Clinical Trials at The Center for Gene Therapy
Muscular Dystrophy Care at Nationwide Children's
OSU/Nationwide Children's Muscle Group
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Guest: Paul T. Martin, principal investigator, Center for Gene Therapy and Neurosciences Center, The Research Institute at Nationwide Children’s Hospital.
Access an abstract of this month’s featured research article: AOverexpression of Galgt2 in skeletal muscle prevents injury resulting from eccentric contractions in both mdx and wild-type mice. American Journal of Physiology - Cell Physiology. 2009 Mar, 296(3):C476-88. Epub 2008 Dec 24.
Dr. Kevin Flanigan: Welcome to this month in Muscular Dystrophy. I'm Kevin Flanigan 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 treatment of these diseases.
Today, it's my great pleasure to have back Dr. Paul Martin, Professor of Pediatrics, Physiology and Cell Biology at the Ohio State University and an investigator here at the Center for Gene Therapy.
Dr. Paul Martin: Thanks, Kevin.
Dr. Kevin Flanigan: We're going to discuss the paper today that's not brand new. It's from 2009 and just a reminder to our listeners there's a link to this paper on our website at Nationwide Children's Hospital.
The paper is "Overexpression of Galgt2 in Skeletal Muscle Prevents Injury Resulting from Eccentric Contractions in Both MDX and Wild-type Mice.... That's the title of the paper.
So Paul, maybe you could start by discussing a little bit about this, the paper explores potential surrogate gene therapy approach to Duchenne Muscular Dystrophy. Maybe we could start there. What is a surrogate gene therapy mean?
Dr. Paul Martin: Well, Kevin, a surrogate gene therapy is, I mean the word surrogate is really is in the same way as we use anywhere in the English language, it means a substitute or somebody, something that's compensating, for something that's missing in this case, you have a gene and children with Duchenne Muscular Dystrophy called dystrophin which has mutation or deletions that prevent gene from making a protein called dystrophin.
And... so these strategies for gene therapy look to induced genes that make proteins that can basically do the same job as dystrophin, so that the muscle cell can function normally even when dystrophin is absent.
Dr. Kevin Flanigan: So some of our listener's heard, I'm sure you're going to podcast here or else you've heard about efforts to put the dystrophin gene back in. In this case, it's another gene, to substitute in. Some of our listeners have probably heard some of this expose, like utrophin is considered perhaps a surrogate gene therapy.
Dr. Paul Martin: That's right. Yes, utrophin. There are number of these genes that make proteins that are now known to have beneficial effects even in the dystrophin deficient muscle cell and this would include things like the gene we study galgt2 but also integrin alpha 7, utrophin, sarcospan and there are several others.
Dr. Kevin Flanigan: Let's talk about the galgt2 gene. What does galgt2 do?
Dr. Paul Martin: Well, you know all genes are abbreviated. So galgt2 is just a bunch of letters that really are short for GalNac glycosyltransferases which is the gal is the GalNac and the gt is the glycosyltransferases.
You know the longer name is the beta1, 4-N- acetylgalactosamine galactosyltransferase. So, you're actually pretty happy to just say galgt2 after a while.
Dr. Kevin Flanigan: OK. Let's stick with that one.
Dr. Paul Martin: Is the gene that is expressed in skeletal muscle in a very specific place like many of these surrogate genes. It's highly concentrated in a small region of the muscle where the motor nerve touches the muscle called the neuromuscular junction.
Dr. Kevin Flanigan: That's the point right where the electrical impulse gets
Dr. Paul Martin: that's correct.
Dr. Kevin Flanigan: transmitted in chemical fashion 03:35
Dr. Paul Martin: and that region of the muscle is obviously very important and defects in that region will lead to lots of other kinds of neuromuscular disorders but in our case we were interested in understanding what these gene did to control curtains in the muscle and so what we did with that is we did what we call a gain of functional experiment where we overexpressed the gene in skeletal muscle cells in the mice and that led to the glycosylation of a very specific protein in the muscle called alpha-dystroglycan which is a part of the dystrophin glycoprotein complex.
