the periphery as soon as we can. A subretinal injection will not allow you to do that. The approach we take is focus on the most common mutation, which is this, deletion of a G in Exon 13. When that's deleted, there's a frame shift, and a new stop codon is created. Our goal is to come in and skip out Exon 13, thereby creating a functional protein. Here's the data we've generated so far. In the upper left, you're looking at, say, a 10,000-foot view. Do we skip exons?
Here, compared to a reference compound, a stereorandom reference identified in the patent listed here, we have a 4-fold shift in potency for exon skipping. 10,000-foot view, we've skipped. We want to understand what did we skip? Let's zoom in, 5,000-foot view. We took this skip product and looked at exons 8 to 17 just to see the size. This is a gel shift assay. What you see is in the compound 1 treated samples, we're missing about 640 nucleotides. That's the size of exon 13. All right. What about street level? What we did is we took these transcripts and did a procedure called RNA-Seq.
You fragment all of your RNA, you sequence all of those RNAs, and bioinformatically put it back together to look for the reads, look for the location, so you can join up exon 1, 2, 3, et cetera. If we just focus in on exon 12, 13, 14, and 15, you'll see in the PBS treated, that RNA-Seq allowed us to see very clearly exon 12 connects to 13 to 14 to 15. What's highlighted in the box here is a histogram of all the reads around exon 13. In the PBS treated, there are plenty of reads at exon 13. There's lots of them there. When we treat with our compound, you'll see the number of reads at exon 13 has gone down. The amount of exon 13 present transcripts has decreased. Importantly, what you'll notice is a new junction.
Exon 12 now extends Exon 14. At the ground level, at the transcript level, we've confirmed that we've skipped the exact product that we're interested in. We wanted to understand, well, how does this translate into large eye? We took on an exercise where we've generated an ex vivo system. We get cadaver eyes, human cadaver eyes, non-human primate eyes. We dissect out the retina, we section the retina, and plate it in a 96-well dish. Everything you've heard today from Chandra is genetic delivery. We plate cells, we put our oligo on it, no Transfectory agent, nothing. We did the same thing here. We treated sections of the retina into the wells, applied oligo genetically, and in non-human primate, we have dose-dependent skipping up to 75% skipping of the proper exon. We did the same experiment in human.
At the same concentrations we achieved similar skipping. We have upwards of 75% skipping of exon 13 in the human retina. Today, we know we skip a non-human primate, and we know our oligo skips in human. We have ongoing in vivo studies, we're excited to provide updates on that as we continue. Utilizing what we learned in our allele selective approach for Huntington that you heard about today, another area of interest for us is autosomal dominant retinitis pigmentosa. In this case, ADRP is essentially a group of virgin eye diseases around retinitis pigmentosa. One of the key mutations specific to cause ADRP, about 10% of all cases, is a P23H mutation. In this case, what happens is it's a dominant negative effect, a gain of function. What happens is that the mutant rhodopsin is generated.
It gums up the works and prevents the wild type rhodopsin from getting to its site of action. Its site of action, again, is here in the photoreceptor. We want to get rhodopsin to these outer segments. There's about 1,800 addressable patients in the U.S. Allele selective approach would be very beneficial here. Again, on the left, what you're looking at is reporter assay system where there's a stereorandom molecule that's been identified from the following patent, it's a reference compound. You'll see that it is not allele selective. In a dose-dependent way, it knocks down black, the mutant, but also the wild type allele in blue. When we make a stereopure molecule, our own version of this, we have dose-dependent knockdown of the mutant, but we do not touch the wild type allele at all. The in vivo work around ADRP is ongoing.
We have collaborations in place for us to do the evaluation of transgenic human P23H pig model. Our stereopure compound is selective here. In summary, our compounds we know are potent and durable. They are lasting up to nine months. We have productive exon skipping with our U.S. program. We have confirmed that skipping at the transcript level. We have ongoing in vivo efforts to identify the skipping in non-human primate, large animal. We have discovery work underway for a second program in ADRP. Our IND-enabling studies we expect to begin next year.
Thanks, Mike. Well, I know this was an intensive day, and we took you across from crystal structures and chemistry through new areas of biology. What we think is important is the consistency with which we've seen that transition. What we know now is with the right rational drug design, we can alter stability, durability, potency, characterize safety, and ultimately see programs in biology transition to the clinic. On behalf of everyone at Wave, we're excited about our first three clinical programs. First two data readouts, as we said in suvodirsen and in PRECISION-HD2 will be this year, followed by PRECISION-HD1. Subsequently, as we laid out, our progress with Exon 53, with C9orf72 for ALS/FTD, with our SNP3 program in HD and USH2A. What we continue to do is move things forward.
