Taysha Gene Therapies, Inc. (TSHA) Earnings Call Transcript & Summary

Good morning, and welcome to Taysha Gene Therapies Rett Syndrome Investor Day. [Operator Instructions] Today, we will provide an overview of Rett syndrome, hear from our patient advocate and discuss the TSHA-102 program and clinical development strategy. Joining the call today is RA Session II, Taysha's President, Founder and CEO; Dr. Jeffrey Neul, our key opinion leader, guest speaker from Vanderbilt University Medical Center; Monica Coenraads, Chief Executive Officer of Rett Syndrome Research Trust; Dr. Steven Gray, Associate Professor in the Department of Pediatrics at UT Southwestern and Chief Scientific Adviser at Taysha; and Dr. Suyash Prasad, Taysha's Chief Medical Officer and Head of R&D. Next slide, please. Before we begin, please note that this presentation will include forward-looking statements within the meaning of the safe harbor provisions of the Private Securities Litigation Reform Act of 1995. These statements may include the expected timing and results of clinical trials for our drug candidates and the regulatory status and market opportunity for those programs as well as Taysha's manufacturing plants. Please see Slide 2 of the accompanying presentation and Taysha's SEC filings for important risk factors that could cause the company's actual performance and results to differ materially from those expressed or implied in these forward-looking statements. Taysha undertakes no obligations to revise or update any forward-looking statements to reflect events or circumstances after the date of this conference call, except as may be required by applicable securities law. I would now like to turn the call over to our President, Founder and CEO, RA Session II.

Next slide. Thank you, Kim, and good morning, and welcome, everybody. Next slide, please. At Taysha, we are developing AAV9 gene therapies to eradicate monogenic diseases of the central nervous system, or CNS. As you can see, we have a robust pipeline of innovative and potentially disease-modifying therapies. But today, we will focus our attention on Rett syndrome. Next slide, please. We hope you find this very -- we hope you find this a very informative day and look forward to you joining our future Investor Day events on Angelman Syndrome on October 26. Next slide. We are excited to do a deep dive into Rett syndrome, a severe neurodevelopmental disorder for which there are no approved treatments. We will also discuss in detail TSHA-102, our promising gene therapy drug candidate for the treatment of Rett syndrome. We are honored to begin our event with a presentation from key opinion leader, Dr. Jeffrey Neul, an internationally recognized expert in genetic neurodevelopmental disorders at Vanderbilt University Medical Center, who conducts clinical research and clinical trials on Rett syndrome, genetic research to identify other causes of neurodevelopmental disorders and translational research. Dr. Neul will discuss the clinical features and genetics of Rett syndrome. His overview will provide a deep understanding of disorder and help set the stage for our treatment approach with TSHA-102. Following Dr. Neul, we will have a very exciting -- we are very excited to have a presentation from Monica Coenraads, Chief Executive Officer of Rett Syndrome Research Trust, a nonprofit organization that funds and spearheads global scientific and clinical activities to advance the most promising curative approaches for Rett syndrome. Monica will provide a patient and caregiver perspective on the burden of disease, which will give real-world context to some of the therapeutic endpoints and current management approaches. We will then transition into our therapeutic approach for Rett syndrome with our promising TSHA-102 program, an AAV9 gene replacement strategy that regulates expression of MECP2 through a novel miRNA responsive target sequence called miRARE. Dr. Steven Gray, Associate Professor in the Department of Pediatrics at UT Southwestern and Chief Scientific Advisor at Taysha will discuss miRARE and review the preclinical and pharmacology data generated to date for TSHA-102. Dr. Suyash Prasad, our Chief Medical Officer and Head of R&D, will follow with a discussion on the clinical development strategy for TSHA-102 and will provide a regulatory update. We are very excited about the therapeutic potential of TSHA-102, which has already been granted orphan drug designation, rare pediatric disease designation, as well as -- from the FDA as well as orphan drug designation from the EMA, which we announced this morning. We expect to initiate Phase I/II clinical trials for TSHA-102 by the end of this year, with preliminary clinical data expected by the end of 2022. With that, I will now turn the call over to Dr. Neul to provide an overview of the clinical features and genetics of Rett syndrome. Dr. Neul?