Dr. Kevin Flanigan: So, can I just step back one minute for others in the show, the galgt2 you mentioned glycosylation of these protein. So, it's an enzyme, it does something to these other protein. Is that right if glycosylation
Dr. Paul Martin: That's right. Yes. So, what galgt2 does is it puts a single sugar called galNac or N-acetylgalactosamine on the certain kinds of proteins. And so what sugar or carbohydrate or saccharide are often used interchangeable in the medical and biochemical fields.
Dr. Kevin Flanigan: So you mentioned that if glycosylate, one of these target is dystroglycan, go ahead with that.
Dr. Paul Martin: So dystroglycan is a membrane protein in the muscle that binds to dystrophin and so in the skeletal muscle membrane, it is part of a complex of proteins anchored by dystrophin that help to maintain the muscle cells stability when it's moving because these muscle cells of course are twitching to move and so the cell has to move around quite a bit.
So at the neuromuscular junction there are different set of proteins many of these surrogate proteins we just mentioned like utrophin are localized to these one very small part of the muscle cell normally and what we discovered was that when we overexpressed these galgt2 gene many of the proteins that are normally expressed only in the small synaptic area are called the neuromuscular junction were now expressed everywhere and these include the utrophin, some extracellular matrix, proteins which bind to dystroglycan at the neuromuscular junction called like agrin and synaptic forms of laminin.
And we have known from the work of others in particular K-Davis for utrophin, for agrin, Marcu Ruegg that when you overexpressed the genes that makes these proteins that you can inhibit various forms of muscular dystrophy that arise from loss of these proteins in the extra synaptic regions of the muscle cell.
Dr. Kevin Flanigan: So, did put it another way may be if I have it right then would be thinking in a way you've made the dystroglycan complex throughout the muscle appear in some ways molecularly like it usually does at the neuromuscular junction. Is that fair to say?
Dr. Paul Martin: That is fair to say with the added benefit though that the proteins that are made by this new complex of proteins are not by enlarged encoded by genes that are caused forms of muscular dystrophy.
So they are really surrogate genes there. Genes that encode proteins that can functionally compensate for this proteins that are missing this diseases but they're just not in the right place in the muscle cell normally to take on that job.
Dr. Kevin Flanigan: So when you did that your first paper some years ago or first papers on this, what did you find? What did you do to the muscular dystrophy in mice when you overexpressed it?
Dr. Paul Martin: So, one of the first experiments that we did after we realized that this was going on in the muscle cell was to test this idea that this complex of proteins could compensate for the loss of dystrophin in the way we did that was to take this mice and breed them to a mouse called the mdx mouse, which is a mouse model for Duchenne Muscular Dystrophy.
So these mice don't make the dystrophin protein much like kids with DMD don't.
Dr. Kevin Flanigan: So, this was a breeding experiment, you took mice that were... you hadn't delivered this but these were mice that were overexpressing it because you'd created these mice. It's over expressing design.
Dr. Paul Martin: Yeah.
Dr. Kevin Flanigan: OK.
Dr. Paul Martin: And the very surprising wonderfully surprising thing we discovered by doing that experiment was that these mice even though they lack these transgenic mice with too much galgt2... not too much but enough galgt2 in their muscle to have a functional effect on these proteins even though they were missing dystrophin did not develop muscle disease and that was true pretty much for the entire life span on those animals.
Dr. Kevin Flanigan: That's a pretty spectacular result in a lot of ways.
Dr. Paul Martin: Well, it was a number of years in the analysis but it's very much true and we published that in PNAS tonight in 2002. So this paper in many ways has a functional follow up to that paper where we've developed tools that we can bring to the clinic and tested those as well to look at how overexpression can be accomplished in a way that we can deliver as a medicine.
Dr. Kevin Flanigan: Well, let's talk about that. What are those tools that can bring us to the clinic and how did you use them in this paper?
Dr. Paul Martin: Well, we're basically utilizing technologies that have been developed by others in the gene therapy field over many, many years and we are grateful to many, many people for pioneering these approaches. You're going to talk to one of them in the next podcast. Jeff Chamberlain who's here on the campus this week.
But the vector that we use is called adeno-associated virus or AAV. This is a nonpathogenic viral vector it's a Nate little tool, it's basically kind of like a 20 nM nano particle to deliver genes to cells.
