Guest Host: Scott Harper, PhD, is a principal investigator in the Center for Gene Therapy in The Research Institute at Nationwide Children’s Hospital.
Guest: Kevin Flanigan, MD, principal investigator in the Center for Gene Therapy, is also an attending neurologist at Nationwide Children’s and professor of Pediatrics and Neurology at The Ohio State University College of Medicine.
Dr. Scott Harper: Welcome to this month in Muscular Dystrophy. I'm Scott Harper, guest-hosting this podcast today from the Center for Gene Therapy at Nationwide Children's Hospital in Columbus, Ohio. Each month on this podcast, we invite authors of recent publications to discuss how their work improves our understanding of inherited neuromuscular diseases and what their work might mean for the treatment of these diseases.
Today, our guest is Dr. Kevin Flanigan, professor of Pediatrics and Neurology at the Ohio State University, and principal investigator at the Center for Gene Therapy here at Nationwide Children's Hospital. Kevin, welcome.
Dr. Kevin Flanigan: Thanks, Scott.
Dr. Scott Harper: We're here to discuss your recent paper published in the very prestigious journal, Nature Medicine, entitled "Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice".
For the listeners, I'll note that a link to the abstract of this paper is posted on our website.
The title of your paper mentions the term 'dystrophinopathy'. Let's start today by talking about the disorder dystrophinopathy refers to and what are the clinical features of those diseases.
Dr. Kevin Flanigan: Sure. Well, thanks. First, I want to thank you for guest-hosting and inviting me to speak.
Dr. Scott Harper: My pleasure.
Dr. Kevin Flanigan: It's funny to be on this side of the questions, but I'm happy to talk about the paper.
The term dystrophinopathy refers to those disorders that are associated with either absence or dysfunction of the dystrophin protein, and really those are the clinical disorders Duchenne and Becker muscular dystrophy. Duchenne is the more severe of those. Most of our listeners are probably really familiar with Duchenne dystrophy, where children are typically diagnosed before the age of 5 years old, and historically, go off their feet by 12 years old. Although with current steroid use, many boys walk longer than that.
Then, Becker muscular dystrophy, which is a milder version, in which typically in children, may present later in childhood and usually stop walking after 15 years old, with some people walking way out into their 5th or 6th decade even in some unusual versions of Becker muscular dystrophy. So, Becker muscular dystrophy is a milder spectrum of dystrophin-related disease.
Dr. Scott Harper: So these disorders arise from mutations in the same gene, the DMD gene, which is also known as the dystrophin gene. So can you tell us what the DMD gene is and how do mutations in this gene lead to muscular dystrophy?
Dr. Kevin Flanigan: Sure. So the gene encodes this protein dystrophin, and that protein is a really important protein in muscle particularly and a few other tissues, but particularly in muscle. It's a multi-component structural protein that really serves as a bridge between the interior cells skeleton or what we call the cytoskeleton and the membrane of the muscle fiber.
So it links at one end to these parts of the cell skeleton, really what's called actin -- filament is actin-- and then, at the other end of the protein, it links to dystroglycan, part of a complex of proteins on the muscle membrane. And then through that complex, links to the extra-cellular matrix or sort of the grizzle of muscle, you can this of it a little bit as, but the connective tissue of muscle.
One of its really important roles, in between that frontend and the backend, there's somewhat a spring-like mechanism consisting of where repeats of a feature called the spectrin repeats. So many of these repeats are there. One of its important roles is serving as sort of a stabilizer. It helps during the force of muscle contraction, as muscle fiber distort. It helps transmit that force from the interior of the cell to the exterior of the cell and stabilizes the muscle membrane.
We know when it's gone, we know the muscle membrane is fry-able. It doesn't work as well. It's easily torn and broken. And we know this because of, for example, the serum enzymes or the enzyme that's usually inside the muscle called creatine kinase is released that into the blood stream, into the serum. So that tells us there's a leaky muscle fiber. So that Serum CK test is how many boys with muscular dystrophy get diagnosed.
