and 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. And 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. And 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 HGT to assess wild type. While that's all about the clinical data, today is really about research.
And research and discovery has been what's driven this company forward. It's what supported the initiation of suvodirsen and our 2 SNP programs, our Precision HD1 and Precision HD2 programs. But it's also what powers everything we do. It's what's bringing the next wave of programs forward. We'll talk about exon 53, C9 for ALS, our USH2A program in ophthalmology.
But it's also been about what's accelerated the platform. And so we're excited today to introduce what we've been doing around the world of RNA editing and our ADAR platform as well. And so 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 2 other features to it. So while today is not about HD in terms of our program, we are fortunate enough to have 2 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 chirochemistry and how we can 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. So 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.
So 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. And 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. So if you look at the difference of the now versus the different resolutions we can see in this case of different screw, 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.
And 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. So 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. And 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. So this is not a delivery problem, being able to deliver our nucleic acids across a broad range of tissues.
While genetic medicines 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? And in most of these diseases, there's one of 2 things: either the patient has this disease because they're missing a protein. So 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. So 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. And so that's how we approached DMD. It's what not just led to our program 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 a function of how do we think about proteins that shouldn't be there, so where there's a toxic protein. And how do we do that in a way where we can be very specific? So we call that allele specificity. And 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 potency, maintaining durability and maintaining that specificity, we'll show you as we 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 neuromuscular system and thinking about diseases like inherited retinal diseases. And 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 and, in addition, a silencing context. But 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 DISTANCE-fifty one study. We were 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 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 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. And so 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. And 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. So the agenda today will be followed by Doctor. Greg Verdeyn. Greg will speak about the chirality in stereochemistry.
We'll then move to Chandra Varghese. 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 then shift gears and focus on programs and biology. And so we'll talk about the biology of the healthy protein, both Doctor. Cantonello and Doctor.
Sidhu, and then move into our SNP3 program with an update on our in vitro and in vivo data in SNP3 targeting HD And then transition to ophthalmology with new work that's been done by Mike Byrne from our ophthalmology group, and then we'll close at the end. So to transition, I'd like to introduce Doctor. Verdine. Doctor. 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. Doctor. Verdine currently serves as the CEO for FOG Pharma and LifeMind Therapeutics, which was born from his own new modality scientific work.
As Irving Professor at Harvard University and Harvard Medical School, he invented staple 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 to Wave, including Veragenics, Inanta, 11 Bio, Tokai, Eliron and Gloucester Pharmaceuticals, which was acquired by Celgene. And these companies have succeeded in achieving FDA approval for 3 marketed drugs. He has served on the Board of Directors of Wave, Enanta, Warp Drive, FOG Pharma and LifeMind Therapeutics. Doctor.
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 Verdiem.
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? So you guys have to tell me where I'm at on that spectrum. But 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.
And 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. So 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. So first, I'm going to show you a molecule, which is known as carvone.
This was first isolated from the leaves of spearmint. And 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. So 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 4 Seasons scent.
It was our peppermint or our spearmint. Now, Carbone was then isolated separately from Caraway Seeds, And it has the version isolated from caraway seeds has the same structure in 2 dimensions, properties except for one property, and that is its interaction with biological molecules, specifically olfactory receptors. So this is the version now that comes from caraway seeds. And you'll notice that it doesn't smell anything like peppermint, and yet these 2 have exactly the same chemical structure. There's obviously something missing from the chemical structure that doesn't describe its full structure.
And in order to get to that last element, you have to now allow a three-dimensional world. So this is a two dimensional world. In a two dimensional world, they're identical. But in a three-dimensional world, these actually have specific shapes and the way that atoms in the molecule project from the structure. And it's very much like hands.
You have 2 hands. They look the same. They're actually mirror images of each other. They're not the same because you can't superimpose them. So you can do the superimposition test, and you can see these are mirror images.
Mirror image so molecules also have this mirror image property that's called chirality. And the way chemists like to describe this is using these kinds of wedges and dashes. And all this wedge means is that this group of atoms is pointed out at you and that group of that atom is pointed backwards. And they can occur in 2 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 1, 2, 3, 4 different things attached to it.
So whenever you have this kind of a core atom that turns out to have tetrahedral geometry with 4 different substituents on it, that gives rise to chirality. And because the olfactory receptors in your nasal cavity are chiral, They interact differently with chiral objects, that is they interact differently with carbone. One smell receptor interacts with our carvone. R stands for rectus or right, S stands for sinister or left. Left, this is sinister.
This one is rectus. So they're handed. So what does this have to do with drugs? Well, all 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 interact and all drugs that interact with proteins have chirality is important for their interaction.
And the most notorious example of this in the drug world is thalidomide, where thalidomide, one of the forms of thalidomide, is shown here. This molecule was prescribed for the treatment of morning sickness in pregnant women. And 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 1, 2, 3, 4 different substituents on it, so it has handedness.
And one of the so it exists as a mixture of what are called stereo isomers. These are mixtures of the 2 mirror images. And 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.
And 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 sterile isomers. And it was the one area in which it was not true that one need to get needed to get the stereochemical purity in order to take forward a molecule into human clinical testing. And it seemed to me that this field shouldn't be any different than the rest of drug discovery. Drugs should be stereochemically pure, period. Now what's going on in Oligonucleotide Therapeutics?
So I'm just showing you the fundamental building block here on the left of Oligonucleotide Therapeutics. It has 2 elements of nucleic acid with a phosphate connecting them, 2 nucleosides connected by a phosphate. And these are strung together in a repeating sequence where B changes from 1 unit to the other unit. Now I want to draw your attention to this phosphate. If you look at this phosphate, it turns out these 2 oxygen atoms are equivalent.
So there are only 3 substituents around this phosphate. It's not chiral. What the industry learned early on so these are 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.
And the introduction of that sulfur atom breaks the symmetry in the molecule such that you now have 1, 2, 3, 4 different substituents. And you all know what happens when you put 4 different around a chiral, a tetrahedral atom, you get stereochemistry. So every time you drop this sulfur atom into this molecule, it provides 2 different stereo isomers at each phosphate that is substituted. So 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 2 sterile isomers.
So there's an RP isomer and an SP isomer. P just stands for around phosphate. So this is now moving into a schematic diagram from a structural diagram where these circles just represent these nucleosides and the carrots represent the phosphate. When there are 2 of them, this means there's a mixture. So when you make a nucleic acid drug, an antisense oligonucleotide, these units are strung together with phosphate connecting them in between.
And 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. So therefore, it generates a mixture of 2 sterile isomers each time you do the substitution. So 2 times 2 times 2 times 2 times 2 times 2 times 2 times 2 times 2. You see how you get up to a horrendous mixture. So if there are 15 of them, that's almost 33,000 stereoisomers.
So 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 1,000. So let me just what I want to do is just provide a visual so you can see that these sulfur atoms are different. So this is showing an oligonucleotide 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. So if you were an object and you were coming up to grab this DNA, you'd notice they look different. That the pattern of sulfur atoms looks different from there.
So that creates the basis of what you just perceived with Spearman versus Caraway, that biology perceives these isomers as being different. Now 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 Bose drug in terms of its stereochemical program. So 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, then this is over 500,000 individual isomers. As you go up to larger N, ADAR, these are adenosine deaminase targeting oligonucleotides higher. Now you're getting up to $30,000,000,000 and then CRISPRs are on the orders of 1,000,000,000,000 of individual.
Every single molecule is different. They're not the same. And biology perceives them as being different. Now let me just show you so why don't we just separate them? The simplest thing would be to say take the 30,000 and just separate them.
But of course, you'd imagine that you couldn't possibly separate 1 out of 30,000 components. First of all, you'd be throwing away 29,000 whatever and then keeping the 1, but it's physically impossible to separate them. They're physically impossible. Just imagine 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.
So what this shows is that clinically approved drug, nipomercen, and this is kind of a sleuthing technique where you try to separate things. And if they separate, you see individual peaks that correspond to individual components. And here you can see there's just there's no feature at all. There's no basis for separating these molecules physically. So separation is out just as with paint.
But what you could do as with paint is make the individual colors individually. So 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 that 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 Lewising technology, if you now have an individual composition of stereoisomers, this is from Mipomercen, a clinically approved molecule, you see that one of them SLEUIS is here, another one SLEUIS is later, another one SLEUIS is later. And these are 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 Mipomercen 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 and so on and so on. Every aspect, stereochemistry affects every aspect of the molecule. So over here, these are trackable, they're optimizable and so on and so on.
So that's 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. So 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 2. And once these 2 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.
Arnaeus H then does a cut on the red strand. It literally chops it into 2 pieces.
Now
in the past, where these are first of all, where these are not substituted with sulfur, they're unstable, as I mentioned. But RNA SeH couldn't recognize any feature of this molecule. Everything kind of looks the same. So it would slide along, and it would not make a kind of defined complex. That's the same thing with stereo random oligonucleotides.
If you have 500,000 different isomers, RNase H interacts differently with every single one of those. So you have this ensemble effect where you can't really understand what's going on at the level of each individual drug because there's so many, there's no way to deconvolve them. On the so 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 at 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. So 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. But 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 2 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 sisyl phosphate. And there is a precise interaction of each phosphate. You can see this phosphate has the sulfur pointed out at you, that's phosphorothioate.
So that's one stereochemistry. This one has the sulfur pointed back. That's the other stereochemistry, S. And 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 guide. I'm coming down to the end of my talk, so 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. So what that means is when you're dosing a patient, every time it has an R configured an R configuration in it, that's a site of metabolic liability. And so if you have a lot of them, those drugs are arriving themselves dead on arrival. What WAVE can now do is judiciously put only the right number of R's into an otherwise S oligonucleotide, again, allowing optimization.
