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: Jeff Chamberlain, PhD, McCaw Chair in Muscular Dystrophy and Professor in the departments of neurology, medicine and biochemistry at the University of Washington.
Access an abstract of this month’s featured research article: Gene and cell-mediated therapies for muscular dystrophy. Muscle Nerve. 2013 May, 47(5):649-63. Epub 2013 Mar 29.
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.
It's my great pleasure today to have us our guest Jeff Chamberlain, Professor of Neurology, Medicine and Biochemistry, and holder of the McCaw Chair in Muscular Dystrophy at the University of Washington.
Dr. Jeff Chamberlain: Thank you Kevin, it's great to be here.
Dr. Kevin Flanigan: Jeff was invited to give a big lecture here at Nationwide Children and we've taken advantage of that to have him talk to us a little bit about his recent paper, a review article entitled "Gene and Cell Mediated Therapies for Muscular Dystrophy.
And to our listeners we have a link to the abstract of that paper on our webpage.
Jeff your paper really --- we might not stick exactly to the paper. Today, your recent review really is a terrific overview of the field today and some historical view points as well about the development of gene therapies and cell mediated therapies. And I think many of our listeners probably know the central role you've played for more than 20 years now in the development of these therapies.
So it's really great to be here with us. May be we could start by saying a little bit about your perspective, how do you feel the field of shaping up today for therapy for Muscular Dystrophies?
Dr. Jeff Chamberlain: Well I think the recent developments of the field are extremely exciting. It has been very gratifying watching the progress over many years. When I first started working on muscular dystrophy, it was the question of what causes this disease.
How can we understand? How these genes work? What are the events that happened to cause a terrible disease? And the idea of developing therapies was really coming in at the back of our mind but it is a very remote thought.
And now as we're approaching what can one do, where is the progress going; it's completely different change in mind set. We're no longer taking about, eventually what can happen but what can we do here now?
There is so many different strategies emerging, the ways to treat a Duchenne muscular dystrophy as well as many other types of muscular dystrophy that are going to be, I think spend all largely work coming out of the Duchenne field. It becomes more of an issue of which one of These strategies is going to have the biggest impact.
First which ones are going to follow up? What is perhaps the best sequence of events to apply these everything, and obviously not everything is working at, there's still a lot of experimental things out there but there are so many exciting developments that it's going from, maybe someday, to when is this going to happen and which strategy is going to be the best.
And I should qualify that by saying, it's probably not going to be one strategy that is the best, but hopefully several strategies will come in together and combine to have a major impact on your muscular disorders.
Dr. Kevin Flanigan: Or maybe we could take a minute actually with your long perspective on this. Let's step back a minute maybe you could tell us a little bit rather your personal, about your initial work in muscular dystrophy, and how you got in to the field.
Dr. Jeff Chamberlain: Well, from my own personal view point it was a slow evolution. I started out being interested in muscle biology and molecular biology, how the genes work, how were different genes switch on and off in liver or muscle and I originally started research in laboratory that was studying a muscle development.
How does muscle form in developing embryo. And we became interested in genes. How were these genes turned on? Why do have genes that are only active in muscle as opposed to other tissues. And we became very interested just in the basic biological questions of gene switching.
Around that time after I was finishing up my Ph.D. I was looking up for something else to do and I thought about getting into things that might be medically more relevant. And around that time the muscular dystrophy Association had made an announcement that we need to find the genes that are defective in these different diseases.
At that time, we did know what cause any of these diseases. And I was contacted by a laboratory that wanted to something to come in and know lot about muscle and muscle genes to begin working on the Duchenne muscular dystrophy gene and the gene had just been identified by Louis Kunkel's lab at Harvard. So we knew what was out there but we didn't know anything about that gene did or anything like that.
And I thought that would be an exciting opportunity. I thought that I would go off and study genetics for two years and figure out what I wanted to do when I grow up, that type of thing, but I really enjoyed the work and it was a fascinating field, a lot of different from what I had been doing previously and I've stayed with it since then.
