Channel: Stanford Cardiovascular Institute clear
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| 1 | Stanford Cardiovascular Institute | "Cardiac Cellular Reprogramming: Doing the Unthinkable" - Todd K. Rose... | 610 | 9 | 47.6 | 52:32 | Stanford University so it's my real honor to introduce my close friend dr. Todd Rosengarten dr. Rosengard did his undergraduate medical school with honors at Northwestern University and did his general surgery training back when we had general surgery training as part of our pathway did this at NYU and then he went up to Cornell in New York City to do his cardiothoracic surgery training and during that time he became involved with a very famous research scientist Ron crystal and the two of them actually pioneered a lot of the work in cardiac gene therapy and the world's first the u.s. is first trial looking at veg F gene therapy in the heart in a clinical trial for myocardial ischemia he's gone on to pioneer several new things in cardiac regenerative therapies and not only has he continued to be active in the surgical arena but has maintained a very active clinical practice in valve surgery and coronary surgery despite being a chair at two programs dr. Rosengard became the chair of the Department of Surgery at Stony Brook and then most recently became the DeBakey chair of surgery in Houston at the Baylor College of Medicine and among some of the people that he has the privilege of working with on a daily basis include names for those of you in cardiac surgery familiar with these names people like Denton Cooley and bud Frazer and Joe coselli and a variety of other luminaries who have published really a lot of the history and the development of cardiac surgery these are the people that dr. Rosen Gard has the opportunity to work with on a daily basis now so dr. Rosen Gard also has had a very viable and continuously funded research laboratory funded from the NIH with a tremendous publication track record studying gene therapy and cellular reprogramming and other regenerative therapies and is here today at our invitation to educate us on novel reprogramming technologies and trying to do the unthinkable thanks Johanna while we're getting us started up first of all my thanks to all Joe and and Joe and a great old friend and a great new friend and it truly is a pleasure I don't need to of course tell anyone here how significant and memorable Stanford is but by coincidence I will mention that one of my other side jobs is editing one of the cardiac surgery journals Seminoles own thoracic and cardiovascular and we recently ran a special issue on great institutions in cardiothoracic surgery and the the very first article was Stanford a hundred years of history here that Jo Jo Lou surgeon contributed and of course truly a remarkable place that we all from the outside you lose perspective on on who you are and what you're doing when you're at a place for very long but of course needless to say stamp Stanford is a a very very special place in all of our consideration so I'm going to talk to you today a bit of science but perhaps equally importantly especially for the younger residents and our PhDs and our scientists in the room the concept of thinking the unthinkable and really most of what I'm going to talk about about my 30-year journey in terms of cardiovascular medicine and science and discovery is really unthinkable and as we've heard this morning most of what we're talking about today if you talk to some of the older people in the room would really be close to heresy to suggest for example that you could grow new blood vessels or let alone take a fibroblast and turn it into a cardio myocyte you would literally have to be committed you would be banned from future science and the like but the again the theme of what I'm hopefully will convey again especially to the younger scientists is if the data are there and you believe in what you're doing think the unthinkable and go for it and again I think a lot of what has been ah so wonderful and I'm so grateful about it now my career is having had the opportunity to do that so the theme of what I'd like to talk about today is the treatment of a heart failure as many of you know this is remains a very very significant problem in a merican medicine five million Americans have heart failure current medical and surgical therapies are still associated very high mortality limited of applicability as we'll talk about 300,000 patients or individuals annually required transplant or VADs but less than 5,000 transplants or VADs are performed annually of course Stanford is a truly the epicenter and the home of certainly heart transplants and of course the VAD program here is a second to none so in many ways there are probably at least a million individuals in the United States annually who are candidates for something other than having their heart cut out heresy to say it here but it's true or having a machine put in their chest to take over heart function wouldn't be it nice if there was a biologic way of repairing someone's own heart so we didn't have to undergo such extreme surgical procedures again hard to believe a surgeon would be saying those things but it's true again as I mentioned even under the best of circumstances Marc Marc slaughters data from New England Journal in 2009 there's still a 20 or 30 percent two or three year