Welcome to our session on add-on therapies for the treatment of spinal muscular atrophy. My name is Mitha Kardon, and I will start by reviewing myostatin inhibitors. Following this, my colleague, Dr. Stanley Copeland, will discuss small molecule treatments in preclinical and clinical studies. And finally, Dr. Klotz will update us on new uses of antisense oligonucleotide treatments. We will take questions at the end of the presentation. My disclosure is unrelated to the topic of this session, but we will discuss treatments still in clinical trials, which are not FDA approved. During my presentation, we will orient to the need for add-on therapies in SMA. We will learn the endogenous function of myostatin and some strategies for inhibition. And we will explore the effect of myostatin inhibitors in animal models and in ongoing trials in patients with SMA. Because of our target audience today, I won't spend much time on the pathophysiology of SMA. It's the most common motor neuron disease in children with an incidence of one in six to 10,000. We will focus on 5q-SMA, which is attributed to a homozygous pathogenic, or homozygous pathogenic variants, mostly, most commonly homozygous deletions in the SMN1 gene. The SMN2 gene is a phenotypic modifier with fewer copies associated with more severe symptoms, and more copies associated with moderate to milder symptoms. Currently, there are three FDA approved treatments for SMA. Nusinersen is an intrathecally infused antisense oligonucleotide treatment, which increases SMN2 production. Onasemnogene abiparvivec is a one-time IV infusion of gene therapy, which replaces the SMN1 gene. And ristoplam is a once daily oral liquid medication, which also increases SMN2 production. Now, although these treatments significantly reduce morbidity and mortality, there still remain symptoms of disease. Based on the 2023 survey from CureSMA, the majority of teen and adult survey participants had utilized at least one of these FDA approved treatments. However, patients still required assistance for ventilation, for sitting, and for ambulating. Survey participants, regardless of the severity of their symptoms, all had higher rates of fatigue compared with the general US population shown in pink. The survey concluded that individuals with SMA and their families viewed gaining muscle strength, achieving new motor function, improving daily functioning, reducing fatigue, and improving swallowing and communication as their top priorities for future research and treatments. So let's now understand how myostatin came onto the scene. In the late 90s, myostatin was found to be a negative regulator of skeletal muscle growth. Myostatin is also named growth and differentiation factor 8, or GDF8. It's in the TGF beta family and is expressed primarily in skeletal muscle. Mice with homozygous GDF8 deletions had larger skeletal muscles than their wildlife, or sorry, their wild type counterparts. So less myostatin resulted in greater muscle growth. Muscle biopsies demonstrated muscle hyperplasia and hypertrophy. This was demonstrated in larger mammals such as racing dogs and cattle, and conversely, high levels of circulating myostatin was associated with muscle wasting in cancer and HIV, in people with cancer and HIV. At the turn of the century, a baby was born with striking muscle hypertrophy and definition. Here's his muscle ultrasound with a schematic compared with a controlled patient. He underwent extensive testing and was found to be homozygous for a myostatin splice site variant, which was associated in cell culture studies with myostatin mRNA degradation and reduced myostatin protein production. So then followed the question, can inhibition of myostatin in people with SMA promote skeletal muscle growth? Let's learn more about how myostatin works. Promyostatin is cleaved to latent myostatin and then finally to its active or mature form. Active myostatin binds to the active in receptor type 2B or R2B, and this causes it to co-localize with its type one receptor counterpart, which is called active in like kinase four and five or ALK four and five. This type one active in receptor has a glycine serine rich intracellular domain, which phosphorylates several transcription factors, including those of the SMAD family. These SMAD transcription factors then go inhibit genes responsible for myogenesis. This is a normal process, which keeps muscle growth in check. In patients with SMA, there is insufficient SMN protein production leading to motor neuron degeneration and secondary muscle atrophy. FDA approved treatments restore myostatin, or sorry, restore SMN protein production, which helps maintain motor neuron function. However, because the myostatin pathway is undisturbed, there's less opportunity to overcome muscle atrophy. Various strategies to inhibit the myostatin pathway have been explored, with the ultimate question of whether the potential for greater muscle mass and strength could be realized in patients with SMA who've already participate in current SMN treatments. So let's look more closely at these strategies of myostatin inhibition. First, a general rule of thumb First, a direct antibody to the ACT-R2B receptor was trialed, but intolerable off-target effects like epistaxis and telangiectasias were observed. Next, inhibitors of active or mature myostatin were explored. I'll focus in on this anti-myostatin adnectin. Adnectins are an engineered class of proteins that have binding capabilities similar to antibodies. They have customizable components that can target, in this case, active myostatin. Finally, antibodies were designed to bind the inactive precursors, pro-myostatin and latent myostatin. And these are the three myostatin inhibitors that we will explore, epidigramab, Taldefgirbep, and GIN329. Let's start with epidigramab. This is a fully human monoclonal antibody which binds to both pro-myostatin and latent myostatin, reducing the production of active myostatin. Because epidigramab is a human-derived antibody, there has been optimism for fewer infusion reactions and hypersensitivity, as well as fewer anti-drug antibodies, which could neutralize potential therapeutic effects. Antibody bound to latent myostatin can be measured in serum and is shown to increase in people receiving this treatment. And active myostatin levels can also be measured in serum and is reduced in people on treatment. Epidigramab is administered via a one- to two-hour IV infusion every