NAIR
MuscleChemistry Registered Member
Okay fellas, this will take a long time and it takes a while to get interesting but about 15 minutes into, when it gets into IGF and its role in muscle regeneration, it get so damn exciting you'll pee yourself. Read it when you get the chance, well worth it.
Also, I must give props to the original poster: bigdaddyhd
Session 7: Enhancement 5: Genetic Enhancement of Muscle
H. Lee Sweeney, Ph.D.,
Professor and Chairman of Physiology,
University of Pennsylvania
CHAIRMAN KASS: All right. I know there are some counsel members with planes to catch, and I don't want us to waste any more of Dr. Sweeney's time. We're delighted to welcome Dr. Lee Sweeney, who is Professor and Chairman of the Department of Physiology at the University of Pennsylvania, who has done just a significant amount of outstanding work on muscle physiology and who is going to speak to us today about the genetic enhancement of skeletal muscle and its performance.
Thank you very much, Dr. Sweeney, for being with us.
Push your button there. There we go.
DR. SWEENEY: Thank you.
Yeah, I'm going to just try to give you in half an hour or so background about some of the work we're doing, and then I'm hoping just to allow you to drive a lot of the discussion because I'll set up some of the issues that I see, but as a scientist, I'm afraid I don't think some of the ethical ramifications through quite to the extent of the discussions I've already heard this morning.
My interest is in disease states of both skeletal and cardiac muscle. I'm going to restrict it to skeletal muscle because I think it gives you a good example of really how gray the boundary is between therapeutics and enhancement when one is starting to think about what can be done with genetic manipulation in adults.
Skeletal muscle is a big target in a person because it makes up the majority of the mass of their body, and it's an interesting tissue in that it has built into it cells called satellite cells which are not differentiated muscle cells, but which are actually cells that are called upon to divide and differentiate and regenerate the skeletal muscle.
So they're not really a resident stem cell population because they're not pleuripotential like a true stem cell, but they're an uncommitted set of cells that can with the proper stimuli be induced to become skeletal muscle. They can become other types of tissue as well, but a fairly limited repertoire.
Now, my interest in this began with years ago the beginning of the whole promise of gene therapy, and of course, the idea at the beginning of gene therapy was really to tackle the simplest of genetic diseases, those that involved single genes and usually the genes being missing or at least defective, which was the cause of a large number of diseases, most of them fairly rare diseases.
The issue, and still the issue, the recent gene therapy really hasn't as quickly progressed as we all would have hoped it would have ten years ago, is because the problems are really finding the right vector for a given tissue, that is, a delivery device to actually get the genetic material into the adult tissue, and then also figuring out how to get it there, the delivery.
So these are still the key stumbling blocks in gene therapy today, although great inroads have been made.
Now, in terms of muscle what is sort of emerging as the best sort of ways to deliver genes to muscle are sort of listed here, and this is as of today. Muscle is actually quite good at taking up so-called naked DNA or just plasmid DNA. This is DNA that is not encapsulated in anything.
When I say quite good, I mean it does it with some efficiency. It's perhaps one or two percent of the cells if you inject DNA into a given muscle would take something up.
So this is not an inefficiency that's useful in correcting a primary genetic disease of the muscle itself. However, this would be useful in terms of getting the muscle to produce a substance that would then be secreted into the blood.
And, in fact, this technique has been shown to be successful in an agricultural setting where a colleague of mine has taken DNA that codes for the growth hormone releasing hormone. This is actually a protein which stimulates the release of growth hormone, and in doing so, he can demonstrate that the pigs will now secrete much larger much larger levels of growth hormone.
Obviously one of the other problems with it is it's transient, but in that sort of setting perhaps it's useful to be transient just to sort of give them a growth boost through some period, and then it will go away, but obviously not something one could think of for permanent sort of genetic correction, and especially not a genetic correction of the muscle itself.
Now, viruses are still the preferred gene delivery vehicles in terms of efficiency. Getting genes into a given tissue are what viruses are engineered to do, and so they can be reengineered to deliver therapeutic genes instead of viral genes.
And probably the best vector for muscle is a virus that's known as AAV, which stands for adeno-associated virus. It's a virus that's unrelated to adenovirus, which has been used, as you're probably aware, in gene therapy trials with complications, and the most severe which took place at my own institution.
But this is a different virus that actually does not cause any known disease in humans. Often it can be found in 20 percent or so of the human population that have been infected by AAV with no consequences that can be demonstrated.
And in the last maybe two years a number of different sort of variants or serotypes of the virus have been isolated, and at least two of them are extremely efficient at targeting skeletal muscle. And so there are now gene therapy trials that have begun and many more that are about to be proposed and started that use this virus to try to get genes into skeletal muscle or in the liver. It turns out that this virus is also very good at targeting the liver, which would be very useful in diseases where one wants something secreted into the blood, like in the case of hemophilia.
There's an ongoing trial now with AAV targeting the liver to get the liver to secrete in this case Factor 9 for patients with Factor 9 deficiency.
Adult stem cells obviously are useful in sort of the idea of helping regenerate the tissue, although I must say that although there are a number of types of adult stem cells that can become muscle, again, efficiency becomes the problem, whether they're muscle or bone marrow derived stem cells. It's very difficult to get a large percentage of the muscle rebuilt using this approach in animal models where it's been attempted.
Nonetheless, we and others see the ability to use adult stem cells now as not in the stem cell sense of rebuilding the tissue, but perhaps in the sense of viewing them as a vector where one would take the adult stem cells in the laboratory, put new genes into the stem cells. That would then allow the tissue that they've incorporated in to secrete a substance that would then affect the surrounding tissue or if it's designed to go into the blood.
So you could sort of put adult stem cells then not only in the tissue regenerating sense, but in the sense of being a vector to carry genes into a tissue, to incorporate into the tissue, then to produce something else in that tissue.
And the advantage that they may have over viruses is one of the easiest systemic delivery. In many disease situations it may be that one could simply put the adult stem cells into the blood and they would home in on the tissue. They would home to the tissue that was actually being damaged by whatever the disease process is, and so it would be a very efficient way of targeting the tissue, which with viruses is more difficult at least at this point in time.
So I'm going to give you an example then of using the adeno-associated viral mediated gene transfer, but the example is actually one that we're now trying to do with adult stem cells, which is why I brought this up, because we think we can accomplish the same thing and deliver it much more simply using stem cells that are bone marrow derived from in this case we're working with both adult human cells, as well as adult rodent derived cells.
Adeno-associated virus, as I said, has the huge advantage for skeletal muscle in that it readily infects it. In fact, this may be the preferred tissue target of this virus at least for various forms of this virus.
It's limiting in that the size of its entire genome is only on the order of about 4.7 kilobases. So this is very small and really smaller than most genes in the human, and so one can only make synthetic genes that code for relatively small proteins. And so one has to be judicious in the choice of what one can attempt to do with this virus.
Delayed onset of expression, perhaps more so than some of the other viruses, but nevertheless, with the more robust infection that one gets with serotype one and five, expression commences within a week of injection of the virus into the tissue or systemic delivery of the virus into the tissue.