mortality associated with even even the best of mechanical circulatory support devices and then again of course I'm having to do heart transplant is is not a trivial undertaking recent story as we're going to talk about we're getting ready to undergo begin a angio genic gene therapy trial and I was sitting on the medical review board over Texas Heart just a few months ago there's a patient with end-stage angina and literally had failed all medical therapy no bypass no angioplasty operation and they were literally talking about this patient who said we're going to do a heart transplant as nothing left to offer this patient normal ejection fraction just advanced engine oh yeah sheepishly raised my hands and fretted in the room with all these heart transplant surgeons I said before you cut this patient's heart out maybe we can inject some genes into the patient's heart and let him grow his own bypasses and by the end of the meeting I had about seven cardiologists lined up wanting to enroll patients in the trial clearly underlying the fact that there's a tremendous need for what not needs to come next other than such aggressive surgical therapies so I'm going to talk about this morning and I will get to this towards the towards the end is something very different and this will be a little heretical then what is customarily thought of the cell therapy for treating advanced heart disease most of the time an important that we're clear on this definition and depicted on the top right is a obviously a cross section of this experimental animal of an infarction the area of a thin tissue over here most of the time when we're talking about stem-cell therapy and again I'm probably gonna offend a number of people in this room right now what we're really talking about is depicted over here this is again an experimental model and we're taking a large area of infarction and we're judging a bunch of exogenous cells stem cells mesenchymal stem cells embryonic stem cells iPS cells even taboo area to a be critical of and what we're doing is something that ends up looking like this and if you talk to most stem cell scientists they will say yeah that's what it looks like so you have this giant area c of fibrosis and you have islands of stem cells working there and then these scientists say well this is great this improves heart function dramatically and I and others will argue and the clinical trials will support it really does not makes much sense that these islands and stem cells sitting in a sea of fibrosis are really impacting global heart function so we'll get back to that we're going to talk about this after afternoon is incite to sell you're reprogramming this sort of crazy idea that you can actually take some of these fibroblasts in the area of my myocardial infarction actually convert them by direct reprogramming back into a cardio myocyte phenotype and thereby improve function so let me put it that aside for a minute and go back about 30 years it's hard for me personally to believe that I'm telling a story that's thirty years old but it does happen to all of us and I guess I'm there and talk about the problem of advanced coronary artery disease so again as with heart failure still a very significant problem despite the fact that we do over a million a coronary stents almost half a million coronary bypass procedures annually but the secret truth be told is that about in half the cases we don't completely revascularize a provide blood flow to the entire myocardium that said risk because of ischemia or poor blood flow because of coronary disease in fact through a number of large studies that have just been done in the last few years syntax freedom among others we now know and we didn't know this up until about five or ten years ago that if you don't completely revascularize myocardium the mortality risk is about double compared to patients that are normal or completely revascularize so again a tremendous opportunity for something other than bypass or and your power angioplasty to improve things so we are going to talk about having the heart grow its own bypass is biologic bypass or angiogenesis and that's going to take care of everything so let me go back thirty years I was a resident working it down at the NIH my mentor Wayne ice and was the chair of us or cardiac surgery at Cornell said I didn't know what to study or what to do research on it showed me two angiograms one of the patient who had a totally occluded a coronary artery the other had grown would appear to be collaterals they said go and figure out how to grow those collaterals i saluted dr. Reisen and i marched off to the nih and you come back when I have it figured out of course that did not happen and back in the 1980s the only thing we knew about any of this this is truth was that work by Tom meshach and others who were just beginning this is true to learn how to grow endothelial cells and culture this is 1980s that's when this whole story what Tommy Jack realized and showed in this paper in science in 1982 is that if you would drew a culture medium support protein called at a time heparin mind in growth factor-1 or acidic FTF today you grew these structures in vitro and cell culture that sort of look like capillaries that was it that was the entire initial field of angiogenesis these funny things in cell culture that look like capillaries we were talking