Guest: Thomas Voit, medical and scientific director, Institut de Myologie, Paris, France
Access an abstract of this month’s featured research article: A phase I trial of adeno-associated virus serotype 1-γ-sarcoglycan gene therapy for limb girdle muscular dystrophy type 2C. Brain. 2012 Feb;135(Pt 2):483-92.
Kevin Flanigan: Welcome to this month in muscular dystrophy. I'm Kevin Flanigan from the Center for Gene Therapy in 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.
I'm very pleased to have as our guest today, Professor Thomas Voit, Professor of Pediatrics at the University of Pierre and Marie Curie, Paris 6, and Scientific and Medical Director of the Institute of Myology in Paris. Thomas, welcome.
Thomas Voit: Hello. Good morning.
Kevin Flanigan: Thanks for joining us. Today we're going to talk about a recent paper from your group, from your center, regarding phase I trial of gene therapy of adeno-associated virus serotype 1 to transfer the gamma-sarcoglycan gene in limb-girdle muscular dystrophy. I'll just remind listeners that a link to the abstract of this paper is available on our website, the website of this podcast.
Thomas, let's talk first about the idea of limb-girdle muscular dystrophies. What are the limb-girdle muscular dystrophies?
Thomas Voit: As a group, the limb-girdle muscular dystrophies are some 40 to 50, and probably even more genetically different disorders that lead to a progressive weakness notably of the limb-girdle muscular group but eventually, of all muscles.
Kevin Flanigan: So the limb girdle, shoulder girdle, and the pelvic girdle.
Thomas Voit: And the pelvic girdle. This disease usually affects ambulation first, and at a later stage may also affect the use of the arms and the shoulders.
Kevin Flanigan: So 40 or more different types, quite a bit of variability in the genetic background.
Thomas Voit: Very huge variability; there are recessive diseases, there are dominant diseases, and the clinical spectrum is also very wide and that is all those that may include the heart with the encephalopathy or the stoned, the disorders that start in the elderly and are very mild and are compatible with maintenance of ambulation, and the severe disorders where an adolescent will go off his legs and be in a wheelchair.
Kevin Flanigan: The disease that you treated in this trial was limb-girdle muscular dystrophy type 2C. We often call this LGMD for short, so we don't have to keep saying limb-girdle muscular dystrophy here on this podcast. So LGMD 2C, that means it's a recessive type first.
Thomas Voit: This is a recessive type to start with, and that is a disease that is particularly common in some ethnic communities. The specific mutation we've treated here is very common to the Magribian areas, so the north of Africa, but also, of course, in the south of France in Italy, for example.
Kevin Flanigan: That's quite the answer. A recessive disorder means you have to have two bad copies of this and in your paper, this mutation that was present was inherited from both sides of the family - from mom's side and dad's side.
Thomas Voit: Yes, yes.
Kevin Flanigan: All right.
Thomas Voit: In that case, the patients were actually homozygous for the same mutation. The mutation background was the same in all patients when we treated.
Kevin Flanigan: So that makes it a very nice, at least the same mutation for all of the patients that had gene transfer. The mutation results in the missing protein, gamma-sarcoglycan, what do we know about the function of gamma-sarcoglycan?
Thomas Voit: Well, gamma-sarcoglycan is one of the sarcoglycan proteins. These are proteins that are part of the dystrophin dystroglycan protein complex, and this is a complex which is essential in skeletal muscle but also in heart muscle to construct this scaffold that connects the contractile elements of skeletal muscle across the membrane to the extracellular matrix.
One important protein is dystrophin. If dystrophin is mutated and missing, it results in a severe disease, Duchenne muscular dystrophy, quite well known because it's a sort of emblematic disease from neuromuscular disease. But the sarcoglycans are very closely associated to dystrophin in this complex and each of the four sarcoglycans are the alpha, beta, gamma, delta, if mutated on both, gives a disease which is almost as severe as Duchenne muscular dystrophy or can also be a little bit milder.
For this mutation, it's actually quite a severe disease and the patients are usually in the wheelchair by the age of 20 years.
Kevin Flanigan: In many ways, similar to Duchenne or Becker muscular dystrophy, although, this affects, again, both boys or girls, males or females. The sarcoglycan complex, you mentioned it functions as a complex, and one of the features I think is if you're missing one of these complex, the entire complex is missing.