There's no viral gene expression. This is one of the big advantages of using this type of vector. You can make the synthetic vector without any viral genes, and because of that, there's no immune response against the virus itself.
Obviously there could still be immune response against whatever the product that you're causing it to make, but an example I'll give you, which is one of the advantages of it, what we're making is not something that's missing from the body, but something that we're just trying to get the body to make more of. And so there's nothing for the immune surveillance to pick up on, and so no possibility of immune response.
It integrates at a low frequency, which is both useful, but also a point of some concern in sort of the regulatory and side effect case. The integration, with any integrating virus there are two things to worry about. One is the possibility of oncogenesis being initiated by an integration event.
We have not seen this in our animal models, and other people have not seen this. So the possibility with this virus seems relatively low because there's some evidence that the integration events of AAV are somewhat site specific, and so not very likely to induce oncogenesis.
The other problem, of course, is whether or not one could get germ line transmission, and compared to something like lentivirus, the ability to do germ line transmission is not zero, but it's fairly low probability of germ line transmission, but again, this depends on the route of administration.
It would be more likely that you might get germ line transmission if you're doing a vascular delivery than of direct injection into the tissue, and the duration of expression, because of the integration, essentially is the life of the nucleus that you infected. And so as long as that cell in that nucleus exists, one will get expression. And so for the animal models that we look at, it's the life of the animal essentially.
Just to show you what efficient means, here's a cross-section through a muscle. So these are now -- if you think of the muscle fibers and muscle cells as very long cylinders, this is now slicing through them so that you basically just see their circumference and not their length.
And as you can see in this example, this is now using AAV-1. Essentially every muscle, well, every muscle in the field is now producing a protein that gives a color and an enzymatic reaction that's developed in the laboratory, a bacterial protein that can be used to give this colormetric readout, and you can see that every muscle fiber in the field is blue, and in fact, one can do vascular injection with this virus, and every fiber and every muscle virtually in the leg of the animal will be blue after vascular administration of a large enough dose.
So the efficiency is extremely high if one puts in enough of the virus.
So potential applications, which is what got me interested in using this in the first place, obviously the initial goal of all gene therapies of this sort was primarily genetic diseases, and for muscle that would mean Duchenne and Becker muscular dystrophy is the most common, but also others, such as the limb-girdle muscular dystrophies, myotonic muscular dystrophy, and whatnot, where one can point to a genetic defect in a single gene as the cause of the disease.
A more difficult problem, but actually in some ways, I mean, biologically a more difficult problem, but in fact, the problem that we focused on initially, which is a very real problem in this society where the society is living to be older and older, is the fact that as we get older our muscle function, our skeletal muscle function diminishes both in size of the muscle as well as the relative strength of the muscle and this is a big problem not only from an ambulatory standpoint, but also from a whole body metabolic standpoint.
If the mass of skeletal muscle drops below a critical threshold, then the whole body metabolism is no longer supported properly because the muscle actually functions not only to move the body, but as an important metabolic organ within the body.
Then the last sort of issue, which is actually a trial, trials have been ongoing in this area, is the use of gene transfer into muscle to get therapeutic proteins in the blood, such as Factor 9 deficiency.
So the initial hemophilia trials with Factor 9 were trying to actually get muscle to secrete Factor 9, but now they've shifted to liver because the liver is just a better organ for secretion into the blood than muscle is, although the muscle is capable of it.
So I want to first tell you about where we started some five years ago, which was looking at this problem that the NIH has coined sarcopenia. I think they coined a term to sort of make it sound more like a disease, probably for congressional purposes, but basically what they're really talking about is this progressive loss of muscle mass in force that essentially begins in the fourth decade life in humans and then progresses throughout.
It's slowed, but it's not prevented by exercise, and obviously as I've already mentioned has negative impacts on health and quality of life. It occurs in all mammals, which is useful because that means all of the laboratory animals one works with undergo the same process, and since they live for a much shorter period of time than humans, their life spans are much contracted.
The whole sort of progression occurs on a time scale that one can approach in the laboratory, and our hypothesis back in '96 or seven when we began this was that in large part we thought that what it was really due to was not inactivity. There had been a lot of discussion that as people got older they were just inactive and that's really the main thing that drove it, but we really thought there was a more fundamental cause, especially since there were studies showing that exercise could slow it down but not stop it.
And that was the fact that the repair mechanisms of skeletal muscle decline as you get older, and this causes the muscle to lose function because it's essentially not being repaired properly, and this goes back to what I said at the beginning, that it has within it a resident population of cells, these so-called satellite cells, that when the muscle is damaged -- and muscle is always damaged as you're using it -- are called upon to repair the muscle and rebuild it.
So this sort of rebuilding process involves some sort of damage signal coming out of the muscle which then activates the satellite cells to begin to proliferate, and they proliferate, then they make the commitment to be muscle, and then they either fuse with the existing muscle to repair it, or if the muscle has been severely damaged, they form new muscle.
So what is involved in this are a number of growth factors, some that drive the process, some that inhibit the process in sort of a yin and yang, but the one that we felt was really the most critical and the one that might be the candidate for what's going wrong in aging is a growth factor called IGF-1, which stands for insulin-like growth factor-1, which in normal muscle is involved in growth. It drives protein synthesis, and it decreases protein degradation, and importantly form the repair standpoint, it stimulates this population of satellite cells to both proliferate and differentiate.
And this is an important fact that it can do both because many of the growth factors will drive proliferation but block differentiation, and so increasing their levels could actually interfere with repair, which has been shown in some cases, but here you have one that has a little built in clock. It will drive proliferation for a while through one pathway, and then it will drive through this pathway, and then it will drive differentiation through another pathway, which it turns on with the delay.
So just the sort of thing you might want to try to drive more successful growth and repair. And the reason we thought it might be a problem in aging is because it's really part of the whole growth hormone IGF-1 axis, which as you know, the signals from the hypothalamus, the growth hormone releasing hormone, that are then taken to the anterior pituitary to stimulate it to produce growth hormone go down with aging.
The levels of growth hormone in the blood go down. The levels of IGF-1 produced by the liver; the liver produces all of the IGF-1 that circulates in the body. All of these levels go down with aging.
And what that means is that the IGF-1 levels in the various tissues of the body will also be diminished with aging.
Now, tissues like muscle and other tissues of the body have two sources of IGF-1. They make it themselves under conditions of either injury or rapid growth, but also they have an IGF-1 input that comes from the liver that's ongoing throughout their life.
And so it's this component in particular that's being lost in the aging animal. And so we sought to essentially replace it by supplementing the amount of IGF-1 that the muscle itself could make.
So the strategy was quite simple that we took. We would use gene delivery into the muscle to give it a synthetic gene to have it produce more IGF-1 so it would not be particularly dependent on the liver for a source of IGF-1.
And then the question was: would that then in muscle promote growth and regenerative pathways and would that, in turn, allow the muscle to function throughout the life of the animal without the aging related loss?
So it's a very simple synthetic gene that we put together using a muscle specific promoter driving the rodent IGF-1, and then it's flanked by the viral ITRs and packaged into the AAV viral capsid, and then just to inject into the animals either a vascular delivery into the leg or a direct injection into specific muscles.
