Good morning, everyone. Thanks for coming. My name is Noelle Tiongson. I'm from Children's Hospital Los Angeles. We have a group of four doctors today who will be giving talks on updates in ped's neuromuscular research and clinical trials, as well as an overview of currently approved therapies, and then some translational science from bench to bedside. So we'll be opening up between me and Dr. Fay, who will be doing clinical trial work, and then Dr. Alan Sharpley and Dr. Nelson will be talking about their work in the translational space. So we'll start with Dr. Fay. We'll be talking about currently approved therapies for DMD and SMA, as well as the evidence that brought them to market. All right, good morning, everyone. I'm going to be sticking to DMD and SMA monotherapies, and then we'll pass the mic over to Dr. Tiongson. Here are my disclosures. And there are four areas I'm going to cover for DMD, steroids, mutation-independent treatments, exon skipping, and gene therapy. And for steroids, I'm going to focus on vomerolone, which is kind of the new steroid to come on the scene in the last year or so. I did want to just note that regarding steroid regimens, there are a lot of different steroid regimens out there, daily, 10 days on, 10 days off, high-dose weekend steroids. I think in the last couple of years, some work has been published. You can review the reference below, showing that daily, whether it's prednisone or deflazocort, is superior to 10 days on, 10 days off. I'm not going to talk further about that, but I think that is one important development in that area. So kicking things off with vomerolone, vomerolone is the third steroid we really have now available for DMD treatment. It's a little bit different than prednisone and deflazocort in that it's a dissociative steroid. It does have that NF kappa B inhibitory activity, but less transcriptional transactivation and acts differently at the mineral corticoid receptor. And so that's expected to have some downstream consequences for both linear growth and, hopefully, fracture risk in the future. So this is a phase 3 trial. It's a bit of a complicated study design, but there was a placebo arm, and then there was a prednisone switching to vomerolone group as well. Vomerolone was tested at two different doses, 2 milligrams per kilogram per day and 6 milligrams per kilogram per day. And on the left, you can see that time to stand was superior in the vomerolone at either dose compared to the placebo. And in the second panel in the middle, you can see NSAA scores likewise are superior to placebo. And so I think the take home point for efficacy is that vomerolone is as effective as prednisone. Nobody's arguing that it's a superior steroid in terms of motor outcomes, but like the other steroids, it's clearly superior to placebo. Where it is maybe more unique is in terms of linear growth. So prednisone, well known to slow height growth in boys with DMD, whereas vomerolone at both of these doses, 2 and 6 milligrams per kilo per day, shows an increasing Z score over the 48 weeks of the trial. There is no change in the BMI when we compare the prednisone to either dose of vomerolone. So less slowing of height growth, but no change overall in BMI. And then they also looked at some markers of bone turnover, osteoblast, osteoclast activity, and vomerolone seems to interfere less with bone turnover. And whether or not that's going to actually translate to reduced fracture risk, I think we have to wait a little bit longer to see, but that's the hope. So I think the take home here is we have a new steroid. I've personally used this one in a child who came to me with a diagnosis of short stature before his DMD diagnosis. And certainly, if linear growth is a big concern for your patient or family, this is a nice new option to have. So onto the mutation-independent treatments for DMD, I'm going to focus mostly on the HDAC inhibitor juvinastat. The three below that are in earlier phase 1 and phase 2 trials, and there wasn't really much data for me to review here, so hopefully next year we'll be able to talk more about some of those treatments. And then three at the bottom there, unfortunately, did not meet their primary endpoints. I'm not going to talk further about those, so we will hear about myostatin inhibition in the context of SMA a little bit later this morning. So juvinastat is a histone deacetylase inhibitor, and its primary modes of action are going to be preventing fibrosis in muscular dystrophy and promoting muscle regeneration. So this was a placebo-controlled trial done over 18 months in boys six years and older who were ambulatory. All were on daily steroids, mostly deflazocort. And the primary outcome measure here was the four-stair climb. And as you can see in the panels on the right, the four-stair climb and NSAA total score favored juvinastat over placebo. And another interesting outcome measure at the bottom of that upper panel is the MRS-VLFF. That's MR spectroscopy of the vastus lateralis fat fraction. So that was also favoring juvinastat. And so based on all of this data, the FDA approved juvinastat for boys six and older. The main side effects to look out for here are thrombocytopenia and hypertriglyceridemia. And so this is a nice option for those patients who are not eligible for the therapies I'll talk about in a moment, the exon skipping therapies, the gene therapies. This one is agnostic as to the genetics of a particular patient. So onto the mutation-specific therapies. The first one I'll touch briefly on is Adalurin, which is for patients with premature stop codons. And then exon skipping drugs, we have four currently approved options and some more in the pipeline. So Adalurin is a nonsense read-through drug. So if a patient has a premature stop codon, it will help the ribosome to basically read through that, replace that stop codon, or insert an amino acid instead of terminating translation. And so this drug has been approved for about eight years in the EU, never approved in the US. But there is this stride registry of patients who both participated in clinical trials with Adalurin or were treated with it post-approval in the EU. So taking these 307 patients and age-matching them to the Synergy Natural History cohort can see that there is a modest improvement in later loss of ambulation in the stride registry compared to the Natural History cohort, and also a lower proportion of patients with very low force vital capacity, less than 30%. And so I would say that Adalurin appears to have a modest improvement. But I think that modest improvement is one of the reasons why it's been difficult to get approval in the US. And I've even heard that there's some question of withdrawing approval in the EU, so stay tuned for that story. And then onto the pathogenic exon deletions. So we currently have four approved drugs. This diagram just shows the idea behind exon skipping. So if a patient is missing exon 50, they have an out-of-frame transcript, which leads to a truncated non-dysfunctional dystrophin protein. If you can, however, skip or mask an adjacent exon and restore the reading frame, as on the right here, you can have a slightly smaller but still functional dystrophin protein product that is more like a Becker phenotype. So Ateplersen was the first of these drugs approved back in 2016. It's an exon 51 skipping drug. And the new developments in the last couple of years are that we have long-term data up to seven years. And I think, kind of like with Adalurin, there's a modest slowing of loss of ambulation by about two years on average. And so not insignificant for those patients. And the force vital capacity declines also at a slower rate compared to natural history controls. So I think this does validate the FDA's early approval of this drug. And I think this is not a be-all and end-all in terms of exon skipping, but is allowing the field to move forward so that the next generation of exon skipping drugs have a chance to do better. And just one other point, there is an ongoing study in boys 6 to 48 months of age. And it appears to be safe and well-tolerated so far, but we don't have a functional readout on efficacy. Vildelarsen is an exon 53 skipping drug. That's one of the two available for exon 53 skipping. And we now have long-term data over the last four years from this paper published last year at the bottom. So the two outcome measures shown here are time to rise and time to walk run. And you can see that Vildelarsen is superior to the natural history cohort. The drug is well-tolerated. Nobody withdrew from the study due to any adverse effects. So here's basically where we are now. We have four FDA-approved exon skipping drugs. They are all approved through accelerated approval, which means that they have ongoing phase 3 studies that, if ineffective, the FDA reserves the right to withdraw the approval. And the ongoing trials, there's high dose of Teplersen. There's the PPMOs, which are the antisense oligo conjugated to a cell-penetrating peptide that will hopefully have better muscle penetration. And then there are ongoing trials using different technologies for skipping exon 44 and 50. Those would be new targets. And then some other approaches to exon 51 and 53 skipping. Those are all in phase 1, 2 trials. So again, maybe a year from now, we'll be able to review some of those findings. And finally, gene therapy, I think, is the hottest topic these days in DMD. We have one approved product. That's delandistrazine moxiparvavec. It was FDA-approved last year for boys 4 and 5 through accelerated approval. And then earlier this year, that was expanded to 4 and up ambulatory full approval, and then accelerated approval for non-ambulatory. The fordidistrazine moxiparvavec program, unfortunately, that one was run through Pfizer. It did not meet its primary or secondary endpoints. And there was a death. And so given the lack of clear efficacy and the side effect, or the adverse events, that program was closed down. And there are two other ongoing, well, there are actually more