Vamshi K. Rao, MD

As we embark on further research and therapies, the wonderful and positive message is that there are a lot of players involved. There are a lot of people thinking about this and dedicating an enormous amount of time. It is very heartfelt in everybody who I have met in the field, whether it be a great scientific mind like yourself, researchers who are in the trenches, or patient foundations. We could spend a lot of time discussing all that’s out there in different phases of development. But it would be fair to say that there are multiple angles, it’s a variable condition, it’s progressive, and we have to approach therapeutics with a lot of thought, without neglecting standard of care in the interim.

As people who care for these patients and think about the mechanisms of their disease, we have to constantly keep standard of care at the forefront so we can at least use what we have at this time—you alluded to steroids. We need to make sure we are keeping the child ready for whatever comes along so we can take them to a better place in their natural history, all the while diverging from what we know about natural history from our experience. I want our viewers know that there are ongoing treatment trials and drugs that are approved, but standard of care needs to continue. Dr Hoffman, as we conclude our session, what would you like to add to that?

That’s a terrific summary. I’d like to add this sense of optimism, too. Ten years ago, there were no clinical trials for Duchenne muscular dystrophy. Today, there are about 60 trials listed on clinicaltrials.gov. As you pointed out, the field of trying therapeutics in Duchenne muscular dystrophy has exponentially expanded, and that can only be viewed as very positive. But with that, participating in trials is so important. It’s a tremendous burden on patients and families, but we know and are so appreciative of the efforts of families, physicians, and sites, such as yours, that participate in clinical trials. Keeping that going will hopefully lead to effective therapies. That’s the only way to get there. It’s a really promising time. It’s a challenging disease, but I completely agree that the standard of care and keeping families engaged, hopefully in clinical trials as well, is critical to the future successes.

Thank you so much, Dr Hoffman. Your expertise and thoughtful way of approaching things is very helpful to this community. I thank all of you for watching this 

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Final thoughts from leading experts on the future of treating Duchenne muscular dystrophy and keeping standard of care in the forefront. 

Future of Treating DMD 

There’s a new kid on the block: casimersen. It’s an approved exon 45 skipping drug. It has a very similar mechanism of action in terms of the whole concept of a PMO [phosphorodiamidate morpholino oligomer] with the synthetic chain that skips through exon 45 amenable mutations. Administration is very similar. It’s proven to be very safe. It has also shown dystrophin production, leading to benefit. Like you said, it remains to be seen how efficacious it is over time in relation to real-world evidence or post-marketing.

With casimersen, we’re very happy that we now have another drug that targets a certain group of children, I believe in the 6% to 8% range. As you alluded to earlier, exon 51 amenable is around 10% to 12%. Both exon 53 and exon 45 are in the 6% to 8% range. Is there anything that caught your eye with casimersen that was different from the previous exon skipping drugs?

As you point out, it seems very similar. I was reading the FDA label yesterday, and the amount of drug-induced dystrophin, about 1%, is similar to the other Sarepta Therapeutics drugs. It seems that the delivery, dosing, and the 1% drug-related increases in dystrophin are all very similar between the 3 Sarepta drugs.

 Thank you for pointing that out. Again, I left the number out.

Recent approval of casimersen, its administration, efficacy, safety, and patient selection for treatment of Duchenne muscular dystrophy. 

Casimersen in the Clinical Setting 

We published a paper together on viltolarsen. Could you give us a few highlights about what we saw? I can start by saying that this was a phase 2 placebo study. The placebo was initially for 4 weeks, but then we switched everybody to treatment. There were 2 treatment arms: 40 mg/kg and 80 mg/kg. Could you tell us a little about what we saw in all the ways of measuring dystrophin that you beautifully alluded to before with the study for viltolarsen? What differences do you notice between viltolarsen and golodirsen when it comes to those numbers?

I’m sorry to force you to give us numbers.

 No, I’m fine with that as long as you interpret the numbers with your remarks, and specifically in terms of clinical benefit.