And we could how -- this is just using PCR to detect the existence now of our synthetic gene that four months, nine months, even two years after injection the synthetic gene is present and producing IGF-1 messenger RNA.
So then we asked the question with it: is this going to increase the rate of muscle regeneration and maintain mass and old age? And this is the paper that I included in your packet.
What we showed was that if we injected mice, essentially middle aged mice or late middle age in mice -- mice live to be about 27 to 30 months in age at least in our colonies, and so we injected them about halfway through their life where they were all just beginning to start losing muscle mass, and then we asked, you know, what would their muscle mass in force and force for cross-section look like when they became old.
And so looking at them at 27 months, which was nearing the end of their life, they normally would have experienced in terms of mass about a 15 to 18 percent drop over that age period compared to a six month old mouse when they're sort of at their peak.
Whereas if we had injected them in middle age, they maintained the same mass or even a little greater than they had when they were younger. The same with the amount of force they were able to produce. Their muscles were able to produce normal force instead of showing the decline in force that they would normally see, and the force for cross-sectional area was maintained, as well.
But the speed of the muscle was maintained and the power output was maintained to an even greater extent because one of the other things that happens as the animals get old, as mammals get old, is they selectively lose their fast and most powerful fiber type.
So skeletal muscle is a heterogeneous tissue in terms of it has some of the fibers in it that are small. Some are big; some are slow; some are fast. We lose the very fastest ones as we get older and preferentially replace them with slower fibers. That's one reason why some of the first athletic things that go are your ability to compete in power events or speed events, because that's the first loss that you experience before you really lose muscle endurance or any of those sorts of properties.
And we were able to prevent that totally. The mice did not lose any of their fast fibers, and they had the same speed and power output when they were 27 month old muscles as they did as animals that were only six months of age.
So from that we were able to conclude that IGF-1 over-expression could prevent all of the hallmarks of age related atrophy and loss of skeletal muscle function in mammalian aging, at least based on the rodent model, and now we're hoping to pursue this in larger animal models.
The skeletal muscle regeneration rate is diminished in old animals, and we showed that in another paper other than the one I showed you, and that seems to be the primary problem, that even if you injure the old muscle, it cannot mount a normal regenerative response, but if you maintain IGF-1 expression, it can maintain a normal repair response, and this also, of course, is this hypothesis that we were looking at.
And also it suggests that one could go about this whole pathway as a therapeutic means of maintaining muscle mass either through the strategy that we used so far in these animals, which is to give them an IGF-1 gene supplement or, as I'll mention at the end, one can think about doing it in other ways that might actually be a little simpler to achieve that we're still evaluating.
This also suggested that maybe in dystrophic muscle where the rate of muscle degeneration or the rate of muscle damage is so high that it exceeds the rate of the muscle to repair it, we wanted to ask the question: if you actually increase the rate of muscle repair by up regulating IGF-1 production, could you slow down the damage, the cumulative damage in these dystrophic animals and maintain their muscle mass?
So that's what we looked at, and here is just now comparing a dystrophic muscle where now we've taken a transgenic approach, but we've also done this with virus, showing that -- this isn't projecting very well, at least not from where I am, but this, the IGF-1 producing tissue shows a lot less of degenerative signs than the dystrophic muscle where there's lots of fragmentation of fibers, lots of clumping, lots of regeneration.
There's infiltration from macrophages because there's an ongoing destruction and inflammatory response in the muscles. Even in the diaphragm, which is virtually destroyed by the time these animals reach about 20 months of age, there's been massive sort of hypertrophy and hyperplasia in the diaphragm. So the diaphragm has become much larger and stronger, and interestingly, the amount of connective tissue.
So here one of the big problems is that as the muscle is destroyed, it basically becomes like a rubberband. It's replaced with fibrotic tissue and fat infiltration.
The driving IGF-1 over-expression not only drives more successful regeneration, but it prevented a lot of the fibrosis. So you can see the normal amount of fibrosis is measured by collagen content here in the dystrophic mouse, and we've normalized that and sort of brought that down to sort of normal levels with the IGF-1 over-expression in the MDX mouse versus just IGF-1 over-expression alone or wildtype.
And furthermore, this is now injecting a dye into the blood stream of the animal and let the animal run around, exercise a little bit, and then see if the dye is taken up in the muscles because normally the dye would be excluded because the muscle membrane would be intact.
This is showing that in the dystrophic muscle they're so fragile and being damaged at such a high rate that the dye penetrates quite easily either in the diaphragm or in the muscle that's being used to run at a very high rate, whereas in the same sort of animals, same dystrophic animal that's now over-expressing IGF-1, the fibers are being maintained in such a better state of repair that there's very little dye penetration in either the diaphragm or the leg muscle, suggesting that these muscles are going to be able to be preserved.
And so this is something that we would now like to really look at in large animal models because there are dog models of muscular dystrophy, with the idea of trying to evaluate whether this would be a potential basis for thinking of therapies in humans, and again, either delivery of an IGF-1 gene or some other way of driving this regenerative capacity is a way to think about attacking this general sort of category of human diseases, the muscular dystrophies, and it may not -- you might not even have to understand totally the primary problem if you could just drive the regeneration for some of the muscular dystrophies where it's really still not very clear what the primary problem is.
So this all leads to the idea that this IGF-1 signaling can increase satellite cell proliferation under growth and repair mechanisms that will drive muscle hypertrophy in extreme conditions, even muscle hyperplasia. Hyperplasia means the muscle is actually making more muscle fibers, not just repairing its existing ones or making them larger.
So I addressed these first two issues that we were interested in, but then it also suggests that IGF-1 over-expression should increase the rate and amount of skeletal muscle growth in young animals, and indeed, we showed that early on that that's true.
If you inject one leg of a mouse and not the other leg while it's in its young adult ages, you can actually show that the muscles of the leg get larger. This is, again, looking at the diameter. It's on the order of about 18 to 20 percent larger, and this is a sedentary animal.
And so this is one leg versus the other leg. Just the only difference here is the injection of the synthetic gene to make IGF-1, and if you do it systemically and look at all of the muscles of the animal, you can see here is a forelimb of an animal where there's no over-expression of IGF-1. Here is IGF-1 over-expression in all of the forelimb muscles, and here at the hind limb.
And so you can see there's pretty massive hypertrophy, again, on the order of 20 to 25 percent overall in the adult animal, and the games are even larger during the rapid growth phases. So in a young animal that's growing, it may outstrip its age-matched control by as much as 40 percent at a given point in its life in terms of its muscular mass.
So there are a lot of benefits then from IGF-1 over-expression, but from the standpoint of now I've been talking in terms of trying to use it therapeutically, but obviously from what I just showed you, one could think about it in terms of a gene enhancement, in terms of either an animal or a human.
And in terms of a human, those were some of the relevant papers, but in terms of humans, the question that we were asked so often and that has been really since the day we published our first paper on this on people's minds: could this sort of gene transfer into skeletal muscle actually be used for a genetic enhancement of athletic performance or even just cosmetic purposes?
Just say you'd like your pectoralis muscles to be a little larger because you want to look a little better at the beach. Just take a few injections of the virus, and a month later while you're watching television, your muscles have gotten bigger.
