 Good morning. It's really a pleasure to be here. Thank you for that kind introduction. So I have a tall task today, and that is to discuss treatment of genetic disorders in general. I'm going to approach this by presenting many small vignettes that reveal both the power and pitfall of some new approaches that are being used that have the potential to be very powerful. So when we're first thinking about a disease, I'm sorry, often we feel stranded on a desert island. We really have no compass and we can look in 360 degrees without clear a conviction about what direction to go in. Now genetics has received a lot of hype, appropriately so, for its ability to allow you to make giant leaps in understanding without taking each small derivative step along the way. You can learn about a new disease gene and suddenly be firmly within a pathway that you never would have anticipated. And initially, when you get there, things look very appealing. But once you get your bearings, you often learn that you're no better off than where you started and that it takes a lot of hard work to reach the promised shore. So initially, when people think about treatment of genetic diseases, often gene therapy comes to mind first. It's a fairly straightforward strategy. If the body is missing a gene product because of a defective gene, you simply use a virus to reinsert the normal gene into the cells of interest, which will then make the protein and restore function. Gene therapy can also be used to treat patient cells that have been removed from the body. Again, giving them the normal gene and allowing them to go back into the patient to restore function. There are many obstacles to gene therapy, including an immune response to viral proteins. There have been a number of very catastrophic cases of failure of gene therapy due to this effect. And also because of disruption of essential genes when the virus inserts the genome, I'm sorry, the gene into the host genome, for example, causing leukemia. But despite this, there have been many startling recent successes of gene therapy for conditions such as adenosine deaminase deficiency causing immunodeficiency for leavers congenital amaurosis causing blindness and also for hemophilia B. So I'm not going to spend a lot of time on gene therapy. There is a reference that is provided and will be provided with the slide set that goes over that topic in detail. Rather, I want to cover the basic concept that it takes a village to really bring a new concept to a patient. And that it really requires a confluence of and synergy between both basic and clinical sciences to develop a full mechanistic understanding of a disease process and in that manner to derive novel and rational treatment strategies. So the first story I want to tell you concerns the so-called lysosomal storage diseases. A lot of very well-known names here such as Hunter's disease or Hurler disease or Pompeii disease. All of these conditions are unified by the toxic accumulation of lysosomal substrates due to a deficiency of a lysosomal enzyme. So real progress in this area actually derived from an accident in a laboratory in Liz Neufeld's laboratory. For this particular example, I'm going to show a green cell as a normal cell that has a full complement of enzymes and normal function and various flavors of red cells to show a defective cell lacking a lysosomal enzyme. And you can see in this hypothetical example that in this cell there's no enzyme A shown by a yellow circle causing lysosomal storage disease 1. In this cell there's no enzyme B causing a different lysosomal storage disease. Now the accident came when a technician in the Neufeld lab accidentally mixed cells from two different patients with two different lysosomal storage diseases. And the remarkable result is that when they were mixed, all the cells adopted normal function. Suddenly they worked. Everything worked. So this is called a complementation where one cell will complement the deficiency of another cell. And it really heralded the concept of treatment for lysosomal storage diseases simply by infusing the defective enzyme or the deficient enzyme. So in this example once again, this cell does not make the enzyme shown in yellow. This cell does not make the enzyme shown in blue. What the Neufeld lab learned is that each cell secretes the enzyme that it makes outside of the cell and then the other cell can take up that enzyme and in effect complement its deficiency by taking up the enzyme that its neighbor made. And again this leads to cells that have both enzymes and normal function. So everyone was understandably excited about the prospect of simply infusing enzymes. But then there was a pretty startling and disturbing result that if you made the enzymes that are defective in these patients and simply put them in the culture medium of the cells, the cells failed to take up that enzyme and there was no complementation. And the obvious question is why? What's missing? So the answer came when the Neufeld lab studied a different disease. This disease is called eye cell disease. And what they learned is that cells from patients with eye cell disease make all the appropriate enzymes. They secrete all those enzymes outside of the cell but that other cells lack the ability to take up those enzymes that were secreted. And what ultimately they learned is that in eye cell disease there's deficiency of a specific enzyme called enocidal glucosamine 1-phosphotransferase that modifies all these enzymes by attaching a chemical called mannose 6-phosphate. And it was learned that recipient cells in order to take up an enzyme have to have a mannose 6-phosphate receptor that will then bind the enzymes in its environment and facilitate cellular uptake. So now there was a complete strategy for enzyme replacement therapy. You could synthesize these enzymes in the laboratory but you had to add this modification, the mannose 6-phosphate group. So does it work? The answer is yes. So here's an example in treatment of Hurler's syndrome with recombinant enzyme alpha-L-I geronidase. You can see that all of these patients at the start of therapy had rather high New York Heart Association classifications but during the course of therapy, about 50 weeks of therapy all of them showed a clinical improvement and many of them showed a very striking clinical