four weeks. So let's look at two mouse studies of epidigramab. This study utilized the dexamethasone-induced model of muscle atrophy in mice. I'll draw your attention to the decline in gastrocnemius muscle weight in the control mice in blue here. And we can compare this to the maintenance of gastroc weight in epidigramab-treated mice in orange. Epidigramab was then tested in the SMA mouse model. All SMA model mice exhibited reduced body weight over the study compared with wild-type mice. However, those treated with epidigramab in the dark gray bars here showed a trend toward increased gastroc and TA muscle weights, a trend toward greater muscle fiber cross-sectional area in red here, and increased maximal torque again in red here compared with control SMA mice in black. Topaz was the phase two study of epidigramab. The study was designed right after nusinersen was FDA approved and before other therapies were available. So all of the trial participants were on nusinersen and an IV infusion of epidigramab every four weeks was added. There were three cohorts and two different doses. All of the treated patients demonstrated increased serum concentrations of antibody bound to latent myostatin regardless of the dose they were given, implying reduced active myostatin. Efficacy was measured by the revised Hammersmith and the HF-MSE. And after one year, there was not much change in motor scales in ambulatory and some non-ambulatory patients. The non-ambulatory patients were followed for three years and this is where continued gains, mostly in the revised upper limb module were seen between 24 and 36 months. Importantly, there were no deaths in the study and no infusion reactions. All participants tested negative for anti-drug antibodies. SAFIRE is the phase three study of epidigramab which followed. It's a double-blinded, or it's double-blinded and placebo controlled in patients ages two to 21 years with type two and type three SMA. All trial participants were on either nusinersen or Ristoplam at baseline. And a press release last week cites that the study met the primary endpoint of statistically significant improvement in HF-MSE scores in the 106 treated patients after a year, after 52 weeks. And we hope to see more details on these data soon. The next myostatin inhibitor I will discuss is Taldefgirbep. Taldefgirbep is an anti-myostatin adnectin recombinant protein. Remember, adnectins are those engineered proteins that have antibody-like binding capabilities. This drug binds to the C-terminus of mature myostatin and to the ACTR2B myostatin complex. And it prevents this ALK4-5 receptor from co-localizing with ACTR2B. So you don't get phosphorylation of those SMAD transcription factors and therefore you don't inhibit myogenesis. This study in SMA model mice used an SMN2 splice modulator to permit the mice to survive longer in order to undergo dynamic studies. So the SMA model on its own without an SMN2 splice modulator, the mice don't survive very long. It's a very severe disease in them. This study design can also mimic potentially the human experience of patients utilizing an SMN up regulator and then adding on another treatment such as Taldefgirbep. So this study demonstrated a significant difference in maximum torque seen in green here as well as increased type 2a fiber cross-sectional area also in green in the treated mice. Taldefgirbep was shown to be safe and well-tolerated in two studies of healthy adult volunteers as well as in two studies in young children with Duchenne muscular dystrophy. In the DMD study however, a pre-planned interim analysis determined it would be unlikely for a clinical benefit to be detected in the functional outcomes and the study was discontinued unfortunately in 2019. But because SMA is not a primary muscle disorder, there has been more optimism for the potential use of Taldefgirbep in patients with SMA. Resilient is an ongoing phase three double-blind placebo-controlled study of Taldefgirbep in combination with any of the FDA-approved SMN up regulators. Taldefgirbep is administered via subcutaneous injection once weekly, rotate the sites, arm, thigh, abdomen and the study includes ambulatory and non-ambulatory patients ages four to 21 years and the measured primary outcome is the MFM 32. So hopefully we'll hear from this study and update soon. The final myostatin inhibitor I will discuss today is GYM329. This is an engineered sweeping antibody targeted to latent myostatin. So at a pH of about seven, which is what we expect of the extracellular space, the antigen binding component of the antibody binds to latent myostatin and the FC component binds to a cell surface receptor. This permits it to be endocytosed and brought to the endosome and at that lower endosomal pH of about six, the FAB portion releases latent myostatin which allows it to be degraded in the endosome and the FC portion now has an affinity to the FCRN receptor, permitting it to be recycled back to the cell surface for another opportunity to bind more antigen. So it kind of has this sweeping and, you know, function, I could say. I just think this is fascinating. This is the same cycle which is targeted by F-gartigamod. In that case, blocking of that FCRN receptor so that disease-causing antibodies can't return to the cell surface in the treatment of myasthenia gravis. I love when these things kind of line up from a pathophys standpoint. All right, back to GYM329. So this was tested in mice, including the hindlimb suspension muscular atrophy model and demonstrated enhanced grip strength in the hindlimb suspension mice, similar to control mice. Cynomolgus monkeys treated with GYM329 demonstrated increased muscle cross-sectional area on MRI compared with their open-circle controls here, an increase in overall body weight and a reduction in plasma myostatin levels, again, compared to open-circle controls. Manatee is a phase two, three double-blind placebo-controlled study of GYM329 in combination with oral ristoplam. GYM329 is administered subcutaneously in the abdomen every four weeks. Part one is a dose-finding study with safety and clinical markers, and part two is planned for study in ambulatory patients ages two to 25, with the revised Hammersmith as the primary outcome. For those accessing the PDF of my slides after the presentation, I included this helpful chart, which compares the three myostatin inhibitors in phase three trials, because I think it's a lot to try to keep track