So, you know, a lot of implications in terms of genetic enhancement, and so I'll show you a little bit of our attempts to now evaluate this in a rat model. The rats are easy to sort of train. We didn't have much luck trying to make the mice exercise for us, but rats are fairly cooperative.
So we had the rats climb ladders with weights velcroed to their tails, and so fairly large weights that we would increase over the time of the training, and so this is sort of a progressive weight training protocol for eight weeks.
What we did was we took control rats who were not asked to exercise, and we injected the IGF-1 virus into one leg, but not the other, and then we had weight training animals where, again, we injected IGF-1 virus into one leg but not the other, and then we had another group where we went through the weight training and then let the de-train for three months. And three months would normally be enough time to lose all of the benefits from the weight training. This is one of the depressing things about exercise.
You know, you can work as hard as you want for two months straight and then sit back for three months and do nothing, and it's like you never did anything. So we had the rats go through that, too, because we wanted to address whether the IGF-1 would help maintain the mass once you stopped, which would also be of interest in terms of an athletic population or an elderly population.
So what we saw was in terms of the muscle mass -- this is after the eight weeks -- so this is the average mass of the animals that did nothing. In the leg where the IGF-1 was injected, on average the muscles are about 15 percent larger.
In the weight trained animals, they worked very hard. It was really quite a severe weight training exercise, and we were able to induce about a 23 percent increase in mass, and in the animals in their legs that had the IGF-1, they experienced an ever larger increase in mass, up to about 32 percent.
But in terms of the force output of their muscles, it was even a more striking difference in that the IGF-1 injected muscles with no exercise got almost 16, 17 percent stronger on average, whereas the ones that were weight trained were actually no stronger than the animals that had IGF-1 and sat in their cages for the two months. They were about the same strength, but the muscles that had the combination of weight training and IGF-1 were almost 30 percent stronger.
So the effect in mass is not as large as the effect on the overall strength of the muscle, and the reason for that was, in fact, the severe weight training had lowered the sort of force per unit mass of the muscle in the weight trained animals, whereas we had an enhancement in the IGF-1 treated animals, and sort of an intermediate with the weight training and IGF-1 together, and the reason for that is shown. Again, some cross-sections of muscle.
What happened in the exercised animals, and I'm not sure why my slides aren't projecting that well, but what happened there was the weight training was severe enough that we actually had a fair amount of injury and fibrosis in the muscle, and so that's what happens to athletes that can overtrain.
You know, you damage the muscles and you get fibrosis. Then it sort of works against you. It lowers the sort of strength per unit mass of the muscle, whereas the IGF-1 was so effective at the repair that even though the muscle was being massively overloaded, it rebuilt itself and looks just like healthy tissue and had normal sort of force for cross-sectional area.
So a number of benefits, and then the last benefit was when the animal stopped. You can see during the de-training, the weight trained animals went back down, and then after two months, as I said, they're back down almost to where they started before they lifted a single weight, whereas in the muscles that had IGF-1, the decline percentage-wise was a lot smaller, and they ended up with some gain over weight training alone and certainly a gain over just IGF-1 alone.
And so they were able to maintain some of the weight training benefit at least three months after the cessation of the exercise.
So this is sort of a summary of all that I was saying, but the bottom line, just to speed this along a little bit, is that this approach certainly would lead to genetic enhancement of athletic performance because it would increase the rate in amount of skeletal muscle growth with resistance exercise. It would increase the rate and extensiveness of repair following an injury. So you'd be better able to maintain muscle mass, strength, and speed after the training stops and certainly during aging, as we had shown before.
So tremendous benefits from the athletic standpoint I think not the least of which is how rapidly one could come back from an injury and how well one would sustain an injury and get complete repair of the muscle, not to mention that for speed and strength events, one might not see the precipitous fall in performance that normally comes after age 30 even in a training athlete.
So this little bit was what I presented to the World Anti-Doping Association because they're quite concerned about how close we are of genetic engineering or enhancement of athletes actually cropping up in terms of international sporting events.
And you know, as I said to them, I think the real danger of that -- and this is just to acknowledge some of my colleagues -- the real danger of that is not that it's going to happen any time soon in this country because we're still going at a fairly slow rate of trying to just really assess the safety of some of these gene transfer techniques even for treating, you know, primary genetic diseases, rare diseases for which there are no treatments.
And so the availability of this sort of technology to an athlete in this country is not going to happen any time soon, but on the world stage, in a world where countries in the past have shown that they want their athletes to win no matter what and they will give them any experimental drug that might be performance enhancing no matter what the long-term consequences, one can imagine that with enough money you could put together a program to genetically engineer your athletes and do it in such a way, which is what one is really concerned about that it would be totally undetectable unless you were to remove tissue from that athlete. There would be nothing in the blood, no signature in the blood or the urine to indicate that the tissues had been genetically manipulated.
So this is their concern, certainly not a concern, I think, in this country in the short term, but maybe a concern on the world stage maybe even in the next decade.
Just to let you know where we're going a little bit, I alluded to the fact that, you know, we started trying to intervene in this growth and regeneration pathway for aging by driving IGF-1, but what I didn't mention is in the last few years it has become really clear that there are major inhibitors of this whole pathway that the muscle actively is producing to sort of keep it in check.
And one unresolved question and one we're looking at and probably other people are looking at is whether some of these components also could help drive the repair and aging if you could block them. And so you could imagine there the approach would be either to create a substance in the blood that would interfere with the action of this protein, which has been called myostatin, or you could even imagine a small molecule screen might pick up a selective inhibitor of this protein which is in the -- it's a TGF beta family member.
So this is a target I think you're going to see increasing interest from drug companies, and it may have application in aging. It may not, but it certainly probably does have application in such things as juvenile diabetes and maybe in some of the muscular dystrophies where interfering with the signaling of this protein might allow the muscle to rebuild itself better and stronger.
And that's going to be much easier to implement, and that certainly would be a performance enhancer for an athlete, and those drugs are being developed now, and the accessibility of an athlete to those sorts of drugs might end up in the same sorts of results as what I was showing you for the IGF-1 over-expression.
And certainly in the general population I think this could be used as an instant muscle builder, and the nice thing about a drug is you sort of take until you've got what you want, and then you stop taking it, and it doesn't drive the process indefinitely.
We're going into clinical trial in the next year or so with AAV targeted at a primary muscle disease, a deficiency in what is called the sarcoglycan complex, which causes a form of muscular dystrophy known as limb-girdle muscular dystrophy. These components are all small enough to fit easily into AAV, and so I think the first real clinical test of this virus for sort of directed at a primary muscle problem are going to be in the context of trying to repair this whole structure, which is deficient in limb-girdle muscular dystrophy.
And we'll learn a lot about how easy it's going to be to actually use this gene transfer vector in humans in the process of that, and it's actually a collaboration between myself and groups at Harvard and the NIH and the Généthon in France, which is funded by the French government and the French Muscular Dystrophy Association.
So we're planning to do multiple -- sort of coordinate trials in this country and in France on this disease with the idea of then moving on to other primary muscle diseases and perhaps even looking at the IGF-1 myostatin axis as a possible therapeutic that we could then bring to humans in a muscle disease setting.