Thomas Voit: Yes.
Kevin Flanigan: Is it right?
Thomas Voit: Yes. The slightly different degrees, one of the other may still be there of these proteins but to much less a degree. So delta sarcoglycan tends to stick around a bit. Essentially, if you pull one out, the others go with it.
Kevin Flanigan: The absence of the gamma sarcoglycan, the absence of this anchoring function, then leads them to break down the muscle fibers, deterioration of muscle fibers, and all the things that go with muscular dystrophy.
Well, let's talk now...in this paper, you used adeno-associated virus. Why is that used for gene therapy?
Thomas Voit: This is today certainly the tool, the shuttle, which allows us in the most rational way to bring genes into skeletal muscle for several reasons. Number one, the virus, as a fully competent virus, doesn't make ill.
Many of us are carrying traces of having seen one or the other adeno-associated virus and this is not related to disease in the human. Second point is you can completely get this virus so nothing of the viral genes remains in the production process. What you're transferring is really an empty hull where you put your gene of interest in. This virus cannot function and recombine as a virus at a later stage.
Kevin Flanigan: It can't replicate it.
Thomas Voit: It can't replicate. It's not dangerous for the recipient.
Kevin Flanigan: It's really an envelope then, an envelope.
Thomas Voit: It's an envelope. It's an envelope and an envelope that tags skeletal muscle and also heart muscle with a very high efficiency.
Kevin Flanigan: Some of our listeners, if they listened to earlier podcasts, for example, about mini-distrophy, talked about miniaturized versions of genes, they've heard the idea that the AAV virus have a limit to the size of a gene you can put in.
Thomas Voit: That's right. You classically cannot put more than 4.5 - 4.7 kilo-basis or thousand-basis into an AV shuttle. If you exceed that proteins of the caps, it cannot close anymore.
Kevin Flanigan: You can't shut the envelope.
Thomas Voit: You can't shut the envelope.
Kevin Flanigan: So that's an advantage here for this gene.
Thomas Voit: That's an advantage for the sarcoglycans and this was one of the reasons why we chose one of the sarcoglycans because they're small genes and you can put the whole gene plus a promoter into an AV. When as dystrophin is a giant protein and you cannot even fit one-fourth of the dystrophin gene into an AV.
Kevin Flanigan: You've got the full-length gene in the envelope in the caps at the AAV, and I guess one of the other features is AAV likes muscle - many types of AAV like muscles. Delivery in the muscle has the advantage, it likes to target that over other tissues.
Thomas Voit: Yes, yes.
Kevin Flanigan: Well that brings us up to your recent paper where your group delivered the gamma-sarcoglycan gene to a muscle in nine human subjects with the LGMD 2C. Can you tell us a little bit about this trial, the design of the trial, and what you did?
Thomas Voit: The starting point for this work is that when we started, there was practically no published work out there. In the meantime, of course, Nationwide Children's Hospital has also conducted in another sarcoglycan disease, a phase I trial, but for muscular dystrophy, there had not been any clinical trial. And it's important to understand that because that means if you meet regulators, which is the FDA in the U.S. or IMA in Europe or at the national level, it's AFSSAPS in France, you come to them with the idea of treating a disease where they have never seen a precedent.
You have to explain why you're using this type of shuttle, why you're using this type of gene, and what you want to do. As regulators, for a good reason, tend to be very conservative and their major goal is to keep patients on the safe side and the way from undue threat. They limit what you can do in a first approach.
What they told us is it was out of the question to do many muscle groups at one go like in the local regional perfusion, for example, but we had to inject a single muscle intramuscularly. This means, for the patient, he cannot expect to really get a benefit from that sort of treatment because you're only targeting one muscle.
The study is a phase I study where you administer increasing doses; so one patient will get one dose, the next patient will get the same dose, and once you have three patients with the same dose and this has been well tolerated, you can go to the next higher dose level, and so forth.
The emphasis of the trial is on safety; is this product safe if given to the human? And so, all the investigations are primarily geared towards regarding, is there an undue immune response, is there inflammation elicited by what you're injecting, has the patient got any other problems related to that like local inflammation, pain, loss of muscle function, for example, so the trial has controlled all these things.