of on my own, so I really liked this chart for reference. And in summary, loss of function mutations in myostatin in animal models lead to muscle hyperplasia and hypertrophy. Myostatin inhibitors take multiple novel forms, including engineered proteins like adnectins, sweeping antibodies, and human-derived monoclonal antibodies. Off-target effects were seen in direct inhibitors of active myostatin, as well as the direct anti-ACT-R2B antibodies, and clinical trials of myostatin inhibitors are ongoing. Got some references if you wanna look, and then I'd like to thank AANEM for this opportunity, my co-presenters today, my clinical team at the University of New Mexico, my mentors and colleagues from around the country, and of course, my family. And thank you guys again for being so patient today. So next I'd like to introduce Dr. Stanley Copeland, who joins us from Dell Medical School to talk about preclinical studies, or preclinical and clinical studies of small molecule add-on therapies in SMA. All right, so I'm going to flip the script a little bit. I am going to talk a little bit about some studies in the clinical space, but I also have a couple of slides on preclinical studies as well that may perhaps be available for clinical studies to treat SMA later on in the future, hopefully. I have those two disclosures. One was in regards to SMA treatment, but it does not impact my ability to give this talk. First off, I just want to show, I think this is always really exciting to look back several years ago, nine to be exact. It seems like longer ago than that, but to see where we were in the drug pipeline for spinal muscular atrophy, it obviously is such an outdated look because today, this is the drug pipeline. I checked over the weekend just to make sure they haven't updated it, but this is the most recent one, which was published in April of 2024. Even I have done a couple of SMA talks for med students and just local department, and I had one from January of 2018. Of course, at that time, we only had one FDA-approved treatment for SMA, and now to see three there is really exciting. We all know that there are different parts of the peripheral nervous system that are affected in SMA, not just the motor neuron, which explains why we still see some of the unmet need, as Dr. Carden mentioned, and talked about directly, working on the muscle. Some of the things that I'm going to talk about are in regards to improving the transmission at the neuromuscular junction, and then we'll talk a little bit about some additional studies for drugs that are already approved. Intrathecal, not intravenous, intrathecal OA, or onosendazine abaparovac, has been in studies for several years now. Why would we try something when we already have the IV version? We already mentioned the unmet needs, and the IV version is, of course, only approved for very young children and babies. The hope is that with the ability to give the medication directly into the central nervous system that this would allow for greater neuronal transduction. Both IV and intrathecal administration of OA has been shown to provide systemic distribution in tissues. Now, there's a study published, and sorry, I didn't, it's in my list of references, but this was seen in some human autopsy samples, unfortunately, from some children that died in the earlier studies from, who had received the IV OA. And then intrathecal administration has been demonstrated in a study with the macaques. So we have published the initial portion, the phase one portion of STRONG, which was an ascending dose study of ITOA. There were 11 sites across the United States. Patients had to have homozygous deletion of SMN1 and with three copies of SMN2, but without the genetic modifier that can be seen. Patients had to be sitting, and this was described as sitting for 10 or more seconds, but they couldn't have the ability to stand or walk. This went from six months up to less than 60 months, so that's five years, less than five years. There were three dosing cohorts, and then two groups in, as far as the ages, age stratification. So in the younger group, these were those who were from six months up to less than 24 months, or less than two years of age. And then in the older group, it was those two years to less than five years. So as with phase one studies, the primary endpoints included safety and tolerability. The primary efficacy endpoint for the younger group was independent sitting for three or more seconds, and then for the older group, it was the change in Hammersmith functional motor scale expanded from the baseline, and that was when compared to historic trials, and that was the PNCR group that they used. So they screened 39 patients, and there were some screen failures, so it ended up being 32 patients. You have cohort one down here with three patients, which was the low dose, and then most of the patients, 25, fell into that medium dose cohort two. And then, unfortunately, there were only four in the high dose cohort, and that was because of a partial hold that was placed on this study by the FDA when there came about information in non-human primates showing damage to dorsal root ganglion. That hold was lifted, but then they decided to keep it at the smaller number, so that's why there's only four there. All patients who received the ITOA had one or more treatment emergent adverse effects. Only one of these was serious and felt to be related to the drug, and that was transemination elevations that did resolve. There was no thrombotic microangiopathy seen. There was no sensory ganglionopathy, or no evidence that was seen of that. Now, I would note, they did not do detailed screening exams and electrophysiological studies of the sensory responses, because at the time that the protocol came out, that was before that study in non-human primates showed that damage. And then, of course, there were no deaths. Some of the common side effects, as we see in the intravenous dosing, including low platelets, there were some, of course, other common adverse effects that we see, upper respiratory infection, vomiting, GI upset. So in the medium dose, we had one of 13 patients in the younger group who achieved independent standing, which that was, as we mentioned, for three or more seconds. And then, in the older group, when compared to the historic trials, there was a change from baseline in the HF-MSC that was significant at month 12. The efficacy analysis was not