Clinical Trials at The Center for Gene Therapy
Muscular Dystrophy Care at Nationwide Children's
OSU/Nationwide Children's Muscle Group
Podcast: Click here to Download
Guest: Louise Rodino-Klapac, PhD, principal investigator, Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital.
Access an abstract of this month’s featured research article: EHomologous Recombination Mediates Functional Recovery of Dysferlin Deficiency following AAV5 Gene Transfer. PLoS One. 2012;7(6):e39233. Epub 2012 Jun 15.
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 their understanding of inherited neuro muscular diseases and what their work might mean for treatment of these diseases.
Today, it's my great pleasure to have as our guest, Dr. Louise Rodino Klapac, Assistant Professor of Pediatrics at the Ohio State University and an investigator at the Center for Gene Therapy. Louise, welcome.
Louise Rodino Klapac: Hi. Happy to be here.
Kevin Flanigan: Thanks for joining us. We're going to discuss today your paper hot off the presses entitled "Homologous Recombination Mediates Functional Recovery of Dysferlin Deficiency following AAV5 Gene Transfer". And to our listeners there's a link that links to this entire paper on our website at the Center for Gene Therapy.
Louise, maybe let's start with Dysferlin. What is Dysferlin? And what happens when it's deficient?
Louise Rodino Klapac: Dysferlin is a protein in muscle, it's in normal muscle. And when it's impaired or when it's not present in the muscle, the process of membrane repair is impaired. And so when you damage muscle, normally Dysferlin is one of the proteins that comes in and helps repair the membrane by forming a patch.
And so when you're absent for it and you have a disease where it's mutated or lost, you cannot repair your muscle membrane effectively. And then over time, that can lead to muscle degeneration and forms of muscular dystrophy.
Kevin Flanigan: So some of our listeners might know of these types of muscular dystrophy, there's limb girdle muscular dystrophy, type 2B is one of them.
Louise Rodino Klapac: Correct. And also Miyoshi myopathy is another form. There are two clinically distinct syndromes, but they are both caused by mutations and Dysferlin.
Kevin Flanigan: And this is pretty devastating disease isn't it?
Louise Rodino Klapac: Exactly. So about one third of patients are wheelchair bound within 15 years. There's a spectrum of the disease, but it can be very severe.
Kevin Flanigan: And no treatment at the present time I guess is the other important thing.
Louise Rodino Klapac: Right. There's no current treatment.
Kevin Flanigan: Well experimentally, how do you tell when Dysferlin is deficient?
Louise Rodino Klapac: In a lot of forms of muscular dystrophy, we have mass models, two models of the disease and we're fortunate for Dysferlin there are three different models that we can look at. And we can look at the muscle this mice and then stain for the Dysferlin protein using an antibody and see that it's absent.
Kevin Flanigan: In your paper I think you use also a membrane repair model, is that right?
Louise Rodino Klapac: Exactly. So in Dysferlin Deficient mice they again are absent for dysferlin, and they are unable to repair their membranes correctly. And so we can use a procedure, we extract the muscle to associate the muscle fibers, and then injure them using a laser attached to a microscope.
And then we can follow that over time and look to see whether their repair process occurs or not. So Dysferlin you would see once you injured the muscle that it wouldn't repair, but then if you test a therapy perhaps, you would be able to see that you could repair that process.
Kevin Flanigan: And is the force altered as well? The amount of force that muscle can generate, it's also different in these mice, isn't right?
Louise Rodino Klapac: Yeah. Unfortunately the Dysferlin in mouse are not as severe as human patients, so one thing that we did was look at the diaphragm which is particularly affected and what we saw was that the force was definitely impaired when we use that too as a outcome for our therapy.
Kevin Flanigan: Let's talk a little bit about adeno-associated viruses and if any of our listeners have heard some of these other PodCast, they've heard them discuss a little bit. But for new listeners maybe we can say, what are adeno-associated viruses?
Louise Rodino Klapac: So adeno-associated viruses are AV are non pathogenic viruses, so they're very small viruses. A large portion of the human population have been exposed to it but they do not cause any form of disease. And what we've done is taking this viruses and remove all of the viral genes so they cannot replicate once we use them. And we just use them to deliver our genes, it's kind of a little delivery vehicle to get into the muscle. And that's basically what they are, they are just for carrying the cargo, the gene in this case to the muscle.
Kevin Flanigan: So one thing I know about AAV is there's limit to the size of the package you can deliver. Is that right?
Louise Rodino Klapac: Exactly.