A big reason we can do this as well is we do have a robust collaboration, as we said earlier, with our partners at Takeda, which actually funds a lot of our discovery research capability. We're working with them on new programs. There's a lot of shared learnings, and so we're able to keep a robust discovery organization moving as we move our clinical programs forward. It's an exciting year in 2019. It's not over yet. We're looking forward to 2020. With that, I'll pause, thank our panelists, and open up the floor to any questions. Yeah.
Informative presentation. If you could just comment a little bit beyond what you're thinking here, since I think you clearly identified that there's a role with wild type huntingtin protein and the health of neurons, and that possible deleterious effect of knocking out any amounts of wild type. Did you have any evidence of what can drive that decline, whether it be some marker of activated microglia, whether it's CD68, NRF changes, or anything like that would be maybe a more causative link between the presence of wild type huntingtin protein and neuronal health?
You are asking what is in between the wild type and the neuronal loss? Is this the question? Okay. I'm not sure I understand the experiment. Basically, we don't have data for the moment, but we are trying to modulate the level of wild type in an HD background. Say, pushing the wild type protein toward degradation to see what happens. I'm not sure I understand the question. Yeah.
My feeling, if I may.
Yeah.
Actually, I had the impression that what you were asking is that what happen if you remove huntingtin in term of cellular response? Is that your question?
Microglial activation.
Okay. Well, we don't know about microglial activation.
Yeah.
One thing we could see is that given the role of what's happening in the expression of BDNF and the trafficking of BDNF, what you could imagine is that because you have a decreased neurotrophic support in cells, then you slightly increase the apoptotic level in the cells, which would raise, for example, apoptosis and then death of the cells. That one possible mechanism.
There is one piece of evidence, actually, in the Dietrich et al. 2017 paper, the one from Ioannis Dragatsis that I mentioned, in which they saw some gliosis. Not really microglia. I don't think anyone has really looked at microglia. Yeah, if you remove huntingtin, in addition to the calcification and iron deposition problem, you also have glial activation, also gliosis.
Oh, we have another question.
I don't know whether this has implication for the inflammatory response. I don't think this has been looked at very carefully.
Yeah
consider.
That's great. Thank you.
Hi, my name is Suji Jeong from Jefferies. Thanks for taking my question. I have one question for Paul and another question for the KOL. The first question for Paul is for the Huntington's disease data for the end of this year. Are you planning to show the wild type huntingtin protein level? The question for the KOL is, what is the ratio between the wild type and mutant huntingtin proteins in the brain? If they're not one-to-one ratio, if the company's presenting the mutant huntingtin protein over total huntingtin protein level, how would you interpret that in terms of whether the wild type protein level is touched or not? Thank you.
These two are nicely integrated, which is great. We will be doing the assessment, as we said, the total HTT ratio to mutant to assess wild type function. That's because you can't quantitate in patients the wild type, so you have to do this ratio. It's a dynamic ratio, one of the things that we're doing in advance of presenting data, we'll be doing a walkthrough of the assay and how the assay's implemented. To your point, a lot of it is going to be following, a lot of work that's being done on actually following that ratio over a variety of data sets. It is a ratio, it is not an absolute quantification.
Yeah.
Okay. What is known, we're discussing that at lunch actually, is the fact that the data that are available in mouse and actually also in human postmortem brain, is that basically it's not exactly a one-to-one ratio between the wild type and the mutant. There's probably more of wild type than mutant, so probably cells and neurons, they are already probably trying to turn off or silence the mutant, the expression of the mutant compared to the wild type. There's always a little bit less of mutant compared to the wild type. That's what we observe in the brain.
To know at the messenger RNA level. You're talking about the protein level.
The protein. I'm talking about the protein.
Yeah.
Which will be how we'll be assessing this protein quantification.
Joon Lee from SunTrust. Is there any logic to how some stereoisomers are better taken up versus others, or is this just a random screening process?
No. It does not appear to be random. Actually, an interesting data set we had is when, and it was in a previous version of the poster, so we're happy to always reference that again, is when we took the inverse, we took suvodirsen and we looked at suvodirsen's ability to get into the nucleus of the cell. We saw this preferential trafficking. When we did the inverse chemistry, so the exact same molecule as suvodirsen, but the chiral inverse of that, we saw the drug outside. A lot of what we're able to do, what you can't do with a mixture, is answer the questions like you're asking, which is the work we're doing now, which is actually doing RNA binding protein experiments to look at preferential trafficking in a proactive way.