performed in the low and high doses. Now, the information is there of what the scores were, but these were not adequately powered for the analysis. In general, the intrathecal OA was very well tolerated. The medium dose older patients had improvement in the Hammersmith expanded versus historic controls. Of course, there were some limitations. This was a short post-dose observation period of only one year. And in the low and medium doses, in the high dose, it was 15 months. And then, of course, I mentioned the small size of the high-dose cohort. Now, there is a long-term follow-up. So hopefully, in the future, we'll be getting the updates from those patients who participated in strong. Now, there is a phase three randomized sham-controlled double-blind study known as STEER, and that is looking at the efficacy, safety, and tolerability of intrathecal OA. This is being done in patients who are from two years of age to less than 18, and importantly, they have to be treatment-naive patients with SMA type 2. So switch gears to the chloride channel inhibitor, NMD670. I know that there was a huge session this morning that was put on by Myasthenia Gravis Foundation in regards to their trials, but this is important in SMA because we also want to improve the neuromuscular junction function in our patients. So to back up a little bit and talk about what studies we do have from it, there were some previous findings in rats that Scove et al. decided to conduct preclinical and clinical studies in rat models and Myasthenia Gravis patients, respectively. So the NMD670 is an orally bioavailable small molecule that inhibits CLC1, which the CLC1 comes from the CLCN1 gene. It is a skeletal muscle-specific chloride channel. Its physiological role is to dampen muscle fiber excitability by stabilizing the resting membrane potential. So in these preclinical and early clinical studies in MG patients, they showed that it restored muscle function in the rat models and patients with MG. In severely affected rats with MG, there was improved neuromuscular transmission, muscle function, and mobility. They did a randomized placebo-controlled trial in MG patients where there were 12 people who had mild MG. This was only a single-dose study, three-way crossover. So they received placebo at one time point, 400 milligrams of the NMD670 and 1,200 milligrams of the NMD670. And then they, of course, rotated patient A, got placebo 400, 1,200, patient B, and then kept changing up the order of the doses. So just to make a couple of points on this. On the bottom left, you see that these are in-play potentials in a healthy rat versus a rat with myasthenia gravis. And then this is an MG rat also, but that is treated with NMD670 right here. So as you can see, there's restoration of much improvement in that in-plate potential. And then right here, I know it's a little bit small on the screen, but on the top of this plot, you can see these are the healthy control in-plate potential amplitudes. And then on the bottom, these are the untreated rats. And then in the middle here, so showing some improvement are those treated with the NMD670. We also can see improvement in the action potentials here of those treated, force versus those untreated. And then here's some EMG data, and then again, the force here. So on the far right, the probability of action potential excitation was shown in those who were the healthy controls on top. And the bottom here, again, the diseased and then treated with the NMD670. In the middle here, we see that force is improved in these three different muscles. There's diaphragm. These are all in the rats too, the diaphragm, the soleus, and the EDL muscles. And then nerve simulated force on the bottom before and after doses of the NMD670. Now this was a part of the human study. 42% to 50% of these patients had a total QMG score of two points or more better compared to placebo, which is significant. And on the individual QMG score items, there were four particular measures that showed improvement, including hand grip strength, ptosis, dysarthria, and diplopia. So that is shown over here. So this in the top left is the QMG total score, change from baseline. So going down towards the bottom is better. And then this is the QMG total score. This is the change from baseline versus the placebo, which again, going down here is better. And then these are the individual components. So in C, this is time without ptosis, so going up better. And then here is the dysarthria, so time without going up, showing improvement. The same thing for double vision, and then improved grip strength here. Okay, so there is a study that is currently recruiting for SMA. It's called Synapse SMA. It is looking at the safety and efficacy of NMD 670 in ambulatory patients with type 3 SMA. It's a phase two randomized double blinds placebo-controlled study. It's a two-way crossover to evaluate efficacy, safety, and tolerability. Patients will be given twice daily dosing of the drug for three weeks. These patients have to be 18 to 75 years of age, and they describe ambulatory as walking at least 50 meters without any aids at screening during the six-minute walk test. They are estimating enrollment to be in the 50s, and there's 29 sites across U.S., Canada, and Europe, I believe Belgium and Italy, I know, are two of the countries that are participating. So hope to see some information soon about that. So moving on, this group from Columbia discovered the G470R variant, so where glycine changes to arginine, of the HSPA8 chaperone works as an SMA modifier. So last year they published their findings in Neuron. This is Dr. Manani's lab. They used a mouse model to map and identify this variant and showed that this synaptic chaperone variant suppressed SMA, and it did so in part by altering SMN to splicing. So it showed that there was a modest elevation of SMN protein by inducing the SMN exon 7 inclusion. This also, this variant simulated formation of the tripartite chaperone complex, which is seen over here, and then they were able to demonstrate that the SNARE complex formation, which is abnormal in SMA mice and patient-derived motor neurons, was able to be restored in these modified mutants with this G470R variant. So on the left-hand side of the figure, there's poor neuromuscular junction transmission because there's a high affinity of the HSPA8 wild type with the abnormal SMN, and so because of that, the SNARE complex is not assembled correctly, and