Thank you, RA. Thanks to all the people who have come to steer this presentation. Today, I'm going to -- as set up, I'm going to talk about the clinical features and genetics of Rett syndrome, really with a focus on a lot of the knowledge that we've learned from longitudinal natural history study. Next slide, please. So Rett syndrome is a disease that has a very specific pattern of disease where people have a normal birth and delivery and have an initial relatively normal piece of -- time of development and then have a developmental delay and importantly, have a regression. And they specifically lose the ability to use their hands and they lose the ability to have spoken language. This regression is not relentless. It actually has a finite period and then it stabilizes when they enter a stationary phase. People with Rett syndrome also have gait abnormalities, please play the video on the left side. So you can see this young girl. She has ataxia gait. She's on her toes. She has a stumbling and wandering quality, we call it an [indiscernible]. They also have repetitive hand movements, please play the video on the right. So you see this girl has a clapping and wringing and classically, it's hand-wringing, and this is -- can be relentless while they're awake. This is an excellent disorder. It primarily affects girls and about 1 in 10,000 live female births. Next slide, please. So as I mentioned, a lot of the information I'm going to be presenting here is based on the natural history study. And this is a multicenter longitudinal NIH-funded study that was directed by Alan Percy, and I was the administrative head. It ran from 2003 until just July of 2021. We ultimately enrolled over 1,800 people with 1,250 who have classic or typical Rett. And we ultimately had expanded to having 14 sites across the United States. Next slide, please. Some of the things that we learned were things about the development of milestones and the time that they were acquired. And we were able to look and then this -- the classic or typical Rett syndrome is in red, and look at the onset -- the ability to gain these skills relative to typically developing individuals. Next slide. We also look at the loss of these time, what's the timing of the loss. And again, red is the classic or typical Rett syndrome. So on the left is the people who lost pincer grasp after gaining it and the time that they lost it. On the right is the time that they lost single words that they had gained. Next slide, please. We also know that people with Rett syndrome are typically underweight and short. And so with the natural history study data, we were able to develop growth, Rett syndrome specific growth curves. And this -- the plot on the right side, typical developing individuals are in orange and Rett syndrome individuals are in blue. And you can see that while their height and weight started at a normal level, it falls off the growth curves. Next slide, I mean, next slide, please. And this is true with their head circumference as well. The head circumference is at a normal size at birth, but it fails to continue to keep growing at the same rate. And so you see some individuals actually become, frankly, microcephalic for under the second percentile. But a lot of people fall off. And one thing that's interesting is that the falling off of that growth curve actually can be observed probably as young as 2 to 3 months old. So that's far before a lot of the clinical features are clearly present. So the biology seems to be occurring earlier than a lot of the clinical features are overly present. So with the low height and weight, of course, that oftentimes would reflect in the body mass index. And you see that, yes, the people with Rett syndrome, their body mass index, the 50th percentile is below the 50th percentile of typically developing. But at the top, you can see the 95th and 98th percentile is actually above typically developing individuals. So there are people with Rett syndrome who actually have increased body mass index. Next slide, please. And here's a video of 2 girls who have increased body mass indexes. And the woman at the bottom, she's 30-year old, and that's the same woman that was in the video that I showed previously when she was 2 years old and initially diagnosed with Rett syndrome. Next slide, please. So seizures are a common feature in Rett syndrome. They occur in a very high frequency. This plot is showing a cumulative distributive -- a cumulative plot showing the proportion of people who have epilepsy and it's that middle green line that's important to look at for typical Rett syndrome. And you can see that over time, nearly all the people, at least 95% have had epilepsy at some point in time. So seizures are a significant concern. Now next slide, please. One of the confounding factors is that people with Rett syndrome also have a variety of movement abnormalities and sometimes have paroxysmal or spells of movement abnormalities that we call dyskinesias. Please play the video. And you see this young girl is having -- she has spells, who's having high amplitude, erratic, non-rhythmic movements of all of her limbs. And so as a clinician, I can look at that and say, that's not epilepsy. However, she was being treated with increasing doses of anti-seizure medicines by her treating physician because they thought it was epilepsy. So oftentimes, we have to bring people into the epilepsy monitoring unit and monitor them overnight to determine for sure if something is an epileptic spell or a non-epileptic movement disorder. Next slide, please. So there are a variety of behavioral type abnormalities that exist in Rett syndrome. One of the things is that during regression, autistic features can be present and prominent, but they typically improve after the regression. But we do know that autistic features persist in individuals who are higher functioning. Rett syndrome is considered to -- the people who have Rett syndrome are considered to have a severe intellectual disability. But we really don't know the IQ range, because people who have Rett syndrome really cannot participate in standardized IT testing. And we know for sure that people with Rett syndrome clearly have much better receptive language than expressive language. And finally, there are a number of other behavioral features such as anxiety and mixed behavioral issues that, again seem to be more prominent in people who are younger or less severely motorically affected. Next slide, please. As I mentioned, the hand stereotypies is one of the defining characteristics of Rett syndrome, and we were able to classify what is the proportion of individuals who have the different types of hand stereotypies at different with classic and then even throughout different age ranges. Next slide, please. And people with Rett syndrome have breathing and other autonomic abnormalities. So they have a breathing abnormalities that typically occur mostly during wakefulness, although they are present in sleep. And there they have a hyperventilation with a breath holding and a combination of -- play the video, please? I hope you can hear that she's I mean, having deep breaths and then holding it and pausing and then heaving a big exhale. You can also maybe hear she has teeth grinding or bruxism, which is also common. And you see her hand stereotypies are a little bit different, but they have a characteristic pattern for her. Next slide, please. So these breathing abnormalities are also very common. So in this slide, the blue line going on the top is the class of a typical Rett syndrome. And as you can see that, over time, nearly 100% of the people will have some degree of breath-holding. Now the severity of that may vary. In some people, it's very interfering with function. And in other people, it's mild, although present and doesn't interfere with their function. Next slide, please. And we can look at what the path -- we've looked at what the pattern is over time. And in this graph, the y-axis is the years of time in the study, and on the x-axis are individuals. And on the far right in the big red group, those are people that the entire time that they were involved in the natural history study, always had some degree of breathing problems observed or reported. On the far left is the small proportion of individuals that had no breathing abnormalities reported during the time of the state. Some might have had a history, but currently did not have it. So you see that's a very small fraction. But there is a sizable fraction, probably about 1/3 of people in -- who are showing that yellow and blue area, who have -- they have breathing abnormalities and then it goes away and then it comes back, so were kind of relapsing remitting quality. So it's important to understand how the potential progression of these features occur by the individual. Next slide, please. Scoliosis is a common problem in Rett syndrome. It -- the severity increases with age. We see severe scoliosis in about 1/4 of the individuals. And surgery in almost 20%, 18% have to have surgery to correct their scoliosis. Next slide, please. And as I mentioned, there's autonomic dysfunctions. The cold blue hands and feet, the vasomotor disturbances are some of the things that are most apparent. Please go to the next slide and play the video. As you see here in this video that I took of a girl, she's blanching, it's kind of purple. You could feel it. It would be very cold just so as the ankle dystonia, which is a common problem. Next slide, please. And they have gastrointestinal dysfunction, which is -- can be very severe and incapacitating. I mean this is really from the entire GI system, they have trouble chewing and swallowing. They have trouble with reflux, troubled gastric emptying, trouble with slow transit time, severe constipation. So it affects a lot of the GI system and it can become a significant problematic comorbidity. And people with Rett syndrome have decreased bone density and have a propensity to an increased risk of having fractures and even a culture of unrealized fractures. Next slide, please. And then there's other autonomic abnormalities such as cardiac rhythm changes. So there is increased risk of prolonged QTc with about a 20% having an increased QTc and a change in the B2B variability. Next slide, please. And I mentioned that there's this progression where you have a stagnation regression in a stationary phase. And then later, there's what's called the late motor deterioration, and this seems to occur in the teens, in 20s, where people will have -- become more stiff and have a decrease or loss of mobility and have the onset of Parkinsonian features. And we've been looking at this in the natural history study, and it does seem to be related to menarche, where you have a progression at the top of how much truncal rocking or hyperactive movements that have, a declining around menarche and the amount of slowed movements or difficulty initiated movements increasing after menarche. Next slide, please. Now in 1999, Dr. Huda Zoghbi identified the genetic basis for the majority of cases of Rett syndrome, which is mutations in excellent gene methyl-CpG-binding protein 2 or MECP2, as I'll refer to it. For people who meet the clinical criteria for Rett syndrome, typical or classic Rett syndrome, 95% to 97% of the people have mutations in this MECP2 gene. Typically, these mutations are de novo, meaning neither the mother or the father has a mutation. So it doesn't really recur in families, which makes it difficult because it is sort of a random process. There is, again, about 3% or 5% of people who meet the criteria of Rett syndrome who do not have mutations in MECP2. We've done sequencing projects, and on these people and found mutations and other [ promising regulators ] and other genes that have been implicated in other forms of neurodevelopmental disorders indicating there is sort of an overlap in some of the clinical features. There are boys who have MECP2 mutations. There are typically we thought have had a severe general encephalopathy, so effected very markedly from birth and having a very shortened lifespan with only a couple of years. However, in the natural history study, we found a markedly expanded phenotype of previously unrecognized individuals who are boys, who have MECP2 mutations. And we're very interested in trying to learn more about this range of clinical problems in boys who have MECP2 mutation. Now all of the mutations that cause Rett syndrome that I've been talking about are loss of function mutations. However, there are people who have duplications of the MECP2 locus, so they have more MECP2. And this also causes a severe neurodevelopmental disorder called the MECP2 duplication syndrome. And this is a fairly severe disorder, mostly affects boys. They have more autistic features. They have more severe seizures, absence speech and an increased risk of infection. So it is as severe or more severe than Rett syndrome, I would say. Next slide, please. Now MECP2 is a gene that by its name binds the methylated cytosines in DNA. So it binds to DNA in particular regions. And the classic model is that it recruits histone deacetylase complexes that, next slide, we removed the subtle groups off of histones or chromatin causing, next slide, please, chromatin compaction and local gene repression. Additional work has deterrent found, next slide, please, that there may be an activating function of MECP2 where it interacts with transcriptional activators to increase local gene expression. There's a lot still to be learned about the exact details of how MECP2 works. But suffice it to say, we really do all think of working in the nucleus and it regulates gene expression. Next slide, please. Now with the discovery of the genetic basis, we've been able to identify that there are hundreds of different mutations that in MECP2 that can cause this disease. However, there are 8 common point mutations, which are shown in orange that account for 65% of the disease, 4 of them are what's called missense mutations and 4 of them are what's called truncating mutations. Next slide, please. And because they have these high frequency of these hotspot mutations, we are able to do genotype/phenotype analysis, looking at the different genotype/phenotype relationship of these common mutations and also very large lesions or a molecular group that we call the C terminal truncations. And what we can see, shown on the left, with a rating scale, clinical severity where higher number is more severe, you can see that on the left in the red and pink, there are individuals who on average or the median have a lower severity. Compared to ones on the right, like the orange or the teal or the purple that are more severe. So we can say that there are some mutations that have a higher likelihood of being mild versus being severe. But you can look at the tails of these cumulative distribution plots and see that there are individuals who have the mild mutation or very severe, and there are people who have the severe mutation are very mild. So it isn't -- we cannot say this at an individual level, we can only really say this as a group property. Next slide, please. Now with the genetic discovery, that allow genetic engineering to make rodent models of Rett syndrome. And mice -- mouse models have been generated that we produce a large number of clinical features that are seen in people with Rett syndrome. And over here on the left is just a list of some of the behaviors and physiological features that have been observed that are modeled very closely what we see in Rett syndrome. Nearly all of the different common allele mutations that I mentioned have been generated as mouse models. And they show that same genotype/phenotype relationship that I presented for the human -- people who have Rett syndrome. Now there are ability to condition knockouts and conditional rescue alleles, so you can have a cell -- you can identify the cell-specific role from MECP2 and also understand the temporal requirement for MECP2. And female animals have very similar phenotypes, most of the work had been done in males, but female animals have these phenotypes. They have robust phenotypes that can be observed. And so they really provide the best model of the disease that is most valid. And a rat model has been generated, and it also shows phenotypic abnormalities and allows for more nuanced and complex behavioral assessments than the mouse models. Next slide, please. Now with the generation of these mouse models, very excitingly in 2007 Adrian Bird had done an experiment where he created this conditional rescue allele of MECP2, such that it was off until another gene of transient called CRE was provided, and then it turned the gene back on. And they could combine this with a drug that controlled that Cre and do it in a time-specific fashion. And what they did was let the mice develop to the point that they became clearly visibly sick, and then they turned the gene back on. And what you can see on that top right graph is the survival curve. And the solid line is when they did not provide the drug, so the animals died. These were male animals about 10 weeks of life on average. This is what you see in a [ no-model ] of the disease. However, they turn the gene back on when the animals are symptomatic they did not die. And it also corrected basically all the phenotypes that were present in those animals. And this was also done in female mice, which is on the bottom. So this shows in female mice that was -- it started turning the gene back on when they were about a year old. So it's really -- this is critical in that it gave real hope that Rett syndrome can be -- at least have significant disease-modifying therapies, if not disease-reversing therapies. Next slide, please. And of course, then that raises the question, they did it with genetic engineering tricks. But what about gene therapy? That seems like the fact that the generic tricks work, maybe gene therapy would be a great way to approach it. So there is obviously some challenges. One, with any sort of gene therapy is the delivery for this. This is a disease that is caused primarily from loss of MECP2 function in the central nervous system. And so you really need to try to get this gene therapy into the central nervous system and really probably pretty widely distribute it throughout. In Rett syndrome, there is the other specific issue that we have concerns about overshoot. And that's because what I mentioned before about the MECP2 duplication syndrome that we don't have too much. But more than that, it has to do with some of the biology, the x chromosome abnormalities. So on the top is shown a male cell with the X chromosome in blue with a mutation in red and then the Y chromosome in green. So in males, every cell in the body only has one X chromosome, so they all have the broken copy of MECP2, and that's red, so that would be bad. So if you had this perfect gene therapy that had just one amount of MECP2 to every single cell, you would change all of the cells to have just the right amount of MECP2 and it should be perfect. Females on the bottom are a little different. So females have 2 X chromosomes. And in this case, the one with the red is the one with mutation. Now in females, during cellular -- during embryogenesis, each cell only is -- has 1 X chromosome expressed, and the other 1 is shut down through the process called X chromosome inactivation. So what happens is you get a mixture in -- all females and in Rett syndrome, they have a mixture of half of their cells have a broken copy of MECP2, whereas half of the cells have a normal copy. So now in this perfect gene therapy, you would be able to correct those red cells that are broken and make them blue on the right. However, those cells that were blue, now you might go too much and make a duplication in those cells and maybe cause problems. So that's the concern. Next slide, please. However, that said, there have been a number of papers published selling the ability of using AAV-based gene therapy vectors to correct phenotypic problems in Rett syndrome. Even being able to reduce the size of the MECP2 to a trunk has a very small mini truncated version, but still have the ability to improve features. And finally, I won't steal Steven Gray's thunder, but recently, there have been work that he's done, showing that you can build in sort of auto regulatory information to control the amount of MECP2 being expressed from the viral vector in relation to how much MECP2 is present. And I think that's an important and advance in terms of being able to come -- overcome the concern about the overshoot creating the MECP2 duplication. Next slide, please. So I'm just going to talk a little bit here at the final part about various needs for clinical trials in Rett syndrome. Of course, in a clinical trial, you need an outcome measure. And these need to be psychometrically reliable and valid. And they should also reflect the relevant issues in Rett syndrome. Next slide, please. During the natural history study, we asked the caregiver top concerns at every visit. So we ask them to list the top 3 concerns. And this is how it was presented, they could choose from a list of concerns where they could just add in, they could free text or write anything they want that was concerning to them. Next slide. So that allowed us to create an order of what is the #1 concern broadly and going down as far as the top 25. I mean as you can see that some of the top concerns are things that are very characteristic in Rett syndrome and obviously problematic like effective communications, lack of hand use, difficulty in walking. But you see that like seizures are #2 and you see constipation is #5. So constipation is a significant issue for families with Rett syndrome. So we'd like to have outcome measures that reflect these things that are concerning to the families. Next problem. Next slide, please. So what kind of outcome measures do exist that we've helped developed in the natural history study? Well one is the clinician reported Clinical Global Impression scale. Now Clinical Global Impressions scale or CGI, are the clinicians' overall default view of a participant's global function. And they're split into 2 different scales, both of them on a 7-point Likert scale. One is the severity. So what is the current situation, how severe they are. The other is improvement. So if you see somebody at a later time, have they gotten better from where they were in the past. Now these have been around since the '70s and they've been used a lot in psychiatric disorders, and they are understood and accepted by the FDA and the EMA. One of the issues, though, is that the way these are written are very generic about the severity and also the improvement. So it's very important to have greater reliability established. And one of the ways to do that is have disease-specific anchors, and we develop disease-specific anchors for the CGI. And we also developed rater training systems where we have written vignette with gold standard scoring. And having that rater culture is really critical to be able to have a strong clinical trial. I think that video-based vignettes would actually improve our rater training and reliability assessment. Next slide, please. We also have developed a new clinician reported outcome measure using this natural history study. This was data -- this was based on something called the Rett syndrome motor behavior assessment scale. And we did formal psychometric analysis on this. And that -- with the factor analysis, we created 5 factors that had a lot -- made a lot of sense clinically, if we look at their motor dysfunction, functional skills, social problems, aberrant behavior, Rett syndrome specific behavior. And we created a total store of those 21 items plus 3 other items that didn't load into any factor, but we think were important because there are issues in Rett syndrome like stereotypic hand movements, truncal rocking and seizures. This overall score had reasonable internal consistency and the factors correlated well with parent reports on those clinical domains, so it really showed that had [indiscernible]. Next slide, please. We're able to show on upper left that it has -- it correlates of age, it seems to increase with age. And on the upper right, it correlated well with another rating scale, this Rett syndrome clinical severity scale that we've used in the past and clearly very strongly with that. On the bottom left, you see that -- we could see a genotype/phenotype relationship with the mild mutations having a lower or less severe revised motor behavior assessment. And the severe mutations have a, on average, a higher score. And finally, on the bottom right, we show that it shows a nice progression of that clinical global impression scale severity. And again, in that high numbers are more severe. And you see that the stair steps up as you move up the Clinical Global Impression scale severity as we would hope it would, with it correlated with overall severity. Next slide, please. So we're currently looking at the natural history study data to look at the trajectory of this measure over time. This is -- and especially by mutation. So on the bottom left, this is showing one particular mutation, T158M of all, and you see this sort of progression with an increase but sort of a natural logarithmic progression. And on the bottom right is actually the individual people within a shorter time frame, with that same mutation but showing the individual values. And what you can see is sort of a pattern of growth. And that's what we'd like to do is develop growth curves with confidence intervals for different mutations so we could be helpful for doing power calculations in clinical trials. Next slide, please. And I'm currently working on establishing intra-rater reliability test/retest, external validity. And we're doing this with a video-based system where we're videotaping a structured exam and having people view it to be able to look at that intra-rater reliability. But with that video system, we want to develop a video-based rater training and reliability platform. And of course, an important part is trying to understand the sensitivity and responsivity to establish the minimally clinically important differences. Now that can be done by a distribution base, which is easy, but it's based on assumptions of clinical relevance. Or an anchor based, which is really tied to clinical meaningfulness. And so in the natural history study, we also have the caregiver, give an impression and improvement over the previous 6 months. So that's a good anchor to be able to look at change in scores if the caregiver thinks there is improvement or got worse. We also then look at the changes in the Clinical Global Impression scale of severity or the Clinical Global Impression scale of improvement, which would be during clinical trials, which has been used and the motor behavior assessment has been assessed in clinical trials. Next slide, please. So I just spend the last bit on the idea of biomarkers, which are also extremely helpful for clinical trials. We'd like to have biomarkers that reflect disease severity that might identify treatment responders, or have the early predictive biomarkers of treatment response that can be incorporated in a clinical trial to get an early signal of efficacy. Next slide, please. There are many different ways to approach a biomarker. What I'm going to talk about today is the use of neurophysiology or EEGs, and specifically on evoked potentials. And this is when you provide a stimulus. And in this example, a visual stimulus of a flashing grid, and you record the brain waves, especially in the back of the skull, which is the primary visual cortex. And on the bottom right, what you can see is you get a pattern of a brain way when you provide many, many similar and you average them together. And these have distinct peaks. Next slide, please. So in the natural history study, we did a multisite study of these evoked potentials in a Rett syndrome, using both visual evoked potential and auditory evoked potential where it's clicks of sound. And what we found, if you look on the top left, that's the visual evoked potentials and Rett individuals are shown in blue and typically developing individuals are shown in red, that the Rett individuals had a smaller peak throughout. And this was true in the bottom left of the auditory evoked potentials too. The peaks are smaller, the amplitudes. And this amplitude in both the visual evoked potential and auditory evoked potential correlated with clinical severity, and that's shown on the 2 right-hand graphs. Next slide, please. And -- it's very important to have -- to be able to do this at multiple sites, and we were able to demonstrate that we could reproducibly get good data at multiple sites. But it's also important to know that there is consistency in recording for the individual, for a subset of individuals that came back a year later. And we were able to show that there was a very good reproducibility of this particular waveform. Since on the top, it's the visual evoked potentials. On the bottom, it's auditory evoked potentials. And what you see is in the first year was in black, in the second year is in blue, and there's a very nice overlap for the individuals. So the individual waveforms were the same. So that's very important, again, for a clinical trial because we'd like that to be something that's reproducible by the individual so you could really track it over time. Next slide, please. And my lab has now gone and tried to really characterize very similar type neurophysiologic features in the animal model of Rett syndrome and female mice with a particular common mutation. And this is auditory evoked potentials. And you see on the upper left where the Rett syndrome mice are in red and the wild-type mice are normal mice are in black, that they also have this decreased amplitude. And what we were able to show in the bottom left in the D is that this change seems -- these decreased amplitude was more present in older animals and was not present in younger animals, so it had a progression. It also correlated with the clinical severity shown in Panel E and correlated with the amount of epileptiform discharges. So this is very good, because now this is what we consider a truly translatable biomarker where it's something we can observe in mice and use a very similar stimulation pattern process, and we can do the same thing in humans. So this allow us to do preclinical studies incorporating this to help guide our clinical studies. Next slide, please. And so now we're going forward. Really, the key experiment that we're trying to do is the Adrian Bird experiment when we turn the gene back on after the animals are symptomatic and monitor that neurophysiological features before and during and after the rescue. And you look at how it correlates with the improvement in phenotype -- phenotypic features. Next slide, please. So I'd just like to thank -- I'd just like to acknowledge the people who were part of the Rett syndrome consortia. As I mentioned, Alan Percy, who is the PI at University of Alabama in Birmingham. And I just acknowledge Eric Marsh, who's at Children's Hospital of Philadelphia, who led the neurophysiological characterization I described. Next slide, please. And of course, I want to thank all of my friends, the people who have Rett syndrome who have really taught me what I know about this disease. Thank you, and I'd be happy to take your questions.