this morning with dr. nan and we were saying that you know well how do you do these things and you need to have the proper combination of growth factors it's got to be in the right sequence and in reality if we ever tried to grow a blood vessel and come together with all of the different structures and molecules and cytokines and integrins and the like to make it all happen we all would have quit a long time ago but being dumb surgeons we didn't know what we didn't know and we just said we can take ng jobs such as fgf or in our area of interest veg F and buy some very fortuitous a pathway if you administer those exogenous antigens the entire angio genic cascade will get stimulated and that's exactly what happened we became interested within vascular endothelial growth factor for a number of reasons now none less than there was a very potent angiogenic mediator as I think everyone knows it comes in three different isoforms 121 165 and 189 and essentially all of the work that was done involved one of those isoforms we'll get back to that in a minute as a Joey mention I would had been doing my work you know angio genic therapy collaborations and relationships which you all here at Stanford so fortunate to have or critical I think to a scientific success and I was fortunate to begin a collaboration at Cornell with Ron crystal who is Joe mentioned had been sort of the innovative one of the early innovators and pioneers of gene therapy and sort of like Reese's peanut butter cups when you have the right combination of two things of nothing to do with each other my interest in angiogenesis coincided with our lawns interest in gene therapy and I said I've got this vascular endothelial growth factor I don't know what to do with it and Ron said I've got this adenovirus vector and I don't know what to do with that well so why don't we put them together and we didn't end up with Reese's Peanut Butter Cup but we ended up with a nice collaborative research so ad virus and despite speculation to the other wise is a tremendously effective delivering a fairly large packages of cDNA for to induce transgene upregulation again in our case we are interested in veg F is in particular very efficient at getting into cardiomyocytes ideal for cardiac gene therapy applications and importantly on this is something that is miss no or misunderstood about adenovirus is it is a short-term expression vector last about three to seven days it remains epic chromosomal in a location thereby preventing a mutagenesis and it down regulates not through necessarily through inflammation but probably by means that we don't completely understand so again very simply I think everyone knows that the concept is really just regional pharmacotherapy we are repackaged adenovirus with the aussie DNA in this case for veg F 121 we are delivered to the myocardium in order to turn on a local of fiber cardio myocyte induction of a veg F the concept is the gene therapy as we envisioned it was just a local drug delivery therapy no more no less in order to deliver veg f ng Jen in order to cause new blood vessel growth to revascularize scar my aquarium that could not be treated by standard therapies well back when we started no one knew whether this would work went to a small animal model with AD Jeff's 121 and his concede on the right be able to induce profound angiogenic effect in a rat retroperitoneal fat pad we found very early on that this was just pharmacokinetics you give more veg F or adenovirus you get more of an effect this is a standard pharmacokinetic therapy I mention this because people forgot that this was just a drug and they thought it had magical properties and what ended up happening were applications that were inappropriate and unfortunately it caused the entire field to crash and ended up setting the program back for about 10 or 15 years because people forgot these basics again importantly adenovirus works very very well because it ended up that we just needed 10 days or so of trans veg F trans gene expect expression to have an effect conversely if you used a chronic expression vector like adeno-associated no associated virus as shown in this paper by Lee you ended up with hemangiomas more trans gene more veg F deleterious effects limited expression and preparin as we later showed solid efficacy of all the slides that are and data that we published the one that or we produce the one that we actually never even published is this slide right here probably more important than any work that we did probably cost the gene therapy industry hundreds of millions of dollars in false starts and lack of success including companies so that presumably is a bright as a Genentech and it is all all that that is depicted right here this is a very very simple study anyone's child to grandchild in this room could probably do this study in very early on in gene therapy days we asked a simple question what's the best way to deliver adenovirus you get the trans gene into the heart and get in effect if you injected intravenously should we inject it in the coronaries as she injected directly in the myocardium simple study here that here's the results and if very clear to see you have about a 1 or 2 log fold increase in local myocardial expression we use a beta galactosidase marketing if we went with intra myocardial delivery versus intra coronary delivery or