Dr. Louise Rodino-Klapac Discusses Dysferlin Overlap Vectors to Restore Function in Dysferlinopathies: January 2015

Guest: Louise Rodino-Klapac, PhD, is a principal investigator in the Center for Gene Therapy in The Research Institute at Nationwide Children’s Hospital. She is also an assistant professor in the Department of Pediatrics at The Ohio State University College of Medicine.



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 their understanding of inherited neuromuscular diseases and what their work might mean for the treatment of these diseases.

Today, it's my great pleasure to have as our guest a returning guest, Dr. Louise Rodino-Klapac who's an assistant professor of Pediatrics at the Ohio State University and a principal investigator here at the Center for Gene Therapy. Louise, welcome back.

Dr. Louise Rodino-Klapac: Thank you. It's nice to be back.

Dr. Kevin Flanigan: Today, we're going to discuss the paper from Dr. Rodino-Klapac's lab entitled "AAV.Dysferlin Overlap Vectors Restore Function in Dysferlinopathy Animal Models". This is online at the Annals of Clinical and Translational Neurology, and there's a link to the paper from our website, from this podcast.

So Louise, maybe we could start today by talking about dysferlin. What is it?

Dr. Louise Rodino-Klapac: Dysferlin are protein involved in membrane repair. Normally, there's a set of proteins that when anyone damages a muscles, they helps repair the muscle. When you don't have dysferlin, like in dysferlinopathies, this absence of the protein doesn't allow the muscle to be repaired. Then as a consequence of that, you get muscle fiber breakdown, degeneration, and then eventually inflammation and replacing of those muscle fiber by fat and then fibrosis or connective tissue.

Dr. Kevin Flanigan: So repairs of membrane of each muscle fiber, muscle cells, am I right?

Dr. Louise Rodino-Klapac: Right.

Dr. Kevin Flanigan: Patients who don't have dysferlin, if they get dysferlin deficiency, what are the clinical symptoms of that?

Dr. Louise Rodino-Klapac: We refer to them collectively as dysferlinopathies, but there's really a group of clinical diagnosis. One is limb girdle muscular dystrophy type 2B. That's the recessive form. All of these are recessive. And then, some of the other clinical variabilities are Miyoshi myopathy and distal anterior compartment myopathy. There is some variability in the clinical phenotype with each disorder.


Dr. Kevin Flanigan: With LGMD 2B -- we call this LGMD for sure -- but LGMD is actually pretty common among the recessive limb girdle dystrophies. Isn't that right?

Dr. Louise Rodino-Klapac: Right. One of the most common recessive forms, right.

Dr. Kevin Flanigan: And I know from seeing some of our patients that it often may present fairly abruptly in some patients. About a third of them, I know, leads to being in a wheelchair perhaps by 15 or so years later.

Dr. Louise Rodino-Klapac: Right.

Dr. Kevin Flanigan: Are there other treatments available for these right now?

Dr. Louise Rodino-Klapac: Currently, there are no treatments available. Some of the steroids have been tested but have not shown to have been effective, so there really is a great need for a treatment at this point.

Dr. Kevin Flanigan: Listeners who've heard our podcast before will recognize that in this paper -- like the other ones you're using, your favorite approach -- that this is gene therapy. Maybe you could go back over how gene therapy works in general.

Dr. Louise Rodino-Klapac: Sure. So the type of gene therapy we use, we use a virus called adeno-associated virus. This is just a small virus that occurs naturally. It's not associated with human disease. There's no really no downside of using it. We just use this virus as a little delivery vehicle, a little shuttle to get the gene that we're interested in into the muscle.

There's different versions of the AAV, so they can target different tissues or different organs. We use one that targets muscle very well. We've been using this in clinical trials here, so we have a very nice safety profile and it has really no downside to using it.

Dr. Kevin Flanigan: That's an interesting point since you've looked at this for a long time that this is now mature enough technology that we're really in clinical trials, with this type of stuff.

Dr. Louise Rodino-Klapac: Right.

Dr. Kevin Flanigan: Your approach for dysferlin here is a little different than your approach previously, for example, to dystrophin. Here you use -- and the title of your paper has it -- or you discussed what are called dual vectors. What does dual vectors mean?


