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: Dr. James Ervasti, PhD, Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota
Access an abstract of this month’s featured research article: Hum Mol Genet. 2011 Aug 1;20(15):2955-63. doi: 10.1093/hmg/ddr199. Epub 2011 May 10.
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 have as our guest today, Professor Jim Ervasti, from the Department of Biochemistry, Molecular Biology and Biophysics at the University of Minnesota.
Dr. James Ervasti: Thank you, Kevin.
Kevin Flanigan: It's a pleasure to have you here and we're looking forward to discussing your paper from 2011 in Human Molecular Genetics entitled, "Internal Deletion Compromises the Stability of Dystrophin".
Before we begin, I'll just remind our listeners that you can find the link to the abstract of this paper on our website at Nationwide Children's Hospital.
Well, Jim, maybe we could start with the idea of protein stability. What does that mean?
Dr. James Ervasti: Well, Kevin, protein stability, we all are, I think, familiar with the or comfortable with the idea that genes are the blueprints themselves that encode all the information required to make you or I. And really, the molecules themselves that converge that information into actual work are proteins.
Now, the thing that most people don't appreciate for proteins is that they are three-dimensional structure; they have folding in shape just like we do. And in order for those proteins to maintain those folded shapes, they have to have stable interactions between different portions of the protein.
And so, when we talk about protein stability, we're saying, "Oh, well, it's a protein-able, how capable is it of maintaining its three-dimensional shape even in the onslaught of stress such as heat or oxidated damage, for example?"
Kevin Flanigan: And if it can't maintain that well, it means it's degraded earlier? Is that the idea?
Dr. James Ervasti: Certainly, one consequence of protein instability is that proteins become misfolded and, well, one consequence of misfolding could be more rapid degradation deterioration, but also in many cases in disease, misfolded proteins or unstable proteins become aggregated and those aggregates tend to clog up cells early and make them functionless as well.
And so, in many diseases including possibly Duchenne muscular dystrophy, it's not only unstable proteins lead to degradation, but also to aggregation of protein.
Kevin Flanigan: So there's a pathway I gather for proteins to normally be cleared out of cells as part of their life cycle, and in that latter case, they can't do that.
Dr. James Ervasti: Quality control is really an important aspect of normal cell function. We have what we call a cellular garbage disposal known as the ubiquitin-proteasome pathway, which is designed to identify proteins that are improperly folded or misfolded and to degrade those into the constituent amino acids. And those amino acids then can be used to rebuild or synthesize new proteins.
We also have other pathways. There's at least three different quality control pathways within cells. But if we are generating or synthesizing proteins in large amounts that are unstable or misfolded, we can also overwhelm those pathways and leading to disease as well.
Kevin Flanigan: So how does one, before we get into your paper, how do you study, what sort of methods can you use to evaluate stability of proteins?
Dr. James Ervasti: Well, studying that in cells is a fairly challenging endeavor. We are continuing to work on that. There's a number of interesting methods. I think in a cellular context, something that we're trying to develop is really using the cell's own response to misfolded proteins as a genetic read-out for misfolded proteins in vivo because I think what we really care about is how are these proteins behaving in live muscle cells as we try to deliver them for patients that lack a particular protein.
Really right now, our forte in my laboratory is to study proteins individually. We can actually express these proteins, purify them, and so we study them as single molecules and we're using very sophisticated spectroscopic techniques where we can either shine various forms of electromagnetic radiation.
And this can be simply light or X-ray beams on to the proteins and we can use the response of the proteins to those forms of electromagnetic radiation as read-outs of whether they're folded or not and how they respond to changes in temperature. I think we're all comfortable with the idea that think about a raw egg and we all have a good visualization of what a raw egg looks like with the very clear white and the yellow yolk.
Protein denaturation are unfolding if we cook that egg and turn it into the very white cooked white. That's very similar to what we're doing in the test tube, is we're monitoring the denaturation or the unfolding of proteins and temperatures are very useful and easy to replicate parameter to stress the protein.
We can also use very fairly toxic agents as well to partially denature or unfold the proteins. But the biggest thing is that we're using various forms of technology called spectroscopy to monitor features of the protein as they fold or unfold.
Kevin Flanigan: I see. So you can take, for example, in a test tube a version that's mutated or not mutated of a protein and compare the curves you get off these --
Dr. James Ervasti: Really. I mean, that's how we got started in this. I was certainly not an expert in protein folding when we started this that we had initiated through our interest in the protein dystrophin which is missing in children with Duchenne muscular dystrophy. We had become interested in cases where very small mutations, where single amino acids in these very large proteins were changed, we wanted to see what were the consequences of those changes.
And we had a very clear hypothesis or predictions on how those changes would affect protein function. And when those hypothesis turned out to be wrong, we actually - the student, all the credit is to the student, Dr. Davin Henderson, he was able to investigate and learn how to study protein folding and stability.