So this just shows the differences in rates of cleavage of a stereo pure oligonucleotide versus a stereo random. Interestingly, even though there are fewer cut sites, the enzyme is faster. The enzyme prefers a stereo chemically pure. And what's not shown here is that the overall extent of cleavage is also dramatically increased. So 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. And now this is looking at intraocular injection. The thing is all of when you have all these R configured stereo phosphorothyoids, they create instability in the molecule. So when you concentrate S configuration, you increase the stability of the molecule biologically in a very rational way. So what you can see is by intraocular injection, there's a roughly fiftyfold this is now looking 1 week after intraocular injection, There's a 50 fold increase in potency.
So there are other properties that are not shown here, for example, like TLR activation and cellular penetration, etcetera, etcetera. But, stereochemistry is a complete game changer for being able to rationally discover drugs. So I want to I'm going to stop there at the I'll be happy to take questions at the very end and to introduce to the podium Chandra Varghese, 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. So 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? So 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 steviochemistry. And this actually helps me to introduce our platform. And like Greg pointed out, chirality is important in biology. So really what we have uncovered at Wave is the use of chirality or use of stereochemistry to design our oligonucleotides.
So what we call our platform is Prism. And I want to explain, walk you through some of the concepts as Prisms and then review some of the advantages of using this across multiple modalities. So 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. So, Prism, here, what we are talking about is when we start designing or when we start thinking about generating oligonucleotides, we look at target indications and then we start generating the target that we want to transcript that we want to attack and then put the product profile in place. So 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 either of any of these modalities to cause to make a therapeutic. So then we come to the middle point where we call our platform as Prism, which takes into account not the 2 dimensional interactions of Watson Crick base pair, it actually incorporates the stereo chemistry so that we now have, to Greg's point, a 3 dimensional interactions. So we take into consideration the sequence and the chemistry, which you all know in oligonucleotides, there are 2 prime modifications. And so we could use all different chemistries. And that's not enough, and we have to introduce the chirality, that is the stereochemistry.
So with all these three things in place, now we have a platform that actually can construct specific stereo pure molecules. So you can control stereo chemistry at every position wherever we incorporate a heteroatom in the phosphorus. Phospholothiodes is an example, but there are also other heteroatoms like Marcellino's, which is exactly the same. It's a mixture of several diastereomers. So for us, we can control.
We can use our technology to control each of these parameters. So at Wave, the scientists are now developing stereo pure molecules using this technology and trying to understand about different parameters of the drug very specifically. So in order to design, we also need to have assays in place to characterize each one of these isomers. So what we have introduced internally is developing cell lines that can take up the oligos genetically. So one thing that is forgotten in the industry is that we use transduction agents or electroporations to introduce oligos into the cells because all of the nucleotides are heavily, negatively charged.
So they do not enter the cells, but we force them to enter the cells using transfection agents and electroporations. So what we decided to do is to mimic closely and to actually eliminate all the false positives, we need to have oligonucleotides that enter the cells by itself by gymnotically or free uptake just like small molecules. So that means we need to develop in vitro systems that can take it up readily. So 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. And so we are developing therapeutics, which has got all the potentials, which contains the potency, stability, immunogenicity and also having very good safety profile.
So we can characterize the molecule precisely with these individual constructs. So 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 are available. In some cases, we may not find animal models, especially in rare diseases. So in that cases, we can actually move on to looking at the PK properties in animals and then use that to drive our clinical potency calculations. So we could actually do a validated in animal model and move on to generating clinical candidates.
So 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 stereo chemistry, and we have developed the knowledge base. So using this knowledge base, now we can create sequences that are also useful for finding additional target sequences. So 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. So in order to do that, we create oligonucleotide in stereo random formats and do a screening across the transcript purely to select the sites. So if we do that, what we identify is that our hit rate is only 10% to 15%, meaning like 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.
So this hit rate is actually fairly low. But if we incorporate all the knowledge based screening using our stereo pure format, what we observe is that the hit rates actually dramatically increases. And with the knowledge base that we have developed over time, so 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, which clearly shows that with the stereo random format, we are not able to access all different sites. And to Greg's point, we cannot optimize a stereo random molecule and we cannot get target sequences that are amenable for biological activities. So 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 have before.
So this is still advancing and we create we actually have knowledge base that we are developing and we are incorporating every day to improve our screening technologies for purely identifying target space. So 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. So in order to do that, I want to walk you through a surrogate sequence. So our targets predominantly what we are targeting resides in the nucleus, primarily pre mRNA and intronic sequences are present in the nucleus. So what we decided to do is to use a target that is present in the nucleus.
So we have been using VALAT-one as a surrogate target. This is a long non coding RNA, which resides in the nucleus and it's ubiquitously expressed. So 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, Malat-one target. So the first question that we asked is, what are the implications of using Stereopyr Malag1 oligos? So what we find here is, with a single IV injection at 25 mg per kg, which is equivalent to roughly 1.6 mg per kg, what we see is a very broad distribution of person generated malignant molecules.
We do have 2 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. And so this provides evidences for intracellular delivery into the nucleus. And secondly, the target knockdown that we see is also substantial and it persists for up to 8 weeks. And this data, this type of data is unprecedented in the case of oligonucleotides, especially when they give a systemic delivery and looking at not liver, not kidney, looking at multiple target tissues that we are getting continuous knockdown.
And 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. So clearly, what we see here is we can develop drugs that target multiple tissues. And similar point is true when we go into CNS space. So since we have a number of neurology programs, we also want to understand after an ICD injection, how are these oligos distributing.
What we clearly see here is the malignant knockdown is actually present throughout the brain in multiple regions, including cortex striatum, cerebellum, hippocampus and brainstem, everywhere. And 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. So again, to the point that our steno pure oligonucleotide generated by PRISM is capable of entering into multiple cell types, which is very important for CNS programs. So now that we have seen proof of concept using MALEK-one, we wanted to switch over to other programs, including C9orf72 for ALS and FTD.
So in this case, what you're looking at is a hexanucleotide repeat and the transcript is actually producing it's a generic cost for ALS and FTD. So in this particular case, we want to knock down the hexanucleotide repeat, which produces toxic dipeptides that causes progression of diseases. So 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, that the C9 protein is very much important for the neuronal strength. So here, I'm showing a lead compound, which we targeted specifically to knock down the repeat containing transgene.
We see substantial knockdown with a single dose of all of this. And what we also see here is that we are still maintaining the protein, the C9 LRf72 protein that is required. So it's a different kind of allele selectivity where we are only targeting a specific transcript. And we are doing currently several in vivo studies and we expect to be in clinical development by second half of twenty twenty. So 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 are also have a good tox profile. When you're making a compound with 1,000, 100 and 1000 of stereo isomers, it's impossible for us to understand about the toxicity of each one of the isomer. And so you get a whole reading, but it's not easy to characterize. So when we see tox, we wonder what is causing the toxicity or is it a class effect. But the answer is that that's not very simple and it's not true.
Here, what I wanted to show you is 2 molecules, which have got exactly the same composition, exactly the same chemical modification. It's the same sequence. And in vitro, it has the same potency in vivo, it has the same potency and it's conjugated to galact. So it gets delivered to the liver very effectively. So 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. And 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 1 isomer and the other isomer is quiet. So with just 2 isomers and with the small change, we can see drastic difference in toxicity. This is very clear because we're able to make stereo pure molecules.
However, when you do a stereo in a molecule and if that was our lead, we would not be able to tell which one caused toxicity and which one is silent. So 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. So this is the power of Wave's technology. Now 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.
And we were wondering why these oligos get in. And so we did some experiments to understand where how these oligos distribute. So on the left hand side, you can see the myoblast 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, they are all the oligonucleotides. So we counted the number of foci and plotted this against to understand about how many molecules are present in the nucleus. And you can see with the stereo random oligos, we see minimal amount of molecules in the nucleus and with stereo pair oligos, we see substantial copies in the nucleus, which clearly might show that why our oligos get much better or why our oligos are more potent.
And we also see the same phenomena when we move into in vivo in mice. So we injected a single dose and looked at the tissues here. We are looking at muscle tissues What we see here is a very good distribution of oligos in the muscle tissues. And what you can clearly see observe is the nucleus, the myonucleus or the central nuclei is 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 stereo pure oligos have a completely different intracellular penetrating properties.
And this could be the reason why we see very good skipping efficiency with the exon 51 oligo with our sordecin molecule. It's an exon 51 targeting exon 51 and a 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. And you can see that from Paul talks, 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.
And 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 twenty twenty. So now I'm going to switch over to another new modality, which is ADR mediated RNA editing modality, which is actually a pretty exciting field. Now this is emerging to be another oligonucleotide modality that is very versatile. So besides silencing and mRNA splicing, there are other targets that we cannot target with regular ASO or skipping oligos.
So with ADAR, we could actually expand our targets profile and we can get into new targets. So 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. So this I is equivalent to a G. So 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. So some of the missense mutations and nonsense mutations are not amenable for splicing.
So you cannot use our splicing technology, but you can use ADAT to restore the protein expression for these mutations. Or on the other hand, we can also cause mutations to alter the protein function or to increase the protein expressions. So ADAR is so versatile. And unlike CRISPR, where we are editing the genome, here we are editing the RNA. So we will not cause any damage to the genomic DNA and this is not inherited.
So and it's also reversible. So this is going to be a versatile tool for us to use in the future. So now we also approach targeting ADAR in the same way as RNase H mechanisms. What Greg pointed out is understanding about the stereo chemical interactions. So when we look at this crystal structure of ADAR, you can see ADAR enzyme binds to the double stranded RNA at several 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. So that means we can work around ADOR using our present technology to optimize for backbone interactions and sugar modification and nuclear based modifications. So 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 ADOT editing oligos. So now we use we generated about 1,000 oligos or so in the last year to develop an FAR. So in order to do that, we need to understand about 2 prime modifications.