Conversely, in the other direction, when the muscle membrane is leaky, things can get into the muscle and sort of institute patterns of muscle degeneration, and that muscle degeneration leads to inflammation and attempts at regeneration of muscle. As that cycle continues, muscle fibrosis in replacement of muscle by fat and connective tissue.
So the big interesting thing about the DMD gene and its relationship to Becker versus Duchenne muscular dystrophy is in most cases, the determination of whether you get Duchenne or Becker muscular dystrophy is determined by what we call the 'reading frame rule'.
The reading frame rule is the relationship between mutations and the severity of disease. The dystrophin gene itself is made up of 79 different pieces or what are called exons. These individual exons take up a huge region of the X chromosome. They all get stitched together to make one final blueprint for the protein.
If some exons are missing as people often have with mutations, the way the genes still get stitched together can be in a way that still allows for some protein to be made or translated. Or it can be stitched together in a way where no protein gets made, and that second way, if it's stitched together in that pattern, we say it breaks the reading frame. It interrupts the reading frame of the final blueprint of the protein.
So different kinds of mutations can interrupt the reading frame and those generally result in no dystrophin whatsoever. Those can be, for example, nonsense mutations or those can be what are called out-of-frames deletion of exons. Then, conversely, you can have mutations where what remains still make, say, a partial protein, allows a partial protein to be translated, an in-frame deletion.
In almost all cases, that rules. If you break the reading frame, you get Duchenne. If you maintain the reading frame, you have a partially functional generally slightly smaller protein but one with function, and you wind up with Becker muscular dystrophy.
Dr. Scott Harper: OK, so the study we're talking about today, initiated from a key observation you made several years ago that sort of goes against that reading frame rule. In this study, you were investigating a group of Becker muscular dystrophy patients that had a unique dystrophin gene mutation.
Dr. Kevin Flanigan: Right, yeah.
Dr. Scott Harper: Can you describe what you found in those patients?
Dr. Kevin Flanigan: Sure. We were doing a large survey of patients with muscular dystrophy, Duchenne or Becker muscular dystrophy, and we found that there was a group of patients who had a mutation in the very first exon, in exon 1, that would have predicted what's called a nonsense mutation. And we would predict that from that point on -- because the reading frame was interrupted -- there would be no protein manufacture, it would be severe Duchenne muscular dystrophy.
But it turned out that we found that in quite a few patients, in fact across the country, from six families across the country that we showed were in fact related by many, many generations back, they have a what we call a founder allele. We found that muscles from those patients actually contains significant amounts of a nearly normal dystrophin molecule, dystrophin protein that began its translation somewhat downstream of this mutation. Instead of starting translational protein at the usual spot, it translated it downstream.
Then, we found other examples of that, of patients with mutations in exon 2 and a couple of other kinds of mutations that broke the reading frame within the first exons but really had very mild disease.
Dr. Scott Harper: So this finding then, in this initial discovery, the dystrophin protein that was produced was actually smaller than the normal full-length protein that's normally found in muscle.
Dr. Kevin Flanigan: Right.
Dr. Scott Harper: So, you were interested in investigating how the smaller and functional protein was being produced. And since, as you mentioned, since the mutation in exon 2 -- or even before in exon 1 -- would have prevented any functional dystrophin from being made and you hypothesize that there must be some other mechanism for how this smaller, but still mostly functional dystrophin protein was being made, and that was sort of the jumping-off point for the current study and the paper we're discussing today.
Dr. Kevin Flanigan: Right.
Dr. Scott Harper: So this actually allows us to bring into the conversation other unusual and key term in the title of your paper, and that is IRES or I-R-E-S. So this is an acronym. So what is an IRES and how does it relate to your study?