Thank you, Dr. Neul for your presentation. I will now open the call over to our Q&A session. Your first question comes from Sami Corwin of William Blair. Given Rett isn't a neurodegenerative disorder, to what extent do you think Rett symptoms could be reversed? And what improvements or developmental retention would be the most meaningful to patients and caregivers?

Yes. That's a great point. It is not neurodegenerative as far as we can tell, that we don't see any signs of neuronal death in Rett syndrome. And the animal work suggests that there's a great potential for reversing symptoms. As I mentioned, they -- in the male mice, when they were symptomatic, they turned the gene back on and they did not die. And in the female mice, they turn the gene back on even at a year of life, and they got better. Now of course, until we start moving into human work, we're not going to really know how wide the window of opportunity is, right? And we hope that the window of opportunity is very wide, meaning even adults with Rett syndrome would be able to show clinical improvement if we could provide something like turning the gene back on or a gene therapy or other approach to restoring function. So I think we're hopeful that it may be very wide. But until we really do the work, we are not going to know for sure. Now with regards to -- what would be the most important things, I think that from my perspective as a clinician, I have my own ideas, but I think that those top concerns are a very good starting point to think about what would be matter to the families. And I think the next presentation from Monica Coenraads, which is going to be talking really from a parent/caregiver perspective, really is very important to understand. That's important for us to know, because I think that's what really guides what we want to improve is what the families find most meaningful.

Thank you for that. Your next question comes from Gil Blum of Needham & Company. Are there any particular genetic risk factors which may contribute to the appearance of Rett considering it appears de novo?

That's a great question. And as far as we know, there are not. It seems to have a very consistent epidemiology across the world when it's been studied well in various populations. It doesn't seem to have any bias by any racial or ethnic or regional differences. So I think that most of the mutations, especially those common mutations, the molecular mechanism is a very common mutagenic mechanism that occurs during spermatogenesis. It actually occurs very frequently for spermatogenesis across the genome. It just when it happens in the MECP2 gene, it causes Rett syndrome, which is problematic. So I don't think there are increased genetic risk factors. I think there may be genetic factors that might modify the degree of severity that is observed, but that's a little different than increasing the risk of getting the mutation itself.

Okay. Thank you. Your next question comes from Kristen Kluska of Cantor Fitzgerald. When looking at the specific genotypes of the condition and the mild and severe classifications, do the mild patients tend to show most of the clear disease stages but a lot to a lesser degree? Or are there specific phenotypes they typically don't experience?

So when I talk about that, that's all talking about people who have classic or typical Rett syndrome. And people who have that, they have the same progression. The degree of severity of some of the problems may be different and not as marked. And one of the things is that people who are most more mildly affected in that sort of global fashion, which is oftentimes reflected on how much language or hand use or walking the [ ramp ], the more motor function aspects actually display higher levels of the behavioral problems. And so whereas people who are very impaired don't have the same degree of motor problem -- I mean, behavioral problems. So that's one thing that is different. And there is some differences in terms of how marked -- how severe the seizures are. They seem to be more severe in certain mutations than others, but that doesn't mean that the people who have those other mutations don't -- aren't at risk or have a high degree of seizure burden because overall, they do. But let me just -- sorry, let me finish this off. So as I mentioned, that's classic or typical Rett syndrome. There are people who have disease-causing mutations in Rett syndrome who don't quite meet all of the clinical feature -- the clinical criteria for classic or typical Rett syndrome and are considered to have atypical Rett syndrome, and they're typically mild or more severe than a typical Rett syndrome. And then there are also people who don't meet the criteria for Rett syndrome. They didn't ever have a regression. But they might have just autism or pervasive development and disability, not otherwise specified. I mean we don't really understand that degree of the variation of what might be contributing to that amount of variation.

Your next question comes from Eun Yang of Jefferies. Clearly, early the prevention, the better outcomes. However, with the different stages of disease progression, can you discuss what would be the most clinically meaningful endpoints for each stage, development stagnation, rapid regression, stationary stage and late motor deterioration.

Yes, that's a good question. And I didn't show this, but we've looked at those top concerns, broken out by age. And there are some differences in what the families report as top concern. The very top level concerns are pretty consistent across, but -- so seizures are something we don't really observe until more school age, 5 to 12, for an onset and then may be worsening during the teen years, but then lessening in the adult years. So seizures is something that changes its ranking based on age structure. So there is some differences that concerns based on age or developmental -- those stages, I mentioned. But some of the top ones remain consistent like communication, hand skills date.

Great. Thank you. Your next question comes from Raju Prasad of William Blair. From a natural history perspective, what clinical endpoints would be the best to determine benefits over the course of 12 to 14 months? Also given the number of dysfunctions that onset in the 2- to 5-year time frame, is there a cadence of age enrollment groups you'd suggest?

Yes. So I think that sort of getting back to what we're saying about the outcome measure exactly, I think that outcome measures that we'll hit upon the top concerns and especially some of the functional aspects of the disorder like the hand skills and the spoken language and the walking, probably are the things that should be built into clinical trials. Just -- remind me of the second part of the question.

Yes. Given the number of dysfunctions that onset in the 2- to 5-year time frame, is there a cadence of age enrollment groups you would suggest?