than two other ongoing programs. But I've listed two of the ones here through SOLID and Regenexx Bio. These are in earlier phases. All these programs are designed around AAV-based vectors. The vectors are slightly different for each of these programs. And the transgenes are all slightly different. It's not clear yet what these differences are going to mean in terms of clinical efficacy and outcomes. But there are some important differences that I don't have time to go into today. Another note at the bottom right is that one patient, a 27-year-old young man, was treated with a CRISPR-based gene therapy approach and sadly died very soon after being treated from what appears to be a massive immune activation event. And so I think that's just a warning for the field that these can be powerful therapies, but also. So Dell Industry Moxa Parvivec was initially approved based on the North Star ambulatory assessment data which showed a clear difference from natural history in the four to five year olds but not in the six to seven year old group and that's why the FDA granted this narrower approval last year but if we look at some of the more fine-grained motor outcomes such as the 10-meter walk run or the time to stand there was actually superiority of the patients treated with gene therapy compared to control groups across the age ranges studied which was four to seven. So this this drug is now approved for four and up I think as we're all probably experiencing FDA approval is not equal insurance approval and so that's an ongoing challenge but to be eligible for this gene therapy patients have to have a low or absent titer of the anti-AVRH74 antibodies and they have to not have an exon 8 or 9. So, here's the data that we have in the published literature up to this point. So, we have a cohort of 21 boys treated over 12 months. You can see the NSAA scores increase in the first 8 to 12 weeks and stay steady, perhaps some gradual increase up to the 52-week cutoff. We also see improvements in the time to stand and 10-meter walk-run outcomes. And then for a smaller cohort of boys, over 4 years of time, we see the NSAA scores increase in that first year, less so in the second and third year, and seem to remain more or less steady. I think the long-term concern is but at least at the four-year mark there seems to be clinical stability. So adverse effects, I mentioned the death in the Pfizer trial and the CRISPR trial. We also have gene therapy, you know, experience in other diseases like SMA, which I'll get to in a moment, and the MTM1 trial, which Dr. Tiongson will mention later. So we know that early on there can be nausea and vomiting, kind of a GI illness type picture. We've seen liver enzymes and cardiac enzymes increase in that early period, the first one to two weeks. Thrombotic microangiopathy is one of the most feared complications that has been seen in several trials and clinically dosed patients and is basically a massive immune activation that leads to thrombocytopenia, hemolytic anemia, and can be fatal if not treated aggressively with complement inhibitors and plasma exchange. And then a side effect, kind of unique to the DMD trials, is this myositis, which seems to be an immune activation against the transgene product because it is recognizing the transgene as foreign. And so some of these patients have had quite severe immune myopathies, ultimately treatable, but that is the rationale for removing some of those mutations that are most prone to immune myopathy. So those are the exon 8 and 9 deletions. So on to SMA. I'm going to just go briefly through the three therapies that we have approved now. So two of them are SMN2 targeting, nusinersen and rizdiplam. We have SMN1 gene replacement. So, Nusinersen was the first approved SMA drug. This is an intrathecally delivered antisense oligonucleotide designed to increase SMN2 expression. And the trial updates for Nusinersen are that we have five-year data for the NURTURE trial. This is a trial of pre-symptomatically treated children with either two or three copies of SMN2. And the take-home point here is that there is continued improvement in motor development over the five years post-treatment. The shaded areas on the right are normal Hammersmith motor scores or normal motor development over the five-year period, and the spaghetti plots are for individual subjects in the trials. On the bottom, you have three copies of SMN2, and on the top, you have two copies. So, what you can see here is that most patients are ending up within the normal motor developmental range. That's more true of the kids with three copies of SMN2 versus two, as you might expect. Other things going on in Nusinersen is the DEVOTE trial. So, this is a higher-dose trial to see if giving 28 instead of 12 milligrams is more efficacious. It appears to be safe and well-tolerated, but we don't have the efficacy outcome data yet for that. And then, there's also a trial, so far as I know, not yet enrolling, but where the Nusinersen can be delivered through a lumbar port rather than through serial lumbar punctures. So, next on to gene therapy on a 7-a-gene, abaparvivec was the second drug approved for SMA. We now have long-term data for IV treatment. This, as you may recall, is restricted to children under two years of age in the U.S., but we have several cohorts published from different centers. The one I referenced here from Ohio showed no SAEs and no cases of TMA, fortunately. However, about 80 percent of the patients after receiving gene therapy elected for an add-on therapy. So, this tells us that patients are still looking for more, even after a treatment that has been really transformative for the field, and Dr. Tianxin will talk more about those add-on therapies. And then, you know, of particular interest in the SMA type 1 population is bulbar function, and it's good to see that bulbar function seems to be improved across the board in patients treated with gene therapy. And then, another way of delivering the gene therapy is intrathecally. This is being investigated for older patients. The STRONG trial was a phase 2 and has fully enrolled, and there have been no cases of TMA or sensory ganglionopathy. That's one of the adverse effects seen in non-human primates with intrathecal delivery of gene therapies. And the take-home here is that there appears to be improved Hammersmith motor scores compared to natural history controls over time, still waiting on the final data readout here. And there is an on the STEER trial, which is a phase 3, that we have not seen results for yet. But I think based on all of these data, we'll probably know within the next several months whether this will be a viable option for patients older than 2 through the intrathecal route. And finally, RISDEPLAM. The SUNFISH study had some newly published data last year for type 2 and type 3 patients. One of the nice things about this study is it has a very broad age range from 2 to 25, and even with that broad age range and the diversity of type 2 and type 3 disease, the broad spectrum of severity there, they were able to show stabilization or improvement across motor outcome measures, particularly in the younger patients. And so Dr. Tiong-Sen is now going to take over and talk about dual therapies and a couple other topics. I'll be back in a moment. We'll hold the questions until the end, so I'll continue on from Dr. Fay's talk. All right. So Dr. Fay did a great summary of what's going on with DMD and SMA-approved therapies, as well as some discussion on upcoming and emerging therapies. I came into this session with wanting to know about what's out there, especially with all the named SMA therapies, add-on therapies, and ongoing clinical trials. So I'll be going through that first, and then also talking about what's going on in congenital myopathies, and then discussing also the MTM-1 trial. I have nothing to disclose. This is the overview, and this is what we'll talk about. So this is not an exhaustive list by any means. I did a lot of digging in clinicaltrials.gov, as well as other support websites for SMA. Not an exhaustive list. There are lots of detail about these other studies that other speakers have already or will talk about. I also liked to get the branding pictures, because I thought they were really, like, fun and cute. So I'll be going through these. So these are clinical trials in SMA going on with nusinersen. So the first one here is the ASCEND trial. This is the higher-dose nusinersen in patients who switched over from ristoplam. So this would be the 50-milligram loading dose times two, instead of four loading doses. And then you'd go 28 milligrams higher-dose maintenance every four months. And this is for patients 15 to 50 years. So it does include pediatric patients. It is ongoing. And then the DEVOTE trial, which Dr. Fay mentioned. This is higher-dose nusinersen in three cohorts. So the first cohort would be later-onset SMA, two to 15 years, using the 28-milligram loading dose. Part B would be infantile-onset SMA, two to less than 10. And then Part C would be patients who are already on the 12-milligram dose transitioning over to the 28-milligram dose. There was an update last week on data from the trial so far. And they're showing some modest lowering in the neurofilament levels, signaling less degradation of the motor neurons. As well as the stable motor improvement seen in CHOPin10, the Hammersmith, and the revised upper limb module. So more to come on that. The RESPOND trial is gene therapy-treated patients. And then by three to nine months, if they have a suboptimal status as determined by the physician, get treatment with nusinersen. There's also measures on motor function scales as well as neurofilament light chain measurements, among other PK and PD measurements. So more to come from that as well. And then there's also a Phase I trial. I included this. This is an adult study, but it is a modified ASO for longer interval dosing. Possibly, like, every six-month dosing so that it's only one to two doses per year. So that's currently in Phase I. And then I remembered all the fishes from when I heard about RISDAPlam. So I'll just go through these briefly as well. So Rainbowfish is an ongoing trial in pre-symptomatic SMA. And they are doing this in one day up to six weeks. And they just had one-year data in October. So just this month. And these are in babies with two copies of SMN2. So they're saying in the one-year cutoff, they have babies sitting without support for 30 seconds and then looking at also CMAP amplitudes. So this also looks encouraging so far. There's also the Firefish study, which Dr. Fay mentioned. There's the five-year data that came out in the summer. 