We incorporated that. We incorporated backbone studio chemistry and backbone modifications, and we looked at various sizes and structures and modified nucleobases. So now the team has developed an SAR so that you can generate a single, stereo peroligonucleotide to cause editing using an endogenous ADAR. So we believe that Wave's RNA editing platform is much superior than the existing technologies in a way that we have developed a fully modified, chemically modified stereo pure oligonucleotide that can be taken freely without any involvement of an AAV or a lipid nanoparticle. So these oligos, as I showed before, they enter cells very easily.
And 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. So with all this advantage, we have shown in vitro high level of RNA editing. So in this case, what you're looking at is editing an UAG site in an actin mRNA. It's an endogenous mRNA present in all the human primary cells. So what we are looking at is a GalNA conjugated ADAR editing ASOS or ADAR editing oligonucleotide and we see here in human primary hepatocytes a dose dependent increase in editing efficiency.
And 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 editing efficient RNA editing. So now we also took the same molecule and took away the galMag and put it in the gymnotic delivery in different cell types. So here again, what we see the stereo pair oligos gives you editing in hepatocytes and also in bronchial epithelial cells. We see editing. However, the same oligo, which is stereo random, 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. And we are pretty excited about the progress the team has made in RNA editing. So now I'm giving back to Paul.
Well, as you can see, we're pretty excited about the chemistry platform. And 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. So 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 500,000 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 modification 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.
And so this idea that we've built fundamentally and foundationally, that rational drug design shouldn't elude the nucleic acid space. So 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 in the array of tools, meaning single strand RNAi, antisense, splice correction and now with RNA editing in ADAR, let's 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.
And so bringing that convergence between a chemistry platform and a biology platform, where do we apply it? So 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, I mean, the MALA-one study where we could show at a human equivalent dose of 1.6 mgs per kg, durable distribution, so it was 8 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. So 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.
So 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. So 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 then 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. And so opening up and broadening those definitions is because we're getting access to more and bigger sequence space. And then we'll talk soon about our excitement about the emerging areas of ophthalmology. You a little bit of the in vivo data that's getting us to that point.
So 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, so increasing the ability and our engine across drug discovery. With that, the next transition in really thinking about the portfolio discussion is now to talk about where are we going. And so I'd like to introduce 2 amazing KOLs who've really been instrumental in the field of Huntington's disease.
We'd oftentimes hear from clinicians who are in the space, and we often get questions about, well, what about this protein? What does this protein do? Why is it important to selectively silence it? And we're very unfortunate to have, 2 KOLs who are going to talk to us today about Huntington as the protein, both the mutant and the wild type. So it's my pleasure to introduce Doctor.
Elena Cantanello. Doctor. Cantanello 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. Doctor.
Cantonello 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. And at MIT, she studied neural stem cell differentiation and its association with neurogenerative 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 Doctor. Cantonello's social merit led the President of the Italian Republic, Giorgio Nafoletiano, to appoint her as Senator for Life in August of 2013. In addition today, we're also joined by Doctor. Fredrik Sidhu. Doctor.
Sidhu he will follow Doctor. Cantonello. Doctor. Sidhu served as a hospital professor at the University of Grenoble LP and CHU Director of Grenoble Institute of Neuroscience. He's also the group leader of the team, Intercellular Dynamics and Neurodegeneration and Director of the Grenoble Center of Excellence in Neuro Generation.
He undertook his thesis at the University of Stroudsburg with Professor Rene Hen on serotonin receptors and completed his 1st postdoctoral fellowship in Strasbourg with Professor Jean Louis Mandel in Human Genetics. And then 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 Huntington protein function and dysfunction in intracellular trafficking. In 2014, he also received the Richard Lunsbury Prize for Medicine and Biology from the French and U.
S. National Academies of Science. So with that, Doctor. Contadagno.
Thanks for the opportunity. And as you see from my title, I would like to lead you through the 1,000,000,000 year long history of the healthy Aliyah. We want to know why it came to us. And I actually want to know why we carried the gene in our genome, given that it can be so dangerous. And so I will deal with this.
But 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 disease. It was cloned in 1993. And it is a big gene, 67 exons. And we know that in exon 1, we have this CAG repetition.
And we also know that the normal population has a number of CAG repeats below 35, while when the CAG gets above at 36, of course, this is a disease range. And we also know that the longest is the CAG, the earliest are the symptoms. The CAG is translated into glutamine. So the protein, whose name is huntingtin, is fairly large protein, 3 40 kilodalton protein. And again, I mean carrying this elongated polyQ in the pathological range.
As a consequence of that mutation, we have neurodegeneration in the brain. And I just want to highlight that neurodegeneration not only hits the striatal neurons, but also there is cortical atrophy and cortical degeneration. And at the end stage, of course, there is a general atrophy in the brain. So the gene, the protein is broadly expressed in all cells of the body, but 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. So in the cortical neurons, actually, that project to those striatum.
And this very large protein is also the subcellular localization is quite complex because you can find the Huntington, of course, in the cytoplasm bound to micro tubules with a role in intracellular trafficking, exocytosis and endocytosis, but it is also associated with many other organs, including the mitochondria, the endoplasmic reticulum and the lysosomes, synaptic vesicles, the Golgi complex and the nucleus. And 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 the vascular structure. Having said that, these are the three points that I want to cover with you today. So yes, so Huntington is expressed everywhere, but I would like to give you my view of, let's say, the 3, 4 key experiments, and Frederic Sadu will add a few more, really highlighting how exactly mutant antigen becomes toxic to neurons. And I would like to start with 4 pieces of data.
The very first one is this one. And this is an old study, but I would say this is a seminal paper really showing that, okay, if you block mutant huntingtin expression, so these are animals that express mutant huntingtin starting from postnatal day 0, okay? And then expression of mutant huntingtin is maintained until week 18. And then at that time, you administer doxycycline, so this is an inducible model, and you turn off mutant huntingtin, and then they've been waiting for another 16 weeks. And basically, the message is that there is a reversal of many or basically all phenotypes.
And I think this is really a key message for all of us, certainly for me, because it says that turning off mutant hunting is sufficient to reverse the disease in mice, okay? This is the first piece of data. Then the second piece of data is, well, we would like to know, I mean, how exactly mutant antigen exerts its toxicity now that we know that we can reverse toxicity. And this is a very nice piece of data coming from 2 groups and 2 papers, 2,0092010. And basically, in this paper, they are focusing on the cortical striatal synapse, okay?
The striatal neurons die in Huntington disease, but the cortex, I mean, the afferents from cortex, of course, are very important for the normal functionality of the striatum neuron and as you might guess also for the dysfunction of the striatal neurons in Huntington disease. And one first evidence of this is from this paper. And here, basically, they show that, yes, at the level of the post sign ups 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. But then, I mean, in this paper, they deal with the extra and NDA receptor because we learned that the extra synaptic and the NDA receptor exerts toxicity.
So an excitation of the activity of this extra synaptics receptor can be toxic to those striatum neurons. And the paper suggests that the balance between synaptic and extra synaptic NMDA activity is critical in determining neuronal survival in Huntington disease. And they proved that in a very nice manner by administering 2 cells in vitro or 2 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.
And by blocking this extra synaptic receptor, of course, you rescue the pro survival pathway and you reverse the disease. So but the key message is that there is something happening at the cortical striatal synapse, which is very important and we have to look at this compartment, I mean, very closely. There is a second paper that goes in the same direction by using, I mean, a completely different paradigm. This is William Young study. And here, basically, what they did, they here, the full length this is another in vivo mouse study.
And here, the full length mutant huntingtin was genetically reduced in striatum with this 3 mice recombination that targets specifically the striatum. So they were able to reduce mutant antigen only in striatum or with this other version, they were able to reduce mutant antigen only in cortex or I mean, with this final mice, they were able to reduce mutant antigen in cortex and striatum. So they were able really to dissect out, I mean, what is the role of the 2 compartments, is there a role for the cortical afferents? And the result to me, I mean, are really striking because basically they show that if you reduce mutant antigen in striatum, so you are looking at the cell autonomous effect of mutant antigen, you are able indeed, I mean, to restore some activities of the postsynaptic compartment because you are restoring in these mice when you remove mutant antigen the activity of these molecules, okay? So there is cell autonomous toxicity of mutant antigen in striatum.
But then, I mean, when they look at reduction of mutant antigen in cortex with this other mouse line, basically they were able to show that they could restore all they could restore some cortical function. But quite remarkably, I mean, removal of mutant antigen in cortex was beneficial to the striatal neurons because they were able also to restore parameters associated to the post synapse with the post synapse density. So the message is there are also noncell autonomous toxicity of mutant hunting in striatum. And in the final portion of the paper, they were able to show that in the mice in which mutant antigen was repressed both in cortex and striatum, there was basically, I mean, total recovery of several functional parameters. So the key message is, to me, I mean, if we want to cure HD, I mean, not only we have to save the striatal neurons, but we also have to work on the cortical neurons.
And then the 4th piece of data that I want to show you is we have been discussing about neuronal toxicity, right, striatal and cortical toxicity due to mutant hand tinting. But antigens is not only expressed in neurons, it is also expressed in astrocytes. So what is mutant antigens doing in astrocytes? Well, for example, here we were able to show that mutant huntingtin in astrocytes suppresses the production of cholesterol, turns down the transcription of many cholesterol or many genes implicated in the cholesterol biosynthetic pathway. Not having cholesterol or having a reduced activity of this cholesterol biosynthesis pathway is very, I mean, problematic because cholesterol the cholesterol that we have in brain is synthesized locally.
It doesn't come from periphery. So there is a local synthesis of cholesterol in the brain in the astrocytes. The astrocytes are the producer of cholesterol in the brain. And of course, this cholesterol, as you may imagine, is very important for neuronal function. And once again, I mean, for the corticosteriorital sign ups where because, of course, the cholesterol and many and also many metabolites and catabolite participates in many different function in the brain.
