23 Apr 2013 ... Dr. Philpot published a very high profile paper in Nature in December 2011 ... It is
a $2.2 million project that Dr. Philpot is doing together with.
Gene Awakenings for the Treatment of Neurological Disorders April 23, 2013 Ben Philpot, Ph.D.
Monica Coenraads: Dr. Philpot is associate professor in the Department of Cell Biology and Physiology at the University of North Carolina Chapel Hill. He earned his PhD in psychology and psychobiology, I don’t think I have ever heard of psychobiology-‐-‐ you’ll have to tell us what that is, from the University of Virginia and he did his post-‐doctorate training in the lab of Mark Bear, who is also doing a Rett project right now. Dr. Philpot published a very high profile paper in Nature in December 2011 where he was able activate a silent gene in Angelman Syndrome and he is going to talk about that project and a project that is being funded by RSRT. It is a $2.2 million project that Dr. Philpot is doing together with Bryan Roth and Terry Magnuson. We are excited about it and the goal here is to do similar things as what he did in the Angelman and activate the MeCP2. He is going to tell is how he is doing on the project and how he is doing on the Angelman project since that served as a model for the Rett project. Thank you very much for taking the trip and coming. Dr. Philpot: First of all thanks Monica for inviting me up. It’s a real pleasure to be here and thank you all for coming and also thanks to the Simons Foundation for having us and allowing us to be in this wonderful auditorium. I also want to reiterate that for those of you here in the audience, to ask questions and interrupt me at any time. I really want to keep everyone with me and understanding the entire talk. So please, if there is something you don’t understand and want a clarification on, just stop and ask me, or if you have a question at any time, feel free to interrupt. Now I want to reiterate a point that Monica made that there really is a lot of reason to be hopeful that we may be able to develop therapeutics for Rett Syndrome. One of the reasons she mentioned is that it is a single gene disorder and the critical gene MeCP2 is known. This is really important because if you think about it, the human genome contains about 25,000 genes and we know that these genes are found in 23 chromosomal pairs and the chromosomes contain DNA, the genetic material that encodes for proteins that perform cellular functions throughout your body. Most neurological disorders are caused by many genetic mutations, which here I represented on the chromosome with little red dots [points to slide]. In schizophrenia, there are thought to be many mutations that are thought to make someone susceptible to getting schizophrenia. From a scientist’s point of view, this makes it very difficult to tackle this disorder—you have to try to figure out what genes you want to focus your efforts on and what combination of genes might be important for causing schizophrenia. Now in Rett Syndrome, it is caused by the disruption of a single gene. We know from the very seminal work of Huda Zoghbi in 1999 that this is MeCP2. So just knowing what the gene is gives us a tremendous advantage from a scientific perspective in trying to develop therapeutics. We know what gene to study and what to go after. Another advantage for Rett Syndrome is the critical brain circuits remain grossly intact. You can think that this is very different from the situation in late onset Alzheimer’s for example. If you compare a
healthy brain to a brain from an individual with very advanced Alzheimer’s, you can see that three is a lot of necrosis in the brain in the individual with Alzheimer’s. You can imagine that overcoming the loss and physical damage of a brain is very, very difficult. So that is why for Alzheimer’s, the focus is on early identification. But in Rett Syndrome, the gross brain circuitry appears to remain grossly intact. Here are brain images. One is from an individual with Rett Syndrome and one is from a neurotypical individual. These are called diffusion-‐tensor imaging. They show fiber tracks in the brain and I would challenge anyone here to be able to tell the Rett brain from the neurotypical brain. So it turns out this one is the Rett brain [pointing to slide]. There are certainly differences that have been quantified. This is group out of Johns Hopkins and they have quantified differences in fiber tracks in individuals with Rett Syndrome versus neurotypical controls, but it is still safe to say even though there are things like microcephaly that individuals with Rett Syndrome have, the gross brain circuitry remains intact and we don’t see large amounts of neuro-‐degeneration that you see with things like late onset Alzheimer’s. So this is another advantage that we have. With this in mind, and also with the important discoveries from Adrian Bird showing that even in an adult model of Rett Syndrome you can reverse the symptoms of Rett Syndrome by activating MeCP2, we took on a drug discovery approach to try to find therapeutics for Rett Syndrome. This is a very large-‐ scale operation that taking investigators and their lab members with very different expertise. My expertise is actually electro-‐physiology, another member of the team is Terry Magnuson, he is an expert in genetics—he is one the foremost leaders in the work on the X-‐chromosome and the X-‐chromosome inactivation. Another team member is Bryan Roth from the pharmacology department. Bryan Roth is the director