The viltolarsen studies looked at biopsies after 6 months of weekly treatment. There was very clear evidence that the drug was hitting its target. You could see the alternative splicing happening in the muscle very clearly. That translated into easily detectable dystrophin that was drug-induced. The FDA said in a public letter that the dystrophin data are very clear. The levels varied from patient to patient, generally from 1% to 10%, with a mean of about 6% normal levels. As we talked about earlier, you’d love to get up to about 10% of the abnormal dystrophin, but 6% is certainly getting there. If you look at clinical outcomes—it’s open-label, you can speak more to the clinical outcomes than I—it seems that patients were responding, at least compared to natural history studies.

There were significant improvements in clinical outcome, but a very small number of subjects. There were 16 subjects, 8 at each dose. These are not big studies, and they’re not powered for clinical benefit. Those in an accelerated approval have to be done post-marketing. In fact, the company, NS Pharma, Inc is doing a larger placebo-controlled study post-marketing with the major outcome of clinical benefit. That trial is not measuring dystrophin levels, because it is just for approval under an accelerated pathway. That’s good for the patients because you don’t want to take a lot of biopsies from the patients.

In terms of benchmarking viltolarsen to golodirsen, which is the Sarepta therapeutics drug, you can just look at how much dystrophin they’re making. What’s published in the FDA materials for golodirsen is about 1% drug-induced dystrophin, whereas for viltolarsen, the FDA documents and publications say about 6% increase in dystrophin. At first blush, it seems that the 6% is probably on average better than the 1%, but all these have to be borne out in clinical studies post-marketing. As I said before, we expect different patients to respond differently. You can’t directly equate a number of dystrophin with clinical improvement. You’re going to find some patients in whom 6% dystrophin has an enormous clinical benefit, and some in whom it may not show much.

That’s a very important point. You could have dystrophin levels that are lower with a higher benefit, and higher with a lower benefit. That connection is very difficult to make. I can tell you from personal experience that talking to families about numbers and clinical outcomes is a very nuanced conversation.

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Viltolarsen phase II study data is discussed as well as a comparison of viltolarsen to golodirsen for treatment of Duchenne muscular dystrophy. 

Viltolarsen Phase II Study  

 Let‘s switch gears to something very positive going on in the field: the availability of exon skipping treatments. As we all know, the first treatment to be approved was an exon 51 skipping agent, which was later christened as eteplirsen. We subsequently had approval for 2 drugs that are exon 53 skipping agents: golodirsen and viltolarsen. Very recently, the exon 45 skipping drug casimersen was approved. Can you talk a little about exon skipping? You alluded to it earlier, but can you talk a little about the differences of these different agents? At the synthetic level, what is the difference between these agents and their backbones?

Sure. Exon skipping is a fascinating area of therapeutics. It’s one of the first drugs meant to help the gene fix itself. It’s a small snippet—about 20 bases of sequence—that’s delivered IV [intravenously]. It goes all the way into the nuclei of the muscle. As the dystrophin gene is transcribing from DNA to RNA and then to protein, it binds to the RNA. In Duchenne muscular dystrophy, the gene is inactive. It’s trying to make RNA, but its RNA is nonfunctional. This snippet comes in and basically splices and helps repair the RNA to make it like in Becker muscular dystrophy, where it can make dystrophin. The real-time fixing of the gene mutation as it’s being transcribed and translated is fascinating. It’s also fascinating that it works to some extent, because getting those oligonucleotides through the bloodstream, throughout the muscle, and to all the nuclei and RNAs is a tour de force.

As you mentioned, there are 4 drugs. That’s because you have to tailor the drug for the gene’s mutation. To fix the mutation, you can’t use any generic place in the gene. You have to target exactly where that patient’s mutation is. Right next to it, the drug helps fix it or skip over the mutation, which is why it’s called exon skipping. About 10% of kids with Duchenne muscular dystrophy have mutations that are amenable to exon 51 skipping, or the eteplirsen drug. The other 90% don’t, because their mutations are not amenable. Likewise, 8% or 10% of patients have mutations that are amenable to exon 45, with another 10% amenable to exon 53. These different drugs target different subsets of patients who have Duchenne muscular dystrophy.