91% are alive, 81% without a vent, and 96% with improved motor function, swallowing, and 80% without tube feeds, and 59% not sitting without support. So there's a lot of great data. There's data collection for safety ongoing, but also very encouraging data. And that's in SMA Type I patients. The Sunfish trial is in the later-onset SMA Type II. And this is from 2 to 25. They have four-year data that came out in April of 2023 with stable MFM32 scores, revised upper limb module increase at 24 months, which was sustained at 48 months. And then the Hammersmith, which contrasts with the natural history with more normal development. And then there's an ongoing trial called Jewelfish. And this is any type of SMA on RISDiplam with any previous treatment. And this is showing great safety data in a wide age range, so from six months to 60 years, and motor function stabilization across testing motor function as well as types of SMA. Continuing with Morefish, these are ongoing trials as well. So there's a Pupfish trial. And they are enrolling babies at less than 20 days of age to receive RISDiplam. And this is one dose daily for 28 days to evaluate the safety in this group. So that's currently enrolling. And then you may have also heard about these other two trials called Hinalaya. Hinalaya 1 is RISDiplam after gene therapy in type 1 SMA in babies less than two years. So this is like after gene therapy within three to seven months to see if there is an add-on effect. And that's currently enrolling. Hinalaya 2 would be intervention with RISDiplam after a plateau or decline after receiving gene therapy. And this would be also in age less than two years, but that they would have a decline as determined by the physician. And then lastly, I was interested to hear about the Marlin trial because a lot of our pediatric parents, the parents of the patients that we treat are worried about long-term fertility outcomes. So they're doing a questionnaire to adult males who have received RISDiplam or ever received RISDiplam. And then also assessing like if they are trying to conceive, have they conceived? Were there any difficulties with conceiving? And this would be important for our families to know. There is also a question, is it from SMA itself that there are fertility problems or is it from the treatment that there were fertility problems in the animal studies? So moving on to two other trials going on. Evers-D currently is a liquid that must be kept in a refrigerated state. And so this may be difficult for people who are traveling or people who do not have access to refrigeration for some reason. There is a clinical trial going on regarding tablet forms of RISDiplam. And so they're testing the relative bioavailability and bio-covalence of two tablet forms of RISDiplam as well as effective food and if you're taking omeprazole. So they'll compare these tablet doses in comparison to liquid dosing of RISDiplam as well. And as mentioned by Dr. Fay, there is currently undergoing a STEER trial. This is Novartis's intrathecal omnisemna gene. And this is to two less than 18 years and it's sham controlled. So they described it as they will do a pinprick on the shams and then the actual gene therapy on the real ones. I don't know how exactly it's like equal. I think they're sedating them probably. And then there was a hold because of the DRG toxicity seen in the animal studies. But that hold has since been lifted because of the encouraging data from STRONG. And then the PIER trial is the implantable intrathecal catheter and SC port. So this is a joint between Alcion and Biogen using a intrathecal port to deliver nusinersen and measuring whether the delivery concentration is the same between that and lumbar puncture. Or is there a difference? And so that is not yet enrolling but the technology is being developed for that. I looked into other clinical trials on clinicaltrials.gov for other ongoing gene therapy trials. And there are some international ones that didn't have any published data anywhere else. So I just wanted to mention they're also doing gene therapy in Russia and gene therapy in China with their own products. All right, moving on. There is a series of trials for myostatin inhibitor through Scholarock. And these are in later onset SMA. And they are IV infusions every four weeks. The patients are allowed to be on background therapy. And this is an anti-myostatin monoclonal antibody that blocks both pro-myostatin and latent myostatin. The TOPAS trial, this was done when we only had nusinersen, so they were on a background of nusinersen. So this was a proof of concept study phase two for safety and efficacy. It was well tolerated and motor improvements that were seen were well sustained. The ongoing SAFIRE trial, these are in type two or three non-ambulatory adjunctive therapy for SMA patients on a background of risdoplam or nusinersen. And there's a dose escalation in this. And so the primary endpoint would be the change in baseline on the Hammersmith. And secondary endpoints are greater than three point gains in the Hammersmith revised upper limb module and the milestones. And so far there was an update on the 7th of October that they met their primary endpoint at week 52. And then in 30% of patients they had greater than three point gain versus 12.5% in the placebo. So this was very encouraging news, late breaking. There is a planned trial called Onyx. I love how everyone has a theme. So there will be a phase four trial in Onyx and these will be long term extension studies, open label extension for patients from both the TOPAS and SAFIRE studies. So they'll be on active drug. There's more myosatin inhibition trials in SMA. There's the resilient trial through Biohaven. And this is any type of SMA can have this. And this is ages four to 21. This is instead of an IV, this would be subcutaneous administration. They're using two doses, 35 and 50 milligrams based on weight. And they have a mechanism that inhibits both myosatin and active N2B. So you have regulation of both muscle and adipose tissue. So you have increase in muscle bulk, but also decrease in the fat that replaces it. They named their penguin Resi for resilient. He's flexing because it's muscle building. And I thought that was really cute. And then there's also the Manatee trial. This is a trial through Roche. They're doing Rizoplam plus their myosatin inhibitor called GYM329. And this is going to be done in the, this is done in ambulatory SMA ages two to 10. And they will, they're assessing safety, efficacy of the dosing of this. And this is going to be subcutaneous as well on a background of oral Rizoplam. So that's all the named SMA trials we'll be going to. And I'm switching gears into congenital myopathies. And so previously before we were doing genetic testing for these myopathies, we were doing a lot of biopsies to find out what kind of myopathy they had. So we would classify them based on central core, multi, mini core, other like descriptions of the muscle itself. But now we're like looking at more of a genetic basis. And so we're also looking on the biopsy, is this dystrophy or myopathy? That would be another reason why we would get a biopsy. And the difference would be in myopathy, we'll have defects in the contractile matrix or the force or the force generation that you can do. So the muscles don't necessarily break down versus the sarcolemel integrity that can be affected in the dystrophies, which is why we see lots of the muscle membrane breakdown, release of intracellular contents, and then all of the sequelae from that. And so now that we're getting all of this gene testing, we're having a lot of our myopathies genetically characterized. And so these are targets for gene therapy now. And we're also discovering, oh, you know, instead of that kid being the slow one in class, we found out that he actually has a congenital myopathy. Or this whole family that just wasn't athletic. They actually have a inherited myopathy. And also our late onset adult patients may have had something all along. So clinical trials in congenital myopathy, not as numerous, of course, as SMA DMD, but there are a few trials going through the clinicaltrials.gov lists. There is one ongoing trial for oral salbutamol for congenital myopathy. And this group was not very strict in who they would, like, who would they allow into the study or inclusion criteria for the study. It would be a clinical diagnosis with biopsy or genetics of congenital myopathy. And this is six-month placebo-controlled versus two milligrams of salbutamol with a crossover using MFM32 as a primary outcome measure. Only in Sweden, I didn't see any published data or results yet. And then there was a trial for congenital muscular dystrophy, which we grouped some of our collagen 6As into myopathy or dystrophy, which is why I included this. And so this is a small molecule therapy for, that showed decreased apoptosis of muscle cells in mouse models. And so they included patients five to 16 years with a genetic diagnosis of either collagen 6-related or Lama 2-related myopathy or dystrophy. There were safety endpoints that were met, but unfortunately, there were no clinical changes on their motor function tests from baseline. And then there was actually a recent trial for RYR1 using N-acetylcysteine as an antioxidant therapy. And in RYR1, because of the calcium influx, there's thought to be a lot of reactive species