Dr. Kevin Flanigan: Sure. IRES stands for 'internal ribosome entry site'. The existence of this IRES and dystrophin actually was a hypothesis of a long-standing colleague of mine, Mike Howard, from the University of Utah. What IRESes are a different signal for initiating translation of a protein. So we talk about this blueprint or the gene. What happens is when a final blueprint is put together in what we call a messenger RNA, there's a signal, a place, a signal that tells the translation machine we have a cell to assemble on that messenger RNA and move down the messenger RNA to make a protein.
In almost all cases, in a million cells or for example, this translation occurs because there's a signal called the cap put on the messenger RNA. So that cap helps to assemble the ribosome complex that then moves down the gene.
Dr. Scott Harper: And the ribosome complex are the set of proteins that actually help to make other proteins?
Dr. Kevin Flanigan: Right. They're the actual, the machinery that does the translation and makes the protein, exactly. That's exactly right. And IRESes are a little different. IRESes are first really described in viruses that don't use cap-dependent translation. They can take a messenger RNA and in the absence of this cap, they can recruit the assembly of the ribosome complex, those important protein complex. They can recruit the assembly of that to a specific site within a gene and initiate translation from that site.
So we turn these different things cap-dependent translation and cap-independent translation. So IRES is a mechanism that uses this alternate machinery.
And more of these have been described in mammalian genes and they're conserved through evolution and so on, and we have to understand why they're there and what they do. That's one of the challenges for us, scientifically, but we're able to clearly show that it exists within the dystrophin gene.
Dr. Scott Harper: And that was beneficial for this family of patients that you identified with these mutations in the front part of the protein.
Dr. Kevin Flanigan: That's exactly right. Because of these IRES, these patients now started translating. Instead of starting the translation in exon 1 like dystrophin usually does, these patients were starting translation in exon 6. So they had an open reading frame from exon 6 and they made a protein that was entirely normal from exon 6 all the way to the end, but was missing the part of the protein that was encoded before exon 6.
We know clinically from those patients, it was sufficient to rescue them from disease. Because I didn't mention, those families that I mentioned that carry this exon 1 mutation, we identified entirely asymptomatic people who were 70 years old in that family. The most severely affected person I know lost the ability to walk at 63, and other people have no symptoms at all well into their 7th, their 8th decade.
So we know that this protein that misses the first part is a highly-functional protein. That's what got us interested in trying to see, could we use this in some way as a therapeutic option for example.
Dr. Scott Harper: That's really amazing. The beginning part of this protein that is missing in this family, what exactly does that do, normally?
Dr. Kevin Flanigan: Well, that's one of the interesting things about this. So the first part of the protein, actually from exon 1 to the exon 11 and 12 and codes what's called the actin-binding domain 1. So it's known as the portion of the protein that's really critical for binding to actin the cell cytoskeleton. And really, our model has largely, I would say most experience to date suggest that you need this entire actin-binding domain when they have dystrophin function.
Now, some other people, other labs, have pointed out the importance of another actin-binding domain called actin-binding domain 2 that's within the central rod region of the dystrophin protein. And our results would suggest that actin-binding domain 1 is perhaps less critical than we thought it was. So that it teaches us something... Let's say, it raises new hypothesis we have to test relating to dystrophin biology itself.
Dr. Scott Harper: That's very interesting finding. So let's next turn to discussing the implications of this sort of basic biology findings and how you've used those to develop potential therapies for DMD. In this basic paper in particular, you're focusing on something called an exon skipping therapeutic strategy as a potential treatment for DMD.
So I thought before we discuss your therapy work in this paper, it would be helpful to describe what exon skipping is.
Dr. Kevin Flanigan: Sure. Exon skipping is the therapeutic approach that takes an out-of-frame mutation and makes it an in-frame mutation. That's the simplest way to think of it. The easiest way to think of it is to say, well, for example patients who have a deletion of just of exon 50 -- they're only missing exon 50 -- when you stitch the remaining portion of the gene together, 49 to 51 join together in a way that the reading frame is disrupted and those patients don't make dystrophin, and they have Duchenne muscular dystrophy.