intrude intravascular delivery much more efficacious to go with direct intra myocardial delivery we'll come back to that point in a few minutes so we had our vector adenovirus short term expression that's all you needed we didn't need to form hemangiomas we had our transgene veg F we had shown in vitro and in small animal model was effective went to a large animal study and this was sort of our Eureka moment back in the mid 1990s we took Pig standard model we put a circumflex amyloid constrictor around it tied off the coronary artery just as if you were having a heart attack we treated the animals either with the null virus without veg F or virus with veg F 1:21 and as exactly as we saw in human angiograms we saw that we were able to grow a plexus of collaterals and literally reconstitute the circumflex coronary artery just as if as a surgeon I was doing a bypass operation on the basis of this large animal data we went to FDA in the early 1990s we said we wanted to treat patients with end-stage coronary disease we're just going to take this syringe injects this solution to the area of an farc Shen grow new blood vessels and the FDA said you have to be kidding that's the craziest thing we've ever heard of we're not gonna let you do it and I don't care what kind of data you have this is very bizarre and you all should leave and we really were up against a complete dead stop all we wanted to do is take a tuberculoma syringe inject 1/10 of a CC and of this veg F solution into the heart and we really didn't know what to do so for those of you who have been around for a while this around this time in the mid nineteen ninety there was a technique called TMR trans myocardial laser revascularization and some crazy of physicians and surgeons had the idea that if you took a laser beam and drilled a series of holes in the myocardium you could reproduce a reptile heart and the heart would profuse itself directly from the chamber well that for some bizarre reason had already gotten FDA approval but the key point of the story was that we happen to have a video from the local Channel 2 News in New York of Craig Smith and well-known surgeon using the TMR laser beam to drill those holes in the heart with this fda-approved trial but more interesting the local channel 2 reporter thought that it would be dramatic if he took the same laser beam that they were drilling holes in the patient's hearts and took a 2x4 and drill took the laser and drilled holes to the 2x4 so we're at lunch break at the FDA and really we're about ready to go home and I said Ron you know Ron crystal my partner Mabley she showed the videotape but the 2x4 that's a little funky you know I don't know and I said let's give it a shot so we put the video tape in I was back in the days when I still videotape FDA panel 20 people in the room turn on the video laser goes on on the video two-by-four smoke flames fire everything and we said you guys just to prove this last year all we want to do is inject the solution in the heart we got approval and now we're off to our clinical trial great science at work absolutely true story so the first trial was done in December of a 97 at Cornell as Joe mentioned first time anyone at least in the United States had ever inject a virus into the heart a little bit scary we found a lawyer from Westchester to be our first subject and we went we went on and did a series of patients literally just injecting a grid of a sort of gold elack's a little red riding-hood with a breadcrumbs in order to inject it really did not know much of what we were doing our most famous patient was columnist for time Meaghan magazine named Lance Morrow Oh get that that back to that in a second but after a phase one dated we had encouraging a preliminary data our patients seem to be feeling better there's a lot of controversy that this might be a placebo effect but we continued on and this went on to a faith to study in Canada we were not involved in this basically looked at an FDA approval endpoint which is time to one millimeter ST depression and what was shown here with a very small and 26 patients looking out to 26 weeks after therapy patients either getting the adenovirus with veg F or a medical control group - statistically significant improvement in evidence of EKG changes not just angina with this a very very small group so pretty encouraging look pretty good and in fact in that study there was one patient who died for unrelated reasons they're able to get an autopsy this is a alkaline phosphatase stain that's brown from blood vessel this is an untreated myocardium you can see a couple of blood vessels this is treated myocardium pretty impressive looks like where the bed jeff was delivered significant increase in angiogenesis and Vascular ization but then as I mentioned and go back to that pharmacokinetic study that I showed you early on interesting thing happened we were going but direct myocardial injection again this is what our data showed us we found it that we and others who had used direct my connection found that there was appeared to be a significant improvement in terms of exercise treadmill based upon EKGs but then there were a number of trials the vive trial again this was a Genentech trial Nicola Napoleon for our brilliant scientist Chiron to study with basic fgf and in these placebo controlled trials again these were in Turkana a