of the Institute of Mental Health Drug Discovery Program. So he comes with an incredible amount of expertise and facilities that are normally only found in a pharmaceutical setting, that we are fortunate to have at the University of North Carolina. Now before I tell you about the approach, I need to give you a little genetics 101 lesson and I will remind you that for everyone in the audience that the genetic material in each one of your cells in your body is identical. So a cell in your heart, a cell in your brain and a cell in your eye… all those cells have the same genetic material but obviously you know that a cell in your heart versus your eye versus your brain… are all very different. And the reason they are different is because there is something above the level of genetics; it’s called epigenetics. Only certain genes are expressed in these different types of cells. So you might have one complement of genes that are expressed in a heart cell, while other genes are turned off and a different complement of genes that are expressed in your brain cells. So there are mechanisms in your body to turn on and off the different expression of genes. Now in the case of the X chromosome, things are a little bit different. That is the case of 22 out of 23 of your chromosomal pairs. But for the X chromosome, as Monica mentioned, there is such a thing as random X-‐inactivation. So you could look at two cells in the brain and in one cell, you may have one chromosome that is inactivated, and in another brain cell, you can have the other chromosome that is inactivated. And obviously this has a huge impact when you start having mutations on the X chromosome. Is that clear to everyone before I go on? Ok.
With this kind of genetic biology in mind that set up therapeutic strategy we are using to try to develop therapeutics for Rett Syndrome. So I will remind you in a neurotypical individual, MeCP2 will be expressed off an active chromosome, but it doesn’t matter what chromosome is active and which is inactive because both copies of MeCP2 are functional. So you have MeCP2 expressed from a cell regardless of which X chromosome is inactivated. Now in the case of Rett Syndrome, as you know, there is a mutation in the copy of MeCP2. So there one copy mutated in every cell. I am depicting the mutation [in the slide] with an X. Now in some cells it doesn’t matter because that is the inactive X [pointing to slide where mutation is on inactive X]. The non-‐mutated MeCP2 is active, so we will get normal MeCP2 expression. In the other cells the functional copy of MeCP2 is silenced and the active copy is the mutated copy so you don’t get any MeCP2 expressed. It’s in these cells that you don’t have normal MeCP2 function, and then do not have normal neuronal properties. So the therapeutic strategy is to find a way to activate the inactive MeCP2 (the one that is silenced). So it won’t make a difference to normal cells, they’ll still express MeCP2 but in the ones that have a good copy silenced, we’ll now unsilence it and allow MeCP2 to be expressed in those cells as well. In that way, we’ll reinstate normal function in those cells. So that is the basic strategy behind our whole drug discovery platform. Is that clear to everyone? Now can the inactive MeCP2 be awakened as a treatment for Rett Syndrome? Well we don’t know yet, but I can tell you by analogy to work we have done in Angelman Syndrome, that there is reason to believe that it may be. I want to give you a little bit of background on Angelman Syndrome and that disorder and some of the progress we have made there, because we are using an almost identical strategy and applying it to Rett Syndrome. This is the face of an individual with Angelman Syndrome, a cute little kid here with jam smudged on his nose, and of course there are many faces of Angelman Syndrome. Immediately you can see one of the characteristics of Angelman Syndrome is this happy disposition. So one the characteristics of Angelman Syndrome is a happy disposition. Many of you may know that there is often a misdiagnosis between Angelman Syndrome and Rett Syndrome because they share a lot of phenotypic similarities. Some of those are shown here. Individuals with Angelman Syndrome have movement disorders, severe developmental delay, strikingly there is a severe speech impediment so 70% of individuals do not speak a single word in their lifetime, there is a high incidence of seizures, they have abnormal EEG patterns and they have severe sleep disorders. So as you know, many of these features are shared with Rett Syndrome. So there are a lot of phenotypic overlap. Just to bring you a little closer to the disorder, I wanted to show you a few videos that were pulled off of YouTube, so you can understand a little bit more about the disorder. This is a little kiddo who is undergoing some drop seizures. You’ll see that there are some stuffed toys judiciously placed around him that will come into play as he has his seizure, and is falling over. In Angelman Syndrome seizures usually manifest at around 3 years of age and then there is a little bit of improvement with age after that. So you can see that seizures are quite prevalent. Another individual with Angelman Syndrome is shown here and you will see a normal stereotypy with his hand flapping, you’ll also appreciate the happy disposition of these individuals and the lack of verbal communication. So I think this [video] captures the hand flapping and really happy disposition of individual with Angelman Syndrome.
