Dr. Louise Rodino-Klapac Discusses Alpha 7 Integrin As A Therapeutic Approach to Muscular Dystrophy: March 2013

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: AAV-mediated Overexpression of Human a7 Integrin Leads to Histological and Functional Improvement in Dystrophic Mice. Mol Ther. 2013 Mar;21(3):520-5. doi: 10.1038/mt.2012.281. Epub 2013 Jan 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 our understanding of inherited neuromuscular diseases, and what their work might mean for the treatment of these diseases.

It's my great pleasure to welcome back today, 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: Thank you. Good to be back.

Kevin Flanigan: We'll remind our listeners that there's a link to your very recent paper, the one we're going to discuss today, that's available on our website. Your new work is entitled "AAV Mediated Over Expression of Human Alpha-7 Integrin Leads to Histological and Functional Improvement in Dystrophic Mice."

In this work, you say that you're developing a surrogate gene therapy approach to Duchenne muscular dystrophy. What does this mean, surrogate gene therapy?

Louise Rodino-Klapac: Most of the therapies that have been focused on Duchenne or DMD are focused on delivering back the dystrophin gene or the DMD gene. And in this case, we're delivering a gene that could functionally compensate as a surrogate gene for dystrophin.

Kevin Flanigan: So not replacing dystrophin itself but something that makes up for it?

Louise Rodino-Klapac: Right.

Kevin Flanigan: I know some of our listeners know about things like utrophin. It has been a long-term sort of target for therapies. Is that right?

Louise Rodino-Klapac: Right. There's some issues with replacing dystrophin in the case of gene therapy because of its size, and then, also other issues for potential immune response to the dystrophin gene. And so therapies such utrophin and, then now, alpha-7 integrin are having advantage over that.

Kevin Flanigan: Let's talk about alpha-7 integrin. What are integrins and what is alpha-7 integrin?


Louise Rodino-Klapac: Integrins are large group of proteins. They function as both structural proteins and also signaling molecules. Alpha-7 is expressed in muscle. Specifically, it's enriched normal muscle at the tendon, where the muscle meets the tendon, and also where the nerve meets the muscle.

Kevin Flanigan: So, not across entire membrane. This is sort of specialized areas, I guess.

Louise Rodino-Klapac: Right. So we're hoping that by forcing it to be expressed all over the muscle, then we can strengthen it in the absence of dystrophin.

Kevin Flanigan: So what does it actually do?

Louise Rodino-Klapac: As I mentioned, it has a structural component which links the actins of the skeleton to the actins on the inside of the muscle membrane to the outside the extracellular matrix. And it forms a structural component to the muscle.

Kevin Flanigan: So it connects that cell skeleton to the outside. And muscles fibres, of course, are under a lot of stress when they contract muscle cells. Probably uniquely in the body, deform a lot, so they're under a lot of muscle stress.

Louise Rodino-Klapac: Right.

Kevin Flanigan: And this plays a role, I guess, at least in part.

Louise Rodino-Klapac: Right. It could help stabilize the muscle.

Kevin Flanigan: And you mentioned the signaling role, what does the signaling role mean?

Louise Rodino-Klapac: It's been linked to a variety of different proteins. Some of those are called talin, vinculin, integrin-linked kinase. These are all sort of just throwing names out there but these have known to play a role in development of muscle size and then, also, roles in information. Some of these work is new and upcoming and not entirely understood.

Kevin Flanigan: But all things, it might be beneficial in thinking about muscular dystrophy, I guess.

Louise Rodino-Klapac: Right.

Kevin Flanigan: Yeah. What's the evidence that lead you and your lab to try to deliver it as a therapy?

Louise Rodino-Klapac: The first evidence that alpha-7 may play a role or be beneficial for Duchenne really came from the Berkin Lab. And they looked at a transgenic mouse model of alpha-7. They overexpressed alpha-7 in all cells in a dystrophin-utrophin double deficient knockout mouse.

Kevin Flanigan: That's your model where both of those proteins are missing particularly bad in muscle pathology.

Louise Rodino-Klapac: Right. And it showed improvement in that mouse model.


And then, also, a second line of evidence was really another mouse model where they deleted both alpha-7 integrin and the DMD gene and that mouse was particularly worse. So again, providing evidence that alpha-7 is playing a role or is compensating for dystrophin in some way.

Kevin Flanigan: So boys themselves, boys with Duchenne muscular dystrophy are not missing this alpha-7 integrin?

Louise Rodino-Klapac: Right.