So that's my background on what we're doing, and I'd be happy to answer questions about where it's going.
Also, I must give props to the original poster: bigdaddyhd
Session 7: Enhancement 5: Genetic Enhancement of Muscle
H. Lee Sweeney, Ph.D.,
Professor and Chairman of Physiology,
University of Pennsylvania
CHAIRMAN KASS: All right. I know there are some counsel members with planes to catch, and I don't want us to waste any more of Dr. Sweeney's time. We're delighted to welcome Dr. Lee Sweeney, who is Professor and Chairman of the Department of Physiology at the University of Pennsylvania, who has done just a significant amount of outstanding work on muscle physiology and who is going to speak to us today about the genetic enhancement of skeletal muscle and its performance.
Thank you very much, Dr. Sweeney, for being with us.
Push your button there. There we go.
DR. SWEENEY: Thank you.
Yeah, I'm going to just try to give you in half an hour or so background about some of the work we're doing, and then I'm hoping just to allow you to drive a lot of the discussion because I'll set up some of the issues that I see, but as a scientist, I'm afraid I don't think some of the ethical ramifications through quite to the extent of the discussions I've already heard this morning.
My interest is in disease states of both skeletal and cardiac muscle. I'm going to restrict it to skeletal muscle because I think it gives you a good example of really how gray the boundary is between therapeutics and enhancement when one is starting to think about what can be done with genetic manipulation in adults.
Skeletal muscle is a big target in a person because it makes up the majority of the mass of their body, and it's an interesting tissue in that it has built into it cells called satellite cells which are not differentiated muscle cells, but which are actually cells that are called upon to divide and differentiate and regenerate the skeletal muscle.
So they're not really a resident stem cell population because they're not pleuripotential like a true stem cell, but they're an uncommitted set of cells that can with the proper stimuli be induced to become skeletal muscle. They can become other types of tissue as well, but a fairly limited repertoire.
Now, my interest in this began with years ago the beginning of the whole promise of gene therapy, and of course, the idea at the beginning of gene therapy was really to tackle the simplest of genetic diseases, those that involved single genes and usually the genes being missing or at least defective, which was the cause of a large number of diseases, most of them fairly rare diseases.
The issue, and still the issue, the recent gene therapy really hasn't as quickly progressed as we all would have hoped it would have ten years ago, is because the problems are really finding the right vector for a given tissue, that is, a delivery device to actually get the genetic material into the adult tissue, and then also figuring out how to get it there, the delivery.
So these are still the key stumbling blocks in gene therapy today, although great inroads have been made.
Now, in terms of muscle what is sort of emerging as the best sort of ways to deliver genes to muscle are sort of listed here, and this is as of today. Muscle is actually quite good at taking up so-called naked DNA or just plasmid DNA. This is DNA that is not encapsulated in anything.
When I say quite good, I mean it does it with some efficiency. It's perhaps one or two percent of the cells if you inject DNA into a given muscle would take something up.
So this is not an inefficiency that's useful in correcting a primary genetic disease of the muscle itself. However, this would be useful in terms of getting the muscle to produce a substance that would then be secreted into the blood.
And, in fact, this technique has been shown to be successful in an agricultural setting where a colleague of mine has taken DNA that codes for the growth hormone releasing hormone. This is actually a protein which stimulates the release of growth hormone, and in doing so, he can demonstrate that the pigs will now secrete much larger much larger levels of growth hormone.
Obviously one of the other problems with it is it's transient, but in that sort of setting perhaps it's useful to be transient just to sort of give them a growth boost through some period, and then it will go away, but obviously not something one could think of for permanent sort of genetic correction, and especially not a genetic correction of the muscle itself.
Now, viruses are still the preferred gene delivery vehicles in terms of efficiency. Getting genes into a given tissue are what viruses are engineered to do, and so they can be reengineered to deliver therapeutic genes instead of viral genes.
And probably the best vector for muscle is a virus that's known as AAV, which stands for adeno-associated virus. It's a virus that's unrelated to adenovirus, which has been used, as you're probably aware, in gene therapy trials with complications, and the most severe which took place at my own institution.
But this is a different virus that actually does not cause any known disease in humans. Often it can be found in 20 percent or so of the human population that have been infected by AAV with no consequences that can be demonstrated.
And in the last maybe two years a number of different sort of variants or serotypes of the virus have been isolated, and at least two of them are extremely efficient at targeting skeletal muscle. And so there are now gene therapy trials that have begun and many more that are about to be proposed and started that use this virus to try to get genes into skeletal muscle or in the liver. It turns out that this virus is also very good at targeting the liver, which would be very useful in diseases where one wants something secreted into the blood, like in the case of hemophilia.
There's an ongoing trial now with AAV targeting the liver to get the liver to secrete in this case Factor 9 for patients with Factor 9 deficiency.
Adult stem cells obviously are useful in sort of the idea of helping regenerate the tissue, although I must say that although there are a number of types of adult stem cells that can become muscle, again, efficiency becomes the problem, whether they're muscle or bone marrow derived stem cells. It's very difficult to get a large percentage of the muscle rebuilt using this approach in animal models where it's been attempted.
Nonetheless, we and others see the ability to use adult stem cells now as not in the stem cell sense of rebuilding the tissue, but perhaps in the sense of viewing them as a vector where one would take the adult stem cells in the laboratory, put new genes into the stem cells. That would then allow the tissue that they've incorporated in to secrete a substance that would then affect the surrounding tissue or if it's designed to go into the blood.
So you could sort of put adult stem cells then not only in the tissue regenerating sense, but in the sense of being a vector to carry genes into a tissue, to incorporate into the tissue, then to produce something else in that tissue.
And the advantage that they may have over viruses is one of the easiest systemic delivery. In many disease situations it may be that one could simply put the adult stem cells into the blood and they would home in on the tissue. They would home to the tissue that was actually being damaged by whatever the disease process is, and so it would be a very efficient way of targeting the tissue, which with viruses is more difficult at least at this point in time.
So I'm going to give you an example then of using the adeno-associated viral mediated gene transfer, but the example is actually one that we're now trying to do with adult stem cells, which is why I brought this up, because we think we can accomplish the same thing and deliver it much more simply using stem cells that are bone marrow derived from in this case we're working with both adult human cells, as well as adult rodent derived cells.
Adeno-associated virus, as I said, has the huge advantage for skeletal muscle in that it readily infects it. In fact, this may be the preferred tissue target of this virus at least for various forms of this virus.
It's limiting in that the size of its entire genome is only on the order of about 4.7 kilobases. So this is very small and really smaller than most genes in the human, and so one can only make synthetic genes that code for relatively small proteins. And so one has to be judicious in the choice of what one can attempt to do with this virus.
Delayed onset of expression, perhaps more so than some of the other viruses, but nevertheless, with the more robust infection that one gets with serotype one and five, expression commences within a week of injection of the virus into the tissue or systemic delivery of the virus into the tissue.
There's no viral gene expression. This is one of the big advantages of using this type of vector. You can make the synthetic vector without any viral genes, and because of that, there's no immune response against the virus itself.