you get low transmission across the synapse. When you apply the HSPA8 G470 variant, this becomes a low affinity interaction, and there's a stabilized and restored SNARE complex formation here, and so this allows the neurotransmission at the NMJ to proceed normally. Nifedipine is a calcium-channel blocker that we know is used to treat high blood pressure. There is a group that have looked at its effects on SMA neurons and culture, and also at the neuromuscular junction in animal-type neurons. So these studies, the SMA neurons and culture were shown to have increased frequency of spontaneous calcium transients. They were also able to demonstrate increased growth column size of the neurons, increased cluster-like formations of the CAV2.2 channels, and normalized axon length. So this figure over here is showing some of that. So this is showing the defective SMA mice without nifedipine, and then the calcium transients once treated with nifedipine. And again, this is without nifedipine, and then treated with nifedipine. Here you can see, I highlighted the growth columns. Down on the bottom, the growth column size without nifedipine in the diseased mouse versus treated with the nifedipine. Here in the middle is the axon length restoration, where it's decreased and more similar to the healthy controls. So at the NMJ, they were able to show that it increased evoked and spontaneous release at low frequency stimulation in controls and SMA mice. But at high frequency, it was not shown to increase the size of the readily releasable pula vesicles, only in the control did it show increased size. Another bit on calcium. So this is a small molecule calcium channel modifier, which is GV58. O'Hala et al tested its effects in an SMA mouse model as well. They combined it with 3,4-DAP, which is a potassium channel antagonist. They actually studied its effects alone, and then its effects in combination with the 3,4-DAP. Both the GV58 alone and in combination with the 3,4-DAP was able to show improvement. So a bath application was shown to restore neuromuscular transmission in two different muscles, one of which was a mildly vulnerable forearm muscle, but the other of which was a strongly vulnerable trunk muscle. Acute subcutaneous administration was shown to increase behavioral measure of muscle strength. And so on the right here, part of this, this is just showing some of the characteristics of the wild type and diseased mice. So the diseased mice, the SMA mice, show lower body weight, they show much decreased riding reflex score, and then decreased grip strength. So this was looking at the control population, this is change in grip strength, and this is looking at just the vehicle versus the GV58 versus the combined calcium channel modifier GV58 in the 3,4-DAP. So these are all pretty similar, and then when you get to the, excuse me, the change in grip strength of the SMA mice, you see that there's improvement with GV58, and then even a little bit more improvement when those two compounds are combined. So RISDAPLAM is being looked at following other SMA treatments. The interim analysis from Jewelfish has been published. This was a phase two study to evaluate safety tolerability, PK and PD in RISDAPLAM in previously treated patients. These were ages six months to 60 years, all SMA types one through three, and prior treatments included the drugs mentioned there. The RG7800 is essentially RISDAPLAM. This was the drug before RISDAPLAM became RISDAPLAM, with similar SMN2 splicing modifier. So 174 patients were treated with RISDAPLAM, 174 patients were enrolled in Jewelfish, of which 76 had received nusinersen, 71 olisoxime, that was the cholesterol-like compound that was treated, but not found to, unfortunately, not found to show improvement, 14 had received OA, and then 13 of those patients had been participants in the Moonfish study. The median age was 14 years, with a medium weight of almost 40 kilograms. Of those ages two to 60 years, 63% had a baseline, Hammersmith expanded of less than 10, so these are very affected patients. That's a very low score, and almost all of these, more than 80% had scoliosis. The most common adverse effects were upper respiratory tract infection and pyrexia. 30 patients each had upper respiratory tract infection and pyrexia. The most common serious adverse effect was pneumonia. This was only seen in four patients. There was shown to be an increase in SMN protein and blood across all ages and body weight quartiles. These findings were consistent with treatment in naive patients treated with Risdoplam in the Firefish and Sunfish studies, and hopefully, Jewelfish will be wrapping up in the near future, so we'll be able to get the final analysis from that study as well. There are my references. So Dr. Jenna Klotz is gonna be talking to us about antisense oligonucleotides. All right. Thank you, everyone, for having me. So I'm gonna be giving some updates on ASOs for SMA. I have no financial disclosures, and then remember to share your feedback and claim your credits by October 19th. For the talk, we're gonna briefly review the basics of ASOs for SMA, talk about the clinical trial data and then real-world data for nusinersen. Then we'll review what we know about gene therapy plus nusinersen, talk about the high-dose nusinersen regimen that's currently being studied, a couple additional novel ASOs that are in development, and then an alternative delivery mechanism for intrathecal ASOs. So antisense oligonucleotides are single-stranded DNAs or RNAs that bind directly to target pre-mRNA via base pairing, and then from there, they can have a few different mechanisms. So one is they can activate the RNases and then induce RNA degradation. They can also be a steric block against ribosome assembly and inhibit translation that way. Or they can modify splicing by activating or inhibiting different splice enhancer or splice-inhibiting sequences. And this is the primary mechanism that nusinersen uses. Because ASOs have poor cellular uptake and an inability to cross the blood-brain barrier, and because our target cells are the alpha motor neurons who have their cell bodies in the intrathecal space, that requires intrathecal administration for the ASOs. And then just a quick reminder about SMN1 and SMN2. So these are the two genes that predominantly determine the phenotype in SMA. So in SMA, you have biallelic variants or deletions in SMN1 so that you are not able to make