Yes. So that is a good point that there is concern -- a lot of clinical trials that have been done so far in Rett syndrome have gone over 5 years. And part of that is because the timing of things and during that regression phase, a lot's changing. So that's why a lot of clinical trials in the past of that have happened in Rett syndrome have generally started after that regression phase. When we start thinking about marked disease reversing or modifying therapies, there definitely is a thought that eventually, we definitely like to try to get the center early age. And so we have to think about what would be in a situation where things are changing more dramatically what would be an outcome measure. And that's one thing I think I sort of touched on who was -- came from the natural history with the developmental history kind of aspect of what's the expected time that skills are might be gained in Rett syndrome? And what's the expected time that skills might be lost? So can we think about if you're very early -- if you do a very early intervention that the expectation is that you might be losing skills at this time rate. And you say, well, now we can say, if we haven't lost them by this time, we've modified the progression of the disease.

Great. Thanks for that. Your next question comes from Raju Prasad of William Blair. Are there some MECP2 mutations that are more prevalent than others or even one that is predominant?

Yes. So there are the 8 hotspot mutations, and they account for 65% of all disease cases of typical Rett syndrome. And within them, there is a ranking order. The most common mutation is R168X, one of the mutations. And the next one, very close is T158M. So we do know that pretty well. There's -- as I implied, there's a much longer tail after you get past that initial 65% at the top 8. But you have another group that are large deletions and they account for 10% of people with Rett syndrome. And then there are a number of mutations that affect the later end of the gene or the carboxy-terminus. And when we group those together, they all are generally frameshift mutations that accounts for almost another 10%, too. So between the common and the large lesions and the C-terminal truncations, we -- that basically accounts for about 85% of the disease cases of Rett syndrome.

Great. Your next question comes from Evan Taddeo of Guggenheim Securities. Given the difference in clinical severity based on mutation status, would the underlying genetics alter the treatment paradigm in Rett's, for example, timing and dosing?

Potentially, there is some aspect of that and there's been papers published, including by myself, that some of the common mutations which retain some of the MECP2 function might be more susceptible to the overshoot when you add the normal copy. I think that, that having the built-in sort of [ rear step ], the auto regulatory thing, is a key part to help overcome that sort of challenge of these partial loss of function mutations that retain some function being more susceptible that now you can sort of titrate the amount of MECP2 function a little more precisely.

Okay. Thanks for that. Your next question comes from Gil Blum of Needham & Company. Are there areas in the brain that require more MECP2 expression than over to reduce than others to reduce phenotype or is broad and uniform expression better?

Yes. It's a great question. And there's been a lot of work done doing conditional knockouts and conditional rescue of the MECP2 gene in different brain regions and different [ fixed ] cell types. And it does seem that MECP2 is predominantly required in the central nervous system. Its highest level of expression is in the neurons, but it is expressed in the astrocytes, too. And there is a role from MECP2 in the astrocytes. And within the neuronal populations, people have shown a whole variety of different phenotypes being associated with loss of MECP2 function in particular cell types or restoration. Probably, there seems to be a special role from MECP2 in the GABAergic or inhibitory system, but there is clearly still a role for MECP2 in the excitatory or the neuromodulatory neurons, too. So I think that you might be able to get some targeted reexpression in specific regions that might improve some things, but I think if you want to see an overall improvement, you probably need a fairly wide distribution of MECP2. Now what I mean by that, it doesn't mean that every single cell in every region, but because it probably is the amount of cells you can get some cells that have a normal amount or some amount and that might improve the overall neuro circuit function. Now if you got all the cells, it'd probably be the best, but I think you still can get benefit by having some cells.

Okay. Understood. Thank you. Your next question comes from Yun Zhong of BTIG. Some clinical studies have used Rett syndrome behavior questionnaire and Anxiety, Depression and Mood Scale. Are there still -- are they still suboptimal efficacy endpoints?

I think that they are -- they have been shown to have validity in Rett syndrome. They -- the ADAMS was mentioned, that is some anxiety, depression, mood and those are issues in Rett syndrome. But as I mentioned, there are really primarily issues in the less severely motorically affected individuals. In the Rett syndrome behavior questionnaire, despite the fact that it has a behavior in its name, it isn't only behavior questions, but it is more biased towards behavioral questions. It does have some motor function, communication aspects of it, but it's more biased towards the behavior issues. As I pointed out in the top concerns, the behavior -- I didn't get to it, but behavior issues are lower on the top concerns. So I think the issue is that you really like to see something that will capture more of those things that are top concerns. I think Clinical Global Impressions Scale when used correctly, with good rater culture and reliability has a good opportunity to capture a very broad aspect of things and hopefully capture really the key things that are issues in Rett syndrome.

Great. Thank you. Your next cotton comes from Laura Chico of Wedbush. What proportion of your patients would be candidates for gene therapy? What qualities would make them most suited for this type of treatment?

If the treatment works and is safe, probably all the patients would be -- all the patients who have MECP2 mutations, right, and so that would be 95% to 97% of the individuals with typical Rett. So I would think all of them would be candidates. In terms of are there specific characteristics and features, part of that is going to be driven by the risk-benefit ratio. So there may be a different calculus that goes into somebody who is very, very mildly affected. So there are some individuals with Rett syndrome. These are exceptional, but actually can speak in sentences and can tell stories and can write their name. They had a loss of skills, but they have a lot retained or regained. And so if that's what I mean about the risk benefit, if there is still risk involved, there may be a question for somebody who is doing relatively well, will they tolerate risk. Where as opposed to the people who are more severely, if I could, which is, by far, the majority of the people with Rett syndrome, I think the benefit probably outweigh the risk in those cases.

Thank you for that. We'll take 1 final question from Yun Zhong of BTIG. Given the heterogeneity in mutations and genotype phenotype correlation, do you see the need to specify mutation types in patient population in clinical studies to increase the chance of success?

Well, it's kind of what I was getting at, and we haven't done it yet, but I'm hoping we will get this data, some of these things done soon. I'm working with RTI in North Carolina. And it's those mutation-specific growth curves of that specific measure, the revised Motor Behavior Assessment score because I think that by having that and being able to put confidence intervals around that, that gives us the ability to know what a natural trajectory would be and then be able to say what would be better. If we move that, what percentiles movement would be better. And that might be driven by those differences in the different mutation groups that have a different pattern. So I think that could be very helpful. Now we can really only do that well for those common mutations or the large groups of mutations together. So I'm talking -- I think I'm answering your question, but not exactly get into it, but I think that the -- I don't think it would mean that you wouldn't want to include people with other mutations. I think it just means that you might want to make sure you're capturing enough people with the type of mutations that we can have that information on, which based on just the epidemiology, the prevalence of them, they're going to be the more common people who are present in trials because they're just more common individuals who have Rett syndrome.

Great. Thank you, Dr. Neul, for your incredible insights and for participating here.

Thanks for having me.

Yes. Thank you. I'd now like to turn the call over to Monica Coenraads, the CEO of Rett Syndrome Research Trust, and she will be discussing -- giving a patient and family perspective on disease burden. Monica?