Obviously there could still be immune response against whatever the product that you're causing it to make, but an example I'll give you, which is one of the advantages of it, what we're making is not something that's missing from the body, but something that we're just trying to get the body to make more of. And so there's nothing for the immune surveillance to pick up on, and so no possibility of immune response.
It integrates at a low frequency, which is both useful, but also a point of some concern in sort of the regulatory and side effect case. The integration, with any integrating virus there are two things to worry about. One is the possibility of oncogenesis being initiated by an integration event.
We have not seen this in our animal models, and other people have not seen this. So the possibility with this virus seems relatively low because there's some evidence that the integration events of AAV are somewhat site specific, and so not very likely to induce oncogenesis.
The other problem, of course, is whether or not one could get germ line transmission, and compared to something like lentivirus, the ability to do germ line transmission is not zero, but it's fairly low probability of germ line transmission, but again, this depends on the route of administration.
It would be more likely that you might get germ line transmission if you're doing a vascular delivery than of direct injection into the tissue, and the duration of expression, because of the integration, essentially is the life of the nucleus that you infected. And so as long as that cell in that nucleus exists, one will get expression. And so for the animal models that we look at, it's the life of the animal essentially.
Just to show you what efficient means, here's a cross-section through a muscle. So these are now -- if you think of the muscle fibers and muscle cells as very long cylinders, this is now slicing through them so that you basically just see their circumference and not their length.
And as you can see in this example, this is now using AAV-1. Essentially every muscle, well, every muscle in the field is now producing a protein that gives a color and an enzymatic reaction that's developed in the laboratory, a bacterial protein that can be used to give this colormetric readout, and you can see that every muscle fiber in the field is blue, and in fact, one can do vascular injection with this virus, and every fiber and every muscle virtually in the leg of the animal will be blue after vascular administration of a large enough dose.
So the efficiency is extremely high if one puts in enough of the virus.
So potential applications, which is what got me interested in using this in the first place, obviously the initial goal of all gene therapies of this sort was primarily genetic diseases, and for muscle that would mean Duchenne and Becker muscular dystrophy is the most common, but also others, such as the limb-girdle muscular dystrophies, myotonic muscular dystrophy, and whatnot, where one can point to a genetic defect in a single gene as the cause of the disease.
A more difficult problem, but actually in some ways, I mean, biologically a more difficult problem, but in fact, the problem that we focused on initially, which is a very real problem in this society where the society is living to be older and older, is the fact that as we get older our muscle function, our skeletal muscle function diminishes both in size of the muscle as well as the relative strength of the muscle and this is a big problem not only from an ambulatory standpoint, but also from a whole body metabolic standpoint.
If the mass of skeletal muscle drops below a critical threshold, then the whole body metabolism is no longer supported properly because the muscle actually functions not only to move the body, but as an important metabolic organ within the body.
Then the last sort of issue, which is actually a trial, trials have been ongoing in this area, is the use of gene transfer into muscle to get therapeutic proteins in the blood, such as Factor 9 deficiency.
So the initial hemophilia trials with Factor 9 were trying to actually get muscle to secrete Factor 9, but now they've shifted to liver because the liver is just a better organ for secretion into the blood than muscle is, although the muscle is capable of it.
So I want to first tell you about where we started some five years ago, which was looking at this problem that the NIH has coined sarcopenia. I think they coined a term to sort of make it sound more like a disease, probably for congressional purposes, but basically what they're really talking about is this progressive loss of muscle mass in force that essentially begins in the fourth decade life in humans and then progresses throughout.
It's slowed, but it's not prevented by exercise, and obviously as I've already mentioned has negative impacts on health and quality of life. It occurs in all mammals, which is useful because that means all of the laboratory animals one works with undergo the same process, and since they live for a much shorter period of time than humans, their life spans are much contracted.
The whole sort of progression occurs on a time scale that one can approach in the laboratory, and our hypothesis back in '96 or seven when we began this was that in large part we thought that what it was really due to was not inactivity. There had been a lot of discussion that as people got older they were just inactive and that's really the main thing that drove it, but we really thought there was a more fundamental cause, especially since there were studies showing that exercise could slow it down but not stop it.
And that was the fact that the repair mechanisms of skeletal muscle decline as you get older, and this causes the muscle to lose function because it's essentially not being repaired properly, and this goes back to what I said at the beginning, that it has within it a resident population of cells, these so-called satellite cells, that when the muscle is damaged -- and muscle is always damaged as you're using it -- are called upon to repair the muscle and rebuild it.
So this sort of rebuilding process involves some sort of damage signal coming out of the muscle which then activates the satellite cells to begin to proliferate, and they proliferate, then they make the commitment to be muscle, and then they either fuse with the existing muscle to repair it, or if the muscle has been severely damaged, they form new muscle.
So what is involved in this are a number of growth factors, some that drive the process, some that inhibit the process in sort of a yin and yang, but the one that we felt was really the most critical and the one that might be the candidate for what's going wrong in aging is a growth factor called IGF-1, which stands for insulin-like growth factor-1, which in normal muscle is involved in growth. It drives protein synthesis, and it decreases protein degradation, and importantly form the repair standpoint, it stimulates this population of satellite cells to both proliferate and differentiate.
And this is an important fact that it can do both because many of the growth factors will drive proliferation but block differentiation, and so increasing their levels could actually interfere with repair, which has been shown in some cases, but here you have one that has a little built in clock. It will drive proliferation for a while through one pathway, and then it will drive through this pathway, and then it will drive differentiation through another pathway, which it turns on with the delay.
So just the sort of thing you might want to try to drive more successful growth and repair. And the reason we thought it might be a problem in aging is because it's really part of the whole growth hormone IGF-1 axis, which as you know, the signals from the hypothalamus, the growth hormone releasing hormone, that are then taken to the anterior pituitary to stimulate it to produce growth hormone go down with aging.
The levels of growth hormone in the blood go down. The levels of IGF-1 produced by the liver; the liver produces all of the IGF-1 that circulates in the body. All of these levels go down with aging.
And what that means is that the IGF-1 levels in the various tissues of the body will also be diminished with aging.
Now, tissues like muscle and other tissues of the body have two sources of IGF-1. They make it themselves under conditions of either injury or rapid growth, but also they have an IGF-1 input that comes from the liver that's ongoing throughout their life.
And so it's this component in particular that's being lost in the aging animal. And so we sought to essentially replace it by supplementing the amount of IGF-1 that the muscle itself could make.
So the strategy was quite simple that we took. We would use gene delivery into the muscle to give it a synthetic gene to have it produce more IGF-1 so it would not be particularly dependent on the liver for a source of IGF-1.
And then the question was: would that then in muscle promote growth and regenerative pathways and would that, in turn, allow the muscle to function throughout the life of the animal without the aging related loss?
So it's a very simple synthetic gene that we put together using a muscle specific promoter driving the rodent IGF-1, and then it's flanked by the viral ITRs and packaged into the AAV viral capsid, and then just to inject into the animals either a vascular delivery into the leg or a direct injection into specific muscles.
And we could how -- this is just using PCR to detect the existence now of our synthetic gene that four months, nine months, even two years after injection the synthetic gene is present and producing IGF-1 messenger RNA.