 Let’s start by talking about one of these drugs. As we look at these drugs, these synthetic oligonucleotides, the way the body eliminates them is through the renal system. Safety-wise, one of the things we are all looking for is to monitor renal function, although most of these trials haven’t shown a serious effect on the renal system, particularly the kidney. Overall, these drugs have been very safe. They have been tolerated well. Most of the safety signals have been at the level of adverse events rather than serious adverse events. There have been no deaths reported. Needless to say, these exon skipping drugs are very safe. All of them are administered IV weekly. There is a lot of work going on to see how they can be altered so they don’t have to be administered every week. Once a month would be wonderful in terms of patient compliance and overall convenience.

As we think about these drugs, let’s look at the 2 exon 53 skipping drugs: viltolarsen and golodirsen. Based on the clinical trial data, what differences have you seen in what the publications have shown? You and I have been intimately involved in the exon 53 viltolarsen study. What is the difference between golodirsen and viltolarsen?

Your description is very accurate. A lot of the drug hits the kidney and is excreted right away. A very small fraction ends up actually hitting the gene target in the muscle or the messenger of any target. There’s so much room for improving this, as you outlined. The drug load to the kidney is significant because it is so rapidly excreted.

We say it’s a snippet of DNA or RNA, but interestingly, it’s a different chemistry called morpholino chemistry. It is not biological per se. It’s a highly modified chemistry. If you look at it at first blush, it looks like DNA/RNA with the bases and ribose backbone. But the ribose backbone is replaced with a completely alien structure called a morpholine ring. That turns out to be a huge advantage because the body does not see it as something biological, or worst-case scenario, as a virus. Most of our exogenous DNA or RNA coming at us is a virus, and our body freaks out and wants to get rid of it as soon as it can. These morpholinos do not invoke any antiviral or innate immunity and they’re not metabolized. They stay intact, which is good. But then the body says, “This is just garbage, let’s get it out in the kidney,” and that’s why it ends up in the kidney.

Any efforts to try to keep targeting, to get more into the muscle and have it stay there longer, are really important. There are lots of efforts underway to try to do that. Even how it gets into the muscle is interesting. It seems to be brokered by macrophages. The macrophages may take it up to try to protect the body, and then in remodeling dystrophic muscle, degeneration/regeneration, seems to release it back into the muscle. There’s a recent publication on that. It’s all very complicated with a lot of room for improvement.

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A review of exon skipping agents, their function, mechanism of action, and the different drugs used in clinical practice to treat Duchene muscular dystropy.    

Exon Skipping Agents Used in DMD 

One of the things I always think about and talk to parents about is trying to tackle this very progressive condition from different angles. There are therapies targeting different parts of the mechanism of progression or destruction, as you alluded to. Your analogy about having a bird in hand, in terms of having available therapy right now, could maybe alter the progression positively. Maybe down the line, if you’re not using a therapy that would be banned in the future or barred in some way, we could always come back with another mechanism. Could you envision combinations in the future with steroids somewhat being the cornerstone and the other therapies adding on top of one another?

 Absolutely. Most of the people in this field probably imagine that the therapies that will really slow or stop the progression of this disease are going to be combination therapies. If we use gene therapy as an example, that’s still semifunctional dystrophin, and the exon skipping is still Becker dystrophin. All those conditions are still progressive disorders. With Becker muscular dystrophy, most patients lose ambulation. It’s just slowed. We need therapies that attack different parts until the we can deliver lots of full-length dystrophin, but there are currently no therapies even envisioning doing that. It’s so large and hard to work with. No clinical trials have attempted to introduce full-length dystrophin. I don’t know of any clinical programs even trying. If we’re stuck with this semifunctional dystrophin, we still need supportive care and supportive therapeutics, as you said.