that get created. And so the point of this was to reduce the oxidant effects of RYR1. And so this was placebo-controlled. They had oral tablets of N-acetylcysteine. Unfortunately, it didn't meet its statistical significance in their markers of stress. They measured metabolites in the urine to make sure that it was actually working. And this was not reduced significantly, reduced a little bit. And then six-minute walk test as their motor measure was not significantly increased in the meters walked. So unfortunately, negative study, but somebody is looking at RYR1. Moving on to the MTM trial. So this was done in the early 2000s. The publication is dated 2023. So they have in their discussion kind of reflections on the trial overall. So this is a trial for X-linked myotubular myopathy. And this is in kids less than five, including ventilated children versus a natural history control. And they were running that natural history control at the same time. And so it was a two-part study with a low-dose and a high-dose cohort. And the primary efficacy outcome was a chained baseline to week 24 and hours of ventilatory support. So if you've met patients with X-linked myotubular myopathy, they are very weak and have a lot of bulbar issues. Other secondary outcomes would be motor milestones. Unfortunately, as you've heard in the other talks, as well as just history, there were unexpected deaths in this study. This kind of, oh, it looks bigger over there. So there was one death in the high-dose cohort. And if you look on the right side of this screen, they had enrolled patients. So at the upper left is the control. And their control natural history group was called Inceptis. And then they screened patients from that natural history study to come into the treatment arm. And so they randomized them into cohorts for high-dose, low-dose, and then a delayed treatment. So it'll be natural history, then crossover into treatment. Unfortunately, in the higher-dose cohort, there was a death. And so the death had the trial on clinical hold in 2020 in May. And the FDA said you can continue, but just on the low-dose. Two additional deaths occurred from the high-dose cohort in the meantime. And then upon continuation in the low-dose cohort, one more death occurred. And then there's a clinical hold from September 3, 2021, which is still in effect. However, there are some very notable results. The top graph here is the percentage time from baseline in ventilation hours per day. So lower towards the bottom and higher towards the top. The low-dose cohort is that blue line that you see, the light blue line. And then the higher-dose cohort is the red in the middle, and then natural history control is the top. And so what was great about that, there was a reduction in ventilation hours. They measured it as a 77% reduction in least means, squares mean hours per day of ventilator support. And some actually became ventilator independent, which is amazing. So that has never happened before, as they noted, where you give a therapy and they come off the ventilator. And then there were some of these patients who were able to sit independently, stand, walk independently, and climb upstairs as they got older after they were dosed. So that's the bottom graph. It's a little small, but they put the motor milestones using these symbols. And these children, once they started the therapy, looked like they were gaining these skills, which is amazing. And so why did these deaths occur? So they found that the patients who died had evidence of pre-existing hepatobiliary dysfunction. And this existed even before they received the gene therapy. And then also, like in this child, they tried to ameliorate the massive immune response with immunotherapy, steroid dosing, other immune therapies, but no response in the markers. So then, and then also ultimately, the deaths involved cholestatic liver failure. And then when they list the causes of death, they died from sepsis, severe immune dysfunction, septic shock, GI hemorrhage, which was also seen. And then control patients also died during the study because MTM-1 is very serious. And they also had this condition called hepatic piliosis, which is hemorrhage within the liver, as well as aspiration pneumonia and cardiopulmonary failure, which can happen with natural history as well. And so what the authors concluded at the end of this was, consider investigation into that pre-existing cholestatic disease in MTM-1. And when they retrospectively look back, they're like, oh yeah, they did have hyperbilirubinemia at the time of enrollment. And then ultrasound findings showed abnormal liver ecogenicity. Other gene therapy observations from this trial, as we know now, now that we're dosing more and more gene therapy, there was troponin I elevation, as well as transient thrombocytopenia that resolved. But what was cool, they did muscle biopsies during this trial, and they saw that there were improvements visually in muscle fiber size in organelle organizations, since we know what the changes are on biopsy in MTM-1. But they didn't see the change in centronuclear, so you still see the centronuclear part of the centronuclear myopathy. There was another trial called DMN-excite, and this is a trial for genetically confirmed DMN-2 or MTM-1, both centronuclear myopathies, I think also for gene therapy, but currently on hold for elevated LFTs. So it's still something that we're trying to get to, and then Dr. Shea mentioned in his talk yesterday, they're looking at other vectors that may not affect MTM-1 patients, like this one, which was, I believe, AAV-8. So in summary, exciting time for pediatric neuromuscular medicine for the congenital myopathies and dystrophies, the MTM-1 trial is on hold, but given those amazing results, hopefully we'll restart with safety measures in place and alternatives in place. And then in spinal muscular atrophy, we have gene-targeted therapy with SMO, small molecule and gene therapy options, as well as add-on adjunctive therapy with myostatin inhibition or combinations of all of the listed medications there. And then many long-term extension studies are ongoing. And how to keep up, I had to look at the clinicaltrials.gov. The SMA parent organizations are great at keeping track of all of these and actually have all the links for the parents to click so that they can enroll if they see that their child is eligible. And then the major manufacturers also have research portals for parents or interested people to look at. Those are my references. Morning. So just to correct a little bit Noelle's introduction earlier, I'm not talking about my own research today. I actually work on stem cell modeling of congenital muscular dystrophy and motor neuron disease, but I'll be telling this story about myostatin. I think it's one of those sort of obscure to most people. What happened to that myostatin? So we're spending a little bit of time on this story. I have no disclosures and so I'm just going to go over some of the ideas behind myostatin inhibition, how some of these strategies were employed to try and translate into clinic and help patients, and then what that means in the current landscape of pediatric neuromuscular disease. But I want to start with this slide because myostatin deficiency exists in nature and there's a pretty consistent animal phenotype. Long before Dr. Sijin Li introduced us to Mighty Mouse in 1997, after he discovered this gene, I'm just trying to get my laser pointer. Long before we met Mighty Mouse, there were cattle that were being very skillfully selected by farmers who had this double muscling phenotype, very consistent. They could breed these cattle and then after myostatin was discovered, these were quickly genotyped and found to be myostatin deficient. These mutations are present in some high-performance dogs. I'm showing a whippet here that shows a dose-dependent increase in muscle bulk based on whether it's missing one or two copies of the myostatin gene. The Department of Fisheries took advantage of this, created transgenic trout that have more muscle, more meat, feeds more people. So it's, you know, it has already been used to help to better humanity. And then there are rare reports of humans with myostatin deficiency. That's not to say there aren't more of us out there, but it's just not something we test for. So these two that you can find, one in the literature of a baby who had large muscles and seemed to be stronger than a typical baby, and then a world's strongest toddler documentary. They just happen to be connected at academic institutions that like to go through these academic exercises and genotype their patients and found that they were myostatin deficient. So what is myostatin? This is a figure that Dr. Sijin Lee actually published a few years ago in blue showing things that are enhancing muscle growth. And this is very overly simplified, but I like that it kind of points out some of the important factors that have been manipulated. So folistatin is one that I want you to notice in blue. And then in orange you can see factors that limit muscle growth. So blue enhances muscle growth, orange limits muscle growth. You can see that myostatin is inhibited by many different factors. It's a member of the TGF beta super family. These are also called growth differentiation factors. There are many of them. They have very close homology with each other. It's the only one that's specifically known to inhibit muscle growth. It's present in embryogenesis, it's present in developing and mature skeletal muscle. And it's interesting in that it's secreted by muscle, goes through the blood, comes back and hangs out in the extracellular space of muscle as an inactive form, and then magically when needed is activated and negatively regulates that muscle's growth. So it's like a break signal to the muscle. So autocrine and paracrine signaling. And these mechanisms aren't completely clear, but nature does this very well. There are multiple proteolytic processes needed to activate myostatin, so this is another important fact