So and this story is so strong that there are 2 therapeutical strategies actually going on, 1 in my lab and 1 in French lab, where we are trying to restore cholesterol biosynthesis and cholesterol level in HD. This is to tell you that this corticosterietal synapse with the other cellular elements are, I think, critical in HD. So what is the first conclusion? Mutant anti toxicity causes nuclear cytoplasmic and mitochondrial pathology. It affects neurons and astrocytes.
It acts in a cellular autonomous and non cellular autonomous manner. Toxicity can be reversed by turning off mutant hunting. And in a disease modifying therapy, we have to cure not only the striatum, but also cortex, the cortical neurons. Now second part of the talk, what is the evidence that white type of antigen is important? And we have also been working on that.
We are cell biologists. And we really I mean, when I started on HD, I mean, I started with the idea of really learning what is the normal function of wild type antigens. And I want to review this data, okay? So what is the evidence that wild type anti tinctin is important? I would say there are 2 type of evidence.
One evidence I called nature's evidence, which is evolution of the gene, genetics and some human studies. And then the second one is the experimental evidence, mainly in mice. Okay. So what is nature's evidence in favor of why type hunting team being so important? Well, this is a protein.
This is the hunting team protein. And you see up here the polyQ, okay, that we want to address. And this is a human protein. It is 3 40 kilodalton protein, 67 exons. But now, I mean, if we turn our eyes to $1,000,000,000 year ago, this is how the protein is in dictiosteum disquideum, okay?
There is no queue. And actually, Dicty is the 1st species to carry the hunting gene and Dicty is the 1st pluricellular organism, right, that has appeared on Earth. Before Dicty, you had yeast. Yeast has no untinting. Dicty has untinting, but with no Q, with no CAG repeats.
And this is why I mean, I used to say that when the gene was born, it was born innocent, okay, with no CAG repeats. And then, of course, we were curious to know, I mean, what happens exactly, I mean, from Dicty to, I mean, human because we know that CAG is in our gene. And to make a very long story short, and of course, this is something the lab is really fascinated by this story and that we're doing a lot of work. To make a long story short, This is a story of the gene, okay? And the gene was born with no CAG repeats.
And then you have the 2 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 2 CAG repeats. Instead, I mean, if you look at the due to Rostom branch, so to our branch, we discover that the urchin is the 1st species to carry a CAG repetition in the gene and we found that 2 CAG repeats.
And by the way, the CAG repeat in searching is found exactly in the same position where the CAGR gene is in our gene. And the CAGR gene, of course, is a fantastic species also because it is the 1st species to carry a very primitive ring of a nervous system. So Dicty has no CAG repeats and no nervous system, okay? C. Urchin has a very primitive nervous system and it has 2 CAG repeats.
And then, I mean, if
you look at the next step, I mean, 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. And the other amazing thing and actually this is the protein version of the story. And in genetics, these kind of repeat that is elongated during I mean, in more about species are called dynamic mutation. And we know that dynamic mutation represent critical point in evolution. And basically, a dynamic mutation is, I would say, is a heritable element associated with the phenotype whose expression is a function of the number of copies of the mutation.
So the suggestion here is that not only hunting Tintersays being conserved during evolution, But that during evolution, the CAG repeat track has elongated probably for a good reason, probably, I mean, to finally tune some function of antigen that we believe have to do with the nervous system formation and function and activity. And to prove that, we did an experiment in vitro. Now we have more ongoing. And we are stem cell biology. Actually, we use stem cells to address questions related to normal and mutant hunting.
And 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. And here you have the lumens. These are also these are the neural cysts. So this structure really mimic aspects of brain formation and maturation.
And we can use, I mean, this structure and this essay to address questions related to Huntington and CAG function, but also related to any huntingtin modification. So here is the experiment that we did. We took, I mean, this cell culture system and basically, we removed the normal un tinting. And you will see later on in the presentation that in the absence of un tinting, you cannot form these rosettes, okay? So these rosettes form only if you have, I mean, the 2 copies of the wild type huntingtin gene, I mean, suggesting that huntingtin is important for cells, hunting theme, heterologous hunting theme from different species, okay?
From VICTI, from Sertin, from Ciniona, from zebrafish and so on and so forth. And here is the result. While the result shows that in the white in the normal condition, when you have the 2 copies of white typantin, you have plenty of rosettes and they're really beautiful. If you remove white type antin, black column, basically the rosettes are gone. You let the cells, but they don't talk to each other.
I will come back to this in a second. And then the cool thing is that if in this minus minus cells, you express anti TMT from DICTI, from drosophila or Dicty or strongylocenterologist, which is the urchin, sayona and the antioxin and so on and so forth, you see that as you go in, I mean, we heterologous hunting team that carry more CAGR peat. I mean, basically, you are restoring rosette formation. And this is, I think, the first experiment suggesting that the CAG repeat in hand painting is not neutral, but it's functionally relevant, I mean, for the activity of the protein. And on the other hand, I mean, it's nice to see that if we do another type of experience, instead of looking at the neural rosettes, we look at the apoptosis.
And we know that once again in the plus plus cells or 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 colonies in the absence of huntingtin, there is more cell death. What happens if we express in this cell once again the same heterologous huntingtin as done here, nothing happens, suggesting that the CAG elongation probably instructs some neural function, I mean, to impose some neural function to the protein, and there is no influence on other activity like the anti apoptotic activity of antignting. So this was from the evolutionary side. I think the message is quite relevant.
And then I will say, I mean, this is another piece of data from Michael Hayden in which now, I mean, he looks at the human allele, the healthy allele, okay? And basically, what this graph shows is that the healthy allele show an abnormal distribution. So now and there are one there is 1 in 17 individuals, so probably 3 of us in this room, carry an intermediate allele. So this is a normal range. I mean, again, I mean, within the normal range, but very high, between 2735.
As if I mean, 1 in 17 is a lot. As if I mean, evolution keeps pushing, I mean, toward probably adding more and more CAG repeats in our gene because probably it is functionally important. And if we would do an MRI scan of all of us, I mean, in this room, I mean, each of us with a different CAGR repeating the gene, by the way, of course, it is polymorphic. And this is another question, why is it polymorphic in human? Why the CAG in our gene isn't just a fixed number, 17.
Mice has 7, okay? We look to many mouse strain, I mean, not only, I mean, from Charles River, but also, I mean, wild mice. And that has fixed number of stages. So why are we polymorphic? Anyway, if we would do, I mean, an MRI scan of our brain in this room, we would discover that those of us that have more CAG repeats in the normal range also have more grain matter in the global palladium, okay, which is involved in anti intent disease, suggesting that maybe there is some functional correlation between also in human between the more CAGRPs in the normal range and some parameters related to brain function.
I don't know whether more gray matter means more intelligence. But there are groups that are working on that, and I will skip this slide because the key message of this second piece of data is that, well, I would say that for a 1000000000 years, nature has not eliminated Huntington, but implemented its function by lengthening its CAG repeat. And I think we don't want to reduce wild type antitankin as well. So what is the experimental evidence? Experimental evidence means, I mean, what are the what happens, I mean, if you increase white type huntingtin level on a normal background?
What happens if you reduce white type anti tinting level? And what happens if you do this modification of white type antitankin level on an HD background? So I will review this data. And while we know that white type of anti inflammatory is important during developments and also depending on the dose, the dose of white type of anti inflammatory is very important. And Frederic Sadu, I think, will talk more about this.
I want to focus on whether there is evidence that white type amphetamine continues to be required throughout life to support neuronal function. And I would like to review what happens if we reduce white type antenna in an HD background. So I think I have 5 pieces of data that I want to show you. First one, this is a very old, but I would say quite a nice experiment. These are YAK18 mice, okay?
These are mice that overexpress wild type huntingtin. The mouse has the 2 normal allele and then on top of that, there is human untending. And then, I mean, this group, what they've done, they have induced a ischemic lesion by middle cerebral artery occlusion. And as you see here, the mice, the YAK18 mice overexpressed in white patent painting show a reduction of lesion volume, suggesting that maybe white type unpinting is neuroprotective, at least in this essay. There is a second essay that was done by another group, and they used, again, the YAK18 mice.
First, they've done this experiment in vitro. So they prepared neuronal primary cultures, triatal cultures. And in black here, these are the YAK18 primary cultures, which were exposed to the excitotoxic and to this toxin NMDA. And basically, they were able to show that this yacatin neurons in vitro were resistant to cell death, okay, again, neuroprotection. And then they did this other experiment in vivo, again, the YAK18 mice, which were injected with kinulinic acid, which is a vision model for Huntington disease.
And as you see, the YAK18 show a reduction in vision volume and also an increase in the survival. I cannot read very well here, but it was an improvement in neuronal number. And this effect is teen dosage dependent, because they were able to show in other version of the YAK18 mice that depending on the dose, the more hunting thing you have, the more protection you have in your mice. So this was the second piece of data. There is a third piece of data which is very interesting, more recent paper from Ioannis Dragatis.
So basically, they here they conditionally inactivate the wild type anti TIN gene in this estrogen receptor transgenic line. They use tamoxifen to induce removal of white ipantintin at 3, 6 9 months. And in this case, this is the experience deals with the long term global, okay? It is not brain specific. They are removing Huntington from the entire organist.
And they were able to show, I mean, very strong data about so you remove Huntington and there is a reduced longevity. Not only that, there are motor and behavioral deficits, which are quite strong. And in addition, they were able to show that there is brain atrophy in the absence of hand tinting, calcification and ion depletion. So removing hand tinting in the adult brain causes all these problems. And the other important message about this paper is that regardless of the time of huntingtin elimination, okay, if you eliminate at 3, 6 or 9 months, basically you go through the same step and all mice develop all this abnormality.
And then I want to show you, so all of this was the removal of huntingtin in the or over what happens if you change huntingtin level in the adult mice on a normal background. Now I have 2 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 hunting, and this was published some years ago. If you reduce if you cross again the YAK 128 mice, so these are the HD mice with they have a very clear phenotypes. If you cross them with anti knock out mice to generate mice that have no endogenous mice mouse hunting, so you reduce the level of endogenous white type hunting, basically, you have a worsening of some phenotypes.