improvement down to the best level. If you look at other markers such as liver size, the toxic substrates and lysosomal storage diseases often accumulate in the liver, you can see that liver size decreases dramatically during the course of infusion of the deficient enzyme. And you can also look at skeletal performance. Patients typically would develop contracture and you can see that there's increased movement at the shoulder, at the elbow and at the knee while these children are receiving infusion of enzyme. So I don't expect you to be able to read anything on this slide but the basic concept is that there are now many enzyme replacement therapies for many lysosomal storage diseases that are either in late-phase clinical trial or that are FDA approved to use for treatment of these children. Is this the whole answer? Is this the cure for lysosomal storage diseases? Unfortunately, there are some obstacles. In the example that I showed you, the enzyme with its Manosix phosphate group is free to interact with cells in their Manosix phosphate receptor leading to functional recovery. But it was recognized that some patients over time develop antibodies that attack the enzyme that's being replaced. And what distinguishes these two groups of patients, those without and with antibodies, is the ones without antibodies make some small level of the enzyme that's being replaced. It's not enough to achieve the function that's necessary, but it's enough for the body to say, this enzyme's okay. It's not foreign. I'm not going to attack it. However, in patients that make no enzyme naturally, their body has never seen that enzyme, so it's recognized as foreign and therefore attacked by the immune system. Another obstacle is the so-called blood-brain barrier. The microvessels within our brain contain a very solid barrier that prevents diffusion of multiple substances from the circulation into brain tissue. And unfortunately, this blood-brain barrier is impermeable to all the enzymes used in enzyme replacement therapy. So currently, there's excellent utility of enzyme replacement therapy in conditions such as maritolamy, where there's no central nervous system manifestations. But there's really very poor performance of enzyme replacement therapy in diseases such as gauchee disease type II or III, where there's very severe brain manifestations and the enzyme simply can't get there to help. There are some potential solutions, just like children receive allergy shots to teach their body to tolerate something that's recognized as foreign. Similar approaches are being used to reduce tolerance to enzyme replacement therapy. There's alternative targeting procedures that are being explored that might allow these molecules to get past the blood-brain barrier to get into the tissue. And there's also intensive exploration of complementary therapeutic regimens that use drugs rather than antibodies and drugs have a greater ability to diffuse into the brain. So there are many different classes of drugs that are being explored. In this scheme that I'm showing you here, I show you a normal protein that's folded properly. It's transported appropriately within the cell. It has its intended activity breaking down some substrate into a metabolite. On the bottom, I'm showing you a patient that has a mutation or a change in that protein, causing the protein to fold abnormally. Sometimes these abnormally folded proteins traffic properly to the right place in the cell, but they simply can't work. Other times unfolded proteins are degraded within the cell. So one way to approach this would be to just ignore the enzyme completely and come up with an alternative strategy to remove the toxic substrate that's accumulating. So this is called substrate reduction therapy. Another possibility is to simply block the toxic effects of this substrate that's accumulated within the body, something that's called a pathogenic modulator. There are other flavors of drugs that are being explored. For example, drugs that bind the abnormal protein and allow it to fold properly, something called a chaperone drug. There are also drugs that can cause proper folding and restore the activity of the enzyme. And finally, there are stabilizer drugs that prevent the cell from breaking down abnormally folded proteins. And all of these classes of drugs can lead to some enzyme activity and therefore a reduction in the amount of the toxic chemical. And once again, just for illustration, there are many of these drugs that are in either clinical trial or are already FDA approved for some of these conditions. So now I want to turn to a different disease. That is cystic fibrosis. I'm sure you're all familiar with this condition. These children have mutations in a protein called CFTR. And that leads to things like chronic lung disease. It leads to pancreatic insufficiency. It leads to multiple problems with intestinal performance. So ultimately, these individuals typically die of their condition. It used to be that they would die in early childhood. Now with just supportive therapy, many are living to mid-adult life. But again, there's the question, can we do better? There are many possible problems that can occur from the... if you begin in the nucleus with a DNA that encodes this channel, the CFTR channel, all the way to the protein being at the cell surface and having its function. You can have problems producing the channel protein. You can have problems processing and trafficking the protein within the cell. You can have problems regulating its activity. It's either inappropriately on or off. Or you can simply have problems allowing chloride to move through the channel. So this is perhaps the most exciting story in my mind in therapy for genetic disorders that's come out recently. And it all stems from a public corporate partnership between the Cystic Fibrosis Foundation and a drug company called Vertex Pharmaceuticals. This partnership set its sights high but focused narrow. So what they wanted to do was develop a drug therapy for people with the Class III mutation G551D. This means that the glycine at position 551 in the protein is changed to an aspartic acid. And you might ask, why so narrow? The chance of finding a drug that would address all the problems, all the potential problems in making this channel, trafficking this channel, and