One of the first things we did was we began working with a mouse that had been reported to have muscle weakness and a possible type of muscular dystrophy and we did some of the characterization on that turned out to be the mdx mouse that is still widely use in a mouse model.
Dr. Kevin Flanigan: Standard model really isn't?
Dr. Jeff Chamberlain: Exactly.
Dr. Kevin Flanigan: Yeah.
Dr. Jeff Chamberlain: And we began characterizing the mdx mouse, we started cloning the --- what then became known as the dystrophin gene from mice following up with Kunkel's work with the human gene and you're trying to figure out what can I do to make the contribution to this field and it seemed like this is a great area.
I can focus on the mouse, the mouse genetics, the mouse gene and eventually I took that off into my own laboratory at the University of Michigan where I first started doing research.
Dr. Kevin Flanigan: I'm sorry we don't say the University of Michigan here at Columbus, Ohio. No actually they've --- for our listener's here not in the big 10 area they might not get that but we did. Please go ahead.
Dr. Jeff Chamberlain: I'm sorry. Well I'm trying to make demands by, I had at several former student from the University of Michigan but I'm now back in the University at Ohio State and I hope that wasn't cause on to lose your job.
Anyways, we begins taking the point of view, I was not a physician and I though well I can figure out what's going on the mouse and make a contribution in that regard. And that you know one thing I do another and the next thing I knew I was interested in gene therapy and trying to develop approaches for that and we went through a whole sequence of events from one way to get genes in the muscle to another.
And eventually some people came to my laboratory and said well the gene therapies office is nice with stem cells are really promising as well and they sort of developing some projects. So certainly over the years, we've touched on a lot of different approaches to therapy. We certainly have our favorites, the ones that had occupied the majority of our work but having touched on a variety of approaches.
We felt that might put us in a good position to try to write an overview of different approaches that would as you mentioned would have a bit of a historical perspective but also to hear now what's going on and I had two great post talks in my laboratory Patryk Konieczny and Kristy Swiderski who took this on and largely wrote this paper by themselves with some editing and insight from myself and we try to present it hopefully as a balance perspective on what's out there, where do we come from where we're going, and what's the current status, and that's what we have here.
It took as a while to get this published actually because it was originally targeted for a different source actually a book that was being written and a book project fell apart for various reasons and related to our own input.
Dr. Kevin Flanigan: Well it's a great article. We'll recommend to our listeners. And maybe we'll start by discussing some of the things you address really, we'll start with gene therapy itself that you've mentioned then of course our listeners probably know that your lab has played a really critical role in defining the kind of mini or micro dystrophin constructs that can fit into an adeno-associated virus and if people are regular listeners to your podcast they've heard multiple talks about adeno-associated viruses.
Maybe you could give a little bit about the perspective about other viral approaches that have now led to adeno-associated virus being sort of the favorite of this.
Dr. Jeff Chamberlain: Yes, there have been a lot of approaches, several of which are still being proceeded by some laboratories. I think the adeno-associated virus where the AAV is an interesting story because it was kind of the last system to come along and now does seem to be the favorite than one that's working the best but I can maybe provide a bit of a historical contacts of how we got there.
And at least from my own personal point of view, it started when I set up my original laboratory, again back in Michigan then around 1990s so it's been quite a while but my initial interest was what does dystrophin do? How does this protein work? Why is it when it's missing that you got a terrible disease and the approach we took to that was to use the mouse model, the so called the mdx mouse and we were trying to introduced new versions of dystrophin gene into that mouse and ask, how well do they work?
And the idea was to make normal versions, mutant versions and it's kind of doing some cotton pasting taking out different regions of dystrophin to try to determine what are the most important parts of the gene and the protein that it makes and if some regions are missing does that affects some part of muscle function or other things, it really gave us a way that really start dissecting what does dystrophin do?
The other thing that did was we realize that several of the versions of dystrophin that we can put back into this mouse cure the disease and corrected all the muscle abnormalities and that's what got us into the gene therapy field, and that very quickly led to the idea. Well, OK it's one thing to manipulate a mouse in a laboratory and the genes we're putting in, we're putting into a single fertilize egg. So that's not an approach that you can do in a clinic. So if that well, is there any way that we can try to get this into the clinic?