And it was through those methodologies that he was able to document that some of these mutations might not lead to degradation, but really rather an aggregate of phenomenon that could be affecting at least a small fraction of patients with Duchenne.
Kevin Flanigan: Well, you mentioned dystrophin in being absent in Duchenne muscular dystrophy, and in this paper there's a lot on dystrophin, but also things on utrophin which you've, of course, done a lot of work on over the years. Can you tell us a little bit about utrophin?
Dr. James Ervasti: Well, when we were investigating how mutations in the dystrophin gene created pretty dramatic protein instability that we hadn't appreciated before the study was carried out, we thought that if such small changes in dystrophin sequence could dramatically affect the stability, how would internal deletions such as occurring in exon skipping or in the versions where miniaturized dystrophin are being used for gene therapy? How did those manipulations of the dystrophin sequence, how did they affect stability?
And we've also been interested in the protein utrophin primarily because it has a very strong sequence similarity to dystrophin. And this is like since you're looking at brothers and sisters and saying they have a strong resemblance, there must be some relationship, genetic relationship, and there is again, with regard to utrophin and dystrophin.
Now, what we understand about utrophin is that it is expressed at high levels during embryonic and fetal development and it is down regulated at birth, which is exactly the point that dystrophin levels are elevated. And so, we consider utrophin to be a fetal form of dystrophin that is important during development of an individual mammal, either mouse or human. But given the fact that it has a very strong similarity in terms of structure and amino acid sequence, there is many different therapies that are in development that seek to either upregulate the endogenous of utrophin gene or to deliver it as a protein replacement or gene replacement for Duchenne muscular dystrophy.
Kevin Flanigan: OK. And so, in this context that is protein stability, you've done studies on that as well.
Dr. James Ervasti: Yes. And so, when we realized that dystrophin as a protein was -- the word that we use now was "brittle". It's a very strong protein, but very small changes in sequence can dramatically and negatively impact its stability and likely its longevity in cells.
With that, well, it's important to compare this with utrophin which there was no data for. And very surprising to us, it turns out that utrophin is, well, it's a slightly less-stable protein in absolute terms. It's a very plastic protein in terms of being insensitive to deletion of sequence.
And so, to this point, it would suggest at least superficially, that for therapies that are involving miniaturization of these very large gene products, dystrophin and utrophin, utrophin may be more amenable to miniaturization because the resulting truncated protein retains its normal stability.
Kevin Flanigan: Let's talk about that miniaturization for a moment because that I know it was one of the key reasons to study this and among the therapies. The title of your paper and the idea of internal truncations, some of our listeners to previous podcasts will have heard about exon skipping therapies where the idea is to affect displacing of the genes. So you have a protein is front end and the back end, but a big internal policing which is exactly what you're studying here. And other podcast listeners have heard about micro-dystrophin versions for gene therapy. Those are really the miniature constructs you're talking about, aren't you?
Dr. James Ervasti: That's correct.
Kevin Flanigan: Yeah, correct. So it's got a direct implication for how these currently tested therapies might work.
Dr. James Ervasti: I guess, what I'd like to point out is that, I mean, we're big fans of the beautiful work that's been done over the last 20 years in developing miniaturized or micro-dystrophin vectors for delivery by, for example, adno-associated virus media mechanisms.
It's clear that the current strategies worked very well in mice. And they are certainly very well-warranted and justified to go forward in pre-clinical and clinical trials. But in the case of any drug development, any therapeutic development, rarely do we get it perfectly right the first time.
And so, really the way once you think about this analysis of protein stability, is that we're looking at the next generation of drugs or we can improve them. If we improve the instability maybe we can give less virus to the patient because we'll get more bang for the bond and less virus is going to incite less immune response.
And so, from a perspective of why so much we care about protein stability is that if nine-tenths of the protein that we synthesize from a viral vector is misfolded and degraded or aggregated, we have to make 10 times more protein than should be normal.
And there is also some evidence that suggests that misfolded aggregated proteins tend to be overly stimulatory to the immune system. And so, if we can maybe make a better construct in subsequent trials, this may show to be more efficacious at lower viral dosages. And so, from that perspective, I'm very keen to investigate this.
The most important aspect I think is from a cost standpoint. We could certainly test these things immediately in mice, but as I think the audience and I know Kevin is well aware, these therapies testing in mice are extremely expensive and slow, and we can actually screen for stability in vitro in a high-throughput manner in the test tube in the laboratory and basically narrow down maybe from unlimited or infinite possibilities for how to make a protein like dystrophin more stable; maybe thinking one or two of that are the winners and then testing those in a pre-clinical setting.
And so, the idea is that we can maybe improve on these therapies with minimal cost to the research endeavor and to the funding agencies, and hopefully improve on what are already beautiful starts to therapies for the disease.