Now I want to tell you about the drug discovery approach we used in Angelman Syndrome and to give you a status update on that so you can appreciate how we are applying the same strategy for Rett Syndrome. A lot of the team members are the same: myself and Bryan Roth’s lab. For this project we brought in Mark Zylka who is a real expert in genetics and microbiology and also has a lot of expertise in the spinal cord which comes into play later. Now the gene that causes Angelman Syndrome is known just like the gene that causes Rett Syndrome is known. And the gene for Angelman Syndrome is called Ube3a. Ube3a is not on the X chromosome, but unlike most autosomal chromosomes is only expressed on one copy. So everyone in this room is expressing Ube3a in your neurons at least in the copy that you inherited from your mother, whereas the copy that you inherited from your father is silenced. And Angelman Syndrome is caused when the copy inherited from the mother is mutated or deleted. So there is a loss of Ube3a expression. So the drug discovery approach we used here is similar to one we are applying to Rett Syndrome, which is to try to find some type of small molecular compound that would turn on the inactive paternal copy of the Ube3a so we could get expression from the normally inactive copy to replace the deleted copy and get expression similar to what we would in neurotypicals. Question from the audience: You are turning both copies on but one does not work so you don’t have to worry about turning one off? Right, in this instance we have a copy that is either deleted or nonfunctional so that we don’t have to worry about turning it on. Now you are actually asking a very insightful question. There are some forms of Angelman Syndrome that have mutations and in certain forms, it may act as a dominant/negative perhaps to alter the expression of normal functioning Ube3a it may be more problematic. But in the larger majority of Angelman Syndrome, it is a deletion that is causing it. So there might be certain types of Angelman Syndrome that this strategy may not work in. Question from the audience: So in Rett Syndrome you have different variations of mutations. In Angelman Syndrome, are there a smaller number of mutations? There are a smaller number of mutations that have been identified to date but there is still a lot of work that is going on to do that. But you could imagine in Rett Syndrome that there may be a few types of mutations that might cause MeCP2 to act in a dominant/negative fashion for examples so you might want to repress the expression of the bad one. The hope, the expectation, is that most copies of MeCP2 that are mutated will be non-‐functional so even if they are activated it won’t have a deleterious effect. But that is something that is a very important issue that will have to be addressed. Question from the audience: Is that the difference between nonsense and missense? Because nonsense you still are producing a little bit of MeCP2? I think it would be certain types of mutations that are not just introducing a stop codon for example, but can cause MeCP2 to act in an abnormal function so yes that is basically the point that will be very
important to address. But like I said, the hope and expectation is that for MeCP2 mutations, the strategy that we are using will apply. You can imagine that in the future we can look at very specific mutations of MeCP2 and see if they act in a dominant negative fashion and there are ways to study this in a laboratory. So you can make a construct with specific mutations, express them in cells and see how they react. It turns out that the tools are available in Angelman Syndrome to look at the genetic activation of Ube3a and the tool was developed by Art Beaudet. When he was in Scott lab at Baylor, he made a mouse that had a florescent reporter for gene activation. I am going to walk you through this so hopefully it is clear. They fused to Ube3a a yellow fluorescent protein “YFP”. Think of it as fluorescent tag. You can make it so the mice inherit this fluorescent tag only from their mother, and that is shown here so they don’t have a fluorescent tag on the paternal copy. When you do this, and take a slice of brain, you can see that the neurons are glowing yellow. So it shows you that the Ube3a is activated. Now if you breed the mice so they inherit the florescent copy from their father, then you look at a section of brain, you don’t see any neurons that are expressing UEB3A and that is because that copy is silenced. So by using a florescent reporter for gene activation, we can distinguish between an active copy of the gene and an inactive copy of the gene. And this turns out to be a very, very valuable tool because we can culture neurons from mice that have the florescent reporter inherited from their father. So these neurons are not glowing fluorescent. Then what we do is plate