I think what Chandra mentioned, that based on our capability, when we look at our potential medicines as they're coming forward pre-clinically, we're by nature, because the potency differential, it's free uptake, essentially what's being driven to the top, the molecules that are getting advanced, are those molecules that are preferentially getting to the nucleus. Greg is always, I have this etched in my head, which is we often talk about nucleic acids as a drug in problem. This kind of constant piece around delivery. I think one of the things you saw today, which was compelling, as much as getting the drug into the nucleus where you see potency, is the retention of the drug. Drug out, that the drug is actually staying in the compartment where it needs to be and can therefore be catalytically efficient, which gives the durability.
To Mike's data, where we're seeing 9 months knockdown in the mouse eye, 4 months, and that's only because that's where the experiment was ended. The catalytic efficiency and turnover is as much a feature as what we're getting in terms of the potency and retention. What we can do now, because we can take those differences where you have 2 isomers, same drug, right, that's inverse of one another, and look at why is one preferentially trafficking, the other's not, and that's the work we're doing to kind of deconvolute from, again, from a scientific and research perspective, what is it about trafficking that drives the biological changes we're seeing?
That rule that you're trying to understand how these are being transported, is that something that's applicable across different cell types or tissues?
Yeah. One, we're seeing it consistently across. That's what was so compelling in our minds on the discovery side around the MALAT1 data, where we could look at, here's a drug where we could take same time point, same concentration, minimize as much variability as possible, and by looking at multiple tissues, be able to see the impact of a dose, duration, and potency over time. We can actually see that pretty consistently across those cell types and tissues. Now, there are cell types and tissues where that doesn't go. As we think about where are the opportunities from a therapeutic perspective, that's our driver. Our driver is, the limitation is if a drug is not distributing proactively to a specific cell type, then we don't necessarily need to pursue targets that are within that cell type.
A key driver for us on the therapeutic side is productive distribution to the variety of cell types that we do see.
Yeah. Sorry. No. Go. You have the mic.
Thank you. Thank you for the talk, very informative. I'm Benett from Mizuho and team. I just have a couple of questions from the HTT program. Have you discussed any new micronucleus data with the FDA that you can comment? Has enrollment in the multi-dose cohort started? If so, is enrollment going in line with what you expected?
Yeah. I'll answer your first question first. As it relates to the micronucleus assay, as we put that out in the Q, it does not impact. It's a routine test done as part of development, and we'll provide routine updates as we normally would in the queue. You can follow the next queue for those updates. No change to the current clinical trial. As it relates to the multi-dose portion, I think your question, if it's around the multi-dose study, the multi-dose study is fully enrolled, I mean, sorry, fully enrolled outside the U.S. and global, that's delivering our data by the end of this year. There isn't an opportunity now that the study PRECISION-HD is fully enrolled for U.S. patients to participate. Our goal, as we said earlier, is we're working with the agency.
Again, we'll follow and update obviously our queue. We'll provide updates. Obviously, it's important to us to make sure by the time we finish the phase I, II study to be able to start our pivotal trial, to be able to have that up and running. We'll definitely are working collaboratively with the agency as our peers and other companies did to be able to get the pivotal study up and running.
We have a question for the two KOLs, and Yaron Werber from Cowen. The question, and it's in two parts, is really about the clinical symptomatology of knocking down the wild type huntingtin protein, and how do you differentiate it from the mutant huntingtin? As you think about Roche and Ionis, let's say, are knocking down the wild type. With long-term follow-up, what would you expect to see clinically from knocking down the wild type protein?
Oh, yeah, I have the microphone. I would say, well, if you think of the BDNF, but the BDNF story, we always go back to that, but it is really a very robust story in the field. Well, if you knock down BDNF, I would expect ventricular atrophy, striatal cell loss, and I would expect.
Cognitive deficits.
Cognitive deficits. I would expect that some of this might happen, especially if you hit the cortex with your compound, with your molecule. A loss of BDNF will be already a huge story and a huge problem. This would be, I think, one. It's not only BDNF. If you think of the ADAM10 story and the synapse, it really seems that this cortical striatal synapse is heavily dependent on the full function of the wild type, no? Because we have the wild type impacting on the transcription of the BDNF, the transport, on the function of the postsynaptic density. I didn't mention many other data from other groups. They look at wild type huntingtin function at the level of the postsynaptic density. Clearly, wild type huntingtin interacts with many protein at the postsynapse.