We’ve learned a lot about the progression. We know that dystrophin is missing in fetal life. The first events we see in the pathological events in the patient muscle is right after birth. The serum creatine kinase goes through the roof. The muscle is leaking. It’s disturbed, and with that comes an innate immunity, NF-kappaB pathways. As you pointed out, those corticosteroids are the prototypical sledgehammer to shut down NF-kappaB or the immunity. That’s why they’re used and prescribed in so many inflammatory conditions. As you noted, corticosteroids are considered standard of care for Duchenne [muscular dystrophy]. Unfortunately, they come with a lot of adverse effects, like stunting of growth, mood disturbance, cushingoid, bone fragility—the list goes on. That can detract from patient quality of life.

With any therapy, you have to balance efficacy and safety and try to find a therapeutic window. Because of that, although it might make sense to prescribe corticosteroids from birth, current feelings are that the risk benefit is not worthwhile, so most physicians wait until children are more symptomatic. Although there are lots of trials in younger patients down to infants, as with Anne Connolly and others. Maybe you’ve participated in some of those. It seems to show benefit.

We have developed an alternative modification of the steroid backbone called vamorolone, which is no longer a corticosteroid, but it retains that NF-kappaB anti-inflammatory activity. We’re in phase 3 trials with that. So far, children have not shown the growth stunting or some of the other adverse effects of corticosteroids. That can shift your balance of efficacy and safety and maybe enable earlier dosing to do what you suggested: a multidrug therapy that will really hit the disease at multiple levels and slow it down. 

 I’m glad you mentioned vamorolone, because you’re the thought leader about steroids and alternative steroids, because I was definitely going to bring that up.

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A discussion with parents on treatment options in Duchenne muscular dystrophy as well as the benefit of using steroids combined with other therapies. 

Use of Steroids in Combination With Other Treatments in DMD

 As with anything in statistics, I absolutely agree that there are those 2 standard deviations and outliers, and our quest is always to look for a bell curve. There are 2 points you alluded to that I want to make sure we put front and center. One is the concept of variability and heterogeneity in terms of Duchenne muscular dystrophy as a disease. The other is the concept of normal vs abnormal dystrophin. These are very powerful concepts, not only now but as we think of further therapies.

In line with that, when I talk to patients in clinic about the clinical trials and research landscape, 1 thing that parents always ask me is, “What should I do? Should I wait for a new therapy in the future, or should I participate in something that’s in front of me right now?” It’s a very difficult situation for parents because they know their child is progressing. They see the changes. They may not see them day by day, but they see the changes over months. They know in their hearts that their child is going to progress. Sometimes, we reach the point where parents ask me about something they’re investigating, or a particular therapy that looks like it could change part of the disease. They ask, “Can I wait for when they can give me back all the dystrophin? Can they give me back normal dystrophin?” It’s heartbreaking, honestly.

This is the point where it’s a long discussion about the nuances. What stage is the child at? What age is the patient? What therapy are they talking about? I will usually go over what I know from the various clinical trials and advise them. Most of the time, I lean toward not waiting. But I do acknowledge special circumstances, like when a child is amenable to an exon skipping therapy but the parents are really hoping to do something like gene therapy. What do you think when you look at current gene therapy interim trial results? What would your advice be to these parents?

First, I’d respond that I’m the scientist and you’re the physician, so I respect your expertise more than mine on the subject. But I have obviously worked with foundations and parents and tried to create infrastructure to facilitate therapeutic development for many years. Your points are all outstanding, very important, and very sensitive. I’d like to emphasize 2 things and vicariously imagine myself in your position talking to the patients. One is the progression of Duchenne. It’s a progressive disease, and we’re increasingly understanding the nature of why it is progressive. Dystrophin deficiency initiates the process in fetal life, but you don’t see weakness until 3, 4, or 5 years of age, clinically or detected. A patient doesn’t lose ambulation until 11 or 12 years of age. As I mentioned, dystrophin is made in many tissues, including heart, vascular smooth muscle, and visceral smooth muscle. Why does skeletal muscle show the most rapid progression of the disease?

Great MRI studies have really informed our understanding of this progression recently. A group in Florida with Krista Vandenborne and other groups in the United Kingdom have taken a large series of patients and watched their skeletal muscle over time. They saw that by 5 years of age, just as that patient is showing obvious symptoms, there’s already extensive fibrofatty replacement of the muscle. That fibrofatty replacement is variable. Some muscles seem to be very upset with dystrophin deficiency, while others seem OK. The sartorius actually gets bigger than normal because the vastus will be largely turned to fibrofatty connective tissue by 5 or 6 years of age. This goes to your point of waiting.