And the opposite is also true. If you overexpress white type unpinting on an HD background, basically you have an improvement of, for example, striatal neuropathology. So I would say that there is evidence that in the adult brain, white type hunting continues to be necessary and is neuroprotective, while mutant antigen is unable to support, for example, BGNF production. And I will come to the BGNF in a second. 3rd, this is my final part.
So I would say, yes, white side hunting is important, but can we demonstrate that Huntington disease is possibly also due to the loss of wild type unpinned function. And if the answer is yes, we have to be careful at not, I mean, exacerbating this pathology. So what is the evidence? I will show you 3 data. Again, I mean, this was an older study.
And here, these were cells in vitro. And we were showing that if we overexpress wild type antigens, so these are the overexpressing cells, the cells become resistant to any toxic stimuli, okay? And this was one of the first data that we had I mean, that led us to the idea that huntingtin could be neuroprotective. Instead, if you overexpress mutant hunting, there is increased cell death. So Y type antintin is anti apoptotic, while on the same assay, in the same assay, mutant untinted increases apoptosis.
And if you look at the pathways, they really go the other way around. The absence of untinting 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 that Frederick will also add more. So the BDNF brain derived neurotrophic factor is very important for the survival and the function of the striatal neurons. But the BDNF is not produced within striatum.
90% of the BDNF that you find in striatum comes from cortex, right? So the BDNF is made here and then it travels to the striatal neurons through the cortical striatal afferent. Well, we discover that if you look at cells overexpressing Y type amphetamine, there is more BDNF. Instead, mutant antigen is unable to support BDNF production. This is a knock in series of cell lines with 2 copies of wild type, 1 copy or 0 copy of wild type.
And as you go from 2 to 1 to 0 of wild type anti until you see that BDNF production is progressively reduced. These are mice, okay? These are HD mice. And once again, you take the cortex from the YAK18 mice that overexpressed the Y type antigens and you have a lot of BDNF. Instead, you take the cortex from the mutant and you see that you have much less BDNF.
And this so the idea is that, yes, white type pungentine in cortex stimulates the production of BDNF. And one further demonstration of that comes from this experiment in which we measure BDNF, not only protein, but also messenger RNA, because we discovered that the function of Y type anti anti anti on BDNF is through facilitation, is through increased transcription. And basically, we took a cortex from this conditional knockout mice. So these are mice in which Huntington has been removed in cortex. And 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, I mean, there is less transcription in the absence of antigen.
And 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 white ziplampline on the BDNF gene is BDNF exon 2. And the story is, I would say, quite strong. And the Frederick Sadu Lab has added to the white hypotintin function on the BGNF gene transcription the evidence that white hypotintin also stimulates the transport of BGNF from cortex to striatum. And this is just a list of several papers that have that show this data. And just some recent data show that also neurons, human neurons from stem cells, either IPS or human ES lines carrying different CAG repeats, you see that as you have the mutation that is less BDNF.
And in this set of 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 knockout, in the wild type antigen knockout cells. And the BDNF story, I mean, has continued over the years and really shows that to show that you can rescue HD phenotype by crossing, for example, mice with mice overexpressing BDNF or another beautiful data from another group shows that if you remove BDNF from cortex, so if you do a conditional division of BDNF in Cortex, these are normal mice, and you look at of course, you have stereotypes and clinical symptoms in mice that mimic what you see in HD mice. But then 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 overlaps with what you see in HD mice or human HD brain, really saying that the BDNF and cortex is really important. And then the story is even bigger because not only BDNF is mechanism that regulates through white type anti that regulates the BDNF and also many other neuronal genes.
So white type anti stimulates anti ting is unable to do so. To close, I would like to show you the last piece of evidence of loss of white type Huntington function in Huntington disease. I mentioned this paper, okay, and the beautiful rosette that we get from just regular stem cells and the fact that in the absence of armpitin, basically, you had the cells in vitro, but they are unable to talk to each other. And we discover that they don't fond the rosettes, I mean, in the absence of white type painting because in the absence of white hypothenin, you have no rosettes because there is a hyper metalloprotease ADAM10. So ADAM10 is up in the absence of antientin.
And when ADAM10 is up, it clears several targets, downstream targets, including uncadhering, which is a cell addition molecule. And basically, if you are clear uncaring, I mean, the cell cell content are gone, and this is why you have no rosette. So basically, the function in this assay of white type amphetamine is to prevent ADAM10 proteolytic activity, okay? But this cells in vitro, I mean, is this relevant for HD? Well, we look in vivo, and this is again, y type ampinting.
And again, these are the conditional cortical striatal synapse, and we found the same phenotype. So basically, in the absence of white type antigen in cortex in but now, I mean, we are in mice, there is increased ADAM10 and proteolytic cleavage. And I didn't tell you that, of course, I mean, the story is important because Adam 10 is critically involved exactly, I mean, at the level of to regulate the functionality of this excitatory cortical striatal synapse. And therefore, I mean, ADAM10 activities under white type antigen function. You remove white type and ADAM10 is hyperactive.
And cadherin is cleaved. And at the level of this sign ups, you have really a lot of problems. And then, I mean, the very final piece of data, this is what happens in the absence of anti TINTINO. And then, I mean, we look at the HD brain. And basically, we found that the HD Brain phenocopies what we see in the absence of white titrantin.
Once again, in the HD brain, 2 mouse models, human postmortem material, we always find hyperactive ADAM-ten, cleavage of encountering. And the data were so relevant that we were able to rescue the ADAM-ten hyperactivity either genetically, and we rescue the phenotype in the HD mice, either genetically or pharmacologically with a compound that inhibits ADAM-ten. So to close, I would say that in addition to being neuroprotective, there are definitely phenotypes in Huntington disease, which are due to loss of white type unpatent function. And my conclusion is that we definitely need to preserve white type of antigen function in DHT brain, especially at the level of the corticosterial striatal sinus. And these are the several people in my lab and I want to thank you for your attention.
Good afternoon, everyone. So 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 antigen protein could actually participate to this pathogenic mechanism. So I will briefly the outline of the talks. I will come back to a few points, kind of like key facts about Huntington disease and Huntington 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. So 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. And basically, what is one of the mechanism by which mutant antigen could lead to 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. And what we have at the level of protein, we have 2 proteins, one that is wild type, one that is mutated.
And basically, they are all expressed at the same level. And this will lead to the disease because the mutant form is actually dominant. And in agreement with the gain of a new toxic function for the protein is the fact that the Huntington knockout is on brineclatiles, which is very different from what happened in the patients because, as we said several times and Elena mentioned that, that most of the patients develop the disease around the age of 40 years, 40 to 50 years, which is very different from the complete loss of the protein function, which leads to on chronic fatality at 7.5 of development. So really like mice developed around the age of 18 days. So now in addition to this gain of a new toxic function, we have also the possibility there are several arguments, genetic arguments pointing out, 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 Huntington protein just after birth, actually, the mice actually are going to develop progressively neurodegeneration. So indicating that if you lose the function of the protein, you will actually develop neurodegeneration. The second thing in term of that point 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 no not the second copy of the wild type, so only 50% of the wild type protein, Actually, those mice show cognitive defects and neurodegeneration. And one thing that is very important is that now if you compare to the mutant situation, so when you have the HD gene actually, you have already only 150% of the wild type because the other 50% is mutant, okay? So now there are 2 other arguments I would like to point out to complement what Elena has said is that there are some evidence in human actually patients that the level of wild type in mutant could regulate disease progression.
And the second thing I would like to mention also is that we and other labs have shown that the Huntington has several central function
in the body. So the
first one is the work from BLA, where they identify a SNP called 1310 blah, blah. And then basically, this is a G2A mutation. And basically, this when the SNP is at the A, actually, there is a decreased expression of the handtitin protein. And what is very interesting is that, so 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. So here you have less HD compared to wild type.
And then the H at onset actually, they develop the disease later. And in contrast, if now you look at the population where the A SNP is on the Y type haplotype, actually you see that they developed the disease earlier. So basically, when you have a higher amount of HD protein, the mutant protein compared to the wild type protein, then the disease starts earlier. So 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 level the disease progression. So now let's move to the second part, which is trying to understand more about the function of the protein.
So this is antigen protein. So as Elena said, this is a very large protein. And 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 HIT repeats or has its anti parallel alphylases, which are very important for protein protein interactions. And so there are several of these domains, the seed domains, so there are at least 7 domains, which are going to make intramolecular interactions. So some part of the protein can interact with other part of the protein.
And in addition to that, actually this Huntington protein binds to many different other proteins. And in fact, we identify or we and others have identified more than 400 different protein interacting with Huntington. So you can imagine that actually you have this single protein here that in fact is able to interact with many different factors, many different forming, many different protein complexes that are going to have several different function in the cells. And actually, Elena pointed out already several of these functions. And this is also illustrated here by Clio electron microscopy showing you here that at least 10.13, by interacting with different proteins, different forming different complex, can adopt several confirmation.
And there were more like the 100 different confirmation that could be formed by Huntington. So let's go now from these several functions. I'm not going to describe the 400 different that Huntington is interacting with, and that is the huntingtin and denactin and kinase complex. And this complex actually is extremely important. It's called the molecular motors.
And those molecular motors, that's basically motors that are able to move along a structure and, in particular, along the microtubule, which are the cytoskeleton of the neuron of the cells. And one of the function that is shared by this Huntington's DNA and acting complex is actually the capacity of this complex to transport vesicular 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 sales is going to differentiate or divide. So I'm not going to describe again. I will focus just on 2 of these two functions that are elicited by Huntington controlling this complex. So as I told you, Huntington is interacting with the DNA and Dynacting complex.
And in fact, here, it plays a facilitating role. Huntington acts as like a turbo for vesicular transport, so moving the vesicular on the microtubule. If you have antigen, you go very fast. If you remove antigen, you decrease the vesicle. Now if you have the mutation, the mutation that caused the disease actually leads to a change of confirmation and detaches the motors from the microtubule.