to remember. So what you see here, this is the active dimer of myostatin, but before that you have a propeptide that's inactive that negatively regulates myostatin. And then in between these two there's actually also a latent form. So different enzymatic processes to get from propeptide to latent peptide to active peptide that then dimerizes, binds to the active end receptor that is shared by active end, which is a different GDF. And the canonical transducers of TGF beta signaling are the SMADs. And so this is just a simplified, very typical pathway that is overly simplified, showing that the SMADs are the transducers, and then they travel into the nucleus, hit the target effector genes, and mediate, you know, different cell processes of growth, differentiation. Some act, you know, same molecule can act as a tumor suppressor and an oncogene. They're very complex pathways. And if that wasn't complex enough, this was published the same year, and it's just summarizing at the time, which was a few years ago, the multiple different pathways that these receptors can activate. So here are those SMADs that were on the last slide, but you can see there are multiple pathways in parallel with crosstalk among and between them. And nature has mastered this, right? And so for myostatin, despite the complexity we see with our eyes, the cell knows exactly what to do, and it has to do with activating muscle stem cells, proliferation, migration, alignment, fusion, differentiation of myotubes. And again, the myostatin is the stop signal to that pathway. So of course we're going to try to harness this, right? Let's try to inhibit myostatin to help patients. We know that in nature there are biologic regulators of myostatin other than itself. I showed you that it regulates itself, but there are also molecules, I'll point out again, folistatin, and we've developed numerous compounds including antibodies, propeptides, soluble receptors, endogenous antagonists, and I'm including a slide that summarizes many of those that have been tried. And in animal models, and this has been shown in some other talks this week, you do, the preclinical data are very promising, right? We have a lot of excitement with, you know, the preclinical data and the animal models. You can increase muscle mass and strength and function and bone mass. You can prevent sarcopenia. You actually get a switch of your muscle type to favor type 2 glycolytic fibers. And then, you know, something that Noel alluded to, there is also really exciting evidence that you can change metabolism, increase sensitivity to insulin, and decrease body fat. But for our purposes, these were employed to try and help patients with muscle disease. So muscular dystrophies, sporadic IBM, as it used to be called, and SMA now. This is just a list that was published in 2020 showing at the time what drugs were in trials or completing trials for myostatin inhibition. And I just want to point out the right-sided column here, that time after time, the drugs were not efficacious. They were discontinued due to lack of efficacy or side effects. And when we talk about side effects, it tends to be because of the off-target effects of inhibiting myostatin, because of the homology it shares with other TGF beta family members. And so you get changes in angiogenesis, like bleeding and telejectasias. You get change in bone development. You get spontaneous fractures. And so many of these were discontinued for side effects or just lack of meeting primary endpoints. This was two years later, so the list starts to dwindle, and it is even smaller now. So I'm just going to bring you back to this slide and talk quickly about how this was used in, well, one of the ways. There were multiple trials for Becker and Duchenne, but in particular, I just want to talk about the folistatin that was actually developed by Jerry Mendel, who we also know for developing gene theory for SMA, and now Duchenne as well. This was an intramuscular injection of AAV1-delivered folistatin. So the idea was to try to inhibit, at the muscle level, myostatin by increasing folistatin with this adenovirus delivery. And so they had intramuscular injections, and they did increase muscle mass, as you can see here in this picture, right where the injections were. Some Becker's patients actually did have improved six-minute walk tests, but by and large, when they combined all of the data together, it failed to meet the primary endpoint. And just to point out sort of some subgrouping, patients who tended to have more fibrosis at their baseline tended not to improve, whereas patients who had less fibrosis tended to be the ones with better scores. But when you look at all comers and when this was used in Duchenne patients, there was no significant improvement. And so what are the challenges with muscular dystrophies? You're probably starting to put it together yourselves as well, but there are several reasons. One is that animal models don't always perfectly represent muscle disease. Increased muscle mass doesn't always equal increased strength. And then fibrotic muscle does not appear amenable to improvement. And so what is the best case scenario? Treating pre-symptomatic patients. We're not there yet with muscular dystrophies, right? We kind of, they end up in our clinic when they're symptomatic. Our institution and perhaps yours as well are talking about maybe doing genetic testing on every baby that comes through. Wouldn't that be great? So if we can find these babies before they're symptomatic, then that, you know, expands our potential for treatment. The AAV used by Dr. Mandel, that was an intramuscular. Why intramuscular? We know by now after this week and all the talks you've heard in your own clinics, it's really hard to target muscle with systemic delivery, right? There are side effects. You can't get to the muscle. And we know that many labs are working on improving their capsids with directed evolution and, you know, really potent muscle specific promoters. So stay tuned. I think we will get better at this and hopefully in the near future. So enter SMA. So we have our motor, our motor unit here. We tried treating muscle with myostatin, failed time after time after time. So what about this guy? What about the neuron? We know that you have motor neuron death from loss of SMN protein. The fibers, yes, they atrophy when they're denervated, but they should still be okay and healthy and maybe you can plump them up. Therapies targeted at SMN protein, we know that those provide benefit. Noel and others have shown that so nicely this week. Here are those treatments. So is there a role for adding in myostatin inhibition? You know, we're all worried about this potential treatment plateau with nusinersen and rizdeplam and maybe zolgensma. These patients aren't completely back to normal, right? They don't look exactly like typically developing babies and we know that people with SMA still have muscle fatigue regardless of what treatment they're on. Cure SMA survey showed that 97% of adults, if we had a magic wand, the main thing they ask is to increase their muscle strength. And so that's where we are now with myostatin inhibition, that it's being tried as a dual therapy for SMA patients. Noel showed that. It's been shown earlier this week. I borrowed this picture from Scholar Rock paper. Actually I like that it shows here these different forms of myostatin. So like I said, floating around in the extracellular space is this pro-myostatin and this latent myostatin. So these actually inhibit myostatin themselves. They take different enzymes to make the mature growth factor, which you see here in yellow. This is what has that homology with other TGF-beta superfamily members. And the first generation of many of the treatments, including some of the antibodies that are currently in trial, bind to this this form. So the active myostatin. And then some of the newer ones bind to pro and latent myostatin. And so those are the differences between the different antibodies that are in clinical trials. And just remember that the active myostatin shares 90% homology with activin. And so I'm not sure if that was actually on purpose or not. Noel mentioned that it may also decrease fat, but activin does a lot of other things. And then GDF11 also shares homology, which has very significant downstream pathways. And so these are the trials that have been mentioned here today. And otherwise I just want to kind of point out some of the key differences. So Biohaven with their T-alpha, this is a more engineered product in adenectin. And it binds the active myostatin and then also activin A. It's in phase 2. This was actually the same product that was tried in DMD under a different name and did not work in that context. Genentech has GYM329. This binds pro and latent myostatin. It's in phase 2 and 3. This is fully humanized and it has a cool mechanism. When the antibody and antigen are released, the antibody can come back and bind more products, more, sorry, more protein. So that seems pretty helpful. They're also thinking about treating FSHD, so stay tuned for that. I believe this one is sub-q. It wasn't disclosed. And then Scholar Rock has a pitagromab that also binds pro and latent myostatin. They just finished phase 3. When I made these slides, we were awaiting data readout. And as Noel mentioned, they just put out a press release last week that they have shown benefit. They're also looking at treating cardiometabolic disorders because, again, increases insulin sensitivity. Seems to be helpful in other ways. And this is the one out of the three that's an IV infusion done monthly. And then here's their press release that was mentioned that they showed statistically significant increase in the hammer scores versus standard of care, which was, you know, either Spinraza or, sorry, Nusendersen or Ristoplam. So in summary, it's complicated, but myostatin is a key negative regulator of mature skeletal muscle growth and hypertrophy that nature does perfectly. And we don't do it perfectly yet, but we're working