And really the first paper that came out to the University of Wisconsin another big 10s were there, that showed that genes could be transferred to muscle was to use so called "naked DNA or plasma DNA... in John Welsh Laboratory pioneer that work and starting in about 1991.
And what he showed was a plasma that's basically just a small piece of DNA a circular PcDNA and an advantage of these plasma is there's no viruses involve, there's no toxic materials and they can be grown a bacteria so you can just grow tons of tons of these things and get a very large amount of these DNA that in this instance would be almost entirely a dystrophin gene with just a little bit of extra DNA to allow you to grow up large amounts of it.
In John's Lab show that you can inject right in the muscle and get a fair amount of dystrophin made and so that was very exciting and we started jumping on that band wagon and got some interesting results but the problem with plasmids, it's one of these pros and cons issues, that the pro is very easy to grow, it's not toxic, there's no viral worries.
The limitation though that emerge over many years was efficiency was very hard to get very much and you could target at largely to an individual muscle but it's so hard to get the spread all over the body and it seem like we really have to move beyond that and the method that started taking that over was to use delivery vehicles that are derived from viruses and I tried to make that distinction between a virus and a viral delivery vehicle. It is an important distinction because a virus is something that infects the body.
It spreads and it makes you sick, but the reason that it's able to infect the body is because of the outer coating of the viruses. These viruses have developed over, you know, millions of years, a thousands of years, the ability to enter the body, enters cells and deliver DNA elements and that's the DNA that gets in there and makes you sick, but you can take out the viral elements that makes you sick and replace it with for example a dystrophin gene.
So then, I think if the gene therapy field in general there was a huge amount of work put in to identifying what would be a good virus that we can harness to make a delivery vehicle and three major viruses have emerge so called retrovirus, adenoviruses, and adeno-associated viruses.
Dr. Kevin Flanigan: So among those three, how did AAV emerges the favored really?
Dr. Jeff Chamberlain: Well AAV was --- early on AAV was largely ignored because it's a very small virus and of course the dystrophin gene responsible for Duchenne muscular dystrophy is a huge gene, and it just didn't seem realistic, so people were gravitating towards big viruses that could hold big genes.
Initially a lot of work went in the adenovirus because it will hold before dystrophin sequence and adenovirus got in the muscle well. And so the early on that was the vector system, the way that deliver and it show a lot of promise but the limitation of adenovirus that emerge was again you can deliver to one muscle is hard to deliver to more than one muscle and it had some toxic byproducts that prove to be very difficult to overcome.
People then move towards so called retroviruses, the most commonly used member which is now a lentivirus, which is derived from the Human immunodeficiency virus and the challenge there is to grow those in such a way that you cannot generate a wild type infectious virus, and I think that's largely been overcome and this vector system is now widely use and there's never been a report of any type of natural infection from using a lentiviral vector.
Dr. Kevin Flanigan: They have the risk of integrating in to chromosomal DNA.
Dr. Jeff Chamberlain: Yes.
Dr. Kevin Flanigan: Right, so ,
Dr. Jeff Chamberlain: The biggest risk and in fact in some of the first human gene therapy trials using these lentiviral vectors, several of the patients were cured of their disease and I should upsize that there are now patients that have been cured by gene therapy.
A fair number of them, the number of approaching a hundreds so far.
Dr. Kevin Flanigan: Not would Duchenne muscular dystrophy used to be clear to our listeners.
Dr. Jeff Chamberlain: These are so far they're mostly been blood borne diseases, immune deficiency diseases and all that but several of the early patients even though were cured of their primary disease they developed leukemia and the reason why they developed leukemia was at this lentiviral vectors are able to insert into the chromosome of the blood cells that were being corrected through this approach and inadvertently activated other genes that were nearby that led to uncontrolled growth of those cells and that turned into a leukemia.