so they could use a placebo no significant improvement in terms of exercise treadmill everyone said you see those scientists those guys at Cornell were wrong they were oh this was all placebo effect because it was Rep myocardial administration everyone wanted to feel better when you did the good placebo-controlled intra coronary trials no effect and therefore placebo controlled trials are always better than open label and therefore field doesn't work we of course argued that there was a hundred fold decreased uptake into my car diem with intra coronary fell on deaf ears this is all sort of written off as a placebo effect and then a very significant thing happened again let me go back to an anecdote and admittedly this is an anecdote our most famous patient was a columnist for Time magazine Lance Morrow who had done a story about gene therapy again this is in the late 1990s and said then came back a few weeks later and said you know I did the story but I'm actually need to be in your trial I've had a couple of heart attacks that can barely get across the room anymore and he subsequently went wrote a trial about going back and playing squash and doing doing great in fact we stay in touch to him to this day ten ten or fifteen years later and continues to do well interestingly so again looking pretty good gene therapy seems to be working and in a tremendously a tragic event happen again probably everyone in this room will recall back in the late 1990s a young man named Jesse Gail singer got about a thousand fold excess dose of an adenovirus he was being treated for a benign inborn an inborn error of metabolism of onething trans carboxyl carboxylase was not affecting him at the time and he died from inflammatory effects of a thousandfold XS dose of adenovirus delivered intravenously and basically this put a hold on all gene therapy studies in the United States and FDA moratorium and it literally took about ten or fifteen years for the field to recover again very famous scientist Jim Wilson and his team at Penn had been doing great work in the field and have clearly made an error in terms of administration so then an interest same thing happened we as many others walked away from the field Joe Joe loo surgeon had been involved as well and we all really went on to other things stem cells in particular I at a time had lost left Cornell gone out to a northwestern and then came back to New York about ten years later interesting things started happen one after another a number of these patients who had been in the original gene therapy trial started calling say hey doctor Rosengard I hear you back in town just wanted to let you know I'm feeling pretty good I don't have anymore angina and glad you back in New York great that's really nice second time happened third time happened about the fourth time one of these patients call and I said to myself and sort of almost say that like you know I thought you'd be dead by now it's you know 15 15 years later and you had end-stage Carter just peace you tell me your engine of free and uh you know fortuitous Ness is only useful unless you do something about and in speaking to Ron crystal he said we really need to follow up on this we track down all of our patients about 32 who had received gene veg F in our trial and interestingly just as a data point what we found is that the survival of these patients compared to a TMR control group and TMR again was I got no statistics here but seem to be better certainly ten-year median survival much better than we would have thought in this n stage coronary disease patient and perhaps more interestingly you can think back to the angiogram of the pig here's an angiogram ten years after veg F therapy and one of the patients and we had about three or four of patients with similar angiograms you can see that again that plexus of blood vessels this was exactly where had given our veg F therapy persistent 10 or 15 years later so we got to thinking about it and we decided maybe we need to go back and pick up the work where we left off don't isn't it isn't it the right thing to do and in classic sort of Silicon Valley star is telling the story to a friend actually a gentleman Hudson news if you in the news in the airport and he said you know this is you really are should be ashamed a self Tod you really should continue this work and I said oh you don't understand it's complicated and he persisted and persisted gays gave us some seed funding and we went back to the laboratory and said if we had to do it over again how would we do it differently and better and so one of the parts of the veg F story again as everyone knows is that veg F just doesn't come just as a single polypeptide it actually under those post post transcriptional splicing and ends up is three different major isoforms 121 165 189 again is anyone in the field knows they have different biological functions they do things slightly differently in terms of relevant receptors heparin binding and the like and he said well you know if Mother Nature has three isoforms maybe just delivering one isoform like 121 and 165 how the trials were done is is not the most effective way and our on crystal and his team who are really truly experts and quite innovative created this genomic veg F which we called veg F all that actually expressed all three isoforms so seen here on this Western over