on it. Mutations exist in nature. You can see bigger muscle mass and strength in the absence of myostatin. And the homology to other growth factors and those common downstream pathways that are so important renders it really challenging to manipulate in vivo without having bad side effects. We've tried many times to treat muscle disease, and those have failed time after time. So the current focus in the current landscape is antibody, anti-myostatin antibody therapy as a supplemental treatment in SMA. And I hope that kind of makes sense now as to how we got there. Future focus, we're looking now at cardiometabolic disease. So this was kind of a surprising and helpful turn of events treating diabetes, sarcopenia, and then hopefully someday to treat muscular dystrophies. I think when we get better at identifying muscular dystrophies before patients are symptomatic. That's my last slide. Thank you. Very good, hi, I'm Stanley Nelson from UCLA and this particular project is an equal collaboration between the Micheli Lab at UCLA and the Nelson Lab at UCLA. And I'm going to guide you through some single nuclei data that we're using to try to dissect what's the true status of muscle in Duchenne and in healthy muscle. And a lot of the data, actually all the data that I'm going to show you will become public and searchable. So, just as a reminder for Duchenne muscular dystrophy, the key missing protein is this purple protein here linking the actin attached to the Z disk to the dystrophin-associated glycoprotein complex through the membrane to laminate outside distributing forces and reducing the number of tears that can occur in the muscle cell membrane. So, that critical function has been known for decades. But what does the true sort of environment look like inside of muscle is not clear and has actually remained fairly hidden. So, in Duchenne muscular dystrophy, on the left is normal where a muscle injury promotes an initial first wave of infiltration of immune cells, a second wave of different immune cells, and then a third wave of different immune cells resulting in fixing of an inflammatory environment into an anti-inflammatory environment and resulting in a repaired muscle, which is actually quite dramatically fast and efficient in mouse and in humans. And this is muscle regeneration. That same process is ongoing in Duchenne because nothing is damaged in muscle regeneration in Duchenne. But the normal mechanisms are wildly disrupted because a muscle is being damaged while a different muscle is being repaired and a different muscle is trying to be resolved. So, all these processes are going on, resulting in a rather dramatically asynchronous muscle environment that is definitely inflammatory, it is definitely immune, and it's coupled with sort of this muscle membrane and damage. So, just expanding as opposed to thinking of probably almost all muscular dystrophies is just a dystrophy. It's really a dystrophy on top of inflammatory environment that's been created by that. So, years ago, recognizing where we wanted to get to in the Duchenne space, we repurposed breast cancer biopsy needles to get a sufficient and gently sampled bit of muscle. So, each core biopsy collects about 130 milligrams of tissue. We've done this on two-year-olds to 70-year-olds, two-year-olds not wide awake, but six-year-olds and wide awake with just local anesthesia. And with three core needle biopsies from the TA, gastroc, vastus lateralis are by far the most common muscles we use in six-year-olds and older. We typically collect about 300 to 400 milligrams of tissue of sufficiently high quality for histology sorts of diagnosis, immunofluorescence. And in the iterations that I'm going to show you here, we can culture out cells from this. We can culture out fibroblasts. We can culture out immune cells exposed to the actual tissues and brace. We can culture out immune cell or actually just purify out immune cells and see what their status was at the time that we did the biopsy. And we can culture out stem cells for some of the studies alluded to by other speakers. The stem cells change almost immediately when we take them out. So, this is a complex process, but we can get all those biomaterials. Here, I'm going to focus on a static look, which is when we capture some of the muscle, we can use about 5 milligrams to 30 milligrams to grind up the tissue, make a single nuclei prop. So, it's sampling both the type 1 fibers, the type 2 fibers, the immature fibers, and all the mononuclear cells in muscle. And through a process of creating single nuclei transcriptome libraries, you can generate an individual transcriptome for less than 25 cents per cell these days. And that cost will keep coming down to allow this type of exploration to occur on grander and grander scales. So, we can typically look at 5 to 10,000 nuclear transcriptomes per person from about 1 20th of a typical core needle biopsy. And then the rest of the materials are remaining for further research along the way. We published a pilot project on this in mouse, which is in the lower right-hand corner, which you can find online if interested. So, this is a human data set that I'll describe for you. These are nuclei purified from 32 different individuals. 27 had a clinical diagnosis of Duchenne muscular dystrophy. And when we take the nuclear transcriptomes, we sample about 20 to 40,000 reads. And that measures only about 1,000 to 1,500 genes. But those 1,000 to 1,500 genes are sufficient information, even though they're not looking at the whole transcriptome. They're random sampling of the transcriptome. So, the nucleus expresses titin, but doesn't express an immune marker. It's very likely to be a myofiber nucleus, et cetera. So, you can imagine doing this across all genes. And indeed, the clustering is performed in a non-directed way. And the clustering that occurs here with the multiple colors on the left are identifying the major cell types of muscles and their proportions within individual. And this is the aggregate data set. So, this is about 134,000 nuclear transcriptomes on the left. And then each individual either had a Duchenne state or a healthy state. So, we can track that as well. So, now we have individual nuclei mapping to individual cell types. And then with disease status and from whom they came. We can then further add to this information. So, it's not a mystery to most of you in the audience that many individuals, particularly when we're sampling muscle from younger children haven't truly manifested what their disease course is going to be over their life. So, out of 27 individual DMD muscle biopsies, we could look to see just some of them express low levels of dystrophin. So, think of this a bit like the most common mutation in Duchenne is an exon 45 deletion. And some of those boys skip 44 naturally and make low levels that's sufficient to mitigate their disease. And we can observe it. But we don't predict it particularly well in young children. So, on the left is an example of this. A bit of a different mechanism. This is a nonsense mutation. Many of the boys in this particular study had nonsense mutations. His nonsense mutation was an exon 39. Exon 39 is in the central rod domain. It's an in-frame exon. And if exon 39 were skipped, the message would remain in-frame. And so, that is what happens in this boy. So, this boy happens to have about 4% of his mRNA naturally skipping exon 39 resulting in an in-frame protein. And on the left, we can see patchy staining of dystrophin consistent with the phenotype that would mitigate the disease phenotype. So, this is a somewhat modulated phenotype. So, we're going to categorize those seven as DMD dys low. Don't really think of them as Becker muscular dystrophy. Some of them clinically are going to be extending their time of walking. But they're going to be relatively severe Becker muscular dystrophy individuals. And I'd think of them as more in the spectrum of Duchenne. So, within this dataset now, we're going to create three categories. So, DMD dys negative, red. DMD dys low, green. And healthy individual muscle samples are blue. And all of the individuals in this dataset have been phenotyped. So, we know where they fit in terms of how severe they are at a given age. So, we're scoring where they are in disease severity. And we know where they are in age. So, we know where they are in the process of DMD that's shifting over time. So, with all that information, we're trying to garner, you know, how can we observe this dynamic process with all these static data, if you will. But what we're now able to do is look specifically just at individual cell types. And only do gene expression between those cell types. That is, is a type 2 dystrophic myofiber different than a type 1 dystrophic myofiber? And the answer is yes, they are different. Are there shared similarities? Absolutely. But we can start to dissect those mechanisms. So, with that, in the muscle resident cell populations, we see substantial shifts. And I'm going to guide you to a few combinations of things. So, one is in the myofibers, which are being depleted. Fibroblasts, which are being expanded. Myeloid cells, which are being expanded. So, and then lymphoid cells play a critical role in the story I'm about to tell you. So, DMD myofibers become immunologically alert. They become better poised to be able to present antigen to lymphoid cells. But they critically upregulate a huge amount of TLR4, which is a sort of a danger signal, if you will, for receiving danger signals. And it's upregulated 28-fold in dystrophic myofibers with a wildly significant p-value. And this, if it's signaling through its receptor, would increase NF-kappa B signaling, which you heard about from one of the other speakers. And genes that are turned on by NF-kappa B signaling are dramatically increased within the muscle as are signals for apoptosis or pyroptosis in the skeletal muscle fiber, a mechanism of likely contributing to that muscle cell death. So, on the left is a green TLR4, just showing that the TLR4 is present at