them in a 384-‐well plate. So this is a literally a plate that has a bunch of different wells in it and each one of these wells will hold about 20,000 neurons we take from mice and all these neurons have the fluorescent reporter from their father. The idea then is that we add different drugs, we add them in quadruplicate, we add drugs to all these wells and we look for a set of wells where the neurons are glowing. If the neurons are growing, we know that we have activated the paternal copy of the Ube3a. So this is how we do a drug discovery project. We can screen for many different drug compounds and try to find ones that activate our genes of interest. Now I wanted you to appreciate the technology that goes into these drug discovery efforts. This is one of the many ways we benefit from the laboratory of Dr. Bryan Roth. As I mentioned he is head of the NIMH Psychoactive Drug Screening Center. Not only does he have a lot of genius in this area, but he has a lot of the hardware and software necessary to do these large drug screens. This is a brief movie showing you some of the robotics that we employ for adding drugs to different cultures. You can see with this type of robotics they can pipet a lot of drugs out very quickly and in a very precise manner so that we can create many, many plates with neurons and screen for drugs that are activators. So without these million dollar pieces of equipment and expertise of Bryan Roth’s lab, these studies would be very laborious and time consuming and really not practical to take on. So I think this allows you to appreciate some of the technology that goes in some of this type of research. One concern is that when you take cells out of a mouse and culture them, they might behave differently. So one of the very first steps we did was to take the neurons out of the mice and see if they still maintained the silencing of the Ube3a just as they do when they are in the brain. So this is an example of cultured neurons shown here, the stain on the bottom is a stain for nuclei, so it will detect every single neuron in the dish. The stain on the top is the florescent reporter for UB3EA. When we breed the mice and the UB3EA is on the maternal copy we see a lot of expression. When we breed the mice and
the fluorescent reporter is from dad, even though we know there are a lot of neurons in the dish from the nuclei stain, we know that none of the neurons are expressing Ube3a. So we can culture neurons and we can maintain the ability to see if genes are on or off. So this is our first proof positive that such a screen will work. And in fact with RSRT funding, we initially had a very small pilot project to see if this would be feasible for Rett Syndrome. Now I wanted you to appreciate the raw data of what it looks like when we are doing these types of experiments because I think it is important for parents to know the type of research being done and to even see an eureka moment that we had. If you look at one of these 384 well plates that once again had a florescent reporter inherited from the father, we add different drugs in quadruplicate from the wells, so this is just a blow up of a small number of wells here. We add different drugs, so we might put Drug A here, Drug B here, Drug C there… We always use the first two lanes for something called DMSO, which is just a drug solvent. So just like water can dissolve salt, DMSO can dissolve drugs. So just to make sure that we are not getting anything strange, that is a just a control we always run. The first thing I am going to show you is once again the stain for the neurons, because the what we don’t want is a drug that kills neurons. So this is a stain from a small subset of the plates here and you can see that all these cells are healthy because they still have neurons. These are healthy, these are healthy and these are healthy, but there are hardly any neurons in these so that means that the drug killed neurons. So that means that we don’t want to use these drugs because they are likely to have very high toxicity. That is one of the things we look for. The other thing we look for is just the increase in Ube3a expression, because the only way we can get Ube3a expression from the florescent reporter is if we unsilence this silenced copy. Focus right on here, you can see that there is one set of drugs, one drug that unsilenced the paternal copy of Ube3a. SO that tells us that none of these other drugs work except this one and that is our unsilencing agent. So this truly was our eureka moment and I can tell you in our screen we initially screened 2,306 compounds, that is depicted here, each one of these dots represents a different compound, plotted on the Y axis is showing the increase in Ube3a expression and out of all of these compounds only one of them unsilenced. There were some other ones that look like they might have but we retested them