here and all three about 2 to 2 to 1 121 165 189 I went back to the laboratory looked at a hind limb ischaemia model and what we found basically is that the veg F all was approximately a hundredfold more potent than veg F 121 which was encouraging in terms of a new clinical trial in fact Ron went one step further looked at alternative alterations of the of the proportions of 121 165 189 and we created a veg F all six a plus this has to do with the expression of the six a.m. exon and what he found long story short is that veg f68 plus is just as potent as veg F all but when you look at things like pulmonary edema and even immortality' the Jeff 6/8 plus was safer likely because it was less veg f 121 even though we had no trouble in our clinical trials but because of its ability and induced vascular permeability and the like so we went back to the FDA we saw the TMR tape in our back pocket but at this point interestingly when we went to FDA they had no concerns about safety which is very very significant considering how far the gene therapy field had come but when we proposed to them and received approval from the NIH for comment visor e committee as we said look the biggest problem with the veg F trials back in the 90s was that the ones that worked such as ours we argued um didn't have a placebo control and so NIH and FDA said well not only do we think having a placebo control veg F trial is an interesting idea we actually think it's the most ethical thing to do and the amount NIH recombinant Advisory Committee is depicted here actually approved a clinical trial we had randomized patients three two one two with end-stage coronary disease either get add veg F all or actually get an ADD null vector that might have some inflammatory effects but it would clearly was a negative control and then I had to prove that unanimously eighteen to zero and FDA although they want us to stop in Part A which is the open label trial will hopefully be supportive as well and we're about ready to undergo that trial which we'll look again at the primary endpoint at time to one millimeter ST depression other standard secondary endpoints interestingly we're going to include cardiac pet which we think will be more sensitive and we'll be conducting this study in New York at Cornell with Ron Ron Crist on this group and that the Texas Medical Center which everything's bigger in Texas so it's the world's largest Medical Center and obviously a hopefully a very suitable place to recruit patience so that's where that story leaves off and now let me go back to a little bit of sort of unthinkable science so I think part one is the unthinkable part of what you can do with some some persistence and belief and the basics of the science let me go back and talk again about what I think from a scientific standpoint is many in this room know and have been working on something even more unthinkable which is taking one cell type by direct reprogramming in turn converting into another obviously this field all came about because a Yamanaka is work with the IPS generation is depicted in the box but the problem is I alluded to earlier is that with all stem cells exogenously delivered the problem is how to get past integration into the host genome host milieu the whole host of myocardial matrix in a way that's clinically relevant so deep extra warszawa i've just up the road at UCSF really was on the pioneer amongst other as Eric Olson in taking Yamanaka his work and taking it one step further and basically he said well if you can take an adult somatic stem somatic cell use ox ox klf4 to induce IPS cells perhaps you could take another combination of transcription factors and directly reprogram without going through an IPS stage from one adult somatic cell type into another and what servoz Tova showed in his work that came out about now I think four years ago was that you could take a gatah form F 2 C 2 X and TB x 5f to say and TB x5 which are associated with cardiac differentiation in the embryo logic state using a lentivirus and directly trans trans differentiate fibroblasts and to induce cardiomyocytes and service are in a very elegant papered seem to demonstrate this we quite frankly couldn't believe it you know never having been taught anything like this was possible only repeated on the service of his studies and in fact duplicated what he saw so it's depicted here if you start with cardiac fiberglass in vitro administered GMT you could actually up regulate cardiac specific markers in this case looking at cardiac troponin T and GMT treated fibroblasts versus control fibroblast we can see market a dramatic staining for cardiac troponin T if you look at other markets such as myosin heavy chain 7 or alpha sarcomere actin similarly up regulation of these markers and in fact survived among others went on to show that in vitro you could actually induce contractile cells this creates a sort of - to me is a is a cardiac surgeon in my day job an intriguing possibility which is instead of administering exogenous stem cells and trying to get them integrate into the host myocardium perhaps you could again take these transcription factors just an aliquot of solution injected into a large area of scar and actually trans differentiate scarifier of last insight - so they're already integrated into the host myocardial architecture to induce cardiomyocytes and