So it's like your the train is getting off the rails basically. So the result of that is that the transport is slowing down as well, okay? So you have a reduction in the transport of these vehicles. So what's the impact of these changes? Now we have to go back to this corticostrata projection Elena mentioned.
So Elena mentioned that Huntington has a role in the transcription of BDNF. Here, what we showed is that in addition to that, Huntington is supporting this vehicle that contain BDNF and provide BDNF to the corticostriatal synapse. And again, Elena told you about the importance of this synapse. And this is extremely important because striatal neurons are unable to produce BDNF. They strictly depend on the BDNF that is provided by the corticosteroid that protects neurons.
And basically, what happened is here, we are having a disease less transport of BDNF, then less activation of the TRK receptor that signal within the striall neurons to provide survival, activate specific pathway, ERK, that provides survival of the striall neurons. And basically, what happened here, you have a dysregulation of BDNF Trk B signaling that in turn will lead to the degeneration of the cortical neurons. So to address more specifically this circuit, because it's very difficult to address that in vivo, we were very much interested to reconstitute this corticosterial circuit that is observed in brain on chips. And actually, we used these microfluidic chips that are as big as this and basically in which we can actually reproduce, put cortical neurons in one of the chamber and the on the other chambers, and they are separated by 3 by 3 micrometer channels, if you want to have a look. And basically, this so they will form synapses there, so exactly reconstituting the circuits that Elena mentioned and that I just mentioned previously.
And using this, using neurons from HD mice, we could show that actually the alteration of all the different events, so from transport of BDNF to the synapse formation to the postsynaptic events occurring in the striatal neurons. And basically, this is an example. So then if we go specifically in the cortical neurons and then now we want to look at what happened at the trafficking. So here, what we do is that we express using lentivirus is BDNF, used to mCherry. So that's the vesicles are fluorescent.
BDNF, the Brandera neurotrophic factor is fluorescent. And we use spinning confocal video microscopy to follow-up the trafficking of the vesicle inside the axon. So each channel is 3 by 3 micron. And basically inside, you have an axon, sometimes branches. And inside, the little dots you see moving are corresponding to BDNF vesicle that are transporting along the microtubules.
And you can see them going from the cell body to the synapse and then some of them go back to the synapse. So we can quantify this movement of the vesicles. And basically, here you can see this is a chymograph from Wild Type and HD, so which is a distance of a time representation. And all these little tracks there correspond to the movement of single inside an axon. And 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, so 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. Also, if we look at the number of we see much less moving in both direction. And then now if we do like 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, and less BDNF reaching the synapse. So basically, in disease situation, we have this alteration of BDNF and less BDNF reaching the synapse.
So one thing that is very cool with these little chips actually is that you can play with the because they are fluidically isolated, so you can put different neurons with genetic status different genetic status into this circuit. So here, what we compare first was the wild type circuit, so Cortex and 3 Adam wild type compared to HD HD circuits. So but now what we can do is that we can put cortic anaeron that are wild type, that are connecting to HD threat and neurons or conversely, we do cortical HD network connecting to wild type neurons. And 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. And conversely, if the wild type the cortical neurons are from wild type, they can make straight line neurons making function working correctly.
So indicating that the pre synaptic compartment actually determines the synaptic the integrity of the network. And this has 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. But that's why we came back to the trafficking defect that we observe in disease when we look at the BDNF trafficking. And we use neural stem cells from human embryonic stem cells. And 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.
And these are 2 independent cortical progenitors from HD patients, where you can see the reduction in the trafficking of BDNF. And now we selected a line that is called VUB-five that is actually heterozygous for a SNP at exon 50. And this actually and this was done in collaboration with Vincent Perry and Nicole Deglan, and where they could see that the wild type allele has actually the SNP C and while the mutant allele has the SNP T. So now if you design SH that is specific to for the 50T, you can selectively silence the mutant allele. So then and conversely, you can use SH50C that silence the wild type allele.
And then if you go back now to your cells, looking at BDNF trafficking, so 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. So really, I mean, 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. And so as a conclusion of the first part, I would like to emphasize the fact that defects in axonal transports are potentially clearly component of the AGT pathogenesis that we can modify the wild type and the level of the wild type of the mutant and this has a strong effect on the trafficking.
And this trafficking is important to maintain the corticosteroid al circuitry. And as I mentioned a bit earlier is that we know that the striatum is affected, and Helena mentioned that, but we know also that the cortex has to be considered for her therapies. Now I would like to move to the 2nd step, to the 2nd part of the talk, which is the other example I would like to share with you about the potential role of antitin just because it modulates the dynein dynactin complex, which is ciliogenesis. So in fact, every cells in our body have a cilia, which is kind of an antenna. And some cells are multiciliated, and I will come back to that point.
But most of the cells in our body has a one single cilia, which is important for sensing like the environment, nutrients and everything. And there are specific proteins that are transported to the base of the cilia. And one of the proteins called PCM-one or pericentrheolar molecule 1, and this PCM is actually transported by Huntington and the molecular motors. And to be brief, actually, what we could show is that if we remove of NTT to bring these PCM proteins to the base of the cilia, and this leads to the disappearance of the cell. So no cilia are formed into these cells.
And in contrast, what is interesting is that if now we have the mutant antigen in these cells, basically what happens is that the protein is transported, but unable to go back in the other direction. So make this kind of balance, bringing material and removing some material. And basically, there is accumulation of the mutant sensing there, and this makes longer cilia. So we think more is better, maybe, but not sure. So we check that.
So the first thing is that if we look at absence of amphetamine leads to the loss of cilia in the brain here, in the ventricle of the brain. So you know that we have ventricles where there is cerebral spinal fluid that is circulated inside our ventricles, inside our brain. And you can see that if you have no cilia, basically, you have mice that develop in rocephallus. Now what happened in disease? In disease, this is the contrary.
Actually, we have longer cilia, and we have accumulation of PCM, accumulation of the cilia there. And this is also shown here on the ventricle of the brain here, where you see these stuffs, so those cells are actually multi seeded. They are several it's kind of like a tulip, like a bouquet of tulip. I don't know, you can say we are, I guess. And they are moving like this, and they make the flow circulating in the brain.
I will come back to that point. And you can see here that in disease, actually, they are longer. And this is also true in human. This is a section from Huntington's disease patient individuals there. So to come back, what's the role of the CER?
So in fact, inside the brain, we have these ventricles. So here, this is mice. But basically, there are several of these cilia tufts there 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 5 to 6 times a day, and it's very important for clearing metabolites in the brain, but also to make new neuron, newborn neurons to migrate and then innovate the olfactory bulb. And what we found is that this is a movie of the particles that are moving inside the brain of a section here of wild type mice.
And you can see that basically there is a very homogeneous movement of the particle inside the brain, so which reflects the movement of the CSF in the brain. And 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 in there. If we do if we go to the next slide, what you can see is tank projection. You can see here, this is very constant. Here, this is a camera graph 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, so indicating that the CSF flow is altered in this situation.
So as part of this is my last two slides. So just to conclude on this part, I hope I convinced you that for this other function, there is another function of antigen in ciogenesis and that has important physiological consequences by changing the circulation of the CSF in the brain and with clear consequences for brain homeostasis. And basically, this function is altered in HD situation. So I would like to conclude now, and I hope I convinced you that we have here one protein that is quite complex with many tractors, many functions, many physiological functions in the cells in the organism. And actually, all these so far, all the function that have been described for Huntington for wild type Huntington has been found altered in disease.
And so 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 consequence, and we already discussed that with Elena, that restoring or preserving wild type HD function is essential. And I would like to raise this question. Actually, I think one mechanism by which the mutant Antigena is acting is probably by acting as a dominant negative on the wild type function of the protein. So I'd like to thank the people involved in this work.
I would like also to mention collaboration with the lab of Sandrine Lambert, 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. So I thank you very much for your attention.
Thank you very much, Fred. So we have heard a wonderful talk about the importance of mutant sorry, Walter Huntington's and taking away the mutant Huntington's. So what we are now going to talk about is our program, our continuing to develop our allele specific program for knocking down the mutant Huntington's with another SNP. So we currently have 3 SNPs. We are targeting 3 SNPs.
We've included SNP 3. We do have SNP 1 and 2 in the clinical trials with precision HD. And this is the first time you'll be hearing about SNP 3 for taking away the mutant Huntington's. So we are using ALA specific RNAase mediated silencing of mutant Huntington's transcript. So here is the population genetics.
So with the SNP 1 and 2, we can cover each one of them can cover 50% of the population and the SNP 3 can cover approximately 40% of the Huntington's population. But the patients can have 2 or more of these SNPs associated with the CAGR repeats. So by taking all these 3 SNPs together, we believe that we can cover about 80% of the Huntington's population. So 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. So because we are doing all in specific targeting, we do have a slightly complex problem.
So we need to make sure that we can actually address each one of the questions for ALLETE specific targeting. 1 is trying to understand about the potency of the molecule in certain cell types. And then secondly, measuring the allele selectivity, both by biochemical assays and also looking at the patient derived neurons. And third, in the case of SNP-three, we do have a back HD model where we can actually do the target engagement and durability in vivo. So let's actually start talking about each one of the steps carefully.
So here, what we are looking at is a homozygous iso neuron. So this particular cell line only has SNP3. So it does not have the wild type allele, so you cannot measure allele selectively 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 hand silencer RG-six thousand and forty two. So in this particular cell line, the 2 molecules or the 2 potential leads for SNP-three are more potent than Roche's clinical compound, 6,00042.
This is an analog that we made internally. So in the next slide, I want to show the Alej specific nature of the molecule. So this is a biochemical assay and these 2 are 2 different 2 compounds for the same SNP. So what we have in this assay is the RNA, the surrogate RNA, which is one is wild type and mutant. And using our oligos, what we can silence preferentially is a mutant copy for the both molecules, leaving the wild type in this pretty much intact.