Thank you. Good morning to everyone. Thank you, Kim, and thank you to Taysha for the invitation to participate today. Thank you to everyone who's tuning in for your interest in learning more about Rett syndrome and Taysha's program, and thank you to Jeff Neul for laying the groundwork for my presentation. So my daughter, Chelsea, was born 25 years ago, eagerly awaited after almost 10 years of marriage, first grandchild on both sides of the family. She had a very typical first year of life. And we only started becoming concerned about her development around the first year of life. Next slide, please. I'm going to show you here some of the early signs. We had started creating a real nervous butterflies in our stomach that something really wasn't right. So in the first slide, top left, you'll see that we were trying to teach Chelsea to crawl, and she just wasn't getting it. We spent a lot of time on the floor with her and coaching her to crawling, but she wasn't getting it. The slide next to that one, we're trying to teach her how to take some steps. And again, she just wasn't getting it. She didn't have the coordination. And at times, she also seemed quite scared about taking step that we're not in [indiscernible]. On the bottom left, you're going to see kind of a repetitive rocking motion that Chelsea began to do. And it became quite continuous anytime we put her on the floor, she would do the rocking motion. And you can also see -- saw earlier some of the hands flapping. And then the last video we're going to see the beginning of some of the hands motions that are really the hallmark symptoms of Rett. So by this point, she had lost the ability to feed herself, to pick up Cheerios, she had lost her pincer grasp. She couldn't hold a bottle anymore and she -- her hand function was completely gone by 20 months, and it was never regained. Next slide, please. So she received a clinical diagnosis at the age of 2, the MECP2 gene hasn't been identified yet. So the diagnosis was based on symptoms and presentation of her symptoms. A year later, [indiscernible] identified MECP2, which the cause of Rett syndrome, and Chelsea was one of the first children to be diagnosed to have the T158M mutation, which is one of the most common ones and represents about 10% of the patient population. It's typically regarded as a "middle of the road" mutation, not the mildest but not the most severe. But in her case, Chelsea has a fairly severe presentation. So even with children that have the same mutation, they can present quite differently from each other. So although as Dr. Neul mentioned, Rett syndrome is not considered degenerative, right, brain cells aren't dying, it is progressive. And I'm going to show you over the next 2 slides kind of the progression of Chelsea's ambulation, which has gotten worse over time. She's never walked independently, but she could walk quite well with assistance. So first slide on the left. This is Chelsea at about 3 years, walking quite nicely with a walker. And then the top video, this is Chelsea at around 10. At this point, she could walk around the block where we live, which is about a 20-, 25-minute walk. The last part is uphill, and she can do that quite well. We do it every day. She needed assistance for balance. She never had balance on her own. And then the bottom video is Chelsea at about 15 or 16. She's requiring more assistance to keep her upright. She's wanting to kind of lean forward all the time. Her -- she's become more stiff. She's developed more Parkinsonian symptoms. She's toe walking more. And so overall, it's become more difficult than actually more dangerous because we were worried that we would fall. Next slide, please. In the slide on the left, you'll see Chelsea in a different kind of walker, which is requiring a harness to kind of -- to her upright and there's a little seat there that she's kind of sitting on, but she's still moving her legs on her own although as you can see, it's just -- it's not as natural flowing of the gait that she had when she was younger. And then on the right-hand side, you'll see how we walk with Chelsea now, which is on a treadmill. We actually have to pull her legs with a stair bands around her knees. But it's still a way to keep her upright, to keep her moving and to try to keep her in the best shape as possible while the treatments like pace move forward. Okay. Next slide, please. Dr. Neul mentioned the autonomic issues that many of these children have and Chelsea has disordered breathing, which is marked by hyperventilation and breath holding started at the age of 3. She's had it ever since, although the intensity of it can change over time. And I have 2 videos here that will show the breathing. The first one is more of a hyperventilation, very deep breathing. And you'll also hear teeth grinding, which is not the most [indiscernible] to hear, but -- and then the bottom video will show you a breath hold. So it's one thing to hear about -- to read about breath holding and a science paper or to read about it. It's another thing to hear it and to live it. Imagine breathing like that all day long every day. It makes everything difficult. You can't focus on anything. She had trouble eating. She had trouble falling asleep. It's loud for going to church, going to a movie theater. It's very difficult. Other autonomic issues that Chelsea suffers from is severe constipation. She's had problems with urine retention. She really cannot bite or chew anymore. She has trouble swallowing. Most of her food comes through a G tube. She gets a little bit of blended through just to give her the joy of tasting, but it's become more problematic and more dangerous if she's gotten older because she aspirates more [indiscernible], meaning that fluid retention of the lungs and aspiration, pneumonia. She also suffers from cold feet, cold hands and she has long QT syndrome. Next slide, please. If I had to describe Rett syndrome in 2 words, I think I would describe it as the movement disorder. There are so many movement issues that these children have and the families grappled with. And that often are misdiagnosed and mistreated if you go to physicians that don't really understand Rett syndrome. So I got a couple of examples here. The top one is a tremor that can sometimes be quite severe. And beyond the tremor, if you are sitting next to Chelsea, for example, on the couch and you have her arms around her, you will feel her muscles twitching and spasming almost all the time. The lower left video shows kind of a head nodding, almost Parkinsonian-like tremor, that has also become more prominent and more problematic. And then the right video shows what's called an oculogyric crisis. And it's basically where she loses control of her eyes and the eyes are pulled upwards. And I find this to be particularly difficult for her because one of the only things she does have control over is her eyes and she uses her eyes for communication. She uses them to show what she might need. She uses them for yes, no flash cards, how to answer questions. So to have the loss of eye control, is really pretty tragic. Fortunately, this is not something that happens all the time. Sometimes we go weeks without it, and sometimes she has it every day but not the entire day. Next slide, please. Chelsea started having seizures at the age of 5. We tried many medications. They remain intractable. We had almost grown to just accept them and live with them. They were quite terrible when she was young because she has multiple seizures in a particular day and then spend the next 2 or 3 days in bed recuperating. Now the seizures typically she'll have one, she gets over and kind of continue with her day, and we have become quite just used to them. However, in this last Thanksgiving, she had a seizure like hundreds that she's had before that resulted in respiratory failure. I was keeping her alive by blowing into her mouth while we waited for an ambulance. She ended up on a ventilator for 2 weeks, collapsed lungs. This is a picture from the first night, Thanksgiving night in the hospital, in the ICU where her blood pressure was extremely low and the doctors did know whether she would make it through the night. That has made every seizure that she's had since kind of traumatic because we're just hoping that we don't end up in a situation like we did Thanksgiving night. Chelsea also has scoliosis and kyphosis. She's not needed any surgery yet. She gets a lot of therapies, massage therapy, chiropractor, physical therapy to try to keep the scoliosis from progressing. Her tone has become very high as she's gotten older. She gets BOTOX injections regularly 12 weeks to weaken the muscle or to lessen the tone. She has severe anxiety issues, sleep issues and continues to grind her teeth, which is a noise that after 25 years, I still haven't gotten used to. Next slide, please. In general, individuals with Rett syndrome requires a 24-hour daycare, that includes individuals that would be characterized as mild. They still only help getting dressed, hygiene, feeding and every other aspect of daily life. There are frequent hospitalizations. Sometimes with surgeries for orthopedic issues, things like scoliosis surgery, G tube replacement, feet surgeries, tendon releases. Oftentimes it's for pneumonia or seizure problems. There are lots of medications. They require special education, therapies throughout life when they're in school typically the school districts pay for it. When school ends at the age of 21, so does access to therapies and oftentimes, families are left to pay for these on their own, which in any case is quite cost prohibitive. They require, in many cases, lots of durable medical equipment, wheelchairs, walkers, standers. Of course, if they're in a wheelchair, they need a wheelchair band. Sometimes we need modifications in the home and home health aides and nurses often required as well. Next slide. This is an example of the medications that many of the children are on, anti-seizure meds, reflux, constipation, anxiety, depression, antipsychotic meds, osteoporosis, heart medications and movement disorder drugs. Chelsea is on valproic acid and Lamictal for seizures, primidone for the tremor, propranolol for the lungs. You would see baclofen and BOTOX for spasticity, chloral hydrate and melatonin for sleep, clonazepam for anxiety and more recently, especially since our last hospitalization and prolonged time on the ventilator, she's developed respiratory issues and has lung damage. And so she's now on Atrovents, inhaled steroids in a percussion vest, and that's part of the daily protocol, which has helped. Next slide, please. In terms of therapy, the mainstream ones are physical therapy, occupational and speech therapy, which every child gets in school. And then many families, if they're able to afford alternative therapies, things like craniosacral massage, hydrotherapy, hippotherapy, chiropractor and music therapy. These help -- they help keep symptoms at bay, but they don't really dramatically impact the [indiscernible]. Next slide, please. This is a picture from about 10 years ago, I need to update it. This is Chelsea's stuff in the Chelsea's bathroom, which as you can see in the background, she has raised bathtub and massage table where we change her. I couldn't fit all of her stuff in one room anymore. For many families, including myself, at times it can be quite overwhelming to care for a child with someone with [indiscernible]. Next slide. In terms of education, this is Chelsea, when she graduated high school, pictured with her brother, who's 2 years younger, but graduated at the same time and Chelsea's teacher, who was with her for 7 years. Chelsea did not attend classes for most of the academic subjects. So in her case, her diploma is really a token, honorary diploma in the sense. She did attend some of the more fun classes like music and art, and she is able to learn. But for so many years, she had difficulty, her mental difficulty and it really is difficult to attend school and attend classes and learning the way her typical peers for that. Next slide, please. We've tried to make life as normal for Chelsea as possible, although as you can imagine, it's quite difficult. She's had opportunities to try adaptive skiing, her brothers and her dad are big skiers and that was a highlight for everyone. Adaptive waterskiing, she has an adaptive beach wheelchair. She went to the prom. But to be honest, these pictures show where instances where the planets and the stars align so that she can enjoy these activities. Next slide. Family life, in our case, has been one of conquer and divide. My husband has planned activities and sports, athletic endeavors like competitive skiing and BMX-ing and mountain biking with the boys, and that's meant that Chelsea and I were often home. So not ideal, but what we needed to do to give up our boys each normal life as possible. Next one. This is my final slide. And it's just a term that I'm sure everyone has heard, patients are waiting. Patients and their families are waiting and we're wishing Godspeed for the Taysha program and other programs that are attacking Rett syndrome factor, which they're sorely needed, the unmet need of fixing. Thank you for your attention, and I'll turn things back to Kim.

Thank you, Monica, for really sharing your story and Chelsea's story and journey with all of us and reminds us -- and stories of others affected by Rett, reminds us that Taysha of what we do, why we do what we do. So thank you again. I'd now like to turn the call over to Dr. Steven Gray. He is an Associate Professor in the Department of Pediatrics at UT Southwestern and Head of the Gene Therapy Vector Core and our Chief Scientific Adviser, and he will be reviewing the preclinical data for TSHA-102. Steve?