So then we asked the question with it: is this going to increase the rate of muscle regeneration and maintain mass and old age? And this is the paper that I included in your packet.
What we showed was that if we injected mice, essentially middle aged mice or late middle age in mice -- mice live to be about 27 to 30 months in age at least in our colonies, and so we injected them about halfway through their life where they were all just beginning to start losing muscle mass, and then we asked, you know, what would their muscle mass in force and force for cross-section look like when they became old.
And so looking at them at 27 months, which was nearing the end of their life, they normally would have experienced in terms of mass about a 15 to 18 percent drop over that age period compared to a six month old mouse when they're sort of at their peak.
Whereas if we had injected them in middle age, they maintained the same mass or even a little greater than they had when they were younger. The same with the amount of force they were able to produce. Their muscles were able to produce normal force instead of showing the decline in force that they would normally see, and the force for cross-sectional area was maintained, as well.
But the speed of the muscle was maintained and the power output was maintained to an even greater extent because one of the other things that happens as the animals get old, as mammals get old, is they selectively lose their fast and most powerful fiber type.
So skeletal muscle is a heterogeneous tissue in terms of it has some of the fibers in it that are small. Some are big; some are slow; some are fast. We lose the very fastest ones as we get older and preferentially replace them with slower fibers. That's one reason why some of the first athletic things that go are your ability to compete in power events or speed events, because that's the first loss that you experience before you really lose muscle endurance or any of those sorts of properties.
And we were able to prevent that totally. The mice did not lose any of their fast fibers, and they had the same speed and power output when they were 27 month old muscles as they did as animals that were only six months of age.
So from that we were able to conclude that IGF-1 over-expression could prevent all of the hallmarks of age related atrophy and loss of skeletal muscle function in mammalian aging, at least based on the rodent model, and now we're hoping to pursue this in larger animal models.
The skeletal muscle regeneration rate is diminished in old animals, and we showed that in another paper other than the one I showed you, and that seems to be the primary problem, that even if you injure the old muscle, it cannot mount a normal regenerative response, but if you maintain IGF-1 expression, it can maintain a normal repair response, and this also, of course, is this hypothesis that we were looking at.
And also it suggests that one could go about this whole pathway as a therapeutic means of maintaining muscle mass either through the strategy that we used so far in these animals, which is to give them an IGF-1 gene supplement or, as I'll mention at the end, one can think about doing it in other ways that might actually be a little simpler to achieve that we're still evaluating.
This also suggested that maybe in dystrophic muscle where the rate of muscle degeneration or the rate of muscle damage is so high that it exceeds the rate of the muscle to repair it, we wanted to ask the question: if you actually increase the rate of muscle repair by up regulating IGF-1 production, could you slow down the damage, the cumulative damage in these dystrophic animals and maintain their muscle mass?
So that's what we looked at, and here is just now comparing a dystrophic muscle where now we've taken a transgenic approach, but we've also done this with virus, showing that -- this isn't projecting very well, at least not from where I am, but this, the IGF-1 producing tissue shows a lot less of degenerative signs than the dystrophic muscle where there's lots of fragmentation of fibers, lots of clumping, lots of regeneration.
There's infiltration from macrophages because there's an ongoing destruction and inflammatory response in the muscles. Even in the diaphragm, which is virtually destroyed by the time these animals reach about 20 months of age, there's been massive sort of hypertrophy and hyperplasia in the diaphragm. So the diaphragm has become much larger and stronger, and interestingly, the amount of connective tissue.
So here one of the big problems is that as the muscle is destroyed, it basically becomes like a rubberband. It's replaced with fibrotic tissue and fat infiltration.
The driving IGF-1 over-expression not only drives more successful regeneration, but it prevented a lot of the fibrosis. So you can see the normal amount of fibrosis is measured by collagen content here in the dystrophic mouse, and we've normalized that and sort of brought that down to sort of normal levels with the IGF-1 over-expression in the MDX mouse versus just IGF-1 over-expression alone or wildtype.
And furthermore, this is now injecting a dye into the blood stream of the animal and let the animal run around, exercise a little bit, and then see if the dye is taken up in the muscles because normally the dye would be excluded because the muscle membrane would be intact.
This is showing that in the dystrophic muscle they're so fragile and being damaged at such a high rate that the dye penetrates quite easily either in the diaphragm or in the muscle that's being used to run at a very high rate, whereas in the same sort of animals, same dystrophic animal that's now over-expressing IGF-1, the fibers are being maintained in such a better state of repair that there's very little dye penetration in either the diaphragm or the leg muscle, suggesting that these muscles are going to be able to be preserved.
And so this is something that we would now like to really look at in large animal models because there are dog models of muscular dystrophy, with the idea of trying to evaluate whether this would be a potential basis for thinking of therapies in humans, and again, either delivery of an IGF-1 gene or some other way of driving this regenerative capacity is a way to think about attacking this general sort of category of human diseases, the muscular dystrophies, and it may not -- you might not even have to understand totally the primary problem if you could just drive the regeneration for some of the muscular dystrophies where it's really still not very clear what the primary problem is.
So this all leads to the idea that this IGF-1 signaling can increase satellite cell proliferation under growth and repair mechanisms that will drive muscle hypertrophy in extreme conditions, even muscle hyperplasia. Hyperplasia means the muscle is actually making more muscle fibers, not just repairing its existing ones or making them larger.
So I addressed these first two issues that we were interested in, but then it also suggests that IGF-1 over-expression should increase the rate and amount of skeletal muscle growth in young animals, and indeed, we showed that early on that that's true.
If you inject one leg of a mouse and not the other leg while it's in its young adult ages, you can actually show that the muscles of the leg get larger. This is, again, looking at the diameter. It's on the order of about 18 to 20 percent larger, and this is a sedentary animal.
And so this is one leg versus the other leg. Just the only difference here is the injection of the synthetic gene to make IGF-1, and if you do it systemically and look at all of the muscles of the animal, you can see here is a forelimb of an animal where there's no over-expression of IGF-1. Here is IGF-1 over-expression in all of the forelimb muscles, and here at the hind limb.
And so you can see there's pretty massive hypertrophy, again, on the order of 20 to 25 percent overall in the adult animal, and the games are even larger during the rapid growth phases. So in a young animal that's growing, it may outstrip its age-matched control by as much as 40 percent at a given point in its life in terms of its muscular mass.
So there are a lot of benefits then from IGF-1 over-expression, but from the standpoint of now I've been talking in terms of trying to use it therapeutically, but obviously from what I just showed you, one could think about it in terms of a gene enhancement, in terms of either an animal or a human.
And in terms of a human, those were some of the relevant papers, but in terms of humans, the question that we were asked so often and that has been really since the day we published our first paper on this on people's minds: could this sort of gene transfer into skeletal muscle actually be used for a genetic enhancement of athletic performance or even just cosmetic purposes?
Just say you'd like your pectoralis muscles to be a little larger because you want to look a little better at the beach. Just take a few injections of the virus, and a month later while you're watching television, your muscles have gotten bigger.