and they weren’t real. So out of 2,306 compounds, we found one. Question from Audience How long did it take you to get there? This is two years of very hard work to do that many. One of the things I was talking to Monica about, she is able to piggy back on this expertise that we have already developed so we are now able to move much, much faster than when this original screen was done because we have a lot of the expertise. It turns out one of the critical things is just culturing these neurons to keep them very healthy and to be able to put them in a 384 well format. So originally we only had one technician that was able to do this really well and now we have 4-‐5 that can do it really well. So that was one of the technical obstacles. Now the great thing is that I have learned about drug discovery is that sometimes you only need one hit to make some large inroads into scientific discovery. So it turns out that Irinotecan is what is known as a
Topoisomerase Inhibitor. It is an FDA approved compound and it is used to treat cancer. Topoisomerase Inhibitors are probably these most common chemotherapeutics. So there is good news and there is bad news. The good news is that we already know how well these compounds are tolerated by individuals so their health profiles are very well worked out. We know what doses can be tolerated. We also know from that having the fortune of watching someone go through chemotherapy that they are not without side effects. So these are compounds that do have side effects. The other thing is once you have one compound that works, you can start looking at other compounds that are either structurally similar or that they hit the same molecular target. So even though we only got one compound through our screening process, we now have more than 30 Topoisomerase Inhibitors that are effective at unsilencing the paternal copy of Ube3a. So this is just showing one of them; it’s called Topotechan, it is another FDA approved chemotherapeutic. You don’t see Ube3a expression paternally with a vehicle as a control. When we add this drug, we get huge expression. Don’t worry about this, this is just showing you that this is a compound that is working at a very, very low concentration. And it is and advantage to have drugs that work at low concentrations because they are less likely to have off target effects. Because as you know, if you take enough of anything, there is likely to be negative side effects. Now it is one thing to have a drug unsilence a gene in a dish, but can you have it unsilence a gene in vivo? To test this we injected the drug intrathecally, so into the spinal cord. So if anyone has had an epidural through childbirth for example, this is the same route for delivery the drug. Now this is an image, just a small section of the spinal cord showing you that there is no paternal Ube3a expression, so there is not much fluorescence there. We know that there are a lot of neurons, because if you look at this neuronal marker, you can see many, many neurons. Now I’m going to show you an image after we’ve injected the mouse for the two weeks with the drug. So we have injected the mouse for two weeks with the topoisomerase inhibitor and you can see we have expression of paternal Ube3a. I want to point out many caveats and many things we are still working on, and hurdles to overcome. One thing we looked at was how much expression of Ube3a we should be getting, do we want to target? We can answer that question just by moving the fluorescent reporter so it on the maternal copy. And you can see without drug, this is how much Ube3a expression we should be getting normally. So we still aren’t there yet, but we are moving in the right direction. Question from the Audience: Do you have to continuously deliver it? I will answer that question next. The question was do we have to continuously deliver the drug or was the effect long lasting? That is a great and very important question and I am going to answer it in a slide or two. Any other questions? Of the 2300 drugs or so that you used, were all of those FDA approved? Yes, that a great question. So the question was, for all the compounds that were screened, were all of them FDA approved? Our screening strategy, which is similar to the one we are using for Rett Syndrome, is to preferably screen compounds that are FDA approved. That was one strategy. The other one was to take ones that modify or are suspected to modify the epigenome. The other one is to take drugs that we