therefore regenerate my accordion from areas of scar tissue so again very very important to think about the basics and one of the first questions we asked which sort of befuddled us and really hard to believe we thought we were missing something is that the I don't understand question and against of thinking of this as a cross-section of heart infarct in myocardial here why did the heart become fibrotic it was there it was because it was an inadequate blood supply all of those residents cardiomyocytes died became replaced by fiberglass so why if they're still ischemia if we injected cells into the scar tissue would you get anything other than persistent scar and they injected cells undergo the same fate as the resident cells which would to be to undergo apoptosis or otherwise die in fact work by Maria McGovern showed exactly that if you inject exogenous cells about 98% of them are dead within a couple of days either because of poor delivery or again ischemic loss so we did a series of experiments this again is about five or ten years ago we were still at this time looking at stem-cell delivery and we said okay well since we're angiogenic people everything if you have a hammer everything looks like a nail why don't we create a scar let's previs karai's the scar with veg F so we're essentially fertilizing the soil before we deliver ourselves and we speculate that if you pre vascular eyes provide a blood supply to that scar tissue then inject those cells you'll have a better result than if you just try to get those cells to stay alive in this hostile ischemic milieu and we did some experiments early on and we treat animals with veg F and nothing sells in nothing or a combination of pre vascular ization we actually put the rats on a treadmill this is clearly not a Stanford student in the front and what we showed is that with the pre vascular ization we couldn't significantly enhance exercise treadmill results and that became our standard model of pre vascular ization so come back to that in terms of further studies so if we're going to do insight to celery reprogram again again the same model pre-vet and induce like infarction by coronary ligation pre vascularized wait three weeks which is how long we knew it took for the new blood vessels to grow and then either administer get gado-gado GMT got a form F to CT bx5 or a control vector and then wait look four weeks and look at results and much is savasana and others showed quite dramatically and a tremendous surprise to us if you look at histology this is control bland infarction coincidentally or not and the data is the data and the treated animals we see these distant islands and strands of myosin heavy chain positive staining cardiac myocytes being induced cardiomyocytes and natives couldn't tell for sure looking at trichrome staining for fibrosis control not only is it tremendous thinning actually of aneurysm formation compared to an almost normal-looking cross-section of treated myocardium and then is shown looking at ejection fraction by echocardiography again what appeared to be dramatic improvement and function again looking at is statistically looking at myocyte density statistically significant improvement and induced cardiomyocytes statistically significant improvement in the amount of fibrosis and finally looking at ejection fraction veg F pretreatment plus GMT statistically significant improving an injection fraction at dramatic twenty twenty thirty percent levels with this reprogramming vs. control treated rats interestingly if we were using three vectors and this is sort of confirmation of what we had done previously the GMT we're all being administered with three separate lentivirus vectors if we combine those into a single triplet vector so each cell is not needing to be exposed to three different trans genes but all in one vector at the same time we showed a significant improvement in function this at this point it was a up to a 40% improvement ejection faction using this triplet vector again very very encouraging a data suggests that you could actually reprogram hearts by this technique most recently this is a material that will hopefully be a presenting this year going back to or you originally talked about all of this worked involve Lenti virus or retrovirus chronic expression vectors but most recently with now reduplicated these efforts and show that we can actually use an acute expression adenovirus vector to also induce improvement in a heart function so perhaps most dramatically olson and others have most recently shown that even though we're getting these very very encouraging results in lower order animals such as mouse and rats when you get to higher order animals such as humans the all of the data suggests that ourselves a much more resistant to reprogramming probably because of epigenetic stabilization compared to lower order animals and in fact it adding GMT to human cells in particular would not induce reprogramming and Olson and others have now shown that only by adding multiple of other factors such as hand to Maya carton on top of the GMT could you induce the reprogramming of cells such as human cells so obviously - I study these things we need to needed a good surrogate and most recently and I don't know if this will play but we've actually now been able to go to in vitro Pig cells and as you can see here we've been able to