You are really screwing up, I think, the synapse.
Clinically, if we think about what we had, and we know we had a lot of conversations with a number of you over time or there was always this question of, what do you think the safety signal would be in knocking out wild type? I think we've always said that we have to reframe the discussion of a safety signal or non-safety signal to what does disease progression look like? What changes on the clinical studies? We're going to be obviously seeing that natural history study data readout later this year. I think we have The New England Journal paper supplemental section to look towards to where there's trends. I think a lot of it is just following that. I think it's incumbent upon us to run the experiment where we are doing the human experiment of allele-specific silencing, and we can look at that differential outcome.
Yeah, maybe just to follow on. In that last study, there was, I think Paul just referenced The New England Journal of Medicine paper. There was ventricular enlargement, and there was also NfL elevations. Any thoughts as to were those due to the underlying chemistry of the ASO? Were these due to They wouldn't be due to disease progression, or were they due to a wild type knockdown?
I don't know.
So can you-
These are the easy questions.
Can you rephrase the question?
Yeah.
I think separating it, and we'll take the ASO discussion, and we'll take the biology discussion. I think the question was really, could the knockdown of the wild type protein explain some of the changes in terms of neurofilament light increase, ventricular enlargement over time? What happens when you knock it down? We'll take the second question.
Can I say, about the neurofilament, I think it is too early to say. Of course, and this is something that raised some alarm, the level of the neurofilament. I would really use this statement. It is too early to say, but it is something that has to be monitored.
On the ASO side, I think it's important just to reflect on the Ionis studies. We have examples with SOD1, we have examples with SPINRAZA, where diseases that one does treat with an oligo in the central nervous system, as you're treating, you see neurofilament light trend in the direction of positive outcomes, right? You have the benefit within SMN protein getting produced correlating with an improvement in neurofilament light. The SOD1 study that was presented more recently, you have SOD1 levels decreasing, and you have neurofilament light decreasing as well. I think we do have good examples from peers in the space using oligos in the central nervous system and seeing that trending.
On the inflammation side, it was one of the questions that we went through because obviously Mike Panzara, our Chief Medical Officer, was involved in a number of the development programs in the multiple sclerosis space. There, where you see some changes on inflammation and improvement, you also saw parallel decrease in neurofilament light. I think as we looked at some of these changes both on the cortical side as well as biomarker, I think it's incumbent upon everybody, and I think Elena's right, to just watch the study progress. We're going to have natural history studies. We're going to be able to compare that to the outcomes data on an open label extension study and clinically be able to do that assessment. Right now, we're just focused on generating our data. We'll have their data clinically and be able to look at that comparatively.
Maybe it's a question for Chandra Vargeese, if you don't mind, to put you on the spot quickly on stereopurity. Maybe give us a sense, the interplay between the base and the sugar as to how you actually fix stereopurity. How do you build an assay or system to actually help you understand what's the best specificity? What do you think confirms the better nuclear localization? Because it's not charged, right? How does stereopurity lead to penetration? Thank you.
Yeah, I can actually answer the first question first. We have actually learned over time how to implement stereochemistry in these sequences. Some of the things that we're seeing are something that we didn't expect. When we evaluate stereochemistry, we always make some knowledge-based learnings to implement stereochemistry. That's why when you make them completely stereopure or when you control stereochemistry at every position, you can actually figure out these subtle changes very easily. This is not obvious if you don't make a stereopure molecule, or if you don't control stereochemistry at every position, you cannot determine this, because there is a lot of interplay. They said there's a lot of interplay between 2' modifications and the chirality of these molecules.
They really talk to each other, depending on the sequence, again, there is a sequence component to it. If you don't make them stereopure, you cannot understand the differences between the sequence chemistry and stereochemistry. This is where our breadth of our knowledge is from the platform. We have a knowledge base to actually mitigate some of these issues.
Just to follow on to that, then you can answer the second question, is creating training data sets. We're doing two things. Chandra alluded to that in terms of the probability getting better. Some of it is, what do you design rationally, and then what do you trust to just make to continue to build data sets that you can then put into algorithms? One of the things that we've been keen to do on the data science side, so that we don't take, as I always say, the reason we're here is other people were very dismissive of chirality to begin with and said better just not to focus on it because it's too complex.