Those patients have sense been cured of the leukemia by the way and they remain free of their primary disease, so there's been approaches to try to prevent that from happening and actually in the muscle systems it's less of the concern because muscle cells in general don't grow and spread, you think of a leukemia is a type of cancer were cells divide uncontrollably.
Well muscle cells for good or for bad don't divide; they don't grow anyways so it's a system that's less prone to cancer than many other diseases. However the lentiviral vectors that delivers a shuttles that works so well for blood diseases have some limitations for muscle which again is delivery and efficiency, you can get them in the muscle but they don't spread very far and there was no one has come up of the way to get them to go to muscles all over the body.
And then kind of get us to the adeno-associated viruses or the AAV vectors. The advantage that emerge with those early on that lead people to try to find ways to overcome the smallest of those vectors, it seem to eliminated as a choice for Duchenne muscular dystrophy. Those worries were overcome by an outstanding observation that you could deliver AAV vectors into the bloodstreams and they would home in on muscles all over the body, so it appeared to be the by far the best delivery system and if you could deal with the size issue then that may be the way to go and I think that those size issues have largely but not completely been solved in the intervening years.
Dr. Kevin Flanigan: That was certainly it's moving forward toward therapy at our institution, and your institution, other institutions on the adeno-associated viruses. Maybe let's talk now about cell base therapy, different route. So this is instead of just correcting the gene here, the ideas is putting cells in to repopulate muscles, is that right?
Dr. Jeff Chamberlain: Yes.
Dr. Kevin Flanigan: Can you please give us a little perspective on that?
Dr. Jeff Chamberlain: Yes, so for the muscle diseases and muscular dystrophies in general, stem cell technologies are really a very promising approach, I might qualify that by saying it's probably a long term and promising approach.
Dr. Kevin Flanigan: And when we say stem cell therapies in muscle disease, we don't just mean embryonic stem cells but there's different types of stem cells using a steminess if you will I guess we should say right?
Dr. Jeff Chamberlain: And that's correct, and it's a very good point because when you start talking about stem cells people start getting worried about the politics or the religion... religious issues and all that and it is important to remember that all of the controversy with stem cells was really derived from embryonic stem cells and in terms of the muscle field that is a cell type that has not emerge as a very interesting alternative.
Dr. Kevin Flanigan: Muscle itself contains what are stem like cells right that already committed to the muscle pathway.
Dr. Jeff Chamberlain: That's right, there are many different types of stem cells and an adult or even in kids in growing people there are what we called resident muscle cells a stem cells that are in the muscle and their stem cells because they can produce a tremendous amount of new muscle but that's all they form.
They form muscle they won't form blood cells or things like that and that's also important to remember that stem cells have been widely used in human disease therapy for many years and for example talking about leukemia, we did that a minute ago. The most common cure for leukemia is a bone marrow transplant and as far as we know that bone marrow transplant is a stem cell therapy, you're isolating bone marrow's stem cells and transplanting them from one individual to another but again those are adult stem cells and there have been a lot of attempts to try to use bone marrow stem cells to do bone marrow transplants to treat muscular dystrophy, it has not worked because of efficiency issues, you get very little new muscle formations.
So now the approach is focused on isolating stem cells that are naturally programmed and formed new muscle. Can you isolate those cells from a donor, either healthy donor or the patient themselves perhaps, produce enough of those stem cells in the laboratory and then transplant them back into the patient.
In the attractiveness for muscle diseases is a least with your limb muscles, your breathing muscles, these are muscles that naturally turn over, they don't live long periods of time in an individual with the muscle disease.
The muscle cells breakdown, that's what leads to the weakness but muscle tissues very good in repairing itself and the way it repairs itself is by taking these resident stem cells, kicking them in the action and they grow and make a lot of new muscle so dystrophic muscle is actually somewhat of a perfect environment for transplantation for taking a muscle stem cell transplanting into the muscle and have those cells grow into the muscle and repair the damage muscle that's already there. So I think in terms of stem cell treatments for patients, muscle has emerge as one of the most promising targets for stem cell therapy.