The problem with waiting in a progressive disease is that if the muscle has turned the fibrofatty connective tissue, no therapy is going to bring that back. It’s only going to preserve what’s there. We increasingly realize how aggressive that fibrotic process is in Duchenne and how important it is to intervene as early as possible. My goal is to push toward neonates as soon as possible because the muscle is fine there. It has not entered that fibrotic stage. If you can successfully prevent that switch to fibrosis, you’ll retain so much more function. That’s 1 thing I’d expand regarding your patients, but you’re doing it and not me.

The second point I’d like to make is that a bird in the hand is worth 2 in the bush. A trial that looks promising or a therapy that has some effect is available now. The problem with gene therapy is that, while ongoing trials are very promising, there is no precedent for such large-scale production of virus to deliver 30% of the tissue, and it’s not clear when that will be broadly available. Even when it’s broadly available, will it be accessible? It’s not going to be inexpensive, and we still don’t know the persistence of effect.

That can be a major issue with gene therapy because, at least in current paradigms, it’s not possible to redeliver gene therapy. With an initial delivery, there are neutralizing antibodies. You’re immunized against the vector, just as we’re getting immunized against COVID-19. The goal of immunization is to prevent infection. If you do that with gene therapy, you can’t use the same vector again because you’re immunized against it. Unless that vector shows very long persistence, we’d need to codeliver, which we can’t at this point. There are many questions with gene therapy. It will take a lot of work, studies, and time to figure out before it’s broadly available. I agree with you to encourage them to do things earlier rather than wait because there are so many unknowns at this point.

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Discussion on the importance of early treatment in patients with Duchenne muscular dystrophy. 

Early Treatment of DMD 

How much dystrophin is good dystrophin? How much dystrophin is enough? There’s been a lot of evolving literature about the percentage of dystrophin in children in natural history studies. My understanding is that anything above 0% is good, but how does it translate into a clinical phenotype where you’re seeing meaningful strength measures? There has been literature where even 1% was good. We have looked at literature that has broken it down to 0.5% to 5% and 5% and higher. In the study that we participated in together, we introduced the concept of 3%, and our study showed dystrophin of being higher than 3% or even 5%. Based on all that, why is this so important for us to think about?

It’s very important. Remember that under accelerated approval paradigms, you’re saying that so much dystrophin induced by drug is reasonably expected to confer clinical benefit later. Of course, zero is not enough to be reasonably expected to confer later clinical benefit. The question is: What is that number that’s reasonably expected? As you said, there’s extensive literature going back for years because nature has done many of those experiments for us in the context of thousands of patients with Duchenne muscular dystrophy and Becker muscular dystrophy.

You also mentioned female carriers who sometimes show symptoms. Those have all been studied by international collaborative teams, me and you included. There are some generalizations. One of the generalizations, which is very important to remember in this context, is that patients are variable. Duchenne muscular dystrophy is a variable disease. Becker muscular dystrophy is an extremely variable disease. Female manifesting carriers are even more variable.

The first thing in any discussion like this and trying to find a number, is patients are going to respond to that number differently. You have to realize in that anticipation of clinical benefit, when you pick any number of how much dystrophin is enough, there’s going to be a significant amount of error. With the same number, some patients will do well and some patients won’t. Everybody really needs to acknowledge that. Then you say, “OK, I get that, but still give me a number.”

We’ve studied lots of patients with Becker muscular dystrophy who have very different levels of dystrophin. What has generally been seen is unique. Remember that Becker dystrophin is abnormal dystrophin. It’s not normal dystrophin. With an abnormal dystrophin that is missing amino acids, you’d probably want over 5% or 10% to really slow loss of ambulation beyond 20 years, whereas Duchenne typically loses ambulation after 10 or 11 years. If you want to slow ambulation beyond 20 years for abnormal dystrophin, you’d better be above 5% or 10%. All those biopsy studies supported that.