And you can take the genomic materials out of that virus and added back to cells reproducing the virus and then you could put in a human gene that you think is therapeutic instead and use that virus protein to deliver that to muscle cells and this AAV has a number of different forms and people have engineered it in a number of different ways but it has a number of properties that are just very, very advantageous of this idea of gene delivery.
Probably the most important of those is that the gene itself once the virus delivers it to the cell, the gene doesn't go in to the genome of the patient, in which by enlarge maintain as an episomal piece of DNA so that the gene is translated without mocking up anything inside the cell.
The other nice thing about the AAV vector that we're using here is that we can deliver it via the blood and it will cross the vascular barrier and go into tissues and that allows us to deliver the gene to much larger stretches of muscle tissue than we could by just injecting the muscle directly.
Dr. Kevin Flanigan: When did individual muscles would be almost impossible to inject enough muscle.
Dr. Paul Martin: Well, muscle has huge amount of your body's tissue, so that's quite a challenge.
Dr. Kevin Flanigan: Now, some of our listeners will know from other podcast, we talked about things like microdystrophins or things where... because of the carrying capacity of this AAV analogue, this AAV developing system... we can't fix the entire dystrophin gene and that's not the case with galgt2.
Dr. Paul Martin: Not at all. Galgt2 is a 1.6 Kilobase gene.
So you could probably fit about three of these genes into an AAV vector without too much trouble and since it's a naturally occurring gene, it has the added advantage also that there shouldn't be any immune responses.
The immune system basically knows that this is your protein and your tissues, so that it's not going to respond to it as a foreign agent.
Dr. Kevin Flanigan: Great. Well, that takes us really, I think up to your paper. Can you tell us a little bit about how you did it and what you've done?
Dr. Paul Martin: Sure. There are number of aspects to this paper and it's sort of technically kind of complicated probably necessarily solve for lay reader but it's a physiology journal that we published this in and so there are a lot of technical aspect of the muscle physiology that we go into in pretty gory detail.
But the basic take home message is that we're showing that when you overexpressed the galgt2 gene in mdx skeletal muscles and also in wild-type skeletal muscles, so muscles that have dystrophin that you can prevent those muscles from becoming damage when you subject them to force lengthening during eccentric contractions when that's just sort of a big way of saying mimicking what the leg normally does when it's walking down, for example down the light of stairs.
Dr. Kevin Flanigan: So that's the standard model of measurement of damage to these animal models.
Dr. Paul Martin: It is and then the x-muscles typically very much more poorly than wild-type muscles in this sorts of test but what we've found is that, in the list in the transgenic mice when we do this sort of test either in a wild-type background of an mdx background that we almost absolutely prevent the ability of the muscle to become injured.
The second part of the paper then deals with treatment with AAV vector to allow overexpression of the gene and muscle cells.
And here, we're trying to mimic what we would like to do in the clinical trial when we finally get IND application finish for this AAV vector and that is to deliver it via the femoral artery to the hind-limb muscles.
Dr. Kevin Flanigan: That would be... that's something feasible and people to deliver in to the large vessels of the leg for example.
Dr. Paul Martin: Absolutely, absolutely. And in such a way that... you know... hopefully we can have a clinical impact immediately. And so we replicated that protocol in the mdx mice which is technically quite difficult and this was pioneered by another member of our center Louis Chicoine who's very good with small needles and manage to pull this off in little mouse leg and again what we found is that overtime after 12 weeks after treatment, we could maintain this mdx muscles that have been treated in terms of their absolute force, their maximum specific force and their ability to withstand damage to a level that was at or above what a wild-type muscle normally would have and this was even though we only perfused about 20% of the total muscle during this experiment.
So what that tells us both is that we can get a biological effect that's clinically relevant but also that we don't even need to treat the entire muscle to get a clinical benefit.
Dr. Kevin Flanigan: So, I'm going to just go back for one minute cause you went sort of quickly to it. One of the three things you've mentioned was the protection from damage which is actually interesting.
Many of our listeners will know about numbers for example, like the serum CK level that their sons may have been diagnose with. So the core length to this is a leaky muscle membrane and one of the things is protects from then is leakage into the muscle using your SH4, etc.
Dr. Paul Martin: Yes. You can - it's not so easy and the way we do this physiology to do serum CKs although in transgenic mice that are... the serum CKs are absolutely back to wild-type levels.