functional SMN protein. SMN2 differs from SMN1 by a single nucleotide in exon 7 so that the vast majority of transcripts from the SMN2 gene don't include exon 7, and so you have non-functional SMN protein or the RNA gets degraded before it becomes the SMN protein. Only about 10 to 15% of the transcripts have exon 7 in it, and so you can't compensate for the loss of SMN1 just with SMN2 alone. So ASOs modify splicing of SMN2 mRNA to promote inclusion of exon 7 and increase the amount of functional full-length SMN protein produced from SMN2. Nusinersen is our one FDA-approved ASO for SMA. The dose is the same for all ages and weights, so it's 12 milligrams with each dose. There are four doses given as a loading dose over a two-month period and then a dose every four months thereafter. In studies of other ASOs, there were higher rates of things like proteinuria, thrombocytopenia, renal failure. We still monitor for these with Nusinersen, but they occur at a lower rate. And then we also typically monitor the CSF to look for signs of infection or chemical meningitis, but in particular, chemical meningitis is generally not seen in Nusinersen. There are a couple of studies that are particularly important that led to the approval of Nusinersen for SMA. The first is the ENDEAR trial, which was infantile-onset SMA less than seven months old at the time of treatment. And then the CHERISH trial, which looked at later onset with two to four copies of SMN2 and ages two to 12 years old at the time of treatment. For both of these, they were stopped early because there was clear effect at the interim analysis with improvement in the motor function scales. There was improvement in the motor function scales and the treated arm, and then a loss of motor function scales in the untreated arm. The EMBRACE trial tried to include the kids that did not meet criteria for ENDEAR or CHERISH, but the goal here was primarily to look at safety and tolerability. Initially, this also had a control arm, but that was stopped once ENDEAR and CHERISH resulted. There were no Nusinersen-related adverse events that occurred in this larger and kind of more variable treatment group. They also looked at some secondary outcomes and exploratory measures to look at plasma neurofilament heavy chain, which is a marker of neurodegeneration and axonal loss, and saw that this decreased over the course of treatment. And they also checked to see if anyone developed antibodies to Nusinersen over the course of treatment, and no one did. For NURTURE, this was a test, this was a trial in pre-symptomatic infants with two to three copies of SMN2. The primary outcome measures were looking at death, respiratory support for more than six hours, or tracheostomy. These children so far have been followed out to five years. Some did need respiratory or feeding support over the course of treatment, but none needed permanent mechanical ventilation. And at the last data cut, all 25 were alive and had met motor milestones, though some slightly late. In comparing these ones who were treated before symptom onset versus the kids that were treated in the ENDEAR trial who were symptomatic at the time of treatment, there was a greater increase in the CMAP amplitude over the course of treatment. The most common adverse events in these earlier trials were primarily felt to be SMA-related and not necessarily treatment-related, like fever, cough, recurrent pneumonias, and URIs. The next set of adverse events that was more common were ones that were felt to be primarily LP-related. There have been two recently published studies looking at some larger cohorts of a wider range of patients treated with nusinersen. One includes 237 adults with SME types 1 to 4, followed out to about three years, and the other includes 130 children and adults five years old and up treated, again out to about two and a half years. For the group that included children, they looked for a clinically meaningful improvement in the top 10, which is an increase in four points, and this was seen in about 60 percent of patients around the 14-month mark and 80 percent around two and a half years of treatment. For the Hammersmith, in the group that included just adults, about a third of the patients met that criteria of clinically meaningful improvement of three points, and for the one that included younger children as well, 62 and 71 percent met that criteria over the course of treatment. For the revised upper limb module, about a third of patients, again, showed a clinically meaningful improvement of two points in the adult cohort, and then the one that included children was slightly higher, between 25 to 45 percent over the course of treatment. And then for the six-minute walk test, there was a clinically meaningful improvement of 30 meters in the total distance for about 40 to 50 percent of patients across the course of treatment. It's important to note that some of the patients in these observational studies still declined on nusinersen, so about 20 percent in the adult-only cohort, and then about 8 percent in the cohort that included children. The greatest benefit was seen over the first 18 months of treatment and then stabilization thereafter. Looking at adverse events that we know about for nusinersen, there was a recently published meta-analysis looking at all the data that we have from clinical trials, as well as real-world data on children and adolescents. The majority of adverse events reported are felt to be related to SMA and not to treatment. The next highest level, again, are the ones that are LP-related side effects, and in terms of things that were felt to be definitely nusinersen-related, it was only about .57 percent. Possible nusinersen-related side effects occurred in about 7 percent, and an example would be thrombocytopenia, which was about 2 percent of patients. And then looking at the two trials that we were discussing on the last slide, there were no new adverse events identified across these adult cohorts. The majority of adverse events were felt to be LP-related, with post-LP syndrome and back pain occurring in 19 and 11 percent, respectively. So moving on to the RESPOND trial, this is a trial that's trying to look at the use of nusinersen after gene replacement therapy. This includes kids who are under 3 years old at their first nusinersen dose who have previously received gene replacement therapy and have been determined to have had a suboptimal response to gene