Kevin Flanigan: As a therapy, you would turn it up for them.

Louise Rodino-Klapac: Right.

Kevin Flanigan: What approach did you take in your study?

Louise Rodino-Klapac: We used an AAV approach. So that's adeno-associated virus which is a small non-pathogenic virus which we've talked about on this program before. Alpha-7 is a smaller gene and it's able to fit in the virus particularly well.

Kevin Flanigan: So the whole gene itself. Our listeners may know from previous studies about mini or micro-versions of the gene or from the last time you were here about Dysferlin where you were breaking it up into parts.

Louise Rodino-Klapac: Right.

Kevin Flanigan: This case, it all fits in.

Louise Rodino-Klapac: Right.

And we're also using a muscle-specific promoters. That just means we're expressing it only in muscle cells and that provide an added benefit of safety.

Kevin Flanigan: Won't show up in you liver and any place else.

Louise Rodino-Klapac: Right.

Kevin Flanigan: All right.

And of course, AAV is something you've played a key role in developing in all of the trials for therapies here at the NCH. So, how did you deliver it to mice?

Louise Rodino-Klapac: In the study, we delivered it by two ways. One by an intramuscular injection and then, also, by vascular approach using the femoral artery. We used a procedure called ILP or isolated limb perfusion. And that's just delivering the gene through a blood vessel -- in this case, the femoral artery -- and trying to transduce multiple muscles.

Kevin Flanigan: So the idea is to, in a brief, reach that point, you'd be able to give it to, let's say, a whole leg or something?

Louise Rodino-Klapac: Right.

Kevin Flanigan: And you did this in mice of four weeks of age and looked at it about six weeks later. What did you find?


Louise Rodino-Klapac: What we found was that there was improvement in the integrity of the muscles, so we looked at membrane leak. You can add dyes to muscles and see if they're leaking into the muscle fibers.

Kevin Flanigan: That's sort of the reverse of what we repair, and I know lessons to this. We have a leaky membrane where ZK leaks out into the blood the enzyme everyone test.

Louise Rodino-Klapac: Right. So that was improved. And then, we also looked at muscle physiology and showed that the muscles were specifically more resistant to damage. And they should be beneficial for patients in a clinical sense.

Kevin Flanigan: I see. And thinking about it down the road and thinking about giving it to patients as you mentioned, is there some advantages to giving surrogate genes rather dystrophin related to immunology in boys, for example?

Louise Rodino-Klapac: Right. As I alluded to earlier, in patients that I've never seen dystrophin before, there's a potential for an immune response to the new protein, by giving a gene that's already expressed in the boys that could potentially be circumvented and you won't have that problem.

Kevin Flanigan: They've already get tolerance for that gene, I see.

And what can you conclude from your study?

Louise Rodino-Klapac: The results so far look promising. We're extending this study by doing longer time points and to see so over time, could you preserve the muscle strength that mice in this case have. We also think it has the potential to help treat other forms of muscular dystrophy and so we're doing those experiments now as well.

Kevin Flanigan: In other models.

Louise Rodino-Klapac: Right.

Kevin Flanigan: Can you share what models they are?

Louise Rodino-Klapac: Sure. Right now, we're looking specifically at limb-girdle muscular dystrophy 2D. Or a cerviclike hand deficient, that's one, but it could apply to others and we'll be testing that as well.

Kevin Flanigan: So that's exciting to our listeners. It maybe a candidate therapy for a whole bunch of muscular dystrophies and not just Duchenne.

Louise Rodino-Klapac: Right.

Kevin Flanigan: Great. So, what's the next steps for your lab?


Louise Rodino-Klapac: Just those that I just mentioned, so we're extending this. And also, one of the thing we're doing is looking at a more severe mouse model, the double knockout model that I talked about before deficient for dystrophin and utophrin. It's more severe and so we can benefit that. That would give us stronger indication of how it would react in patients.

Kevin Flanigan: Well, that's very exciting. Thank you very much for coming and sharing with us today.

Louise Rodino-Klapac: Thank you.

Kevin Flanigan: This podcast is brought to you by Nationwide Children's Hospital and the Nationwide Children's Hospital's Wellstone Cooperative Muscular Dystrophy Research Center. You can find out more about the Muscular Dystrophy program and about ongoing clinical trials at Nationwide Children's at our website nationwidechildrens.org/muscular-dystrophy-podcast. You'll also find a link to the abstract of Dr. Rodino-Klapac's paper there as well.

Thanks very much for joining us.