But what we did instead to look at leakiness was to add a dye outside of the muscle cells while we were doing these eccentric contraction injury paradigms and looking at uptake of the dye which is a measure of the membrane allowing a dye to leak in or you can fracture or something like that.
Dr. Kevin Flanigan: You look on your microscope you can see muscle fibers that have that dye, and that was improved with this.
Dr. Paul Martin: Very much improved prior to treatment.
Dr. Kevin Flanigan: And one thing that I've found very interesting about paper was essentially you could get equivalent levels of protection with this as you could by putting in microdystrophin. The kind of dystrophin gene therapy molecule we're talking about, is that correct?
Dr. Paul Martin: That's true. What we did is we design this in a... as a sort of a completely blinded experiment to compare two different genes at the same time.
One is our best case scenario for gene replacement which is the microdystrophin construct and we compare it to galgt2 and the investigators that deliver the gene and analyze the muscles didn't know which one was which but it turns out that the galgt2 is as effective if not better than microdystrophin in this particular assays.
Dr. Kevin Flanigan: So, what can you conclude from all this work?
Dr. Paul Martin: Well, our main conclusion is that this is an approach to gene therapy because it's a surrogate gene therapy that has a potential to treat all patients with mutations in the dystrophin gene.
Dr. Kevin Flanigan: Regardless of their mutation.
Dr. Paul Martin: Regardless of their mutation because we really... what we're doing is up regulating surrogate proteins that are not dystrophin and to compensate for the loss of dystrophin.
So it doesn't matter how much dystrophin you've loss this should have a benefit and the other, I think, big take home message from this is that... because we're actually changing a whole complex of proteins it has a potential to be applicable to other forms of muscular dystrophy as well.
So we've actually tested both the gene therapy and the transgenic sort of experiments and two other models of muscular dystrophy; one is the alpha sarcoglycan deficient mouse, which is a mouse model for limb girdle muscular dystrophy 2D and the others the dyW-mouse which is mouse model for congenital muscular dystrophy 1A.
And both of those models we also see clinical benefit for galgt2 overexpression.
Dr. Kevin Flanigan: That's quite exciting. So development of this in testing in the Duchenne population might led to therapies for this other forms of muscular... less common forms of muscular dystrophy.
Dr. Paul Martin: Well, we're optimistic that that would be the case.
Dr. Kevin Flanigan: That's great. What are the next steps for your group and for your lab?
Dr. Paul Martin: Well, we're trying to forge ahead with this... the development of galgt2 gene therapy to develop a clinical trial for patient with Duchenne muscular Dystrophy.
And we've benefited from funding from the National Institute of Health specifically with the grant called the U54 Grant whose goal is to develop clinical products for rare diseases.
And so we are nearing the end of that process with the FDA and head up what we call a pre-IND meeting to suggest to them what are trial might look like.
Dr. Kevin Flanigan: Sorry, just want to know for our listeners may not know that term IND we've used a few times, it's an Investigational New Drug application and that's the step. The gate keeping step in a way to bringing a new therapy to a child. The FDA has to approve that IND application.
Dr. Paul Martin: Right. I'm sorry I didn't mention that. This is a completely new drug. It's never been used before, and so we have additional levels of scrutiny that we have to pass for safety before the FDA will allow us to go in to patients' and the IND application is the end result of that.
So we are in a middle of doing those safety studies to prove that this gene therapy is safe and our goal then will be to wrap those hopefully by the end of 2013 at which point we would be filing that application.
Dr. Kevin Flanigan: Once that's approved then we can actually think of a trial for boys with Duchenne Muscular dystrophy.
Dr. Paul Martin: Yes.
Dr. Kevin Flanigan: That's great. Well terrific. Well thank you for taking the time to explain all these exciting work to us today.
Dr. Paul Martin: It's my pleasure, thanks for having me.
Dr. Kevin Flanigan: This podcast is brought to you by Nationwide Children's Hospital. You can find out more about the muscular dystrophy program, an ongoing clinical trial at Nationwide Children's at our website nationwidechildrens.org/muscular-dystrophy-podcast.
You'll also find a link to the published abstract of the study we've discussed today. Thank you and we looked forward to you joining us for our next episode.