So clearly demonstrating that we have allele selectivity in this biochemical assays. So next, we also developed a cell line. In this case, this is a patient derived neuron, which has got both wild type and mutant, is shown in this PBX group. And what we have here clearly is the 2 compounds, which are labeled as SNP3 compound 12, and we are measuring Allie specific target knockdown at 2 different concentrations here. So with our SNP3 compounds, compound 12, what we can see is a clear allyl specific differentiation knockdown of the mutant while leaving the wild type pretty much untouched.
And the same thing is true even at the higher concentration, we do see exactly knockdown of the mutant while leaving the wild type intact. So on the other hand, if you take a look at the pan silencing RG642, you can see a very nice potent reduction of both wild type and mutant at the low concentration and 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. So now, we can look at the potency of these molecules in a back transgenic mice. So 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.
So that means you cannot measure allele selectively in this model. However, we can measure potency and duration of activity in this model. Secondly, the transgene here, this is a human Huntington's mutant Huntington's transgene and the transgene is over expressed. So we have multiple copies of the transgene. However, not all copies have got the SNP3 in it.
So that means we cannot silence any of the mutant Huntington's that does not have SNP 3 in this transcript. So 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. So first, we did oligo distribution study, and we injected all the 3 molecules, the pan silencing RG642 along with our 2 SNP3 compounds. What we clearly see is distribution in both Kotex and striatum.
We see our oligos, both of our SNP compounds show oligos and the PAM compound also shows distribution to Kotex and striatum. However, we see more of our molecule present in Kotex and striatum and compared to the pan targeting molecule from Roche. The next is looking at the target knockdown of SNP 3 in this model. So what you can see here is all the 3 molecules were injected at the same quantity. And the one that is written in black is PAN RG642, which and that molecule plus a SYMPT molecule give within 2 4 weeks time point, give exactly the same knockdown in Kotex 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. And at 12 week 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 Huntington's, 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 Huntington transcript, it's a highly expressing Huntington's highly expressed model and all the transcripts do not have snip there are few transcripts that do not have the transcript. So we are at a disadvantage of getting a lower percentage knockdown of the Huntington's.
So 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. So in summary, what we have shown here with our SNP targeting approach is that in a homozygous cell line, our SNP targeting molecule actually has a is more potent than the PAN targeting analog, Roche's analog. And we also see very good allele selectivity with our SNP targeting molecule. And we do see duration of activity in back transgenic mice.
And we also see target knockdown up to 12 each. And we get very good distribution in both striatum and cortex. So we have a plan of doing our IND enabling stock studies by 2020. And with this, I would like to introduce Mike Byrne, who is the Director of Biology, and he is in charge of the ophthalmology program. And he's going to walk us through, Ashut, today.
All right, everyone. Thank you. I understand we're tight for time. So 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 interesting space for oligonucleotides because of the ability for them to be delivered by intravitreal injection. So 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 of it 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. So subretinal injection is essentially, if we use E here in this example, it's formed by a bleb. So they have to make a retinal detachment and inject into that detachment.
The distribution of an AAV vector is limited to where that blood is created. The oligos that we inject intravitually 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.
So what I'm going to walk you through is some ALT-one 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, US2A. What you're looking at here is an extension of what Greg showed very early today.
He showed a potency shift at 1 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 3, 5 9 months after a single intravitreal injection. At 9 months, using our PRISM oligos, we maintained 50% knockdown on MALAT-one after a single injection.
Very importantly to understand is that the back of the eye, the cells of the retina are terminally differentiated. So once you get that delivery, the potency of the Stereopur molecule extends for a long time. Here, you're looking at the PK. So minimal exposures detected at 9 months. Here, we're into the nanogram per gram of tissue.
Yet this minimal exposure is the minimal exposure is fine because we still maintain 9 month duration. So that's the 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. So here you're looking at nonhuman primate data.
So in this case, we've taken the non human primate, we've done a single intravitreal injection again to look at Mal-one knockdown. And we've dissected out the retina itself, just the retina, nothing else. And when we do that, you'll see that greater 90% knockdown is maintained for 4 months. So this is with one of our compounds from prison. So we wanted to understand, well, what does that mean?
Well, our oligo has an RNA species to it. So we can actually look using an assay called vuRNA, which is typically used to look in tissues for the RNA of a gene, to identify a gene. But 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, and I've highlighted the photoreceptors. The blue are the cell nuclei, but the compound is in red.
So the drug is delivered across the retina. And at 4 months, it's still detectable in all layers. So this matches up well with the QPCR data showing that we have knocked out of the target. So what's important to point out is that both of those situations, 9 months in the mouse, 4 months in nonhuman primate, that lends itself for 1 to 2, maybe 3 doses per year. Anti VEGF therapies, depending on the indication, are dosed 8 to 9 times a year.
We're talking about 1 to 2 times a year dosing here. So using that information on durability, understanding what we've done in the Huntington space and our DMD space, we started to look through inherent retinal diseases through the lens of our knowledge base. So 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 4th or 5th decade of life, they have vision loss. The problem in Usherin 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 is not there, the outer segment is 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.
So the approach we take is to look 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. So our goal is to come in and skip out exon 13, thereby creating a functional protein. So 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? So here, compared to a reference compound, a stereo random reference identified in the patent listed here, We have a fourfold shift in potency for Exxon skipping. So 10,000 foot view, we skipped. We want to understand what did we skip. So let's zoom in, 5,000 foot view.
We took the Skip product and looked at exons 8 to 17 just to see the size. So this is a gel shift assay. What you see is in the compound 1 treated samples, we're missing about 6 40 nucleotides. So 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. So 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, etcetera. So if we just focus in on exon 12, 13, 14 and 15, you'll see the PBS treated that RNA seek allowed us to see very clearly exon 12 connection to 13, 13 to 14, 14 to 15.
And 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. 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 decreased.
And importantly, what you'll notice is a new junction. Exon 12 now extends exon 14. So at the ground level, at the transcript level, we've confirmed that we've sequenced that we've skipped the exact product that we're interested in. We then wanted to understand, well, how does this translate into Large Eye? So we took on an exercise where we've generated an ex vivo system.
So we get cadaver eyes, human cadaver eyes, non human primate eyes. We dissect out the retina. We section the retina and plate it in 96 well dish. So everything you've heard today from Chandra's gymnotic delivery. So we plate cells, put a rollover on it, no transfectionery 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 75% skipping of exon of the proper exon. We did the same experiment in human. At the same concentrations we achieved similar skipping.
So we have upwards of 75% skipping of exon 13 in the human retina. So today, we know we skip a nonhuman primate and we know our oligo skips in human. We have ongoing in vivo studies, so we're excited to provide updates on that as we continue. Utilizing what we learned in our leal selective approach for Huntington that you heard about today, another area of interest for us is autosomal dominant retinitis pigmentosa. In this case, so ADRP is essentially a group of originated diseases around retinitis pigmentotal cell.
One of the key mutations specific to cause ADRP about 10% of all cases is a p23h mutation. So this is in this case, what happens is it's a dominant negative effect, again, a function. What happens is that the mutant rhodopsin is generated. It gums up the works and prevents the wild type rhodopsin from get to its site of action. And its site of action, again, is here in the photoreceptor.
We want to get rhodopsin to these outer segments. So it's about 1800 addressable patients in the U. S. So the 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 stereo random molecule that's been identified from the following patent, the reference compound.
And you'll see that it is not a little selective. In a dose dependent way, it knocks down black mutant, but also the wild type allele in blue. We make a sterile pure 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 P2A3H pig model. So our stereo pure compound is selective. So in summary, our compounds we know are potent and durable. They're lasting up to 9 months. We have productive exon skipping with our RUSH program.
We've confirmed that skipping at the transcript level. We have ongoing in vivo efforts to identify the skipping in nonhuman primate, large animal. And we have discovery work underway for a second program in ADRP. Our IND enabling studies, we expect to begin next year.
Thanks, Mike. I know this was an intensive day, and we took you across from crystal structures and chemistry through new areas biology. But what we think is important is the consistency with which we've seen that transition. So 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. So 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 exon53, with C9orf72 for ALS FTD, with our SNP3 program in HD and USH2A. And so 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 capabilities. So 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. But it's an exciting year in 2019. It's not over yet, and we're looking forward to 2020. So with that, I'll pause, thank our panelists and open up the floor to any questions.
Informative presentation. If you could just comment a little bit beyond kind of what you're thinking here since I think you clearly identified that there's a role with wild type Huntington protein and the health of neurons and that possibility is effective knocking out any amounts of kind of wild type. Did you have any evidence of maybe that of what can drive kind of that decline, whether it be some marker of activated microglia, whether it's CD68 NRF kind of change or something like that, it would be maybe a more positive link between the presence of wild type Huntington protein and kind of neuronal health?
So you're asking whether what is in between the wild type and the neuronal loss? Is this the question? Okay. I'm not sure I understand the experiment. So basically, we don't have data for the moment, but we are trying, I mean, to modulate the level of wild type in an HD background.
So, say, pushing the wild type protein to our degradation to see what happens. I'm not sure I understand the question.
So my feeling, if I may, actually, I had the impression that what you were asking is that what happened if you remove NTT in terms of like cellular response? Is that your question?
Our co activation.
Okay. Well, we don't know about microbial activation. One thing we could see is that given the role of what's happened in the expression of BDNF and the trafficking of BDNF, what you could imagine is that because you have a decrease in our traffic support in sales, then you slightly increase the apoptotic level in the sales, which would raise, for example, apoptosis and then death of the cells. So that's one possible mechanism.
There's one piece of evidence actually in the Dietrich et al twenty 17 paper, the one from Ioannis Dragartis that I mentioned, in which they saw some gliosis. So not really microglia. I don't think anyone has really looked at microglia. But yes, if you remove Huntington in addition to the classification and ion depletion problem, you also have glial hyperactivation, so gliosis.