regulating this channel was really low. By definition, a drug that binds to the CFTR and improves the function of this particular mutant form might have potential to bind other mutant forms. So let's start with this mutation but then we can see whether this drug works for many different people with many different mutations. And even at a minimum, a drug for this particular mutation would address about 4% of all people with Cystic Fibrosis. This happens to be a fairly common mutation. So how do they go about finding a drug for a patient with a specific mutation or change in a specific protein? And they used a process that's called small molecule screening. So what you would do is you'd take a plate and you'd put patient cells into each of these welds and you'd also introduce some sort of marker, some sort of often a fluorescent marker that will only glow when activated by chloride conductance, when the performance of the channel has been restored. And then you take a library of small molecules. These could include hundreds of thousands of small molecules, often includes established FDA approved drugs to see if they'll have an effect. And in each well of this plate you'd add a different drug. So you're basically screening all the drugs within this library to see if they have any beneficial effect on restoring how the channel works. You then read the marker, the fluorescent marker for example, and you score the performance of different compounds within this library. You look for the welds that glow the brightest, for example. You might have a reasonable compound, something that's doing pretty well, but not a perfect compound. Then you can take one of your hits and you can tweak that compound a little bit using medicinal chemistry and then put it back through this whole process to come up with the best drug for this particular purpose. And this led to the identification of a drug for this particular mutation causing cystic fibrosis. And again, when I read this paper it was one of the few times that I sat back in my chair and smiled. The results were just so remarkable. So here we're looking at patients who were either not treated with this drug, with CF, or people that were treated with this drug. And you can see that you're seeing changes in the sweat chloride, the amount of chloride that's moving through the channel. So these people that were taking the drug dropped their sweat chloride significantly. And in fact, the absolute sweat chloride level went within the normal range. So it didn't only nudge this in the right direction, it shoved it in the right direction. They then looked at performance of these patients and they saw that by about two weeks there was already this dramatic elevation of FEv1, so a marker of lung function. There was a significant drop in the number of serious events like pulmonary exacerbations that these patients were having. Their respiratory score increased dramatically and they showed abrupt and sustained weight gain over a full year while taking this medication. The one question was, well, did they cherry pick their patients? Was it just the most mild patients that would respond? And the answer was no. The response was dramatic regardless of initial severity, regardless of geographic location, regardless of gender, and regardless of age. So it really seemed to work for the full spectrum of CF patients with this particular mutation. The drug was also tolerated extremely well. There was a significant reduction of serious adverse events when you compared the treatment group to the placebo group. So the conclusions of this remarkable study was that this drug now called IVACAFTOR was associated with improvement in lung function at two weeks and that this improvement was sustained through 48 weeks. That there was substantial improvements also in the risk of pulmonary exacerbations, patient reported respiratory symptoms, weight, and concentration of sweat chloride. And that IVACAFTOR was not associated with increased incidents of adverse events when compared to placebo. This drug was FDA approved on January 31st of 2012 and it represents the first and only drug that has approved for the treatment of cystic fibrosis, currently in children older than the age of six with this particular mutation. But again, the value of this drug is now being explored for other patients. Yes? This is going to pass just for economics. What is it like a bull car? Unfortunately, I don't know that answer, but I can find it out. So I'm going to turn to a different disease now that again a very common condition known as Duchenne muscular dystrophy. These individuals show loss or significant impairment of muscle function usually with a diagnosis at around the age of five. Usually these patients are wheelchair dependent by their teens and death occurs due to respiratory insufficiency by young adult life. In contrast, there's a different condition that's called Becker muscular dystrophy and these people are typically not diagnosed until their teenage years, not wheelchair dependent until mid adulthood and if death occurs it's generally delayed until the fourth to fifth decade of life. Now the remarkable observation is that both of these conditions, very severe and mild, are caused by mutations or changes in the same gene, the dystrophin gene. So an obvious question is, what's underlying this clinical difference? If you look at the muscle and you use an antibody that reacts with the dystrophin protein, in normal muscle you see that every muscle fiber is surrounded by a mantle of dystrophin and Duchenne muscular dystrophy, generally the protein is absent and you can see in Becker muscular dystrophy while not normal, there is at least some preservation of dystrophin expression. What does dystrophin do? Well it serves as a link between molecules deep in the cell and a cluster of molecules that are at the cell surface. Establishing a bridge between what's called the cytoskeleton of the cell and the extracellular matrix outside the cell. In normal people, not only dystrophin, but all the other proteins within this complex are easily seen at the cell surface. If you simply take away dystrophin, you lose this whole complex. The entire complex is destabilized. So you can see from this diagram that dystrophin plays an important bridging role and you might infer from this that