therapy by the investigator. There are a couple subgroups within here, subgroup A and B, both less than 9 months, both with two copies of SMN2, but for subgroup A, it's those that were symptomatic at the time of receiving gene therapy, between 6 weeks to 6 months of age, and then subgroup B was asymptomatic at the time of treatment and had received gene therapy at less than 6 weeks of age. For the interim analysis, which is going to be the data that we look at in the next couple slides, there were two key analysis groups, less than 9 months at first nusinersen dose and then between 9 to 36 months for first nusinersen dose. For the efficacy data, this includes 37 patients who had reached day 302 of the study, and for the safety and tolerability, it was anyone who had received at least one dose of nusinersen, so 46 patients. So over the course of treatment, motor scales improved with nusinersen after gene replacement therapy. The green is the groups that were less than 9 months, and then blue were over 9 months at the first nusinersen dose. The HIN-E2, which is shown on the left, improved by about 9 points for the less than 9-month cohort and about 7 points for the over 9 months. And then similar for the CHOPin10, that also improved by about 9 points for the under 9-month group, and then about 5.5 points for the over 9-month group. They also looked at some biomarkers of SMA and treatment response, including the neural filament light chain level, which again is a marker of neurodegeneration. On the right is the data from RESPOND, and it showed that at baseline there was higher levels of neural filament, which dropped pretty quickly after initiation of nusinersen, and then continued to trend down and stay low over the course of treatment. However, it's important to know that in RESPOND we don't have a control group, and so on the left is some data that we have on neural filament levels after gene therapy, and you can see that after treatment it continues to decrease anyway. And so it's hard to know what is the contribution from nusinersen versus gene therapy alone as you're looking at the treatment over time and that combination in the absence of a control group. In terms of adverse events from the RESPOND data, again this includes 46 patients for the safety analysis, 17 had serious adverse events, but none were felt to be treatment related. There were no significant changes in lab parameters or EKG or vital signs. And then again, the most common AEs were things that were felt to be SMA related, like recurrent URIs and pneumonias. From real-world data we just have a few case series and case studies published on combination therapy, and overall they showed stable to modest improvement in motor function and no new treatment-related effects, or adverse effects I should say. All right. So next we're going to talk about the rationale for the high-dose nusinersen regimen that's being studied. So from ENDEAR and from some of the earlier trials which were called C3A where they were trying to determine the dose for ENDEAR, we know that higher CSF nusinersen levels correlate with lower plasma neurofilament heavy chain levels and a greater decline in the plasma neurofilament heavy chain level. And there's a clear dose response. So the graphs on the right, green is the ENDEAR trial which received the 12 milligram dose, four doses. The next one, the purple dotted line is C3A that got three loading doses of 12 milligram. And then the blue is six milligram doses, three doses. And you can see that higher doses of nusinersen result in higher CSF concentrations of nusinersen and then a greater, which correlates with a greater decline in the plasma neurofilament heavy chain. So then they took this information, they took the CHOPIN-10 scores from ENDEAR, matched them up to the CSF concentration levels and came up with a PK and PD model for this. So based on their model, that predicted that the 12 milligram dose should result in a CSF level of five nanogram per milliliter and that that corresponded to an improvement in the CHOPIN-10 by 7.5 points. And that matched up relatively well with the actual data that they had from ENDEAR. So then to get an additional four points on the CHOPIN-10, the necessary level based on this model is 12 in the CSF for nusinersen. And then to target that level of 12, they came up with the high-dose regimen here, which is 50 milligrams given as two loading doses separated by two weeks and then 28 milligrams every four months thereafter. So the primary trial that's looking at the high-dose nusinersen regimen is called DEVOTE. It's a three-part Phase 2-3 study of nusinersen and SMA. Part A is open label, six participants between age 2-15. And the primary objective here is just to look at safety of this high-dose. Part B is designed to help determine efficacy. And it's a randomized double-blind active controlled trial with some patients receiving the 28 milligram dose, sorry, two 50-loading doses and then the 28 milligram dose maintenance and then others randomized to the standard treatment of 12 milligram. Part C is open label and includes children and adult participants with SMA who have already been on the regular standard dose of nusinersen for more than one year. DEVOTE Part A results have been published. At Day 269, the CSF nusinersen level was 10.2 versus 5.6 at the equivalent time point in ENDEAR. So they did reach a higher CSF nusinersen level. The 28 milligram dose was safe and well-tolerated. The majority of adverse events were mild. Most common were LP-related and none were felt to be related to nusinersen. Motor function was felt to be stable to mildly improve from baseline. But again, because there's only six patients, it wasn't powered to determine efficacy here. And these are the motor function scales. You can see some improved. Some had a ceiling effect because they were already at maximum scores on the revised upper limb module for example. And then there's one patient marked by the asterisk who did have some functional decline but had had a fall with a femur fracture. DEVOTE Part B results, which is the one looking at the high dose versus the 12 milligram standard dose, this data was recently presented at World Muscle Society last week. But there is a press release which gives us some of the information from this trial. What they showed at the six-month time point was that higher dose nusinersen showed statistically significant improvement in the CHOPIN-10 