Dr. Kevin Flanigan: So some of our listeners, I imagine, may have heard of different cell types that are all potentially useful for these kinds of therapies. You've heard probably of myoblast or satellite cells or mesoangioblast, these are all underway I know in laboratories around the world.
Dr. Jeff Chamberlain: They are. Yes.
Dr. Kevin Flanigan: One of the things well you've just mentioned taking a patient's own cells out to do that. That will require a correction step, I believe.
Dr. Jeff Chamberlain: Yes
Dr. Kevin Flanigan: So what are the approaches for that? One might correct the cells in the test tube and put them back in?
Dr. Jeff Chamberlain: That's correct. And so there are really two issues dealing with correcting a patient's own self. One is what is the best source of your cells and as you mentioned there a lot of different sources of cells that might be able to make new muscle. Once you deal with that issue you then have to --- how do you fix that gene?
And there are two ways to do that, one is just to bring out a new gene using a viral vector just like we're talking about earlier. The preference shifts though from whereas the AAV vectors seems to be the clear best choice at this time for delivering genes body wide to the muscles, AAV vectors don't work with stem cells and the reason for that is that they don't merge with the stem cells own genetic material.
Since they're separate from the DNA and your nucleus and when a stem cell divides and grows to make new muscle it loses the AAV vectors. It's not stable inside the stem cell. So that gets you back to the lengthy viral vectors we're trying about a moment ago that do enter the genetic material that become a permanent part of it and so it's an ideal vector system for stem cell because as the stem cells grow and migrate through the body they bring the new gene with it.
So the approach which is sometimes called ex vivo gene therapy is to harvest the stem cell from a patient, bringing a new gene using a lengthy viral system. Grow up those stem cells, the large quantities, perhaps manipulate those cells a little bit to maximize their ability to form new muscle and then find a way to transplant, I mean to the patient to create or rescue the existing muscle.
Dr. Kevin Flanigan: That's why I don't want to just inject one, know the question I think of this. Not the question about what you said but I'm thinking about parents listening to this for example and it's important to emphasize that these are therapies that are shown and promised in the test tube right now and I just have to say and some from the clinic.
I talked to parents in the clinic sometime. There are no stem cell therapies you can go and buy today that will cure a muscular dystrophy. And there are individuals who advertise $10,000 or $20,000 in the Ukraine and or the Dominican Republic or something and we don't know anything about the safety of these cells, we don't know anything about the efficacy in that, we don't know what's being given at all sort.
There's level of even a significant question parent should have, but what you're trying about as real science being done in the test tube that as a potential down the road of actually doing something. We're not in clinical trials with them yet though.
Dr. Jeff Chamberlain: That's right. And it's a very important point you bring up.
In terms of the people that are charging money for trials using stem cells, I think I can say without any hesitation that those are fraudulent, all of them. And the reason I can say that is that while stems cells are showing tremendous potential for therapies nothing is working yet, I mean they're not even working much in a level of a mouse.
They're certainly not working in larger animal models and nothing is even close to working well in human, but that doesn't mean that they're not going to someday. And the glimpses of data that we've seen in the smaller animal models show a lot of promise. You can get the stem cells and they can grow new muscle but the efficiency is way down, you're not getting enough to be therapeutic.
There are issues of a lot of the cells that you transplant don't survive the procedure, they don't last long enough. Sometimes they're kick back out of the body and there's a lot of significant issues that need to be doing with. And that my own personal feeling of the stem cell approach is that down the road maybe 20 years or so stem cell transplants may be the ultimate way to treat muscular dystrophy but right now they're far from clinical utility and many of these other approaches with the small molecules, the anti-stem cell like the nucleotides, the gene therapy vectors are much closer to clinical application than the stem cells which have long term and clear relevance down the road but they are not working yet.
Dr. Kevin Flanigan: Maybe well, I'll take the opportunity here to just ask what's next for you and for your laboratory.
Dr. Jeff Chamberlain: Well, we're trying to take a dual approach. Our main interest is what the gene therapy using the adeno-associated viral vectors and I think that's true for a lot of groups in the field. There are exciting developments in laboratories around the world, in Italy, in France, in England, here in the United States, several groups in Asia of making progress with AAV vectors.