dystrophin needs its head to attach to proteins within the cell. It needs its tail to attach to proteins at the cell surface. But the obvious question is, does it need all of its middle? Is it important exactly how long this middle segment is? So we're going to have to look at this in a little bit more detail to understand what's going on. As with all other protein encoding genes, the dystrophin gene is initially copied in the cell to a molecule that's called precursor messenger RNA. And this precursor messenger RNA includes blocks of sequence that encode the protein called exons and intervening junk sequence that's called introns. And ultimately, all of these introns are removed by a process that's called mRNA splicing. So you end up with all these coding blocks all in a row. And there's a signal that tells the ribosome where to start making protein that's called the start signal. And there's also a signal that tells the ribosome to stop making protein that's called a termination codon. So in this normal example, a ribosome can come along and it's going to read the triplet code of the messenger RNA. Every three base pairs encodes a specific amino acid. So the ribosome will move along. You'll end up with a full length protein and you'll end up with a normal muscle phenotype. Now in Duchenne muscular dystrophy, the most common cause is that one of the exon blocks is skipped during splicing. And the important fact is that in Duchenne muscular dystrophy, the block that is skipped is not an even multiple of three. So now when you splice these two exons together that don't belong together, you're going to shift this triplet reading frame. Everything downstream of that point is just going to be nonsense. And this invariably leads to what we call a premature termination codon, something that will tell the ribosome to stop too early. So again the ribosome will latch on. It'll move along this RNA but it will stop early. And you might guess, well that's going to cause a truncated protein missing its tail. And I've already told you the tail is very important. In fact, when a RNA has a premature stop signal, the most important consequence is that the cell simply gets rid of that RNA through a process that's called nonsense mediated mRNA decay. So you don't have any potential to make protein from these prematurely terminated transcripts. And the answer is you make no dystrophin and you get Duchenne muscular dystrophy. So what's going on in the mild form, the Becker muscular dystrophy? Well here you also commonly have skipping of an exon. But in this circumstance, the blocks that are skipped are an even multiple of three nucleotides. So your reading frame is going to remain preserved. You've got a piece missing right here but you don't have any premature stop signal. The code is all intact. So again, the ribosome latches on. It reads through to the end and it makes a largely normal dystrophin protein that's missing a central block. But it's got both its head and its tail to latch on to the appropriate protein complexes. And that's what causes the more mild Becker muscular dystrophy. So the obvious question is is there anything that we can do to turn this into this? That is a goal. Let's make this more mild. So in order to explain how this happens I have to tell you a little bit more about splicing. And the only important point is that this is a very tightly orchestrated process. It's not random. You have your precursor messenger RNA that has different signals embedded within it that tells protein complexes where to bind. And these protein complexes ultimately define what's the beginning of the junk and what's the end of the junk. So it tells the cell what to get rid of and what to splice together. And after this you end up with the appropriate splicing event joining X on one to X on two and then this continues down the length of the messenger RNA. So what if you wanted to get rid of X on two? You thought that that might be a good thing. So what can be done is you can introduce into the cell a small little artificial piece of RNA or DNA that we call an anti-sense oligonucleotide. You can make it so it attacks across this spot at the beginning of X on two, for example. What would be the consequence of that? Well, some of these protein complexes would continue to bind but the protein complex that was supposed to bind here is blocked. So this is no longer recognized as an important sequence and that would lead to skipping of X on two and then you'd have that messenger RNA having X on one to X on three. That's not that helpful. Why would that be a good thing? I'm going to tell you now about a convention that I'm going to use for the next couple of slides. So these little exons that are shown as puzzle pieces show how the exons are supposed to fit together to maintain the triplet code along the whole sequence. So if the puzzle pieces fit together like this everything is a-okay. You've got a good messenger RNA and an open reading frame for the ribosome and you'll get your protein. Now let's take an example where a patient has skipped X on 45. You can see that when X on 44 splices to X on 46 the puzzle pieces don't fit. You're going to get one of your premature stop codons you're not going to make dystrophin and you'll get dishain muscular dystrophy. So in this same patient that skipped X on 45 what would happen if you used anti-sense oligonucleotides to tell the cell to also skip X on 46? Now suddenly 44 and 47 fit together. The puzzle pieces attach. So you've preserved the reading frame for the ribosome. You'll be missing a central chunk but you'll make a protein that still has both its head and its tail and therefore some function. Now you might say well this is a nightmare. Okay I understand it might work but you're going to have to come up with a different method for every patient because everyone's going to be skipping a different X on. So you're going to have to optimize this for hundreds and hundreds of different circumstances. Well that doesn't turn out to be the case because it's been recognized that about 60% of all muscular dystrophy mutations occur within this central block of Exons from Exon 45 to 55. And someone came up with the brilliant idea of coming up with a method of causing all of these Exons to skip. So you'd use a pool of your anti-sense oligonucleotides targeting