versus the pre-match sham control patients from ENDEAR. But when comparing it to the standard dose, there's not a clear difference yet in the functional outcomes. They noted that there was a positive trend in efficacy measures with the higher dose regimen versus the 12 milligram. And that's more reflective of there was a greater increase in a higher CSF nusinersen concentration level and a greater decrease in plasma neurofilament. But in terms of the CHOPIN-10, the scores were pretty similar between the two groups at the six-month time point. They did comment also on a relative 30% decrease in risk of death or permanent mechanical ventilation in the 50 milligram dose versus, 50 and 28 milligram dose versus the 12 milligram dose arm. There were no new safety events identified so far. The high dose regimen had a slightly lower rate of serious adverse events versus the approved dosing. And the reported adverse events were consistent with those seen in SMA with the known adverse events associated with nusinersen. ASCEND is another clinical trial that's currently active. We don't have data from this yet. But this is looking at the high dose regimen after a RISDA plan. This includes patients who are non-ambulatory between 15 to 50 years of age would later onset SMA. Because it's looking at non-ambulatory patients, the primary outcome measure here is looking at the revised upper limb module and upper limb function. It excludes patients that have trachs or are on permanent mechanical ventilation or daytime non-invasive ventilatory use. There are two cohorts. One is nusinersen-naive and has only been on RISDA plan previously. The second is nusinersen-experienced, now off nusinersen and on RISDA plan for more than 12 months and ready to switch back to nusinersen. One of our ASOs that is now also an active clinical trial is a new one called BIIB115. This is similar to nusinersen but has a modified backbone that may increase its potency and allow for less frequent dosing. Part A is healthy adult volunteers looking at the pharmacokinetics pharmacodynamics. Part B is children with SMA between 6 months to 12 years old previously treated with gene replacement therapy with a suboptimal response. So kind of similar model to the RESPOND trial which is looking at nusinersen after gene replacement therapy. Another ASO that is in the preclinical stages is called E1V1.11. Here is an image of a section of the SMN2 mRNA and showing you that there are two sequences that modify splicing. One is downstream from exon 7 and that's where nusinersen acts. And then upstream from it is element 1 and this is where this new compound acts. Regardless, both of them modify the splicing to try to increase the amount of full length SMN protein and include exon 7 in the transcript. In a mouse model treated with this compound, on the left are some bar graphs looking at SMN expression in the tissues. So on the far left, the light gray, so light gray there is unaffected wild type mouse. These are the SMA mouse models. And then after treatment with this compound showing increase in the SMN tissue expression in the brain and in the spinal cord. And then over here is the muscle biopsy. On the bottom right is an untreated mouse model of SMA. Bottom left is a wild type mouse and then the two upper images are biopsies from mice with SMA that were treated with this new compound. And then lastly, there's in development a subcutaneous intrathecal catheter system to try to help out patients that have severe scoliosis, spinal fusion, or other comorbidities or who have to get sedated every single time for their lumbar punctures for intrathecal nusinersen. So there's a subcutaneous port that's placed and it's connected to a permanent intrathecal catheter. And there's two case series published on this so far. One includes 17 patients, one includes eight patients. In general, it was safe and well tolerated. For the patients, they had that initial surgery to have the port placed. And then after that, further nusinersen administration. They just needed topical lidocaine or topical anesthetic to administer it versus having to go under general anesthesia, which many of our pediatric patients have to do. For the case series that included 17 patients, four of the individuals required revision and one needed removal for infection. They noted some improvement in some of their functional scores and functional testing. But there was no change in muscle force, pulmonary function, or the general motor skills that we typically use in SMA or SMA biomarkers, suggesting that the medication was still reaching our target tissues. The PIER study is trying to look at long-term efficacy and safety of this port. And this is including patients three years old and up with SMA on nusinersen. So in summary, our ASOs and SMA modify splicing to increase the amount of functional full-length SMN protein produced from SMN2. The most common adverse events that we see with nusinersen not attributed to SMA itself are usually LP-related. The need for serial sedation and LPs might be reduced by these novel subcutaneous intrathecal catheter systems. We don't have data yet on the long-term efficacy and safety, and the PIER study is hoping to answer this question. High-dose nusinersen appears to be safe and effective compared with placebo or natural history and results in higher CSF nusinersen levels. But it's unclear yet if there's superior efficacy to the currently approved dosing. Additional devote trial data is pending and hoping to answer that question. The ASCEND trial, again, is the one that's going to assess high-dose nusinersen after rizoplam in older non-ambulatory patients. Nusinersen after gene therapy is safe, but we don't have a control group in the RESPOND trial. So it's still a little bit difficult to differentiate with 100% confidence the clinical improvement we see, whether it's from ongoing effects of gene therapy versus a synergistic or additive effect of nusinersen. And RESPOND is that trial. And then we have a couple additional newer ASOs in development that may allow for less frequent dosing and also help out some of those patients who need serial sedation for their LPs.

spinal muscular atrophy

SMA

myostatin inhibitors

antisense oligonucleotide therapies

nusinersen

SMN protein

gene replacement therapy

RESPOND trial

DEVOTE trial

small molecule therapies

neuromuscular junction

nmj

nmj

neuromuscular junction

spinal muscular atrophy

SMA