Thank you, Kim. And I want to start off by just giving Monica, a very heartfelt thank you. This has been a long journey that I've had, starting with Monica really about 14 years ago when we first started this attempt to try to develop gene therapy. And as Monica says, the patients are waiting. In the case of my involvement in Rett, they've been waiting 14 years so far. And it's -- I think it's very difficult that the road has been this long, but I think we're very excited about the possibility that we're finally getting the reagents and the approaches together to address this disorder. If you can go to the next slide, my role here today is to give an overview of the gene therapy vector design, how it works, what some of the supporting information is that are driving this product forward. And I'll start off here with the -- basically an overview of the vector design. And this is -- it's all packaged in AAV9 capsid. The -- it utilizes a fragment of the endogenous MECP2 promoter to afford some level of cell-specific regulation. But the real kind of important component of this is this miRARE element, which was designed to provide a true feedback mechanism of regulating MECP2 expression. And as Jeff pointed out, the challenge of treating Rett syndrome with a gene therapy approach is to try to treat the null cells, the disease cells while not inducing any kind of overexpression, detrimental effects in the mosaic cells in the patients that are normal. Facing an incredible challenge from a gene transfer perspective, but I think that the miRARE element is really the first instance of being able to provide this type of regulation. And then we package this with a miniature version of MECP2 gene in order to fit these regulatory elements. So if we go forward the slide, I want to -- really this miRARE element is the key component of this. So I want to make sure that it's clear how this works. The miRARE element is a tandem series of binding sites for cellular expressed microRNAs. So there's no expression of microRNAs off the vector. The only thing expressed off the vector is the MECP2 gene. This miRARE element is simply a -- basically a responsive element to signals that are coming from the cell. So if we can think about how this works, the viral genome enters the cell, enters the nucleus and then we can start getting expression of the MECP2 transgene that will include this miRARE element in the RNA message and that's in #2. And then this gets translated into MECP2 protein, the MECP2 protein exerts its effects throughout the cell. And as a normal feature of MECP2 biology in the cells, there are certain microRNAs that are expressed in the cell that end up being that are responsive to MECP2 levels. So this is the case naturally. If you were to compare a cell devoid of MECP2 with a wild-type cell, you would see that these microRNAs are expressed higher in a wild-type cell than in MECP2 null cell. So anyway, MECP2 has expressed these microRNAs in the cell elevate. And then as microRNAs can come back and bind to the transgene mRNA and suppresses expression. And so this would be basically a cellular mechanism that we're taking advantage of to make our transient mRNA responsive to those signals that are coming from the cell. And so you can imagine that this goes into a wild-type cell where you already have MECP2 expressed. These microRNAs are already regulated, and it would inhibit the transgene. But if this went into a null cell, disease cell, then it would permit a level of MECP2 expression. But if that expression ever got too high, then it would suppress the transgene only. So this is a mechanism to regulate MECP2 on a cell-by-cell basis, not -- rather than in some kind of global fashion. So if we go to the next slide, this was a design -- this is a novel design. This is the first time that I'm aware of that this type of design has been created to make something -- have a true feedback mechanism of regulation based on microRNAs. And it was generated through a combination of kind of empirical studies where we deliberately overexpressed MECP2 in mice using AAV and looked for microRNAs that were upregulated. And then this was combined with the bioinformatics approach to try to look for patterns and endogenous gene expression and some of the untranslated regions to look for a degree that this might be, to some extent, a natural regulation in the cells to regulate mRNA expression for dose sensitive genes. So it was really this combination of bioinformatics and empirical investigation that culminated in the selection of these microRNA binding sites. And I should say a couple of features, we kind of screen these and focused the selection to identify microRNA binding sites that are 100% conserved between mice and humans. So this should be a regulation mechanism that would be -- that should function in a similar way in rodents as it would in primates and humans. So if we go to the next slide. If you want to look in more detail on how this miRARE element was developed, it's all detailed in this publication that came out this year in Brain, where we described the development of this. So I'm going to take you through a few slides and kind of show pieces of data from this publication that are being used for ultimately TSHA-102, this vector that's proposed to go into humans. So if you go to the next slide. This is just an initial look to try to see what degree of down regulation that the miRARE element could confer. So this is just a study in wild-type mice, where we used an AAV vector to deliver the mini MECP2 gene. 2 vectors injected in parallel. One has the miRARE element and one does not. And so the left panel is just quantifying the amount of vector DNA that is going to different regions of the nervous system, brain, cervical cord, thoracic, spinal board, lumbar spinal cord. So you -- so both vectors are going to the brain to the same extent as we would expect. But then if you look at the expressed RNA, then you will see that the nonregulated vector in green is expressed at roughly 10x higher levels than the vector with the miRARE element, which is in blue. Showing that there is in wild-type mice, there is substantial down regulation that is conferred by the miRARE element. So if we go to the next slide, that's looking at RNA levels. We wanted to look at this in more detail on a cell-by-cell basis, and this was using immunofluorescence in histology sections. And we took advantage of a myc tag that was added to the mini MECP2 team. This is a little complicated, but it's important data where, again, we had these parallel vectors of one version that had the miRARE element and an exactly similar version that did not have the miRARE element. So -- and then we dose this into wild-type mice, the knockout mice. And again, we're looking for cells that express the transgene. So if we look at the nonregulated vector, which is in wild-type mice in gray and knockout mice in blue, and then you'll see across these different regions of the brain, that we're basically expressing the same amount of MECP2 in wild-type mice and in knockout mice because we don't have this feedback regulation. I'm going to contrast that with the mice that received the miRARE vector. And this is, again, wild-type mice are in green, knockout mice here in yellow. And if you look across these different structures, there's 2 things that I want to point out. One is that the expression in wild-type mice that already have endogenous MECP2, the endogenous microRNAs are already elevated, then you see substantially less cells expressing the MECP2 transgene. So you can see that in the green bar that's lower across 2 different brain regions. We can contrast that with -- if this is dosed into knockout mice where they don't have any endogenous MECP2, then it actually permits the expression of the MECP2 transgene, and we see it expressed in the knockout mice to similar [indiscernible] as we see if there's no regulation of the transgene. So again, simplified the miRARE element permits expression in knockout cells and it suppresses expression in wild-type cells, which is exactly what we wanted. So if we go to the next slide, this is just to give you kind of a visual picture of this, where we're -- this is the types of images that we quantify. And so you can see again in wild-type mice and knockout mice without where it does not have this miRARE element, then we get similar levels of expression. But then the wild type, you see -- you can visually see the repression of the transgene that's occurring due to the miRARE element. So if we go to the next slide. This is -- again, this was a -- the design of this vector was really the culmination of about 14 years of research on trying to develop gene therapy approaches for Rett syndrome. This started with a publication with Stuart Cobb's group in 2013 with some initial vector designs that showed some potential that gene therapy could improve MECP2 knockout mice. We followed this with a pair of publications in 2017 with newer versions, sort of newer iterations and incremental improvements in the vector design, which highlighted -- and these studies really highlighted some of the dangers of associated with gene therapy for Rett syndrome, where we documented a number of adverse effects that could happen if you're driving unregulated expression of MECP2. And then that culminated again in this paper that we published this year, published with Sinnett that details the effects of inclusion of this miRARE element. And this was the first instance where we really saw that we could achieve a level -- a benefit from MECP2 gene transfer that wasn't associated with any strong toxicities. So if we go to the next slide, I'm going to walk you through -- I kind of showed you some of the molecular and histological evidence for how -- and the mechanism for how this miRARE element creates this feedback loop of regulation. But the proof is really in the functional behavior survival studies in the animal models. And so I'm going to start off with safety because I think any approach of gene therapy for Rett syndrome really has to start with safety. If we can't manage this problem of MECP2 overexpression, then I think it's nearly impossible to drive something forward. So this is an initial study in wild type mice. So this is essentially a safety study. And you can see that the -- in blue and in green are high doses of MECP2 vector that does not contain the miRARE element. So it does not have this autoregulatory feature. And in wild type mice, these are dosed about 4 to 5 weeks old. And you see actually death of these mice and ultimately about a 40% mortality with mice receiving high doses of an unregulated MECP2 vector. We can contrast that with the miRARE vector, which is in red and in orange. And this was a version that we published that contains a myc epitope tag. But you see that there's essentially 100% survival of these long term. So from a survival aspect, this vector is safe, whereas an unregulated vector really is not. So if you go forward another slide, this is kind of an extension of the safety study in wild type mice that instead of just looking at survival, we're looking at an aggregate behavioral score. This is a score of Rett-like behaviors. So higher on the scale is bad. And again, you see the nonregulated -- 2 different version of the nonregulated vector in blue and in green, where you see that these mice are elevating on the scale soon after they're dosing at 4 to 5 weeks old. And then they remain elevated on this scale. So we've basically taken normal mice, and we've induced adverse Rett-like behaviors. And we can contrast that with our regulated vector in -- the myc tag regulated vector in orange and red that track similarly to vehicle injection, which is in black. So if we go to the next slide, that's really evidence as far as the safety thus far contrasted against different iterations of unregulated vectors. The readout -- the primary readout with efficacy when you're screening any kind of gene therapy approach or really any therapeutic for Rett syndrome is to look at survival in the MECP2 knockout mouse model. And this knockout mouse model is used because it has a striking phenotype that shows very quickly in the mice. And ultimately, the most striking feature of this is that the knockout mouse model, which is a more severe model of Rett syndrome, has a median survival of about 9 or 10 weeks. And you can see that in the gray line here on this Kaplan-Meier survival plot where untreated mice die with a median survival 9 or 10 weeks. We can -- and so we can see that the miRARE regulated vector in orange gives a nice extension of survival, and that is equivalent to a full length MECP2 vector that we've published on previously in 2017, which is in blue. So again, it provides -- the regulated miniMeCP2 vector provides essentially the same amount -- level of survival as kind of our traditional benchmark of a full length MECP2 that we published on in 2017. So I think the evidence right now is that it's not necessarily more efficacious, but it does provide a clear benefit. And it does -- it provides this benefit in a manner that is coupled with a favorable safety profile, whereas any nonregulated vector that we've looked at really doesn't share that safety profile. So if we go next, then I believe that should wrap up my section. And I'll hand this back to Kim Lee to manage the Q&A.

Thank you, Steve, for detailing the preclinical data for TSHA-102 as well as the miRARE technology. I'd like to now open up the -- for Q&A. Your first question comes from Gil Blum of Needham & Company. "Is there any known role for these MECP2-driven miRNA sequences? Is there any risk in their sequestration?"

Sure. Well, these would be microRNAs that are -- that have various functions in the cell as far as normal cell biology. And -- but one thing is if the microRNAs get elevated in response to MECP2, as long as we regulate and we control MECP2 levels, then those microRNAs -- as long as the MECP2 levels never get above normal levels, then those microRNAs are never going to achieve any kind of supraphysiological elevations either. So as we really look -- you're looking at this kind of homeostatic regulation, where the levels of MECP2 and the levels of the microRNA are going to be -- end up balancing each other out.

Great. Thank you for that. Your next question comes from Raju Prasad of William Blair. "Can you talk about any different dosing considerations with a one-done gene therapy where you are expressing the miRARE elements?"

Well, one of the things if you're doing especially intrathecal administration of AAV9 is that the dosing is going to scale in terms of the numbers of cells transduced. So I think that this is a reason why the dosing gets pushed very high, and it would be pushed very high for things like Rett syndrome and other disorders. And the aim of pushing the dose high would be to deliver the gene to as many cells across the brain as possible because the number of cells transduced is probably going to translate to the degree of therapeutic efficacy. That's certainly what anybody would expect. I think the importance of including this regulatory mechanism is if you're trying to deal with some of these toxicities, these overexpression toxicities by just reducing the dose, then it's very difficult to do that because you're also reducing the number of cells and reducing the potential for efficacy. So I hope and I think what the miRARE element is doing is that it's permitting us to push the dose higher to transduce more cells but to still control for overexpression of MECP2.

Thanks. Great. Your next question comes from Kevin DeGeeter of Oppenheimer. "How should we think about the lack of expression of regulated vector in the hippocampus? And how do you measure the expression in other parts of the CNS?"

Yes. And I have to say, you can get very -- this type of analysis is very time-consuming. And so I just have to apologize from a practical consideration. When we went through the studies that we published, we focused on particular brain regions that would have been important for the disease. And we did a degree of quantification in those areas. But it's an ongoing analysis to expand that really across the whole brain. So I mean, we can look at images across the whole brain, and we can sort of qualitatively see the degree of regulation. But we -- for the purpose of that publication, we're really focused in on these particular areas. So we do get change transfer to the hippocampus. We do see expression in the hippocampus. It's just, I think, in that graph with the scale that, that didn't come out being quite as apparent.

Thank you, Dr. Gray. Your next question comes from Laura Chico of Wedbush. "In the mouse safety study, could you remind us how high the doses are in the mice with early death and how that would potentially translate to a human dose?"

So I believe that in those cases, it was a dose of 1x10^12 vector genomes total delivered into the CSF. So that was a dose per mouse. That is quite a high dose. In terms of translating that to humans, I think that, that's -- it depends on the metrics that you're using and whether you're scaling by CSF volume of brain mass, and there's different ways to do that. So I can maybe ask Suyash Prasad, if he wants, to weigh in on how these doses are being scaled to humans in the clinical trial design, but I might leave it to him.

So Suyash -- I mean, Steve, what I would just say, we kind of consider our approach to scaling from rodents to NHPs to humans as somewhat of a trade of secret. So I think we would probably stop short of answering that question. But Laura, we appreciate the question.

Thanks, Steven. Thanks, RA. Your next question comes from Sami Corwin of William Blair. "Does TSHA-102 have broad distribution throughout the brain, including deep brain regions? And how important is widespread transduction for therapeutic benefit?"

Yes. So I'll just comment on the mouse studies that we've published on that my lab did. And in that case, we do see with intrathecal administration of AAV9 that we do get broad distribution throughout the brain, including deeper brain structures. And in terms of what we need to target, I think Jeff Neul had commented a little bit on this. But ideally, we'd like to get it everywhere 100%. But what we can see, at least conceptually, benefits of targeting the brain stem, different benefits of targeting the cerebellum, different benefits of targeting different areas of the brain. And ultimately, we can kind of theorize about this quite a lot. But what we're trying to do is generate empirical data in the mice, looking at more detailed phenotyping of what's rescued, to what extent. And I think that they -- what we've shown so far in our publication in Brain is that we get extensions in survival and at least some trends towards benefits with certain motor functions. And so I'm going to just kind of stop where the data is that we have publicly right now. But we can certainly see benefits. We can conceptualize benefits based on where we're seeing the vector going. But ultimately, we want the animal models to tell us that and, in the end, the patients to tell us that.