them all. Exon 44 fits together nicely with Exon 56. The puzzle works. So this would be a potential cure for about two-thirds of patients with Duchenne muscular dystrophy. Just a single cocktail of anti-sense oligonucleotides. So how does this work? Well here we're looking at local delivery of anti-sense oligonucleotides by injecting them directly into a muscle group in the foot of Duchenne muscular dystrophy patients. And what the investigators saw is that adding the anti-sense oligonucleotide indeed causes the Exons to skip. In each case you get a smaller product. And suddenly you go from no dystrophin protein to lots of dystrophin protein in all of these patients. And it's not just little tiny spots. If you look at the entire biopsy of the muscle you can see that there's widespread dystrophin expression. So this is looking really good. I'm sorry? It takes somewhere between one to two weeks to see robust expression. Now an important question is how would you really need to deliver this to make a difference to an individual? We can't just inject each muscle group. So instead people have been infusing these anti-sense oligonucleotides into the circulation. These are early days. But in some patients just putting it into the circulation causes significant improvement in dystrophin expression. In other cases there's perhaps a subtle effect. I think that this is still within the phase of optimization. And the very important issues are going to be to address how to best deliver these anti-sense oligonucleotides so they get to all muscle groups. And how do you improve the stability of these small little oligonucleotides in the circulation? Are they simply just being degraded too quickly? But I would say an extremely promising approach for a very important condition. So now I'm going to change gears and talk about a very rare but very interesting condition called Hutchinson-Gilford progeria, a premature aging syndrome that causes young children to start an accelerated aging process in early childhood at around the age three to five or so. They rapidly show loss of hair. They show wrinkling of skin, atrophy of fat, osteoporosis, coronary artery disease, and are basically dead of old age by the time they're about 15 to 20. Really a devastating condition. So Francis Collins' group a few years ago described the cause of Hutchinson-Gilford progeria, and it's due to a single point mutation in a protein that's called Lamin A. So here on top, I'm showing you normal Lamin A. In patients with Hutchinson-Gilford progeria, there's an altered splicing event that causes the skipping of a small chunk of Lamin A shown in green. So the mutant Lamin A protein in these patients has both a head and a tail, but it's missing a central bit. And it turns out that this central bit that it's missing is really important. So normally what happens is when the cell makes this Lamin A protein, that protein is modified by a process that's called farnesolation, and this causes the protein to attach to the nuclear membrane. But that little bit that's missing in progeria is so essential because ultimately enzymes have to come along and cleave the protein right at that site to cause it to release from the nuclear membrane. In patients with progeria, because they're missing that cleavage site, this Lamin A protein remains stuck to the nuclear membrane. It can't get off. And so that led to the question, what would happen if you just prevented this farnesolation process in the first place using a class of drugs called farnesoltransferase inhibitors? If it can't get on, it's not going to get stuck there, and it doesn't matter that it can't get off. That's the logic. So initially there was a need for a marker to say, are we doing something good or not? And the marker that was selected is called nuclear blebbing. If you have Lamin A stuck to the nuclear membrane as patients with progeria have, the nucleus becomes very misshapen. So that was initially used as the readout. So here are cells from patients with Hutchinson-Gilford progeria in the absence of any treatment, and you can see the obvious altered contour of the nucleus. If you give a little bit of a farnesoltransferase inhibitor that prevents the protein from getting stuck, you already see improvement. If you give a little bit more, the improvement is striking. They look like normal nuclei. And here's that quantified. In someone that doesn't have progeria, there's very little nuclear blebbing. In three people that do have progeria, there's a lot of nuclear blebbing. In the absence of drug, give a little bit of drug, it gets better. Give a lot of drug, it gets even better. So as a marker of progeria, this suggests that this treatment is working beautifully. So now there have been trials of farnesoltransferase inhibitors in patients and in mouse models with progeria. I'm going to show you the results from a mouse model with the same type of Lamin A mutation because it's much further along. So here we're looking at both female and male mice. You can see that the patients or the mice that have progeria have very... Sorry, I'm having trouble seeing that. So their body weight is very low compared to normal individuals. If you give them this farnesoltransferase inhibitor, there's a significant increase in body weight. You can also see that mice that are treated with this farnesoltransferase inhibitor, both female and male, show a significant improvement in grip strength of the muscle, that there is delay of death in the mouse model of progeria, and there's also a dramatic reduction in rib fractures. So again, just by understanding where the mutation is, a little bit about the protein, you can come up with a completely novel hypothesis. I mean, no one would have possibly imagined that this class of drugs would have anything to do with treating progeria. What's really interesting is that it's not only relevant to these children with this rare devastating disease. What's been shown is that as we get older, our bodies get a little bit sloppy in how they make lamin A, and some of that lamin A looks exactly like the lamin A in progeria. So the concept is that this accumulation over years and years and years of this mutant progerin protein may be contributing to our aging, and that treatments under development for this rare condition might be relevant to the broader