You cited a really nice recent paper out of France. I don’t remember the author’s name. They found that much lower levels could also be associated with milder disease; even down to 1%. It’s important to point out that this paper studied normal dystrophin, not abnormal dystrophin. They were looking at specific types of splicing mutations that still led to normal full-length dystrophin that you expect to retain more function—more bang for the buck—for normal full-length dystrophin than a semifunctional Becker dystrophin or a gene therapy dystrophin, which is micro-dystrophin. There are so many variables: what you’re looking at, what your denominator is. When you put any number on that, you have to realize that some patients will do well and some will not do well.

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Eric P. Hoffman, PhD, outlines the variables in measurement of dystrophin levels in patients with Duchenne muscular dystrophy and how they translate into clinical benefit. 

Varying Dystrophin Levels in DMD 

As we start to think about all these clinical trials, we are trying to look at biomarkers or what makes a successful clinical trial. One of the key questions that comes up is: How do you measure dystrophin to say that you have actually made an improvement?

That’s an excellent point. The FDA has said that if you can show you’re bringing dystrophin back in patient muscle through a therapy, then that is a surrogate biomarker worthy of accelerated approval. In those settings, which are fairly rare in FDA drugs, you don’t have to show clinical improvement. You can show that later. Instead, you just show that dystrophin is being produced in patient muscle. That is reasonably expected to confer later clinical benefit because dystrophin is the clear cause of Duchenne muscular dystrophy. There’s no question about that, so it seems to make sense that if you can bring dystrophin back, it should help—particularly in muscle.

There are some drugs that are approved for exon skipping that are bringing dystrophin back to some extent in muscle. That has been the basis of accelerated approval. It’s interesting that it seems that the FDA has made a distinction between exon skipping dystrophin and gene therapy dystrophin, or what you call micro-dystrophin. Maybe we should briefly explain exon skipping. There are 4 approved drugs that are small oligonucleotide drugs—little snippets about 20 letters long that, when given intravenously, bind to the RNA of the dysfunctional gene and convert a Duchenne gene into a Becker gene. An inactive, nonfunctional Duchenne gene repairs it at the RNA level into a Becker gene, which is residual semifunctional dystrophin. The FDA has said that when you do that, the semifunctional Becker dystrophin is good for the basis of drug approval. They have not said that for the gene therapy dystrophin. The FDA may believe that’s too far away from normal dystrophin, it’s too small, and we’re not sure how well it will function in the context of a patient. To my knowledge, the FDA is asking for clinical benefit in gene therapy trials. 

That’s a really important point. Many of our viewers and many of us in the field sometimes gloss over that really important distinction, so I’m glad you brought up that point. That also brings up something else. Because there are so many sponsors, players, researchers, and patients involved in clinical trials, because we need lots of people concentrating on and treating this disease and finding a cure, how do you standardize what people are looking at? For example, let’s go back to your point about measuring dystrophin. Clinical trials have measured dystrophin as a marker for what the FDA needs. How do you look across dystrophin in clinical trials and compare 1 against another, if it’s possible?

Dystrophin is a protein and where it should show up to be most clinically beneficial is in skeletal muscle. It would be great if it were in the brain, heart, peripheral nerve, etc, as we discussed earlier, but the first step is to see if we can get it in the skeletal muscle and preserve strength. To see dystrophin coming back, you take a muscle biopsy. These trials that are using dystrophin as a surrogate marker for approval generally have pretreatment and then post-treatment muscle biopsies, often after 6 months or a year of drug treatment. The goal is to see that you’re bringing dystrophin back.

There are a couple of standard ways of measuring dystrophin. One that pathologists are very familiar with is immunohistochemistry, or immunostaining cells or tissue sections and then looking under a microscope to see if it is there or not. Those are extremely powerful pathologic tests. They’re not so great in this instance because they can be very challenging to make quantitative, or to put a number on how much dystrophin you’re actually inducing. There can also be issues with background. How do you really know it’s missing before the treatment and compare that difference? The FDA has not been enthusiastic about considering immunostaining or histochemistry as a valid end point when considering accelerated approval. They want something more quantitative.