Great. Thanks, Steve. Your next question comes from Silvan Tuerkcan of JMP Securities. "Thank you for your presentation. Could you please tell us what exactly makes miniMeCP2 different from the full length gene? And apart from the overall survival data, what other analysis have you done on the truncated gene?"

Well, I'm going to credit Adrian Bird, Sir Adrian Bird in Edinburgh, that it was really his group. And he's one of the foremost experts on Rett syndrome and MECP2 biology. It was his group that developed this MECP2 mini gene, and they published on it initially. They also generated -- well, and it is a considerably truncated gene. And the approach was to look at the -- there was a conserved area in the N-terminus and a conserved area in the C-terminal half of the gene that are -- and this is where the majority of all the mutations in Rett syndrome -- MECP2 cluster and the biology of what's known about MECP2. These are the core functional domains of the protein. And so they really retained those. They removed a lot of the excess material and kind of miniaturized and redesigned a little bit the linker between those 2 domains. So this is all detailed in Dr. Bird's publications. One of the things that they did is they made a transgenic mouse that only has the mini gene and not full length MECP2. And that mouse was, for the most part, normal. So it looks like the miniMeCP2 gene is able to, for the most part, functionally compensate for full length MECP2. I'm going to definitely stop short of saying that it is 100% equivalent and it compensates 100%, but I think what his publications have shown and what our work has shown is that it is capable of substituting for full length MECP2 in most aspects and providing a significant and meaningful benefit.

Great. Thank you. And there's a question from Eun Yang of Jefferies. "Can you compare the size of full length MECP2 gene versus the miRARE transgene? Sorry if I missed it."

Yes. I think honestly, the miniMeCP2 is about 1/3 of the size of full length MECP2. So it is a substantial truncation. But again, if we were to incorporate the microRNA element, the miRARE element is a fairly compact element. But even still, it won't fit if we were trying to package full length MECP2. So we moved to this truncated version of MECP2 because the really -- if you don't incorporate this regulation mechanism, then I don't think you can deliver full length MECP2 safely.

Okay. Thank you. Your next question comes from Yun Zhong of BTIG. "Have you looked at the periphery in mouse studies? Is protein expression in the peripheral organs similar to other IT-delivered AAV9 gene therapies?"

Yes. So this comes from, I guess, kind of a known phenomenon that if you do intrathecal delivery of AAV9, you get a substantial amount of the vector that leaks out into systemic circulation and goes to peripheral organs. So even though we're concentrating the vector in the CNS, you still get gene transfer to liver, heart, muscle, all the peripheral organs. And this may be as much as, say, 1/3 to 1/2 of the vector. In terms of the expression of the miniMeCP2 transgene, this is downregulated by the miRARE element and with -- and also regulatory features in the polyadenylation signal. And we detailed some of that even in our 2017 publications. But overall, I think if we -- what we showed in 2017 is that if you drive unregulated overexpression of MECP2, even using the endogenous promoter, like if you just do very high doses given IV, then you will get overexpression in the liver that can be fatal, rapidly fatal. But we have never seen that with the miRARE-regulated MECP2. It does suppress expression in the liver is where we've looked most strongly. We haven't seen any indication of toxicities stemming from overexpression in other peripheral organs.

Great. Thanks, Steve. Your next question comes from Silvan Tuerkcan of JMP Securities. "What is known about potential differences in the microRNA abundance, localization, et cetera, between rodents and mice that could lead to a potential difference in the ability to regulate gene expression?"

Well, I think that the question -- it's a very broad question on microRNA biology, but I'm going to focus on specifically the microRNAs that we focused on to incorporate into the design of the miRARE regulation element. So all of these microRNAs -- like I said, the sequences, the microRNAs, the sequence of the binding sites are conserved, highly conserved between mice and humans. The microRNAs are expressed in mice and humans. That's been shown in postmortem human tissue. In terms of the exact expression level on a cell-by-cell basis, I mean, that would require very detailed autopsy studies in humans. And then I think that there's some data to indicate that gives us confidence that this regulation system should translate pretty effectively between mice and humans. But it's impossible to kind of have that complete data set, to have that finely mapped out to say that it is exactly the same.

Great. Thank you, Steve, for all your insights in this program. And that concludes your Q&A session. Thank you again. I'd like to now turn the call over to Dr. Suyash Prasad, our Chief Medical Officer and Head of R&D, who will discuss the clinical development strategy and provide a regulatory update. Suyash?