population. I'm going to end by telling you a little bit about my own work, which has focused on a condition called Marfan syndrome. So Marfan syndrome is a disorder of the body's connective tissue, the material between the cells that give the tissues form and strength. It's a very complex and variable condition, but the main features include dislocation of the lens of the eye that shifts out of place, overgrowth of the bones and low fat stores, and also, and most importantly, progressive dilatation of the root of the aorta, just as it's leaving the heart, that will lead to aortic tear, and early death if left untreated. In 1991, we were able to show that Marfan syndrome is caused by mutations in the gene that encodes the connective tissue protein fibrillin-1. So what do we know about fibrillin-1? Well, we knew that it aggregates outside of the cell to form these very complex structures called microfibrils, and that these microfibrils cluster around the maturing ends of an elastic fiber during embryonic growth. So this simple spatial and temporal relationship led to the absolute conclusion that you need a lattice of microfibrils to make an elastic fiber during embryogenesis, and that in people with Marfan syndrome, this never happens. There's inadequate elastic fibers game over. If you think about it, that really boated poorly for the development of these treatments. It suggested that children with Marfan syndrome are born with inadequate elastic fibers and that there's nothing that you could do after birth to improve the situation. So I remember the day and even the moment that I walked into a patient room, saw these exceptionally long fingers just like this and thought to myself, this just doesn't make sense. It just doesn't cause the bones to overgrow. For that matter, why would weakness of the tissues cause the facial features of Marfan syndrome like downward slanting eyes, flat cheekbones, and a small chin? Each of these findings was more suggestive of altered cellular behavior rather than simple tissue weakness. Now to make a long story short, we learned that microfibrils that are composed of fibril in one serve a second important function. They're not just glue, but rather they bind to the inactive complex of a growth factor called TGF-beta. It's a molecule that tells cells how to behave. And what we learned is that in Marfan syndrome where you have inadequate microfibrils, you have failed matrix sequestration of latent TGF-beta, and that leads to too much TGF-beta activation and activity. This molecule is now over-stimulating the cells. This sets in motion a cascade of events inside the cell. One important event, and the only event that I want you to notice here, is that there's a molecule called phosphorylated SMAD2. That's going to be our marker of TGF-beta activities going on. And we were ultimately able to show that this excess of TGF-beta stimulation of cells had consequences in Marfan syndrome including emphysema, mitral valve prolapse, aortic aneurysm, and skeletal muscle myopathy. The way that we proved that is we made Marfan mice that were deficient in fibril in one, still had bad glue between the cells and then injected them with a TGF-beta blocking antibody. And we found that virtually all of these conditions were prevented by simply blocking TGF-beta in Marfan mice. So we then asked, well, is there a drug and even better an FDA-approved drug that might mimic this protection? And our attention turned to a very common antihypertensive medication called lozartan that lowers blood pressure, something we think is good for people with aneurysm, but had also been shown to block TGF-beta in mouse models of kidney disease. So we wondered whether this might be a magic bullet, having two different effects. So we know that angiotensin 2, a molecule that regulates blood pressure, acts by working through both a type 1 receptor and a type 2 receptor. And it's the type 1 receptor that stimulates the TGF-beta pathway. So it's also known that angiotensin 2 working through its type 2 receptor can suppress all the events that are caused by the type 1 receptor. So in this view, AT1 is bad. That's the culprit. And AT2 might actually be protective. So we reasoned that if you used an ACE inhibitor that stopped the production of angiotensin 2, you'd be limiting signaling through both the culprit and the potentially protective pathway. But if you selectively blocked the AT1 cascade with a drug like lozartan, you might actually stimulate signaling through the protective pathway. So we did a clinical trial in our mouse model of Marfan syndrome. Normal mice show very slow rate of growth of their aortic root. Marfan mice treated with placebo show this very accelerated aortic root growth. If we gave a drug such as propranolol that only lowered blood pressure but did not address TGF-beta, the aortic growth was decreased to an extent. But if we treated these mice with lozartan, we saw that they showed absolutely normal aortic growth for a full lifetime. And even more important if you looked at the aorta under the microscope, no observer could distinguish the lozartan treated Marfan mice from normal mice by any parameter. So there's a large clinical trial that's now ongoing but we felt compelled to treat a subset of children with the most severe form of Marfan syndrome. These kids show unrelenting growth of the aorta despite maximal treatment with beta blockers or ACE inhibitors, reaching death or surgical endpoints in early childhood. So here's the aortic root growth curves for the first two such children that we treated with lozartan. And you can see that there was a dramatic plateau, no further growth of the aortic root once lozartan was started. In this child this plateau has remained now with eight years of follow-up. There still are some things that require improvement. We found that some children show a relative response to lozartan but then the aortic growth starts creeping up again. And what we've learned is that other drugs in the class of angiotensin receptor blockers such as herbisartan with ultra high dosing can allow you to achieve a plateau in aortic growth even in these most severe children. We again wanted to try to understand more about this pathway. I've told you about TGF beta activating the so called SMAD pathway. But it's also known that TGF beta can activate