Normally, this is a phase I trial for which then afterwards, you would have to perform a phase II trial and eventually, a phase II-B trial for lots of cohorts where then, in a second and third trial, you could try to go towards a clinical benefit for the patient.
Kevin Flanigan: That's an important point. Many of our listeners, I'm sure you have the same experience, many parents might ask us how come I read about this compound working in a mouse or someone, why can't we just give it to my boy today? So it's worth hearing about this process.
Thomas Voit: They will never let you do this and...
Kevin Flanigan: Right. And appropriately so, it's safety. These are important issues - is it safe? You did look at safety and then you looked at gene expression as well. You biopsied boys after about 30 days of treatment.
Thomas Voit: Yes.
Kevin Flanigan: What did you find?
We eventually found a clear expression of the protein only in the three patients who had received the highest dose. This is not completely unexpected because what happens in the negotiations with the regulators is that we had some animal work suggesting which would be the lowest-efficient dose. For them, this is the highest dose they are prepared to accept and they start with a 10-fold lower dose just to keep the patient on the safe side, and then slowly upgrade.
Eventually, we saw protein and a limited amount of protein expressed in some fibers of the injected muscle in the three patients who were in the highest-dose cohort and we saw RNA evidence in four patients.
Kevin Flanigan: But that's still very good news that after 30 days of expression, the gene you put in was making the protein and so on, and it appeared safe in that as well.
Thomas Voit: It appeared safe and that there was no inflammation associated to that. That's very important because muscular dystrophy muscle in itself is an inflamed tissue. Due to the chronic degeneration there's a higher prevalence of lymphocytes, of macrophages in this tissue, and these lymphocytes are exquisitely sensitive to local alterations.
So if there's one tissue that will easily react with an inflammation towards an external stimulus, it is the skeletal muscle of a patient with muscular dystrophy. The fact that we did not see inflammation related to the administration of the product, is really a very positive point.
Kevin Flanigan: Did you see any other immune responses that worry in this? Any time that we put on a virus into someone, we have to think about that obviously.
Thomas Voit: No, we didn't see any reactions that were obviously related.
We had one patient who seemed to have an intercurrent infection, something like a small flu that coincided and he had a slight fever the day after having received the virus, but it was obviously unrelated to the administration of the product. In the patients we did not have any clear adverse events.
Kevin Flanigan: How about immune responses to the protein itself?
Thomas Voit: We checked for immune responses to the protein itself and did not see immune responses directed against gamma sarcoglycan. What we did see, and that's something one would expect, immune responses against the viral capsid. What we saw, in fact, when we started the trial we had not systematically screened all patients for that subtype of AV specifically and we then quickly saw that patients who were carrying even very low titus of pre-existing antibodies, would probably not be transfused, so that we made an amendment to the protocol and for the later cohorts, only include patients who were completely AV -1.
Kevin Flanigan: Well, that's all sounds very promising and I presume this work is going on. What are the next steps for your center in developing this rather therapy?
Thomas Voit: The upscaling from the industrial production site requires, if you want to extend this sort of approach to say first a lymph perfusion as the next step, much higher production rates off the AV, and in a clinical-grade production.
Kevin Flanigan: Just to give enough. To have enough to give.
Thomas Voit: Just to give enough vector, exactly. This next step of industrial production has been taken by the Genito, which is the non-for-profit company that produces the vectors.
They are now in a position to produce large batches. Probably, within the ongoing program, the gamma sarcoglycan treatment will be promoted but will not be the next trial that starts because the next on the list is Duchenne muscular dystrophy trial, also using an AV, a different subtype AV 8, with a local regional perfusion. Depending on what the results and the tolerance of this much larger approach where you put several locks of virus more in what the tolerance is, we will then go forward for the sarcoglycans.
Kevin Flanigan: Well, that's, again, sounds very promising and we'll look forward to talking another time about the results of that.
Thank you very much for taking the time to chat with us and our listeners today.
Thomas Voit: Thank you very much. Pleasure.
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 abstract that's published regarding the study that we discussed today.
Thanks very much for joining us.
Clinical Trials at The Center for Gene Therapy
Muscular Dystrophy Care at Nationwide Children's
OSU/Nationwide Children's Muscle Group