A nice thing about that is... you know... sometimes people worry about competition versus sharing and all that is. It's nice to have multiple groups trying things because everybody is trying to do something a little bit differently. Most of these groups are talking to each other, sharing data, sharing information and the field as a whole is moving forward quite rapidly and so, I'm excited about the progress.
Our own approach is to try to develop the best possible form of dystrophin to deliver with this AAV vectors because there's a lot --- dystrophin doesn't fit very well and A, B, C. We got to make some compromises and its structure and we're spending a lot of effort trying to deal with that. The other issue is we have some concerns about how the body is going to react to bringing a large amounts of a new vector system, a new dystrophin gene and we're trying to pay close attention to the immune system and do we need to control the immune system to allow these gene delivery systems to work and so that's become a major focus of our work in the laboratory.
So our major effort in my lab at the University of Washington in Seattle where I worked is to start bringing these technologies into the clinic but with the focus on immunology and just long term functionality maximizing the ability of the genes to function well in human muscle. Now where also as I mentioned the future potential of stem cells we're also developing more and more effort to stem cell approaches but I viewed that as much more of a development project something we're trying to bring along.
And we're focusing there on two types of stem cells, one is a cell --- well actually both of them were a stem cell derive from a fibroblast. A fibroblast is a kind of a boring cell type found in muscle, it doesn't normally make new muscle but it provides a supporting role to the muscle environment. And it turns out that fibroblast can be used in two ways to potentially create new muscle; one is that a fibroblast can be converted into a kind of a super stem cell known as an inducible pluripotent cell and these cells can be grown up to unlimited quantities and you can just get how many you need and the trick is to convert them back into a muscle stem cell.
And it's been very exciting over the last year or so that several groups have published new ways to take this fibroblast turn them in to a super stem cell and then get them to turn back in the muscle. There's been progress from Italy, from University of Minnesota, from Australia and from Japan showing a lot of promise to actually take a massively expanded population of cells and converted in to a muscle stem cell.
So we're doing some similar cells with that and I think there's going to be a long term potential. Another thing that I might want to mention here which I think is little unique to one of the things we're doing is that we're trying to use the fibroblast in a slightly different way. And that is to take a fibroblast and directly convert it in to a muscle cell and you can do that by introducing a single gene in to those cells that somewhat of a master regulator of the muscle programming environment.
And we're trying to do this in two ways one is just sort of a proof of principle that we can grow up large amount of fibroblast that are present arising from the mouse model for Duchenne muscular dystrophy. We're putting a couple of new genes and they're using this lengthy virus technology we're then transplanting this fibroblast back in the mice and then we're giving the mice a drug that causes the fibroblast to form new muscle, once they're already inside the muscles of the mouse.
So we're not manipulating or making muscle in the test tube and then transplanting it. We're actually trying to get all of the muscle stem cell formation happen inside the body. And that's a bit of a proof of principle because where we want to go with this long term is to try to find the way to create new muscle from damage muscle. One of the promised you get with Duchenne dystrophy and other forms of muscular dystrophy is that as you lose muscle is replaced by a lot of connective tissue and non-muscle tissue.
And a lot of these cells are these fibroblasts like cells. And we're trying to find the way to actually deliver some of our gene therapy vectors back in to a severely damaged muscle in a way to create new muscle stem cells from within from this field of devastation where there are used to be muscle to take advantage of those cells that are still there convert them back in the muscle and grow new muscle from within.
So it would be a way of not only replacing the dystrophin gene but also making new stem cells to grow new muscle in. in some ways that could be the ultimate form of therapy but it's way, way down the road and it's a bit of --- kind of a dream project in our laboratory and this going to go on for a very long time, I think.
Dr. Kevin Flanigan: Well, thank you for taking all this time today to share with us your perspective in your field and your work. So, really do appreciate it.
Dr. Jeff Chamberlain: Thank you.
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 published abstract of the study that we've discussed today.
Thanks for joining us and we looked forward to talking you again, next month.