So by skipping another exon during the assembly of that, during the splicing process, if you can cleave out or avoid splicing in exon 51, you wind up stitching exon 49, exon 52. That's in-frame, you make a lot of dystrophin and you can really modulate the disease severity. That's what we would predict.
And really, that's what current study suggest. There's ongoing exon skipping studies by a couple of different companies. They have compounds that can induce this kind of exon skipping, an exon 51 in particular.
Now that's works very well for the middle-rod-domain portion of the gene for restoring the dystrophin expression. But we're doing something somewhat different on this end. We're way up at the frontend of the gene. Exon skipping in general is not going to be really helpful here because one of the things we know about is to induce activity of this IRES. The IRES is active when you have broken the reading frame in front of it, when you've interrupted the reading frame in front of it.
Dr. Scott Harper: So mutations in exons 1 through 5 would allow that IRES to be utilized?
Dr. Kevin Flanigan: That's exactly right. That's what we predict. So there's one other interesting clinical correlation to mutation in this region of the gene. We knew that duplications of exon 2 are really sort of common among all duplication mutations in the gene. They're the most common single exon that's duplicated.
Dr. Scott Harper: So when you say a duplication mutation, you mean there's actually another copy of that exon 2 to stitch together side by side with the original exon.
Dr. Kevin Flanigan: That's exactly right. Instead of being absent in the final mRNA, there's two copies in a row, and that induces an alteration of the reading frame. We found something interesting, when we duplicated exon 2 in our experimental modeling of the activation of these IRES, exon 2 duplications interfered with IRES activation.
So we knew that. Then, as we looked at really thousands of reports of mutations, we realized there had never been a report of deletion of exon 2 and it had never been described before. So when we deleted exon 2 in our experimental models of the IRES activation, when we deleted it entirely in a test tube, the IRES was activated.
So we knew that the IRES could be active in that deleted setting. So we predicted that we might find patients now who had deletions of exon 2 but no symptoms and our colleague, Alessandra Ferlini in Ferrara, Italy, turned up one of those patients which helps us support the idea that you can do away with exon 2 entirely and do no harm, because you activate the IRES.
So what we decided to do was to try to induce skipping of exon 2. We really wanted to test it in the setting of a model of the human duplication, too. And you can think of two reasons why we would want to do this. One is we can really think about treating the most common exon duplication, and with an exon skipping strategy. We can do that, and if we do it inefficiently, we'll come up with just a normal DMD gene, final product. We skip it inefficiently, we only get one copy of exon 2. We always got normal dystrophin.
On the other hand, if we're super efficient at it, we cut it out entirely, we have a deletion of exon 2, then we activate the IRES. So we know that it gives us a big potential window of therapeutics. We can't really do wrong by deleting it.
Dr. Scott Harper: That's really a beautiful strategy.
Dr. Kevin Flanigan: Yeah. So we had to make a mouse model proof. We made a new mouse model. It's got an extra copy of duplication, too. So really the first mouse model is category of dystrophin mutations. And the mouse looks very much like our standard models of Duchenne dystrophy, the MDX mouse. Listeners to this podcast would have heard before about the MDX mouse.
We showed, when we skip that exon 2, we use a virus approach, adenovirus approach, which is against something that we've used commonly here, and we've talked about it on other podcast. But we used an AV virus to deliver an RNA that interferes that interferes with splicing exon 2, and we found just that. We found that we can get robust exon 2 skipping, and when we delete exon 2 entirely, we activate this IRES. And when we activate the IRES in the mouse, it's functional. It's highly functional, just like we found with patients. It correlates with what we found from patients who activate the IRES as well.
So we're quite excited by that result.