Kevin Flanigan: So maybe related to that improvement idea, picking the right horse, if you will, I was interested you mentioned one of your outcomes in the paper was that utrophin and dystrophin don't behave exactly the same for stability. The other thing I found interesting was not all internally truncated transcripts behave the same. For example, when one thinks of these exon skipping therapies, there may be reasons based on this kind of data to pick skipping one end of the deletion rather than the other end of the deletion. Is that a correct interpretation?
Dr. James Ervasti: Well, thank you for raising that point. In fact, in response to the publication of the paper in 2011, we were funded by a foundation that's interested in developing exon skipping. To just investigate such a question is that our exon skip proteins that are potentially the products of exon skipping therapeutics for patients with deletions of various exons, are those proteins going to be sufficiently stable to help the patient?
And I think what I can report is unpublished data, and I'm happy to share with you and your audience, is that I think Mother Nature is much better at identifying how to truncate dystrophin than we are. That I'm actually pretty interested in the idea that the exon boundaries that for whatever reason appear in large gene like dystrophin, cutting and pasting together based on those exons might be a really excellent strategy for making the most effective micro-dystrophin gene therapeutic, and that I think it bodes well for the efficacy or the long-term success of exon skipping strategies.
And so, to say that we know all of them work, but I think that's one of the things that in hindsight, I'm wondering why I was surprised to think that the natural exon internal boundaries wouldn't be better than our guesses are based on past patient deletions which are, of course, were identified because they had a form of muscular dystrophy.
Kevin Flanigan: Well, so, what are the next steps then for your group, for your lab, where are you headed to this?
Dr. James Ervasti: Well, we have pretty big plans for this. And, again, when I say big plans, the beauty of the plan is that I think it's a fairly cost-effective strategy, it allows us to explore a fair amount of space, research space, with minimal cost. And in these times of limiting resources, I think these are the sorts of approaches that need to be explored. But we're actually taking a multi-prong approach.
One is we're looking at the structure of dystrophin such as in the internally deleted micro-dystrophin constructs that are being developed here at Nationwide Children's and elsewhere in the country, and trying to use our knowledge of protein structure to guess a better design.
Now, we're not that smart, so I think there's options in using high-throughput random mutagenesis, we call this directed evolution where we can randomly insert different amino acids at the site of junction between the two pieces of dystrophin that are being fused together in a micro-dystrophin construct. And then we're coupling that to a really cool approach where we use a light-emitting protein called green fluorescent protein that is only fluorescent when it is soluble.
And so, if we attach that to an insoluble, unstable micro-dystrophin, we'll so no fluorescence. But as we mutate residues to make it more soluble and stable, we should see increased fluorescence and then we can go and try to engineer those in. I think probably the one that I didn't appreciate is the best approach on first glance, but in collaboration with my colleague, Dave Thomas, is we're going to be looking for the possibility that there are small molecules or drugs that we can apply that improve the folding or stability of existing gene therapy constructs.
The idea here is, again, we have the assays in place to measure protein stability in a high-throughput manner. And if we can try, first of all, all the FDA-approved compounds and then go on, I think if you think about protein misfolding, there are disorders in the neurogenerative field such as Alzheimer's or ALS or Parkinson's disease where protein misfolding and aggregation have been the focus of disease mechanism for years.
And we're going to be able to leverage this entire large research endeavor in the nervous system really as a source of compounds that maybe could possibly stabilize existing gene therapy constructs. And so, I think I'm most excited about that as a cold therapy with gene therapy.
The beauty of that approach, the reason I like that the most now is because think about patients with Becker muscular dystrophy or these missense mutations that started the whole research project, is that could these drugs that stabilized misfolded dystrophin be used to treat those patients for which there really isn't any other therapy that I'm aware that's in development that focuses on their condition.
Kevin Flanigan: That's an exciting possibility for this...
Dr. James Ervasti: And again, it's very early going right now. It's quite possible that we could fail in this endeavor, but the risk-to-reward ratio is, I mean, I think the reward risk is very, very high in this and again, it's the sort of thing that I don't think we need to succeed on all fronts to be successful. Advancing in any one of these areas would be a great movement forward for the field and for patients.
Kevin Flanigan: Wow. Well, that's exciting. And thanks very much for taking the time to share with us your work today and discuss your paper.
Dr. James Ervasti: Well, thank you, Kevin, for having me. I've enjoyed it.
Kevin Flanigan: This podcast is brought to you by Nationwide Children's Hospital and the Nationwide Children's Senator Paul Wellstone Research Center. You can find out more about the Muscular Dystrophy program and ongoing trials and issue on children in our website nationwidechildrens.org/muscular-dystrophy-podcast. And you'll also find a link to both the published abstract of the study we discussed today in a link to Dr. Ervasti's webpage.
Thanks for joining us.