the membrane in these dystrophic myofibers. And then the part that I'll show you on the right, because we're going to juggle a lot of information pretty quickly, is down here we've separated each of the individual main myofiber types so that we can do differential gene expression in a very specific way, and then categorize if there are pathways that are upregulated. So, in muscle, TNF-alpha signaling via NF-kappa B as a pathway is increased, particularly in type 1 and type 2 fibers. Apoptosis is increased. And that's all I'm going to point to here. Myogenesis is also increased. So, hold that in your head for a second. Fibroblasts become wildly more diverse. So, when the dystrophic environment occurs, there's a typical endomesial fibroblast that simply does not exist in Duchenne muscle. So, fibrosis and this sort of the supporting structure has dramatically changed through epigenetic reprogramming. And I'm going to focus you on one cell type in particular, which is fibroblast 6. Fibroblast 5 is the normal fibroblast. Fibroblast 6 is a portion of the fibroblast that get generated. There's actually several different types of fibroblasts that are being created. Fibroblast 6, we think, is going to be critically important because it is mimicking what's been observed in other inflammatory diseases. In this instance, it's analogous to a critically important synovial fibroblast derived from inflamed joints of rheumatoid arthritis that are driving inflammatory priming that are causing this inflammatory environment within the muscle and could be a target for therapeutics. Variety of reasons we know that these exist. This is a gene expression plot. So, blue on the left is higher expression and it's a thigh 1 positive fibroblast. And if you go down that column where I have the arrow here, it has very high expression of TNC. TNC is induced 30-fold in these fibroblasts. And TNC is one of those TLR4 ligands that's a damp so it can signal danger to the muscle and promote this environment in this cycle of inflammation. So, this is a healthy muscle here, almost all five. These are Duchenne muscles over here. No five exists. And multiple other fibroblasts type exist. This is showing all of those original cell types. This is the fibroblast group down here. And this sliver here is very high TNC expression. It actually happens to be the six that are shown in different projections of the data. And TNC can be observed in the plasma as well. So, we can look back at the muscle, excuse me, muscle RNA expression correlates highly with the amount of TNC that we can observe in these patients' plasma. And it correlates with disease severity. So, these gene expression from this fibroblast six of synovial fibroblasts from rheumatoid arthritis, there's a large expression signature for how they've been altered in rheumatoid arthritis. And that signature is replicated well in our fibroblasts in C2, which is here. This is from the frozen biopsies. And then when we culture out fibroblasts, we can reiterate that signature by taking the same fibroblasts chronically stimulating with TNF alpha and generating a similar expression signature that can be squelched or lessened by rapamycin indicating that it's driven at least partially through mTOR. These are many of the genes that are induced include C3, which is dramatically induced by TNF alpha and can also be suppressed in culture by rapamycin. So, rapamycin is something to keep in mind in terms of mitigating this specific portion of the inflammatory response. Again, in other approaches, this is a mouse data down in the lower right by Basel Petrov. And this is showing that there's trained immunity. Trained immunity is teaching the myeloid cells about how to generally be poised for promoting inflammation. So, in the Duchenne mouse, damps in the bone marrow in this particular model are retraining through epigenetic mechanisms, myeloid cells. And we're seeing that in the human dystrophic muscles. So, we can see multiple different myeloid populations. These three here, TAMO, PPAR gamma, and SPB1 positive cells are actually some of these trained immunity monocytes, relatively immature monocytes that are now trained and we can observe them in the muscle. MRC1 is the major population of myeloid cells in the muscles with a different role to play. So, I might just skip over that. So, a final ingredient to throw into this mix are the immune cells. So, we took immune cells, purified them, and did multi-parameter identification with a method that's putting a tag, a sequence tag on. So, we could do 40 different markers, do very effective T cell, B cell, and macrophage delineation. And I'm going to focus your attention here on these CD8 effector memory cells and CD8 central memory cells, which are TEK cells. So, TE effector K, and the K is for granzyme K. And the expression of granzyme K is indicated here in this plot on the right. And granzyme K expressing T cells are capable of creating an alternate complement activation system. So, granzyme K can cleave C4 and C2 to create the C3 convertase, which converts C3 secreted by the fibroblasts into the interstitium to create active C3, which is C3A, which can interact with C3AR, which is present on the MRC1 positive fibroblasts. This cycle is an important cycle to consider, even as we're doing gene therapy, even as we're doing gene replacement, even as we're doing myostatin to try to fix these things. So, this environment simply exists in our dystrophic muscle. And it's not just put the myofiber, or, you know, put the dystrophin back in, but it's mitigate this entire inflammatory environment is going to be critical for ultimate success of all of the therapies that we're discussing. So, this is just a summary of this data. So, in the myofiber, actually, let me start with the fibroblast 6. Fibroblast 6 has developed through mechanisms of inflammatory signaling, particularly by TNF-alpha and interferon gamma. And it has receptors for receiving signals from TNF-alpha, which is coming from the macrophage populations and the lymphoid populations. It, in turn, makes various TLR4 ligands. I'm going to highlight TNC because it's so highly induced. TNC can then go back to interact with cell surface receptors on all of the macrophage. And that further induces NF-kappa B activation and secretion of more TNF-alpha. So, this is a feed-forward loop that's ongoing, regardless of whether we put gene therapy in or not, and is an important one to try to mitigate. Similarly, the TLR4 ligands, again, highlight TNC, and specifically can interact with TLR4, which is not normally on myofibers, but is definitely on in dystrophic myofibers, indicating that they can receive this signal. We have evidence that they do receive the signal because one of the mechanisms that occurs when TLR4 is interacting is upregulation of components for antigen presentation, which is observed in the myofibers. And there's signaling for NF-kappa B gene expression program being observed as well. And then finally, this is the fibroblast-secreting C3, this fibroblast-6 population-secreting C3, which is then interacting to make C3-alpha, coming back to interact with MRC1, and further stimulating this inflammatory response. So, alternate ideas that we would be proposing is, as we move into gene therapy, how do we mitigate these responses? So, gene therapy is a reality. We can put genes in. Exon skipping is a reality. We can observe the therapeutic benefits of these. We can observe the therapeutic impact of these. These are actual data from muscle biopsies that we're doing post-exon skipping and post-gene therapy to now look at the impact of these therapies and what is improved, what's not improved. Post-gene therapy, we're now collecting a group of boys to assess what happened before, what happened after, what got mitigated, what didn't get mitigated, what immune targets, what inflammatory targets remain. And this fibroblast-6 population, we predict, is going to be actually rather important. And this is an example, not showing you all the data behind this for lack of time, but 1576 is a boy who's not doing particularly well post-gene therapy, and he is further expanding the relative importance of his fibroblast-6 population. So, these are data sets that we're gathering post-gene therapy now, and these are individual data points for when we have muscle biopsies collected. And we're inviting a few dozen boys post-commercial gene therapy, post-previous gene therapy, to come in for muscle biopsies. And usually in a boy who's nine, who's not sort of neurocognitively shifted, you know, doing a wide awake in clinic with local anesthesia, core needle biopsy is readily possible. So, we've done, well, actually, we've done hundreds of the biopsies, and we've done about 16 biopsies. So, that data will be aggregated and integrated with what's happening in the peripheral immune system by looking at alpha, beta, a snapshot of that. Already we see massive expansions of specific clonotypes. I'll just highlight this one individual here. 8203 has 6% of one specific clone occupying, this is actually from peripheral blood, and that's a huge expansion of a single clonotype. We also see some evidence that CD4 and CD8 cells can have the same clonotype indicating that they may be transdifferentiating between CD4 and CD8 status, which will be actually quite interesting if that proves to be true from our genomic data. So we're building more models of this, and I think I should pause here and acknowledge all of the many great collaborators on this project. Thanks. So I know it's time. We're running over time, so if you would like to stay, Dr. Fay and I are going to talk about pediatric MG. Otherwise, it is a break. And then we'll take questions at the end, and we'll try to kind of make it fast. So I'll start. So these are just kind of in a similar vein, wanted to go over pediatric myasthenia and clinical trial updates in this space. Nothing to disclose. And so as you've heard in the other myasthenia sessions here, there's so many exciting drugs for adult MG. And so we do not yet