Dr. Scott Harper: So could you describe how you were able to show that it was functional. My understanding is that the dup two mice that you generated actually developed muscular dystrophy developed muscular dystrophy just like the MDX mouse that you mentioned.
Dr. Kevin Flanigan: That's correct. They do. They showed up very similarly to the MDX mouse they mentioned. It's the same degree of pathology with fibrosis of muscle and remodeling of muscle and so on.
We showed it in a couple of different ways. The first is that we showed what we called... We called it the end-truncated. That means that it truncated at the front end. The short end dystrophin goes to the right place of the muscle membrane. It localizes it appropriately, and it recruits all of the binding partners of dystrophin just as we would expect it should. So it puts them in the right place.
And then, we showed it protects muscle from damage. The correlate, if you will, ways to investigate muscle in mice to say the mice muscles take up, die, for example in response to injury. We could show, when we induce exon skipping of 2 and induce this IRES-generated form of dystrophin, it protect the muscle from damage. And then, we show by some measures of physiology as well, that it protect it from... It improve measures of force as well in the muscle.
Dr. Scott Harper: So actually, your treated animals were just as strong as normal mice.
Dr. Kevin Flanigan: That's right. So by our model of this, it suggests that induction of this -- really, essentially by all the measures we can do -- is almost normal for some of the measures. That's right.
Dr. Scott Harper: And as you think about moving this strategy forward, how many DMD or Becker patients do you think this strategy applies to?
Dr. Kevin Flanigan: Well, we think that the patients are actually carry duplication of exon 2, around 1% or less of the patients. But if we take all of the mutations in that frontend of the gene there, it's probably 5% to 6% of patients, when we count them out of the databases. Some of those patients already have milder disease but we think that... I'm sorry, some of those patients have mild disease because they have in-frame mutations.
So they're less severe than the standard Duchenne, but on the scale of the Becker muscular dystrophy, some of them are quite severe Becker muscular dystrophy. So we think even taking everybody together, we can take some of those patients who have even in-frame mutations there and make them do better by using this therapy. So we think it's really 5% to 6% of therapy.
Dr. Scott Harper: That's fantastic. That's a significant amount of patients that who would otherwise may have no other alternatives for treatment.
Dr. Kevin Flanigan: Right. Currently, no trials available to whom where the current therapy should be. That's right.
Dr. Scott Harper: So what's next for your group?
Dr. Kevin Flanigan: Well, we're working very hard in two routes for this. One is we're proceeding with our viral approach. We're developing, bringing forward all the pre-clinical studies that we think will support eventually bringing to a human trial AAV delivery -- adeno-associated virus delivery -- of the exon skipping construct. We have that well under way, sort of figuring out the safety or dose range for that.
We're also collaborating with one of the companies to test in essence all of the nucleotides, which is a medicinal-chemical approach rather than a viral approach to induce exon skipping, as an alternative way to bring forward exon skipping for this.
Dr. Scott Harper: That's great. That's very exciting work. Would you mind mentioning who supported this work?
Dr. Kevin Flanigan: No, not at all. This is really a long standing work. We're dating back almost five years, so there's different groups who supported different components. Our initial work was supported by the National Institute of Health, and much of the subsequent work, particularly the development of the duplication to model was supported by The Cure Duchenne Foundation. And we've received as well from the French organization, Association Francaise Contre Les Myopathies or AMF, as well.
Dr. Scott Harper: Well, as I mentioned, this is exciting. Congratulations. I thank you for taking the time to discuss your work.
Dr. Kevin Flanigan: My pleasure.
Dr. Scott Harper: And I'll remind the listeners that you can find the link to the abstract of Dr. Flanigan's paper and find out more about the muscular dystrophy program at Nationwide Children's Hospital on our website at NationwideChildrens.org/muscular-dystrophy-podcast.
This podcast is brought to you by Nationwide Children's Hospital and the Wellstone Muscular Dystrophy Research Center at Nationwide Children's.