Kevin Flanigan: So some of those genes I know that we put in there are small enough to fit within that packaging limit like you've done work or we've discussed here before about sarcoglycan genes, they fit entirely within it, don't they?
Louise Rodino Klapac: Right. So packaging capacity is about five kilobases. And so for people that are not familiar with that, that's sort of a moderate to small sized gene. Dystrophin is about the gene affected and duchenne is about twice that size. Sarcoglycan is fortunately only one kilobase. So it's much smaller.
So in the case of dysferlin, it is larger than that of 5kb packaging capacity.
Kevin Flanigan: Typical. Well that brings us really up to your very recent paper. So what's special about AAV5?
Louise Rodino Klapac: There was a paper that came out in 2008 which claim that they can package a much larger gene than the normal.
Kevin Flanigan: So more than that 5kb limit?
Louise Rodino Klapac: Exactly. So we thought this was a good opportunity for dysferlin. And so we tried it when we package dysferlin in AV5 and we deliver it to the muscle, we found that indeed we were able to get full length dysferlin. And subsequent to that we found out that the mechanism was a little bit different than we originally thought.
Kevin Flanigan: So that was -you mentioned full length dysferlin, I guess they had been previously a paper as well where they used the many version of dysferlin like some of the many dystrophin work? Is that correct?
Louise Rodino Klapac: Exactly. So there was one patient I was found to have a large deletion in dysferlin, but yet still was had a pretty mild
phenotype or disease. And some researchers modeled a mini version of the gene after that. They were able to find improvements in the repair process, but not in the muscle pathology, so the disease process in the muscle. And we would like to deliver the full length genes of hopefully..
Kevin Flanigan: That's the holy grail isn't it?
Louise Rodino Klapac: And for both of those.
Kevin Flanigan: Yeah. The full length of it. So you found after you put this and then you could get full length dystrophin and you mentioned this special process by which is a curse. What is that?
Louise Rodino Klapac: Well we found after that, that we weren't exactly packaging the entire gene into one virus particle. What we were doing was packaging parts of it, so both the beginning portion and end portion.
And then once we delivered those to muscle through a process called 'homologous recombination', where the middle portion of the gene recognize each other, recombined, and then the full length protein was made from that.
Kevin Flanigan: So this is interesting process. So after the gene is getting expressed in there, they lined up together and link up to make this full length gene?
Louise Rodino Klapac: Right. This is why the normal processes of the cell. So when DNA is injured or impaired in some ways in the cell, this is a process where the cell can repair that and we're basically using the cells on machinery to do this.
Kevin Flanigan: So in your paper, you showed the expression I know by staining and so forth. What else did you find about those other measures that you mentioned?
Louise Rodino Klapac: Well, we used the deficits that we identified in the mouse. So in both them and then repair, and then also in the force generation in the diaphragm. And we found that we were able to restore both those parameters. So we restored the ability to pair the membrane completely and we also completely restored the force generation in the diaphragm as well.
Kevin Flanigan: So again just mention to our listeners anyone who's interested can see the figures that describe this by going to the link on our webpage because it's what's called an open access paper, so anyone can download it. Well, one question I have for you, and I know some of the listeners will have is, well first of, you've mentioned dysferlin is a process that happens this homologous recombination. Is that likely to happen with other big genes? Or do you have a sense of that yet?
Louise Rodino Klapac: We're hoping it does. There are several researchers working on other large genes. We think there might be some things specific about dysferlin and we're working heavily on looking at that. If that's the case we could even learn something from dysferlin that could be applied to other genes if there's something specific about a sequence region that could help us.
Kevin Flanigan: Well then the next question is; what are the next steps for your lab?
Louise Rodino Klapac: We're trying to move dysferlin gene therapy to the clinic. We ultimately would like to use a vascular approach to deliver this. We showed also in the paper that we could deliver AV5 dysferlin through the blood vessels and then transduced all the muscles of their lower leg.
And we would like to take this to patient model because ultimately we would like to deliver to multiple muscles rather than just one by intra-muscular approach. And so we're moving forward to hopefully get this to the clinic.
Kevin Flanigan: Well that's a really quite exciting work and an exciting paper. And thank you for taking the time to discuss it with us today.
Louise Rodino Klapac: Thank you.
Kevin Flanigan: This PodCast is brought to you by Nationwide Children's Hospital and our Senator Paul Wellstone Muscular Dystrophy Research Center. You can find out more about muscular dystrophy program and about ongoing clinical trials at Nationwide Children's at our website, NationwideChildrens.org/muscular-dystrophy-podcast.
And again you'll find a link to the published study that we discuss today. Thank you for joining us. We look forward to being with you again next month.