The most quantitative test, often used by pathology labs, is immunoblotting, or western blot. That’s where you dissolve the tissue, resolve it electrophoretically, and then use antibodies to see how much of the protein—the target protein in this case is dystrophin—is in that biopsy. That’s what the FDA has accepted. All 4 drugs that have been approved based on dystrophin levels for exon skipping in Duchenne muscular dystrophy have all used western blotting as their primary outcome. That’s what the FDA looks at. They really want to see those blots. They want to see the pen in the gel and they want to see it go up after treatment. That’s what all companies have used. Western blotting is a fairly standard approach that has been around for almost 50 years. Of course, the specific method you use for western blotting can vary a little—which gels, which transfers, which antibodies you use—but they should be comparable across studies. There’s no reason to suspect that they’re not.

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Consideration of the measurement and standardization of dystrophin improvement to infer clinical benefit and the FDA distinction between exon skipping dystrophin and micro-dystrophin in Duchenne muscular dystrophy. 

Measuring Dystrophin in DMD 

 Let’s talk about this truncated form of dystrophin, called micro-dystrophin. What do you think about micro-dystrophin in general? How is it relevant in today’s world as we think about dystrophin clinical trials and potential future therapies?

 Because the dystrophin gene is so large, a goal is to put the gene back into a patient. That’s often done by gene therapy. That’s working very well for particularly eye disorders and some blood disorders, but the key to gene therapy and giving the normal gene back is being able to fit the gene inside a viral vector. The 

 gene, the dystrophin gene, is so much bigger than any virus can handle, so there’s been a lot of work to make variants of the gene and variants of the dystrophin protein coded by the variant gene that retains enough of the function of full-length dystrophin to recover the muscle enough.

The current gene therapy trials are using a dystrophin protein that’s only about 30% of the size of the normal dystrophin protein. Scientists have tried to retain the most important parts of dystrophin. There aren’t many proteins that you can cut out 60% of and it still retains function, but it seems possible with dystrophin to at least some extent. The way to visualize it is like a tinker toy or an erector set, where you have the bars and connectors. Dystrophin is a structural support to the myofiber and needs connectors at the end. What scientists have done with gene therapy vectors is truncate much of the rod part but keep the connectors so that the tinker toy or erector set still functions by connecting, but it’s shorter. It’s clearly not normal dystrophin. Nobody should expect this dystrophin, at 30% of its size of the normal protein, to retain 100% of function.

 I like your analogy about the tinker toy. It gives you a nice visual.

 I think the goal here, as we’re thinking about micro-dystrophin and dystrophin, is to at least make some protein so that we are moving beyond no protein or a very small amount of protein that is connected with a Duchenne phenotype. We are trying to ameliorate the phenotype and make it into a Becker muscular dystrophy phenotype, which should inherently improve life span and other organ system functions that are usually the progressive nature of Duchenne.

 You bring up a good point of Becker muscular dystrophy, which is a milder variant of Duchenne that also has mutations within the same 

 gene. Those patients are nature’s experiment of making semifunctional dystrophins that are missing big chunks of the amino acid sequence. Some patients with Becker muscular dystrophy have been found to be missing half the gene, yet they can be ambulatory into their 60s; whereas a patient with Duchenne muscular dystrophy typically loses ambulation at 11 or 12 years of age. 

 has already done, through clinical medicine and these patients, a series of structure function relationships of what parts of the protein seem to be most important. As you said, the goal is to find something that fits in a viral vector and still retains enough of the protein function. One of the advantages of the viral vectors is they can hypothetically make lots of the protein, but gene therapy in Duchenne is challenging because muscle is 30% of your body mass. It’s much different from successful gene therapy already in clinical utilization of an intraocular injection to deliver the retina cells, or in blood cells where you can take out stem cells, put in the gene, and put them back so they populate the bone marrow. That’s straightforward, but if you talk about in situ or in vivo delivering to a tissue that’s the largest organ system of your body, that’s a technical challenge as well.