have these novel therapeutic options, and we do, you can, get off-label use for these. There are ongoing clinical trials in pediatric MG, including FCRN inhibition with F-gartigiamod, both IV and subcutaneous, as well as nipocalumab and rosaniliziximab. And then there's also complement inhibition trials going on and completed. So eculizumab has completed, and ravulizumab is ongoing, as well as the leukoplan for subcutaneous administration. For congenital myasthenic syndrome, 3,4-DAP is on the market under FDA approval, but for adult and pediatric Lambert-Eaton myasthenic syndrome, not congenital myasthenic syndrome. Unfortunately, that would have to be a workaround to get. There is a lot of support for oral albuterol for congenital myasthenic syndrome, and that is used widely. And also, there are preclinical studies in the mouse model of DOC7 for gene therapy, as well as other targets for gene therapy, such as CHAT, congenital myasthenic syndrome. And so there are clinical trials in peds going on for F-gartigiamod. And so there's a phase two, three study for the IV form. Just like the adult form, four doses administered weekly, IV, and then a break, and then administration again. And this is for ages 2 to 18 years. There's also ongoing subcutaneous trial for the subcutaneous version, administered also four doses weekly, and then a break. And then also, there's an ongoing trial for ARGX119. This is a monoclonal antibody for activation of musk to stimulate the stabilization of the neuromuscular junction in DOC7 congenital myasthenic patients. This is an adult study, but hopefully comes over to the pediatric population. Ongoing trial for IV nipocalumab, which is an FCRN mechanism. This is an IV Q2 week infusion. And the interesting thing about this one is that they're doing both acetylcholine receptor positive and musk antibody positive patients. It's 2 to less than 18, or 8 to less than 18 in the US. And in 20, there was just an update presented at this session that there was efficacy in adolescents who received this over a course of six months, as measured by MGADL and QMG. For the complement inhibition space, there is a completed trial in ecolizumab. And this is a Q2 week infusion. There are other pediatric indications for this. This was done in adolescents 12 to 17 years, acetylcholine receptor antibody positive. It is currently approved in the European Union since July of 2023, so not that long ago, for ages 6 to 17. Not yet approved in the US for the same indication. Hopefully, hopefully soon. There is an ongoing trial for ravulizumab. This is the Q8 week infusion. And this is for 6 to less than 18. And the people who were in the ecolizumab trial were allowed to roll over. And then lastly, there's a exploratory study for geferilimab, which is a subcutaneous complement inhibitor that would be administered weekly in a pre-filled syringe. They're not yet recruiting, but they're exploring this for pediatric clinical trials. And so just to briefly go over the ecolizumab trial. I think this data has been shown before, but there was a dramatic change in the QMG and MGADL results. And it's doing this over the 26-week period. By week 4, you're already seeing a drop in the impairment of these myasthenia patients, and then greater than 5-point reduction in MGADL results, which looked great. Other clinical trials in myasthenia, the rosanolixizumab trials for subcutaneous. This is a daily subcutaneous injection by FCRN inhibition, ongoing for acetylcholine receptor antibodies. And then they will be doing a long-term extension trial. And then the Zilbrisk or Dilucoplan daily subcutaneous injection, which would be a C5 inhibition in adolescents, 12 to less than 18 years of age in the US, and then 2 to up to 18 outside of the US, ongoing recruitment. So in summary, the way we currently treat pediatric MG is things that are generally accepted. If it works in adults, why not in peds? We don't actually have a lot of FDA approval for much of what we do in pediatric neurology, but we have options, many of which are listed here. But there's a lot going on. So management of pediatric generalized myasthenia may be catching up to adult myasthenia relatively soon. There are a lot of trials going on in already approved adult medications. Off-label use is possible if you need it. I have a few patients who are on those novel therapeutics. And then if your patients can enroll into these studies, they can get free access to drug, and they're currently recruiting. And so there are also options for congenital myasthenic syndrome on the horizon. Most exciting is that for DOC7, and then gene therapy for congenital myasthenic syndrome. So the insurance may pay for your off-label refractory, but you will not get the patient support from the company necessarily, because it is off-label. And thank you for listening. And those are my references. And then I'll have Dr. Fay finish up. I will be quick, and I'm going to piggyback off what Dr. Tjongsun just talked about with the ongoing trials and talk about real-world situations of what to do when your patient is refractory. And so we don't have approved options. A lot of children, fortunately, respond to IVIG and steroids, but many do not. And IVIG, when it's needed as a long-term therapy, is quite burdensome, both in terms of missed school and missed work for parents, long infusions. And so monoclonal antibodies offer shorter, less frequent infusions. But since we have limited data in children, we have no FDA approvals, what can you actually do in the real world? And so I'm going to talk about a few different monoclonal antibodies that we've used in our most refractory patients. These are the main pathways for monoclonal antibody treatment. There's the B-cell targeting anti-CD20 antibodies like rituximab and obinutuzumab. There's the plasma cell targeting anti-CD38 antibodies. So Rituximab probably has the best data of all the monoclonals in the pediatric literature. Hopefully that is changing soon with the ongoing trials. This is all retrospective from some nice work in a French cohort, and Rituximab performs better than conventional immunosuppression and is even better if you use it first line rather than second line. So this is the first case I'm going to mention. This is a nine-year-old boy with an autoinflammatory syndrome, so a genetic disorder of the innate immune system, who developed myasthenia later, and he had a very clear response to periodostigmine and steroids, but too many side effects, a partial response to IVIG. And so we put him on Rituximab, and it just worked incredibly well. He went into a complete remission. He lives up in the mountains and got back to mountain biking and snowboarding. But then after about six to eight months, it stopped working as well, and then it didn't work at all. And our theory was that he developed anti-Rituximab antibodies. We weren't actually able to send testing for that at the time. He went into his respiratory crisis and stabilized him with IVIG and then plasma exchange. And then together with an immunologist that I work with, we decided to try Obinutuzumab, which is like Rituximab, an anti-CD20 targeting antibody, but has a couple differences. One is that it's humanized, and two is it does not cross-react with anti-Rituximab antibodies, and three, it's thought to have a better tissue penetration. And so we put him on Obinutuzumab, and within about a month of the first dose, he was completely in remission again, and he's now had sustained symptom control. So Obinutuzumab, there's not much literature about it in MG, but it may be an option for patients who respond to Rituximab and then stop responding to it. Next case is a seven-year-old girl with initially seronegative MG, later seroconverted, also with an autoinflammatory syndrome. She had a good response to IVIG but kept relapsing and is one of those patients who is showing up in the infusion center every three weeks. And so we decided to add on Ecolizumab for her, and she actually had very good symptomatic control with Ecolizumab and IVIG intermittently, other than some relapses with COVID. But then there was an insurance delay, and insurance is a recurring theme in getting authorization for these drugs. And she had a terrible relapse then, ended up on BiPAP, and has continued to be responsive only to plasma exchange now, no effect with Rituximab. So we ended up going the plasma cell inhibitory route to Daratumumab and alternating that with plasma exchange. She's had a good response in this diagram here. Plasma exchange is in blue. You can see she's needing these long cycles of plasma exchange. And then we started Daratumumab, and initially were only able to do one or two doses in between plasma exchange. And now we're able to do weekly Daratumumab doses, up to five in between the Daratumumab. And we later started Obinutuzumab, and she just had her best month of the last year and a half on this drug combination. So we're hopeful that by suppressing both B cells and plasma cells together for this super refractory patient, that we're going to be able to better control her disease. And finally, just quickly, a 14-year-old with refractory seronegative MG. Again, sort of similar story, responsive to periodostigmine, steroids, but too many side effects, IVIG, plasma exchange, do help him, but are not a long-term solution. So we were able to get him onto Efgratigimod maintenance, and that kept him out of the hospital at least. He still needed some intermittent therapies, and of course, using IVIG with Efgratigimod, they're incompatible. So we have had to interrupt his Efgratigimod at times to give him some IVIG. But this is a summary of all the monoclonal antibodies we used in our seronegative refractory patients. Thank you.

neuromuscular research

clinical trials

pediatric diseases

Duchenne Muscular Dystrophy

Spinal Muscular Atrophy

SMA

Vamorolone

Exon skipping drugs

gene therapies

Nusinersen

Onasemnogene abeparvovec

Risdiplam

myostatin inhibitors

Myasthenia Gravis

monoclonal antibodies

spinal muscular atrophy

SMA