Great. Thank you, Kim. And thanks to Steve for his wonderful explanation of the science, to Jeff for his clinical perspective and natural history data and to Monica, of course, for sharing the perspective of the patients and their family. I'm now going to talk a bit about clinical development strategy. And as Kim mentioned, a lot of this will relate to our regulatory strategy, and I'm going to share some of the feedback that we've received from our regulatory interactions. I'm also going to talk a bit about our KOL engagements and our engagement with patients and families and how they've informed both our clinical development and the regulatory strategy. Next slide, please. First of all, we're very excited to be able to share with you this morning in a press release that we received orphan drug designation from the European Commission. That is to -- that is on top of the already existing orphan drug designation from the FDA and rare pediatric disease designation from the FDA. And what this does is it does several things. It gives us a greater ability to connect with on a more frequent basis with the regulators just by virtue of having this designation. There are various tax credits and other financial incentives as well. And importantly, it gives us market exclusivity for a period of time after commercialization. So it's a really significant additional regulatory milestone. Next slide, please. So in terms of what we've been doing at Taysha on the 102 program for Rett this year, we've run a number of advisory boards with Rett experts. We've run a number of patient focus groups with patients and families. We've had regulatory feedback from several key regulatory agencies in recent months, specifically on Rett. And in fact, we've had a number of regulatory interactions, I think 7 or 8 now, over the past 3 months across our portfolio of programs. And all of that gives us wonderful insights into how we conduct our programs going forward. We've actually made drug for the Rett programs. All were made due to commercial process. There's a little bit release testing yet to go, but the drug is all ready to go with the clinical trial. And we're actively working on site feasibility for the clinical trial, which we plan to initiate by the end of this year. Next slide, please. So the next 2 or 3 slides will talk about our advisory board feedback, and some of this will echo what you've already heard Jeff talk about. So we've brought together a number of international Rett experts, have several very deep discussions with them. And they've given us great insights on the biology underlying Rett, the natural history of Rett, how we should think about the clinical trial going forward, in particular around patient selection and inclusion/exclusion criteria and outcome assessments. And I know we've had several questions already on some of those points from you all. Next slide, please. So I have 2 slides of specific feedback that -- just some themes that came through from the advisory boards, just to highlight the fact that these are issues that we're aware of, we're talking about, we're thinking about and weaving into our plans for clinical development. So the first group of comments are on MECP2, and we've already heard a lot about MECP2 biology from Jeff and from Steve. And it's clear that small amounts of MECP2 gene expression can lead to significant improvements in survival. What's also clear is that there is a fairly narrow therapeutic window, and we're not entirely sure where that therapeutic window is. We know that when we get to 200% levels of MECP2, i.e., as we've seen, MECP2 duplication that Jeff talked about, you're going to reach toxic levels. I will also note that less than 50% in the X-linked female gives you Rett. So it's between 50% and 200%, but what that range is exactly is not really certain. But our intent is to get to as close as 100% in as many cells as possible. They were impressed. The advisers were very impressed with the miRARE platform and how it really dramatically improves survival and [ function ] in the mouse. And they're really quite hopeful for us being able to be translated into humans. And you've already heard one of the things on this discussion around MECP2. And that is the fact that you may only need a little bit of MECP2 in the mouse cells, but you should try and get that into as many cells as possible. So there is a nuance there to how we extrapolate doses from the preclinical setting to the clinical. So I think getting this balance right between MECP2 levels and the number of cells that get transduced will be critical. On the nonclinical side, there was a feeling that treating younger mice would lead to better outcomes, which is often the case. And that relates once again to the fact they felt we might get better outcomes in younger treated patients. Although as Jeff mentioned earlier, we feel and the key opinion group feels that actually, virtually everybody with a MECP2 mutation would benefit from this gene therapy approach regardless of age, regardless of degree of compromise. There's discussion about doing studies in female mice and also NHPs, which we are currently doing also. Next slide, please. With regard to clinical trial design, there's a lot of discussion. Treating younger patients earlier might yield maximum efficacy, but there's a potential benefit to going earlier in older children or adults really to ensure safe and tolerability in humans. And based on this feedback, based on the discussion we're having with regulators, our initial clinical trial will be in adults with Rett syndrome really because of the desire and the need to explore safety and tolerability first before moving very rapidly into the younger children. There's discussion about doses, higher doses, meaning broader distribution and transduction of more cells. There's discussion about how we should select outcome measures that are really relevant to patients and families. This is very important for regulators. In a few moments, I'll take you through our approach to doing that to make sure we weave that patient and family feedback into our selection. And of course, and has been the case with all our regulatory discussions, there's discussion about DRG inflammation and immunological reactions and how we manage that and how we monitor for that. So as an aside, we are very disciplined on how we look at DRG inflammation in terms of looking at deep tendon reflexes over time and nerve conduction studies. And also from the immunological perspective, we're very -- spending a lot of time thinking about how best to dampen down any immunological response. And we have a specific approach, which is based on what we've learned from the [indiscernible], in particular, while we will use a year's worth of sirolimus or rapamycin and 6 months' worth of prednisolone. And that seems to work very, very nicely in our GAN program. And indeed, that's the impression across the majority of our gene therapy programs. There's a lot of discussion on outcome measures. We've already heard a lot about outcome measures from Jeff, and much of the feedback that we received echoed some of Jeff's comments on scale such as the Rett Motor Behavior Assessment, the CGI-I and outcome measures in communication, for example. There's discussion about EEG and about potentials. Jeff has already taken you through that in some detail. And there's also some interesting discussion about how actually the therapies are effective. We may actually see changes at the anatomical level. As you've already heard, one of the issues with Rett is that these children have much smaller brain than microcephaly. Part of the reason for that is that at the ultrastructural level, what happens is that the neurons lack dendrites, that the connections between the neurons, they have far fewer of those than a healthy individual would have. And the thinking from this advisory board was that actually, part of the reason -- probably what you might see by replacing MECP2 is a regrowth of this absent dendrite. And you might see this functionally and actually physically and anatomically with the growth in the dendritic spine at the microcellular or structural level. We talked also about how we would make sure we have facilities to continue supportive rehabilitation therapies during the clinical trial. As you heard from Monica, obviously, they -- that is critical. It's really important. In general, the KOL advisors were very enthusiastic and very optimistic about our approach based on Steve and Sarah Sinnett's work. And we're looking forward to taking part of investigating the clinical trial. Next slide, please. So we also spend a lot of time talking with patients and families. We have a very active patient advocacy group led by our colleague, Emily McGinnis. And Emily and I work very closely together on really ensuring we can capture insights from patients and families and weave them into our clinical trial designs and into our regulatory interactions. Next slide, please. And for Rett specifically, we've done this in 2 ways, actually. We've done a number of in-depth workshops, which we did for all of our programs. So we spent 2 to 3 hours on a Zoom call. We usually do this in person. But at the moment, we're doing these meetings remotely with 5 or 6 patients and families and parents represented on these calls, where we talk in-depth about their hopes, their concerns. We ask them what they'd like to see from a gene therapy treatment. We asked them how -- if a particular symptom could be improved, what would be the most important symptom that they would like to see improved. And we had some really nice, very valuable feedback, and I'll take you through that in a moment. We were also in partnership with the advocacy organizations in several countries for Rett, ran a confirmatory online survey with over 300 caregivers, which really echoed a lot of the findings in the workshops and actually echoed and reflected some of what you heard from Monica earlier today. Next slide, please. So from the workshops, what we found is that the most challenging symptoms over the years really relate to 3 areas: So the motor function issues, which Monica alluded to; the communication issues; and some of the autonomic dysfunction issues, and I'll split that into 2 categories. One is gastrointestinal issues, and the other is the breathing issues. So of course, with regard to motor function, there's loss of purposeful hand movement, and the lack of being able to walk or move or reposition themselves as time goes on really has a detrimental functional compromise to these children with Rett that causes a lot of challenge for the families. The loss of speech and communication is very, very upsetting because your -- the children will lose their ability to verbalize. They lose their ability to understand the language, although that happens at a slower rate than the expressive language, that's the verbalized language. And also, they have problems with seizures and the ability to move. If you lose your hand movements, then the other thing is that your ability to nonverbally communicate becomes much more compromised. And so you're losing communication capability in many, many different domains, which is particularly troubling for the patients and families. And then on the autonomic dysfunction, we heard earlier from Monica and from Jeff about the breathing issues these children have, the breath holding; the swallowing of air, also known as aerophagia; the alternating rapid breathing and slow breathing that seems to be somewhat also related to anxiety. And this can be happening many, many hours during the day. And then the GI issues, also related to autonomic dysfunction because of stomachaches, diarrhea, constipation and additional challenges, which are very hard to manage from a family perspective and compromise the quality of life quite significantly. Next slide, please. A couple of quotes or several quotes. I won't read them all, but they're on the slide. Have a look at them in detail, but I'll read 1 or 2. The first one, "Not being able to hear what my child says or what she wants from me is truly heartbreaking." "Her lack of speech denies her the ability to let us to know what is wrong, why she's happy, what needs to be changed abilities. It impacts the entire family and her social abilities." That's on the speech and communication. On the GI issues, "I wish her tummy didn't hurt so much all the time, and that she wasn't always so bloated and uncomfortable." So there are really -- there are huge impacts to this particular disease across a whole wide range of symptoms that are particularly impactful to patients and families. Next slide, please. We also asked the patients and families, what do you really want from a therapy? And in general, it was -- there's a desire to treat the genetic root cause of the disease. It really meant that they were very open to a gene therapy approach. The individual we spoke to were very happy to really seriously consider taking part in the clinical trial. They saw gene therapy as a way of really treating the genetic root cause of the disease and, of course, reducing, improving, slowing down symptom progression or disease progression and improvement of overall function, unsurprisingly. Next slide, please. Now in addition to the in-depth patient work group, we ran this online survey of over 300 caregivers across several different countries in the international field. And once again, communication, hand use, GI issues and mobility were really very important aspects of the disease, all of which the caregivers want to see improvement. Based on that, we wove all of these aspects into our collection of end points and outcome measures in the clinical trial. Next slide, please. This is a subset of that patient survey, and we're planning to publish this some at point in the future so you can see in more detail how -- the methodology behind it and the data in more detail. But this was a subset of the 18-year-olds and over in the group of surveyed caregivers. And the reason we pulled this out is just to show the fact that actually, it's true for the older patients as well as loss of speech, purposeful use of hands, motor issues, once again, are what the -- what are the commonly complained about issues. And given that our initial clinical study will be in adults, we felt important to share some of this with you as well. Next slide, please. So we've talked a bit about the key opinion leader input into our clinical trial design and also the patients and family and caregiver input. This is the actual clinical trial design. You can see construct in the middle of the slide. Steve has gone through this in detail. It's the miniMeCP2 gene with the miRARE strip of microRNA binding site wrapped up in a self-complementary AAV9 capsid. We will give the drug intrathecally. As you know, that is at the base of the spine. We will inject the drug probably between 10 and 12 ml. The drug has a slow rate, 1 ml per minute. We'll have the child placed on their side and then tilted up by about 15 to 30 degrees. This is known as the Trendelenburg position. We keep them on that position for about an hour. We do that for 2 reasons: First of all, just to help the CSF flow down towards the brain to encourage transduction of those tissues. Also, it's always done with intrathecally administered drugs simply because the -- keeping them head down for about an hour mitigates that post LP headache that some patients also have. We'll be covering them with our immunosuppression regime of prednisolone, 6 months of prednisolone and then a year's worth of sirolimus or rapamycin. And we have had very nice results from our GAN program with that particular regime. It will be adult females in the study, 18 years and older. Notionally, it's going to be a 5x10^14 dosed for the first cohort and a 1x10^15 dosed for the second cohort. They may alter a little bit as we just refine our titering assays and just going through the multilevel data in more detail prior to the submissions. But that's the -- those are the notional doses. And there's a question earlier about extrapolating from animals to humans and going through the extrapolations. And we do this in a very, very disciplined way. We're very, very particular about it. We've got our approach to doing it that is very accurate, and we feel confident about the dose selection across all our programs. Okay. Next slide, please. So in terms of efficacy end points, you've heard already from Jeff about some of the key ones, and these are the key efficacy end points that we'll be looking at. We are going to look at a whole breadth of end points. And I think we have to move away from this idea of there being a registration-enabling end point for diseases like Rett. It's more about the totality of data. That's the phrase the FDA often comes out with, and it's really what we hear from regulators. They want to see -- in these multisystemic diseases, especially with rare diseases where less is known, they want to see improvement across many, many domains. So we are going to use the revised Motor Behavioral Assessment that Jeff talked about earlier. We will look at hand function with the Rett syndrome hand function scale. There's the ORCA scale, which specifically looks at communication capabilities, expressive language, receptive language and actually nonverbal language. This is really important. And so this will be a key end point for the study. And then the CGI-I, which, as Jeff mentioned earlier, gives the opportunity to look at a broad perspective. And we will be using the version we talked about, specifically using the Rett anchors to give as accurate a reflection of the global change in these children and adults as possible. Next slide, please. And we're also going to look at a whole host of additional end points as well. So it's going to be a very detailed and robust study. We'll be looking at the Rett Syndrome Behaviour Questionnaire. There was actually a question about that earlier. It's a scale actually that is favored by the regulators. So we're including that. We'll be looking at dystonic posturing. We'll be looking at seizure activity. We've got our Rett episodes. In addition to Clinical Global Impression improvement, we'll be looking at Clinical Global Impression of severity, the Vineland Adaptive Behavior Scales. We are going to be collecting CSF and blood samples, which we'll be banking for potential biomarker analysis. But as yet, as you all know, there's no really good blood tests or CSF biomarker that exist for Rett syndrome. We're going to be doing a lot of neurophysiological assessments, constant EEG. We've got potentials that we discussed earlier, brain volume MRI and MRS of the brain as well and then a mixed bag of assessments looking at quality of life and health care resource utilization and caregiver quality of life as well. So a real breadth of assessments that I think will be helpful as the FDA and other regulators look through the data in detail. Next slide, please. So as I say, we've had a number of regulatory interactions specifically on Rett syndrome. We've had a number of regulatory interactions across our whole platform, and there's many key things that come across -- come through across our platform. But specifically around Rett, they -- the regulators very much agreed with our approach, the dose rationale, dose selection and first dose. They felt that the preclinical data package was very comprehensive. As Steve mentioned, he's been working on this for 14 years. And so there's a wealth of historical data. And as you all know, as RA and I have talked about previously, we have a very disciplined and robust approach to toxicology by looking at 3 specific species. So the regulators definitely appreciated that. There was real alignment around safety monitoring, in particular, around DRG inflammation, which, as you know, is a hot topic at the moment. And also, there's alignment around efficacy end points that we've selected. And we will be initiating the clinical trial by the year-end. We're on track to do so. Next slide, please. So in terms of anticipating the next steps, IND/CTA will be submitted in the second half of this year with plans to initiate the study by year-end. As I said, we've already made the material commercial grade. The release testing is underway, and we will continue our engagement with the key opinion leaders through our Rett advisory board and colleagues such as Jeff, with patients and families, with Monica and her organization and others and, of course, with our colleagues and friends at UTSW. And there are the 3 key individuals at UTSW I wanted to mention that we partner with. First of all, Berge Minassian, who is Division Chief of Pediatric Neurology and a Rett expert himself. He's actually published on -- he's described CNS isoforms of Rett. So it's really wonderful to have insight -- his insights in forming the program. Steven, of course, who leads that core facility, is a wonderful partner to us; and Sarah Sinnett, who leads the Rett preclinical scientific program at UTSW. On that note, I'll close. I'm happy to take questions and also happy to hand over to RA to close as well. Thank you.

Thank you, Suyash, for the in-depth presentation. Unfortunately, our Q&A system has gone out. So we will -- please e-mail me with any questions. In the meantime, I'd like to turn the call back over to RA Session for his closing remarks. RA?

Thanks, Kim. Next slide. We've enjoyed sharing with you in greater detail about our Rett syndrome program. As we look ahead, we'll continue to focus on rapidly advancing our numerous pipeline programs with many key milestones to be achieved over the next 18 to 12 months. We've made a significant transition into a pivotal stage gene therapy company and expect to provide both clinical and regulatory updates on our lead program, TSHA-120, by the end of the year. Furthermore, we remain on track to report first-in-human clinical data for our TSHA-101 program in GM2 gangliosidosis as well as initiate our Phase I/II clinical trial in TSHA-118 in CLN1 Batten disease, which currently has an open IND. Lastly, we expect to open an IND or CTA for our Rett syndrome program and initiate a Phase I/II clinical trial by the end of this year. I would like to give a special thanks to Dr. Neul, Monica, Dr. Gray for participating in our event today and sharing your insights. We appreciate you taking the time to provide your expertise and perspective. In addition, we would like to thank the Sinnett lab for all the work done to develop the miRARE platform as well as this Rett syndrome program. And to all of our patient advocacy partners around the world, thank you for your support. Lastly, I'd like to thank everybody for joining us today. And please, if you have any questions, please send those over. And we thank you for your continued interest in Taysha. Have a wonderful day.