Another question.
So I don't know whether this has implication for inflammatory response. But I don't think this has been looked at very carefully. But this is something that actually we should consider.
That's great. Thank you.
Hi. My name is Suji Jeong from Jefferies. Thanks for taking my questions. 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? And the question for the KOL is, what is the ratio between the wild type and mutant Huntington proteins in the brain? And if they're not 1:one ratio, if the company is presenting the mutant Huntington protein over total Huntington protein level, how would you interpret that in terms of whether the wild type protein level is touched or not? Thank you.
So these 2 are nicely integrated, which is great. So we will be doing the assessment, as we said, as total to assess, so the total HTT ratio to mutant to assess wild type function. And that's because you can't quantitate in patients the wild type. So you have to do this ratio. It's a dynamic ratio.
And one of the things that we're doing in advance of presenting data will be doing a walk through of the assay and how the assay is implemented because to your point, a lot of it is going to be following and a lot of work being done on actually following that ratio over a variety of data sets. But it is a ratio. It is not an absolute quantification.
Okay. So what is known, and we are discussing that at lunch actually, 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 like turn off or silence the mutant, the expression of the mutant compared to the wild type. So there's always a little bit less of mutant compared to the wild type.
So, that's what we observe in the brain?
So, no, at the messenger RNA level, you're talking about the protein. That protein protein, Which
will be how we'll be assessing this protein quantification.
Julie from SunTrust. Is there any logic to how some stereo isomers are better taken up versus others? Or is this just a random screening process?
No. So I think one of the things we were it does not appear to be random. And 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. So we saw this preferential trafficking. When we did the inverse chemistry, so the exact same molecule is suvodirsen, but the chiral inverse of that, we saw the drug outside.
So 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 Tuncay mentioned that based on our capability, when we look at our potential medicines as they're coming forward preclinically, where by nature because the potency differential is 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 and I have this etched in my head, which is we often talk about nucleic acids as a drug in problem. So 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.
So drug out, that the drug is actually staying in the compartment where it needs to be and can therefore be catalytically efficient, which gives us durability. I mean, to Mike's data, 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 is not. And that's the work we're doing to kind of deconvolute from a again, from a scientific and research perspective, what is it about trafficking that drives the biological changes we're seeing.
So 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?
Yes. So one, we're seeing it consistently across and that's what was so compelling in our minds on the discovery side around the MALA-one data, where we could look at, here's a drug where you can 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. So 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. So a key driver for us on the therapeutic side is productive distribution to the variety of cell types that we just saw.
Thank you. Thank you for the talk. Very informative. I'm Bennett from Mizuho in Salim Sainte. I just have couple of questions from the HCV program.
Have you discussed any new micronucleus data with the FDA? With a new and you can comment. And has enrollment in the multi dose court started? And if so, is enrollment going in line with what you expected?
Yes. So as to your first question first, so as it relates to the micronucleases, we put that out in the school, 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. And so you can follow the next queue for those updates. But 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 in the U. S. For precision I mean, sorry, fully enrolled outside the U. S. And global.
So that's delivering our data by the end of this year. So there isn't an opportunity now that the study of PRECISION HD2 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 Q. But we'll provide updates with obviously, it's important to us to make sure by the time we finish the Phase III study to be able to start our pivotal trial to be able to have that up and running. So we 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.
And maybe we have a question for the 2 KOLs and Yaron Werber from Cowen. The question, and it's in 2 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. So as you think about Roche and AONIS, let's say, are knocking down the wild type, so with long term follow-up, what would you expect to see clinically from knocking down the wild type protein?
I have the microphone. I would say, well, if you think at the BDNF, the BDNF story, I mean, 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 that cognitive deficits and I would expect that some of this might happen, especially, I mean, if you hit the cortex with your compound, with your molecule. And yes, if BDNF, I mean, the loss of BDNF will be already, I mean, a huge story and a huge problem. So this would be, I think, one.
And I mean, it's not only BDNF. I mean, if you think at the ADAM-ten story, I mean, the synapse, I mean, this cortical striatal. So it really seems that this cortical striatal synapse is heavily dependent on the full function of the Y type, because we have the Y type impacting on the transcription of the function of the postsynaptic density. I didn't mention many other data, I mean, anti function at the level of the postsynaptic density. And clearly, I mean, white type anti interaction interacts with many protein at the post synapse.
So you are really screwing up, I think, the sign ups.
So clinically, I mean, if we think about what we had, and I know we had a lot of conversations with a number of you over time or what there was always this question of what do you think the safety signal would be in knocking out wildtype. 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 is 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.
So 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. Yes.
Maybe just a follow on. So in that last study, there was and I think Paul just referenced in 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? Are these due to they're not they wouldn't be due to disease progression, or were they due to wild type knockdown?
It is all. Can
you rephrase the question? Yes. So I think separating it, and we'll take the ASO discussion and we'll take the biology discussion. I think the question was really, does not could the knockdown of the wild type protein explain some of the changes in terms of neurofilament light increase, ventricular enlargement over time. So what happens when you knock it down?
And we'll take the second question.
Can I say about the neurofilament? I think it is too early to say. So, I would of course, and this is something that raised some alarm, the level of the neurofilaments. But I would really use this statement. It is too early to say, but it is something that has to be monitored.
I mean, on the ASO side, and I think it's important just to reflect on the IONIS studies in a pilot. I mean, 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'd see neuro filament light trends in the direction of positive outcomes, right? So you have the benefit within SMN protein getting produced correlating with an improvement in neurofilament light. And the SOD1 study that was presented more recently, you have SOD1 levels decreasing and you have neuro filament light decreasing as well. So 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 Panzera, our Chief Medical Officer, was involved in a number of the development programs in the multiple sclerosis space. And there where you see some changes on inflammation and improvement, you also saw parallel decrease in neurofinance. So 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 is right, to just watch the study progress. I mean, 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.
Okay. Maybe just a question for Chandra, if you don't mind, to put you on the spot quickly on Stereopurity. So maybe give us a sense, the interplay between the base and the sugar as to how you actually fix there your purity? How do you build an assay or system to actually help you understand what's the best specificity? And then what do you think confirms the better nuclear localization because it's not charge, right?
So how does stereo purity leads to penetration? Thank you.
Yes. I can actually answer the first question first. So the stereo chemistry requirement for some of these profiles are kind of we have actually learned over time how to implement stereochemistry in these sequences. But some of the things that we're seeing are something that we didn't expect. So when we evaluate stereochemistry, we always make some educated, some knowledge based learnings to implement studio chemistry.
And that's why when you make them completely stereopyr or when you control stereochemistry at every position, you can actually figure out these subtle changes very easily. And this is not obvious if you don't make a stereo pure molecule or if you don't control stereo chemistry at every position, you cannot determine this because there is a lot of interplay. They said there's a lot of interplay between 2 prime modifications and the chirality of these molecules. They really talk to each other and depending on the sequence, again, there is a sequence component to it. And if you don't make them stereo pure, you cannot understand the differences between the sequence chemistry and stereo chemistry.
So this is where our breadth of our knowledge is from the platform and we have a knowledge base to actually mitigate some of these
issues. Just a follow on to that and then
you can answer the second question is creating training data sets. We're doing 2 things. I mean Chandra alluded to that in terms of the probability getting better. So 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. And so 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 and we have the luxury now that we can print plates and plates of chirally controlled drugs is this to continue to build training data sets that you wouldn't also inherently design. So 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. Because as Chandra mentioned, one of the things that's really interesting from a computational standpoint is there is an interplay between sequence, 2 prime 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.
So the question number 2 is about the intracellular. So this is also when we started these, we also had some empiricism. And what we actually quickly again, this is to the point that free uptake can actually predict some of these stuff, right? So if you use transfection agents, all these stereo isomers go in at the same time. So it's actually it could be some of these receptor mediated uptake, which is possible.
But what we also learned is that it's independent of sequence. So some of the stereo chemistries that trache us to the nucleus is completely independent of sequence. So, you can transfer these signatures to other sequences. For example, exon skipping oligos, we see this over and over again that using similar chemistry and backbone chemistry, we are able to actually achieve exactly the same thing that we achieved with EXOND-five thousand one hundred and twelve. So that's really what we have seen.
And again, making the wrong isomer or the opposite isomer does not take us through that. So, we are working with several academic collaborators to really understand what is the principle behind this trafficking. Because anyways, intracellular proteins, they're all like chiral. So, there are several aspects, there are several RNA binding proteins that can actually traffic these molecules.
I mean, it's interesting when you look to Chandra's point, you look at the melon1 example today, the exon 51, exon 53, those are 2 different sequences between exon 51 and 53. And then even some of our early posters in HD SNPs 12, where we're looking at nonhuman primate distribution, showing nuclear co localization. And so those are 4 different examples across 4 different sequences that have still the same common traits of nuclear uptake. And so to that point, a lot of the work is, though, what's driving it and really, again, that's kind of 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 out? Suji, John from Jefferies again. For the written to total Huntington level ratio, do you expect that ratio to go down over time? And another question is, do you plan to have another research date to go over the assay?
Yes. So I mean without dictating a research date, but there's definitely continued updates on the assay in advance of data. So we're not going to have data come out into a vacuum. But I think that you heard 2 things that are important. 1, the differentials skewed to the mutant down over the wild type.
So I think our piece there is selective silencing of the mutant over the wild type, I think continues to push that in favorable directions. I think secondly, there is a kinetics piece to it. And so a lot of what we want to make sure is that you aren't all 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. I mean, 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. And so that will be a component of how we use the total assay to assess wild type function.
Hi, Rick Mankowski from SVB Leerink. My question is about the C9orf72 program. I believe you mentioned earlier that you were able to directly target 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 antigen mediated disease such as Huntington's?
Yes. I mean, 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 seropured 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 is going to occur. So 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 2 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. And so 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, 1, 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, 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.