know get into the brain well. So all of those have advantages. If it is FDA approved, we know how well it is tolerated just for health reasons. We also know a lot about the molecular targets. Another reason for using epigenetic modifiers is that they are thought to modify the expression of genes already, so those might be likely drugs to try. And the reason for trying to use drugs that get into the brain well is that there are so many different compounds that don’t get into well, so you might as well start with the ones that get to the brain so they will get to the brain, the place that is most important that we get them. So the one of the ways we shape the drug libraries that we are screening is to pick the ones judiciously, because we can’t go through a million compounds. It’s just not practical with neurons. So you appreciate a limitation, most drug screens that are performed in pharmaceutical industries, they are performed on cell classes that can divide. So they just take cells and they divide and they divide, and divide ….and they get as many cells as they want and they can perform drug screens relatively easily on hundreds of thousands if not millions of compounds. Neurons do not divide so to collect neurons for these studies, the unfortunate part of my job is that we have to sacrifice a mouse and use neurons from mice and we don’t have the luxury of being able to use dividing cells. So you asked the really important question: is the un-‐silencing transient? The question is important for a number of reasons, because if it is transient you can imagine that you have to continually dose an individual with these compounds in order to maintain that gene activation. If it’s long lasting, then you could have a much longer interval between dosing. Especially for compounds that have toxicity, like the topoisomerase inhibitors, it would be completely impractical to give them frequently. This is an important issue. We can answer this in a very straightforward experiment. We take mice that once again have the florescent reporter on the silent copy. We give them drugs for two weeks and then we look immediately after drug, 4 weeks later, 6 weeks later 12 weeks later 52 weeks later, so almost a year later. I should say in these studies we are using a very low concentration of these drugs because it allows us to quantify the neurons more sparsely and in an automated fashion. These are raw data showing spinal cord images where mice were treated with vehicle so there is no un-‐silencing. Here are ones that were treated with Topotechan, either immediately after 2 weeks of treatment or up to a year of treatment. Because this is a pretty striking effect, I am going to show it bigger. That a year after we give mice Topotechan just for two weeks, in a subset of spinal cord neurons, the gene we activated, Ube3a, continues to be activated. Once again, there is reason for optimism and there is reason for caution. The reason for optimism is the best case scenario is we do a drug course for a limited time and we have a permanent affect. The caveats are that we haven’t shown we can have this lasting effect in brain neurons (this is spinal cord; and we don’t know if other classes can have this seemingly permanent unsilencing. Another caveat is that we are certainly affecting other genes; we are not just affecting the gene we are measuring. In the best case scenario we are affecting the other genes transiently and our disease gene permanently. So these are the types of things we are looking at now and we have some insights I can tell you a little bit about afterwards if you are curious. Once again, I want to remind you that there are limitations to this approach and to where we are currently. Some of these limitations we may come up during the discovery for Rett Syndrome, they may or may not come up. But I wanted you to appreciate some of the things that could arise:
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One is that our compounds are not the best at getting into the brain so they do get to the brain but not to the levels we want. So our big push right now is to find compounds that get into the brain better and we are lucky to have a whole host of topoisomerase drugs that are now working. We are kind of marching through those and seeing which ones get to the brain the best. If we don’t get into the brain, it could limit our ability to get a good behavioral rescue. You need to unsilence a large portion of neurons to see a benefit. We need to identify additional topoisomerase inhibitors or other compounds with better brain penetration. We are also continuing our drug screen at least for a little while longer in the hopes we are going to find something that is even more innocuous than topoisomerase inhibitor. The best case is to find something that is as well-‐tolerated as aspirin. We are also working very hard to determine the mechanism of action. So even if the topoisomerase inhibitor might not be useful therapeutically, If we can use this as a tool to figure out the mechanism by which it is un-‐silencing our gene of interest, we as scientist can then design very specific treatment strategies to un-‐silence our gene of interest without affecting other genes. So by understanding the mechanisms, we hope to gain insights.