 That is a great point about the limitations as we’re learning about not just the technology involved in gene therapy, but the ultimate goal of improving the phenotype. As you said, it’s 30% of the body.

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Discussion of micro-dystrophin, its place in gene therapy, and relevance in the clinical setting in relation to Duchenne muscular dystrophy.

Micro-Dystrophin in Gene Therapy

® presentation titled “Dystrophin and its Role in Effective Treatment of Duchenne Muscular Dystrophy.” Today we are going to discuss how dystrophin relates to Duchenne muscular dystrophy, approved therapies, as well as treatments and development. I’m Dr Vamshi K. Rao, an attending neurologist at the Ann and Robert H. Lurie Children’s Hospital of Chicago and an assistant professor in pediatrics at the Northwestern University Feinberg School of Medicine in Chicago, Illinois. Joining me is Eric P. Hoffman, an associate dean for research in the School of Pharmacy and Pharmaceutical Sciences at Binghamton University in Binghamton, New York. Dr Hoffman is also a cofounder and involved in the management of ReveraGen BioPharma, AGADA Biosciences, and TRiNDS, LLC. Thank you so much for joining us. Hello, Dr Hoffman.

 Hi, Dr Rao. It’s a pleasure to be here with you.

 Likewise. Let’s start by talking about the cause of Duchenne muscular dystrophy.

 Duchenne muscular dystrophy is 1 of the most common genetic diseases worldwide. It’s on the X chromosomes. It’s X-linked recessive, meaning predominantly boys get the disease. Mothers can be carriers, but there’s a very high new mutation rate of the gene, so many mothers are not carriers. It’s just a mutation in an egg that leads to a child with Duchenne muscular dystrophy. The very high mutation rate is now understood. It’s all tied to the gene and the mutations involved, which is the 

 gene on the X chromosome, the largest gene ever identified. A typical gene is about 30,000 base pairs, or letters—which is like 1 recipe in 

 gene is 100 times bigger. It’s 2.3 million base pairs. Because it’s so large, it’s a high target for mutation. It’s like hitting the side of a barn with a baseball. It has a very high mutation rate. The types of mutations are often missing pieces, deletions, or duplications. Most patients have a private mutation. Many of the mutations are distributed throughout this enormous gene. In Duchenne muscular dystrophy, the mutations knock out the function of the gene. In knocking out its function, it can’t make the protein product, which is normally dystrophin. Dystrophin is a component of skeletal muscle, heart visceral smooth muscle, vascular smooth muscle, peripheral nerve, and some neurons. When this gene is knocked out by a mutation, that protein is missing from all those tissues in a patient from fetal life onward. Dystrophin deficiency is the cause of Duchenne muscular dystrophy.

 As I understand, the incidence in children is predicted to be between 1 in 3500 and 1 in 5000. There are female carriers, but there are also women with dystrophinopathy, which I believe can manifest symptoms. Dystrophin is in the brain, along with all the other tissues that you mentioned. There’s always the question of how dystrophin functions in the brain. We do know that there are some children, almost one-fifth, who can have an autism-like phenotype along with the Duchenne muscular dystrophy.

 The gene is very complicated. It is normally expressed in these different tissues and there are different types of dystrophin protein in each tissue. It’s clear that the mutations and loss of dystrophin initiates the disease process, but how the different tissues respond as a function of age is unclear. Visceral and vascular smooth muscle is not a primary component of the pathology, but it’s probably there and contributing to it, along with the brain.

We have found animal models that have mutations of the same gene that occur spontaneously. Dozens of breeds of dogs and cats have mutations of the same gene because of the same high mutation rate. A lot of those phenotypes, the different involvements of the different tissues, vary between the animals as well. You’re right that the brain seems to be more affected in some patients than others. In mice, some experiments suggest that might be because of ischemic episodes. Dystrophin deficiency might be particularly susceptible to ischemic insults, and that may be upstream of this variability and brain involvement.

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Vamshi K. Rao, MD, and Eric P. Hoffman, PhD, review Duchenne muscular dystrophy and the mutations involved.