So, you know, a lot of implications in terms of genetic enhancement, and so I'll show you a little bit of our attempts to now evaluate this in a rat model. The rats are easy to sort of train. We didn't have much luck trying to make the mice exercise for us, but rats are fairly cooperative.
So we had the rats climb ladders with weights velcroed to their tails, and so fairly large weights that we would increase over the time of the training, and so this is sort of a progressive weight training protocol for eight weeks.
What we did was we took control rats who were not asked to exercise, and we injected the IGF-1 virus into one leg, but not the other, and then we had weight training animals where, again, we injected IGF-1 virus into one leg but not the other, and then we had another group where we went through the weight training and then let the de-train for three months. And three months would normally be enough time to lose all of the benefits from the weight training. This is one of the depressing things about exercise.
You know, you can work as hard as you want for two months straight and then sit back for three months and do nothing, and it's like you never did anything. So we had the rats go through that, too, because we wanted to address whether the IGF-1 would help maintain the mass once you stopped, which would also be of interest in terms of an athletic population or an elderly population.
So what we saw was in terms of the muscle mass -- this is after the eight weeks -- so this is the average mass of the animals that did nothing. In the leg where the IGF-1 was injected, on average the muscles are about 15 percent larger.
In the weight trained animals, they worked very hard. It was really quite a severe weight training exercise, and we were able to induce about a 23 percent increase in mass, and in the animals in their legs that had the IGF-1, they experienced an ever larger increase in mass, up to about 32 percent.
But in terms of the force output of their muscles, it was even a more striking difference in that the IGF-1 injected muscles with no exercise got almost 16, 17 percent stronger on average, whereas the ones that were weight trained were actually no stronger than the animals that had IGF-1 and sat in their cages for the two months. They were about the same strength, but the muscles that had the combination of weight training and IGF-1 were almost 30 percent stronger.
So the effect in mass is not as large as the effect on the overall strength of the muscle, and the reason for that was, in fact, the severe weight training had lowered the sort of force per unit mass of the muscle in the weight trained animals, whereas we had an enhancement in the IGF-1 treated animals, and sort of an intermediate with the weight training and IGF-1 together, and the reason for that is shown. Again, some cross-sections of muscle.
What happened in the exercised animals, and I'm not sure why my slides aren't projecting that well, but what happened there was the weight training was severe enough that we actually had a fair amount of injury and fibrosis in the muscle, and so that's what happens to athletes that can overtrain.
You know, you damage the muscles and you get fibrosis. Then it sort of works against you. It lowers the sort of strength per unit mass of the muscle, whereas the IGF-1 was so effective at the repair that even though the muscle was being massively overloaded, it rebuilt itself and looks just like healthy tissue and had normal sort of force for cross-sectional area.
So a number of benefits, and then the last benefit was when the animal stopped. You can see during the de-training, the weight trained animals went back down, and then after two months, as I said, they're back down almost to where they started before they lifted a single weight, whereas in the muscles that had IGF-1, the decline percentage-wise was a lot smaller, and they ended up with some gain over weight training alone and certainly a gain over just IGF-1 alone.
And so they were able to maintain some of the weight training benefit at least three months after the cessation of the exercise.
So this is sort of a summary of all that I was saying, but the bottom line, just to speed this along a little bit, is that this approach certainly would lead to genetic enhancement of athletic performance because it would increase the rate in amount of skeletal muscle growth with resistance exercise. It would increase the rate and extensiveness of repair following an injury. So you'd be better able to maintain muscle mass, strength, and speed after the training stops and certainly during aging, as we had shown before.
So tremendous benefits from the athletic standpoint I think not the least of which is how rapidly one could come back from an injury and how well one would sustain an injury and get complete repair of the muscle, not to mention that for speed and strength events, one might not see the precipitous fall in performance that normally comes after age 30 even in a training athlete.
So this little bit was what I presented to the World Anti-Doping Association because they're quite concerned about how close we are of genetic engineering or enhancement of athletes actually cropping up in terms of international sporting events.
And you know, as I said to them, I think the real danger of that -- and this is just to acknowledge some of my colleagues -- the real danger of that is not that it's going to happen any time soon in this country because we're still going at a fairly slow rate of trying to just really assess the safety of some of these gene transfer techniques even for treating, you know, primary genetic diseases, rare diseases for which there are no treatments.
And so the availability of this sort of technology to an athlete in this country is not going to happen any time soon, but on the world stage, in a world where countries in the past have shown that they want their athletes to win no matter what and they will give them any experimental drug that might be performance enhancing no matter what the long-term consequences, one can imagine that with enough money you could put together a program to genetically engineer your athletes and do it in such a way, which is what one is really concerned about that it would be totally undetectable unless you were to remove tissue from that athlete. There would be nothing in the blood, no signature in the blood or the urine to indicate that the tissues had been genetically manipulated.
So this is their concern, certainly not a concern, I think, in this country in the short term, but maybe a concern on the world stage maybe even in the next decade.
Just to let you know where we're going a little bit, I alluded to the fact that, you know, we started trying to intervene in this growth and regeneration pathway for aging by driving IGF-1, but what I didn't mention is in the last few years it has become really clear that there are major inhibitors of this whole pathway that the muscle actively is producing to sort of keep it in check.
And one unresolved question and one we're looking at and probably other people are looking at is whether some of these components also could help drive the repair and aging if you could block them. And so you could imagine there the approach would be either to create a substance in the blood that would interfere with the action of this protein, which has been called myostatin, or you could even imagine a small molecule screen might pick up a selective inhibitor of this protein which is in the -- it's a TGF beta family member.
So this is a target I think you're going to see increasing interest from drug companies, and it may have application in aging. It may not, but it certainly probably does have application in such things as juvenile diabetes and maybe in some of the muscular dystrophies where interfering with the signaling of this protein might allow the muscle to rebuild itself better and stronger.
And that's going to be much easier to implement, and that certainly would be a performance enhancer for an athlete, and those drugs are being developed now, and the accessibility of an athlete to those sorts of drugs might end up in the same sorts of results as what I was showing you for the IGF-1 over-expression.
And certainly in the general population I think this could be used as an instant muscle builder, and the nice thing about a drug is you sort of take until you've got what you want, and then you stop taking it, and it doesn't drive the process indefinitely.
We're going into clinical trial in the next year or so with AAV targeted at a primary muscle disease, a deficiency in what is called the sarcoglycan complex, which causes a form of muscular dystrophy known as limb-girdle muscular dystrophy. These components are all small enough to fit easily into AAV, and so I think the first real clinical test of this virus for sort of directed at a primary muscle problem are going to be in the context of trying to repair this whole structure, which is deficient in limb-girdle muscular dystrophy.
And we'll learn a lot about how easy it's going to be to actually use this gene transfer vector in humans in the process of that, and it's actually a collaboration between myself and groups at Harvard and the NIH and the Généthon in France, which is funded by the French government and the French Muscular Dystrophy Association.
So we're planning to do multiple -- sort of coordinate trials in this country and in France on this disease with the idea of then moving on to other primary muscle diseases and perhaps even looking at the IGF-1 myostatin axis as a possible therapeutic that we could then bring to humans in a muscle disease setting.
So that's my background on what we're doing, and I'd be happy to answer questions about where it's going.