I think in leaning into it and understanding it and developing it, to Greg's point, there was a principle of rules that were established as we looked at the SAR around the crystal structures. One of the things as we go forward are, as in we have the luxury now that we can print plates and plates of chirally controlled drugs, is just to continue to build training data sets that you wouldn't also inherently design. The idea that what could also be built as empiric data set versus a rational data set, which then ultimately, as we're building our machine learning algorithms, give us a vast amount of data that we can continue to crunch.
As Chandra mentioned, one of the things that's really interesting from a computational standpoint is there is an interplay between sequence 2' modifications and chirality, and so the bigger and bigger data sets that we've been building help us to get faster and faster predictive probability. I don't know if you want to answer question number 2.
The question number 2 is about the intracellular setting. When we started these, we also had some emphasis. What we actually, again, this is to the point that free uptake can actually predict some of this stuff. If you use transfection agents, all these stereoisomers go in at the same. It could be some of these receptor-mediated uptake, which is possible. What we also learned is that it's independent of sequence. Some of the stereochemistries that take us to the nucleus is completely independent of sequence. You can transfer these signatures to other sequences. For example, exon-skipping oligos. We see this over and over again, that using a similar chemistry and backbone chemistry, we are able to actually achieve exactly the same thing that we achieved with exon 51. That's really what we have seen.
Again, making the wrong isomer or the opposite isomer does not take us to that. We are working with several academic collaborators to really understand what is the principle behind this trafficking. Anyways, intracellular proteins, they're all chiral. There are several aspects. There are several RNA binding proteins that can actually traffic these molecules.
It's interesting when you look to Chandra's point, you look at the MALAT1 examples today, the Exon 51, Exon 53, those are two different sequences between Exon 51 and 53. Then even some of our early posters in HD, SNPs 1 and 2, where we're looking at non-human primate distribution showing nuclear co-localization. So those are four different examples across four different sequences that have still the same common traits of nuclear uptake. So to that point, a lot of the work is, so what's driving it, and really, again, that's the algorithmic piece of what we're working on is, are there pieces that would accelerate the rational design that's preferentially going to be taken up?
Suji Jang from Jefferies again. For the mutant to total huntingtin level ratio, do you expect that ratio to go down over time? Another question is, do you plan to have another research date to go over the assay?
Yeah. Without dictating a research day, but there's definitely continued updates on the assay in advance of data. We're not going to have data come out into a vacuum. I think that you heard two things that are important. One, that the differential skews to the mutant down over the wild type. I think our piece there is selective silencing of the mutant over the wild type, I think, continues to push that in a favorable direction. I think secondly, there is a kinetics piece to it, a lot of what we want to make sure is that you all are doing homework in advance of the assay, but to put those two pieces out in terms of the kinetics interpretability and that measurement of the retention of the wild type.
There are examples of looking at ratios in terms of as we think about things like cardiovascular disease and other areas where being able to understand that, see that trend over time, and know that you're having a positive impact becomes important. That'll be a component of how we use the total assay to assess wild type coverage.
Hi. [inaudible] from SVB Leerink. My question is about the C9orf72 program. I believe you mentioned earlier that you were able to directly target a disease allele without the need for it to target a SNP. I was just wondering, could you expand about how you were able to achieve this and if such an approach would be possible for another expansion-mediated disease such as Huntington's?
Yeah. What's fascinating about the C9 differential in terms of knocking down the repeat-containing transcript is we identified sequence space that would get us that distinguishing characteristic. What's also unique is, remember, by having a stereopure drug, is Greg put the crystal structure out, and there's that elegant pair of scissors that you kind of see on the outside of it. It's this recognition that when you have a single drug that engages with the enzymatic system, you can know predictably where that cleavage site's going to occur. When you map that cleavage site on top of the sequence space where you want to have that happen, in some cases, it's a SNP because that's the distinguishing feature between the two alleles.
In the case of C9, where we would just want to take down the repeat-containing transcript and let the protein get coded, we could do that by identifying the sequence space, and again, the work we're doing on the algorithm, where we identify the sequence, how we design it, where the cut site happens, and allow that to happen in that position. As you know, we've done more work in improving the potency and durability side, hence the lead that we've selected. We're excited to see this now in the durability studies and, as Chandra mentioned during her talk, see that transition next year. Okay. Well, one, I appreciate everybody, again, taking the time to visit us here in person. For those of you who stayed on to the webcast, thank you. We appreciate it.
As always, you know we're open for questions, and so we're happy to follow up whenever it works, but appreciate your time, and thank you to our panelists.