Dr. Louise Rodino-Klapac: So with AAV, because it's a small virus, you can't fit a very large gene into it. Just to give you an idea, the AAV, the typical capacity is about 4700 nucleotides. That may not mean something to all of our listeners but that's a small gene.

Dr. Kevin Flanigan: So a nucleotides, just for our listeners, are the individual pieces of DNA, the basis in DNA, that make up the genetic code.

Dr. Louise Rodino-Klapac: Exactly. So just for some perspective, dystrophin, for example is about three times as large as that, so that can't fit into an AAV. That's why some other approaches came about -- miniaturizing genes or just using genes that are small enough to fit.

So Dysferlin is a little bit larger than that. It's about 6500 nucleotides. So just a little bit larger and we really wanted to deliver the whole gene, so the way we did it was to use two separate AAVs and then split the gene in two and then deliver it to muscle, and then they recombine to form the entire gene and then protein.

Dr. Kevin Flanigan: So you split it into two bits, sort of the front half and the back half?

Dr. Louise Rodino-Klapac: Right.

Dr. Kevin Flanigan: And you said about a thousand nucleotides have overlapped between them, is that right?

Dr. Louise Rodino-Klapac: So we use sort of homologous recombination in order to reconstitute the gene.

Dr. Kevin Flanigan: So, homologous recombination for our listeners is where aligning two pieces of DNA that are similar, there are mechanisms in cells that allow then to sort of swap those pieces to make one large molecule that are in it. So, so simply speaking, yes?

Dr. Louise Rodino-Klapac: Right. They recognize each other inside the cell.

Dr. Kevin Flanigan: And I should just say for our listeners, we'll throw in one little thing. A little off the track, we keep talking about 6,500 nucleotides or 4,700 or so forth. Often, our listeners, they might hear this referred to in a different way, which is basis, or kilo basis. That these are 6.5 kilo basis is another way of saying 6,500 nucleotides.

Dr. Louise Rodino-Klapac: That's right.

Dr. Kevin Flanigan: I only threw that in because I know it's sometimes referenced differently online.


So other genes you mentioned, dystrophin might has to be sort of miniaturized or truncated to fit in. And of course, your own group has developed alpha-sarcoglycan therapy as well. But that fits, doesn't it, entirely?

Dr. Louise Rodino-Klapac: Right, that's a smaller gene.

Dr. Kevin Flanigan: So what's unique is two pieces of the dystrophin gene and it gets in the cell. And it does recombine?

Dr. Louise Rodino-Klapac: It does. It's sort of amazing how well it works, and we've done studies to look at how efficient it is. Most of the pieces actually do find each other and combine. There's very little that doesn't. And we've done studies to show that, independently, those pieces don't function on their own. So the safety of the approach is very high.

Dr. Kevin Flanigan: So once they recombined to make the full-length gene, the result then, really, is the full-length protein.

Dr. Louise Rodino-Klapac: Right.

Dr. Kevin Flanigan: So you can restore the full-length protein. Now, you've done this previously with one of the other serotype, I know with AAV5. What are you using here?

Dr. Louise Rodino-Klapac: Here, we're using AAVrh.74. That's just the nomenclature or the name that we provided when it was identified here at Nationwide Children's, similar to one that's seen in the literature called AAV-8 are also a resistant. So these are just different versions that can target muscle well.

Dr. Kevin Flanigan: I guess it's worth pointing that's a particular serotype here at NCH that has been well-tested. We have a lot experience with it. It's already leading to clinical trials. So it's interesting that can do it.

So what did you find in this study? When you injected this dual vectors, you put them into mice, I know dysferlin-deficient mice. How did you do the injection? What did you find?

Dr. Louise Rodino-Klapac: So we took several approaches. Really, we wanted to have a platform to take the clinical trial for a multiple delivery methods. The dysferlinopathies, as I said, have a range of phenotype. Some patients just have one muscle that's affected, but a large percent have entire bodies affected -- weakness, generalized weakness.