other pathways. And the one that I want you to pay attention to is particularly this one, the so called IRC pathway. When we looked at the award of our Marfan mice we saw that the SMAD pathway was excessively activated compared to normal mice or what we call wild type mice. But look at IRC activation. It's dramatic in the Marfan mice and almost non-existent in the normal mice. So our attention turned to this significance of this IRC activation. We went on to partner with the therapeutics for rare and neglected disorders program at the NIH. And they were able to provide a compound called RDEA 119 that's a potent inhibitor of IRC activation. When we treated our Marfan mice with this IRC inhibitor not only did we greatly diminish abnormally aortic growth, we actually caused regression in the size of the aneurysm over time. It got smaller over time the first time that we've ever seen that. So now IRC is firmly on the radar screen. You know, finding genes and making animal models allows you to find new therapies. It also allows you to evaluate the performance of existing therapies. And currently calcium channel blockers are considered the second line treatment for patients with Marfan syndrome that can't tolerate beta blocker medications. But there's really not a lot of evidence that they work and there's not even a lot of evidence that they're safe. So we decided to test this in our mouse model. We were initially very optimistic because there was some work in the literature showing that calcium channel blockers can blunt IRC activation and at least some cell type. So we did a trial of amlodipine in our Marfan mice. Here I'm showing you the heart and ascending aorta of a normal or wild type mouse. In a mouse with our Marfan mutation, we see that there is flaring at the base of the aorta. If you give a normal mouse amlodipine, you don't see much. Perhaps the aorta's looking slightly generous, but you know, it's subtle. But virtually a hundred percent of Marfan mice given amlodipine develop these massive ascending aortic aneurysms. And this occurs the aortic size triples within three weeks of starting the medication. And the mice are dying due to aortic dissection within four weeks of starting the medication. If I show you this quantitatively, a placebo treated Marfan mouse shows accelerated growth of the aortic root. Look what happens when you add amlodipine. The rate of aortic growth, root growth doubles. If you look further up the aorta where Marfan mice normally don't have an aneurysm, you now see that amlodipine is causing this dramatic aortic growth. In this Marfan mouse model, shown in red, there's typically no death if you treat with placebo. But if you treat the mice with amlodipine, death due to aortic dissection starts within five weeks and then accelerates quickly, we are able to see that contrary to hypothesis, amlodipine is actually accentuating irk activation rather than blunting irk activation. And now we found the identical results for all calcium channel blockers. This is not specific to amlodipine. It's a class effect. So we want to try to figure this out. And one thing that we did was asked, well, what happens if you give amlodipine but also block irk with our irk blocking drug? And the answer is that you prevent all of this detrimental effect. So it is irk activation that's responsible and gives us greater confidence that this is a great therapeutic target. We also, in our mice that are dying due to aortic dissection with amlodipine, if you give the irk antagonist, you again see no death due to aortic dissection. So not only do you suppress aortic growth but you prevent aortic tear. So I think it's pretty remarkable that seven years ago we had the model that there's weak tissues in Marfan syndrome at birth and there's nothing you can do about it. Currently we have over seven different medical treatment strategies that have shown remarkable effectiveness in our mouse model and that we're moving forward to people just as important as finding new effective drugs is learning about things that might be detrimental such as calcium channel blockers. In the last two minutes I'm just going to tell you that what we've learned about Marfan syndrome is not just relevant to that condition. It extends to many causes of aortic aneurysm. About six years ago now my colleague Bart Lois and I recognized and described a new aortic aneurysm syndrome that has many features of Marfan syndrome like curvature of the spine long fingers and aortic aneurysm but also many unique features like a widely spaced eyes, a cleft palate or a bifid uvula. Most importantly these patients don't just have aneurysms at the root of the aorta but rather all throughout the arterial tree. So a much more aggressive condition. And these aneurysms rupture at young ages as young as six months and at smaller dimensions when compared to Marfan syndrome. So based upon what we had learned about Marfan syndrome we bet that this condition that's called Lois Diet syndrome would also relate to TGF beta. And in fact the very first two genes we looked at are the two genes that encode the TGF beta receptor and we learned that all of these patients have mutations in the receptor for TGF beta. They also show high TGF beta signaling looking at our old friend phosphorylated SMAD2 in the nucleus of cells in the aorta. We went on to make mouse models. We saw that these mice have horrible looking aortas. Their aortas grow really fast but if we treat them with lozartan aortic root growth returns to normal and aortic wall architecture returns to normal. You go from this picture with all these fractured elastic fibers to a very normal looking aorta. Currently there is now a new class of conditions that are called the TGF beta vasculopathies that are shown here. All aortic aneurysm conditions that now have been associated with high TGF beta. So these data suggest that altered TGF beta signaling is a common pathway to aneurysm and that treatments under development for Marfan syndrome may find a broad application. My last slide the conclusions are that the study of rare Mendelian disorders is both an obligation and an opportunity. The obligation stems from the fact that while they're individually rare these conditions are personally burdensome and effectively common. And also that patients with rare genetic disorders