What are we doing for MeCP2? We are nearly doing the identical sort of screen so I will go back to this slide that I showed you before. We are screening for compounds that can un-‐silence the inactive MeCP2 allele. So we are doing the same type of discovery project. So we are fortunate to have tools in place, just like we did for Angelman Syndrome, which will allow us to this. This is a tool that was developed by Adrian Bird who is also supported by RSRT, so once again this is donor dollars doing valuable work. And he developed a florescent reporter for activation of MECP2. So just like UB3EA was fused to a fluorescent reporter, he fused MeCP2 to a green florescent protein. So here are two brains, you would probably be hard pressed to guess which one has a fluorescent reporter, but if we turned out the lights, fluorescently activated and imaged them in a certain way, you can see a brain is glowing. You can see this is a brain that has MeCP2 fluorescent reporter knocked into it. So once again, we can see activation of the gene by florescence. So the screening strategy, and I know this is a busy slide, but I think you can appreciated the take home message, is that we’ll take mice and we’ll mate a male mouse that has the MeCP2 florescent reported. We’ll mate them so the offspring has, remember these are XY and the female is XX, the male offspring of these will not have any florescent reporter. The female offspring from this breeding pair will have the florescent reporter for the MeCP2 “GFP”. So with random X inactivation, half of the neurons will express the fluorescent reporter and in half of them the florescent reporter will be on the inactive X. Because they are only inheriting the reporter off of one X allele. Half of the neurons are expressing the fluorescent reporter and in half of the neurons, it’s silenced. We’re taking this population of neurons, we’re adding small molecules, and we are trying to make the inactive allele active, which we will detect by florescence. So once again, we have a population of neurons that is mixed, that have florescence and don’t, and we’re trying to make it, by adding, that all they are expressing fluorescence. So a very straight forward concept—we are trying to make all the cells green. Question from the Audience:
These still don’t have mutations right? Yes, there is nothing to do with mutations at this point. Right now, we just want to find ways to activate the inactive X, well I should say the inactive MeCP2. What we won’t know, until the later phases of this process is to see how specific these compounds are. So for example we could totally activate the inactive X or it could be very specific for MeCP2. But that is a later stage of the discovery process. The first stage is let’s just find compounds that will be able to unsilence the inactive MeCP2. The dream of what we hope to do is depicted here. You can imagine two unhealthy neurons and they are unhealthy because they have a mutated copy of MeCP2 and an inactive copy of a good MeCP2. What we hope to do is to find compound which we can treat these neurons, the drugs come in like a flurry of snowflakes, and they’ll unsilence the healthy MeCP2 copy and it will make the neuron healthy. So that is really the dream of what we hope to do. I should point out that this work is done by a large team of experts. Most laboratory investigators will tell you that they are nothing without the people in their laboratory, and their collaborations and the same is very true for me. This is my team of researchers in my lab. I have been fortunate to rcieve funding for the Angelman Syndrome project both from the Angelman Foundation and from the Simons Foundation, and this is another example of the importance of parent organizations and private foundations for providing money that can advance research in a really powerful way. For this project I have been funded by Rett Syndrome Research Trust and without that, the project wouldn’t be funded; it’s my only source of funding for this project at the moment. The team of researchers working on this project met just yesterday for an update report. Unfortunately I took the picture after some of the team members had left. These are the team members. Bryan Roth is the director of this drug screening center and Terry Magnuson is an expert in X-‐inactivation. This is the team of researchers that allows us to collect the neurons and to screen drugs for unsilencing. Once we have active hits, we will validate those and try to move forward in a very careful and methodical way. Once again I would like to thank Monica and the Rett Syndrome Research Trust for funding my research and for all of you for supporting that as well. Thank you for your time and attention and I am happy to take any questions.