So we wanted to start with intramuscular injection of a single muscle. That's where clinical trials will start. And those look great. We have mice subjected out to 12 months with very good sustained expression. We didn't lose expression over time. And then, this lead to functional improvement, or restoration really, of the membrane-repair defect in mice.

Dr. Kevin Flanigan: So you have a test to test membrane repair.

Dr. Louise Rodino-Klapac: Right.

Dr. Kevin Flanigan: Just out of curiosity, for our listeners, it's a very interesting sort of assay. Maybe you could describe that briefly if you will.

Dr. Louise Rodino-Klapac: Sure. So we treat the muscle with the vector and then take it out at the appropriate time, and then we can disassociate the muscle fibers and individual fibers, and then use a thing called a multi-photon microscope. We use this, the laser on this microscope, to injure the fiber. So it's just a high-impulse beam on to the muscle, it injures it.

We can do this in the presence of a dye that goes into the muscle only when you injure it, and then follow that over time to see if it's repaired, to see if the dye keeps coming in or if it seals off. And in dysferlin-deficient, you just see continuous dye coming in, but if there's repair, then we can see that.

Dr. Kevin Flanigan: I've seen your data presented, and it's really pretty cool to our listener to imagine you make this wound, you can actually directly see the difference in this dye signal.

Dr. Louise Rodino-Klapac: Right.

Dr. Kevin Flanigan: Then, with direct injection of muscle, you did systemic delivery as well, right?

Dr. Louise Rodino-Klapac: Right.

Dr. Kevin Flanigan: What other measures of repair did you see?

Dr. Louise Rodino-Klapac: So as systemic delivery, we were able to assess different muscles in there. We also looked at diaphragm muscle which can also be affected and saw a restoration of force in that muscle. The mouse model unfortunately isn't as severe as the clinical disease, so that the diaphragm actually is one of the more affected so we can test the effectiveness there, did restore force there.


Dr. Kevin Flanigan: So this is really quite great area, quite good evidence. Not only that you expressed it and localized this, but you have these measures of functional recovery.

Dr. Louise Rodino-Klapac: Right.

Dr. Kevin Flanigan: That's really great. So what's next for your group, and what's next on the table for you?

Dr. Louise Rodino-Klapac: So this project is highly translational. We have the intention of going to clinical trial from the beginning. So right now, we're in a phase of toxicology studies where an offsite organization looks at doses that we would use in the clinical trial by the ways of delivery that we use -- intramuscular for starters, and then systemic -- really to test the safety of the vector, make sure everything is safe prior to applying for an IND Application.

Dr. Kevin Flanigan: So the IND, for our listeners, is what's the call the Investigation of New Drug Application, sort of the final step. That approval of an IND is what leads next to a clinical trial.

Dr. Louise Rodino-Klapac: Right.

Dr. Kevin Flanigan: So you've already have some interactions, I gather with the FDA this year.

Is this applicable to other genes? Are there other genes you're thinking about for it?

Dr. Louise Rodino-Klapac: The dual vector approach and literature has also been done with dystrophin and that's still using sort of a mini-gene on top of other dual vector approach in order to get it in. Some people have actually tried three vectors and a triple approach to get dystrophin, so that's interesting. There are other large genes out there that could be amenable to this, the large genes associated with muscular dystrophies and another one I know that people are looking at. So any gene really larger than the packaging capacity of this could be worked out.

Dr. Kevin Flanigan: That's the name of the gene, the large gene is a large gene.

That's terrific. Well, I want to give you a chance to recognize anybody who supported this work for you.

Dr. Louise Rodino-Klapac: This work has all been supported by the Jain Foundation. They specifically work on dysferlinopathies and they've been extremely supportive throughout this whole process and continue to be so. So we're very grateful to them.

Dr. Kevin Flanigan: That's great. Thank you for taking your time to explain your results to our listeners. That's exciting work. Thank you.


Dr. Louise Rodino-Klapac: Thanks. 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 and ongoing clinical trials at Nationwide Children's at our website, nationwidechildrens.org/muscular-dystrophy-podcast. You'll also find a link to the paper that we discussed today.

Thanks once again for joining us.