have really fueled progress in the field of molecular therapeutics. They've given of themselves. They've accepted risk. They've allowed us to learn. So there's a real personal cost despite a remote chance of personal advantage. The opportunity relates to the single gene basis of the defect. What we know when we find a gene for a Mendelian disorder is that a defect in this pathway is sufficient to cause this condition, this disease phenotype. And therefore it tells us that these pathways are inherently attractive therapeutic targets. If you could nudge them in the right direction they might make a big difference and such therapies can then be explored in more common conditions like emphysema rather than the lung disease in Marfan syndrome. I'd like to end by acknowledging the truly remarkable young people that I have the privilege of working with every day, my collaborators at other institutions and my funding sources, and I'd be happy to answer questions. Thank you for your attention. Yes, sir. Is there any evidence of a immune response to the new program? So again it depends upon whether someone normally has a little bit of dystrophin or whether they're completely naive for dystrophin. But so far that has, to my knowledge, that has not turned out to be a problem. It seems that everyone, even with the most severe form of DMD is making enough protein to elicit tolerance so the immune system doesn't react. Yes. It'll take me weeks to absorb all this, but I do treat osteoporosis and osteoporosis. Is that just disuse or because the bone is much different than some of the other tissues? It is not a disuse situation. We've been able to, at least in Marfan syndrome, tie the osteoporosis to the same TGF-beta cascade. And our collaborator in New York, Francesco Ramirez, in culture systems has shown dramatic responses to some of the TGF-beta modulating agents. So I think it's going to teach us something about common osteoporosis. Yes, sir. Are adults with Marfan at all treatable? Yeah, so that's a great question. The question is, are adults with Marfan all treatable or is the window of opportunity to make a difference over in childhood? At least in our mice, we can allow them to become mature adults. They're sexually mature at about two months by six months of age. They're sort of in mid-adult life and by a year of age, they're old mice. And whether we start treatment right after birth, in the middle of that sequence or at the end, we see the same kind of benefit. So we think that the window doesn't close, that there is an opportunity even later in life. Is there a test that could determine this embryonically? Yeah, so the question is, is there a test that can determine the diagnosis as a fetus is developing? Yes, for all the conditions that I discussed, the gene is known. So you would be able to do prenatal diagnosis and know that a fetus has this predisposition. So you didn't mention the so-called read-through drugs for in-frame premature termination codons. Do you have comments on those? Yeah, so actually I have slides for those, but I just didn't have time to cover them. So these drugs that are called read-through agents, basically they make the ribosome sloppy. So the ribosome reaches a stop codon, but it just marches through and inserts any amino acid at that position. There is a company called PTC Therapeutics that has developed a drug called PTC124 that makes the ribosome sloppy. So the whole idea is that the ribosome will march through and really stop codon and make a full-length protein. There is a clinical trial or was a clinical trial for muscular dystrophy with PTC124, but that trial was stopped because they did not reach endpoints. There was some suggestion that there might have been a subtle benefit at a lower dose as opposed to a higher dose. So that's being explored, but there are many potential problems through approach. One potential problem is that most of the RNAs are degraded by nonsense mediated decay. So there's not many transcripts left that are engaged by ribosomes where you could have read-through. Also the context of the premature termination codon defines the efficiency of read-through, and there's only one context that's really potent that allows a lot of read-through to happen. We also know that inserting any amino acid where a stop codon was is often not good enough. What's needed at that position is the intended amino acid rather than any amino acid for the protein to have its function. So I think it's a really exciting idea. I think it potentially could treat a broad spectrum of genetic diseases, but I think the idea is in need of refinement. And one potential refinement would be to both block this decay pathway, this nonsense mediated decay pathway while also stimulating read-through. And I know that a number of companies are looking at that closely. Yes. In the large data complex is the right state. It's actually a thousand-fold higher than the active form. So what in the absence or in the mutation of curriculum that then releases this large complex that actually activates TGF and could that be a target? So the question is what's actually activating TGF beta that doesn't bind to the matrix, for example, in Marfan syndrome. There are many, many activators of TGF beta, including many proteases, including integrins, including low pH. There's some evidence that in Marfan syndrome a molecule called MMP9 or M-matrix-Matellopyrtonase-9 may be particularly important in activating TGF beta. There are some trials going on with different inhibitors of TGF beta activators, but there simply aren't results yet to share. So it is a developed concept, though, to go after these activators. What do you think about the gene correction using zinc-figured technology? So there are a number of very interesting technologies that are being considered to actually correct the mutant sequence within your gene, within the native gene. Some of them have shown promise in cell culture systems. To my knowledge, none have shown sufficient efficiency to suggest that they may work in vivo. I think it's, again, a very exciting concept. It would be curative for any genetic, if not most, genetic conditions. But I just don't think it's far enough along. There's still some leaps in technology that will be needed to bring that to fruition. Well, thank you again for your attention.