induce beating Pig induced cardiomyocytes these were originally fibroblasts that are now beating based upon administration of reprogramming transcription factors so they're our most latest work and the theme of this could potentially be we could be tadpoles if you'd like is how do we get past this problem of anti plasticity of human cells versus Road in cells and probably and this is an area of a new investigation there's a lot going on in human cells it's not present and rodent cells that are preventing our gene gene act genes from being activated into factors are probably very significant in this that we found out through IPS studies is p53 and p63 so obviously everyone knows that p53 mutations and one of the primary causes of a hunka janessa t but we actually believe we have found that we could actually turn that around and look at it from a therapeutic standpoint and not focusing so much on p53 but it's cousin in the p53 family p63 as much as this when these are down regulated they promote mutations and oncogenesis 'ti essentially by allowing increased cell plasticity we could take that to our advantage in terms of reprogramming and we most recently have shown that if we down regulate p63 this anti plasticity gene we could significantly increase as shown down here our ability to reprogram fibreglass into cardiomyocytes as evidenced by nearly a sevenfold increase in CT expression in these cells so very early work very exciting essentially suggests that there are a lot of ways to enhance the plasticity of mature somatic cells such as fibroblasts for the purposes of reprogramming as suggested by these data so in conclusion really on what has been a incredible hard to believe 30 year journey I think we are back in the gene therapy arena we will be seeing more and more in terms of clinical applications including angiogenic gene therapy trial that we look forward to a beginning this year we think that I'm going back to the basics and the concept of pre-treating the infarcted myocardium is incredibly important in terms of making sure you have essentially all of the principles aligned in terms of ideal opportunities for enhancing these therapies and finally although formidable variant remain and work at places like Stanford and others will inevitably and undoubtedly lead to advances in the field we think that there's an entirely new chapter to be written in the next five to ten years terms are using a cardiac cellular reprogramming to completely rewrite how we treat patients with advanced heart disease so again regenerative therapy is a truly a new paradigm for treatment of heart disease we think it is something that will be as seeing increasingly greatly and I thank you for your time and attention thank you so thank you very much for that amazing lecture showing us both some history and really what the future is going to be I think we have just a couple moments for a few questions from the audience Phil yank yeah so as we're talking our thanks Phil as we were talking earlier I think what's most interesting so this is kind of data you see from your own laboratory I said well what did we do wrong you can't really believe it and one thing that's heartening sort of speaking that is now about four or five groups Olson's Rostova some of Deepak's disciples have now gone back to japan we're all seeing the same thing so the fun part is I think we've got the end point which is there truly is 20 or 30% to give us statistically significant proven and function which of course is a clinically relevant endpoint we actually now need to take a step back and really really understand what's happening and I think it would be foolhardy for us to say we're creating these cells and that's the story there are clearly other things going on for example there's probably an anti fibrotic effect it's potentially gatah for playing a role in that there's probably other things going on and we just don't understand and for anyone in this room who's looking for something to study I think especially because we know that it we don't know but we seem to have evidence that it works it's just a fertile for a lot portunity to go back and start understanding mechanisms that's obviously what we're spending a lot of time doing the efficient so the other thing I would say that again is fascinating this field is it it appears and we're going to talk about this morning that the efficiency in vivo um sort of fortuitously is actually better than the efficiency in vitro and we see that a little bit one of the magic tricks that I didn't fully explain was that on those beating pink cells they only work if you have coal culture with cardiomyocytes so then again we feel comfortable that these truly are the pig fibroblasts but yet you need the co culture and again Deepak has shown that too this is clearly in vivo insight to crosstalk communication hand-wave and we don't understand it that is important to making this more successful more efficient in vivo so wide-open opportunity to try to figure this out yeah so and again I was talking to Joe about this earlier we believe in ad now and one of our greatest frustration we believe in it not just intellectually or emotionally because we've used it clinically we intensely looked at safety parameters we've never had safety parameters issues we know that it works for as long as we need it to we don't want to have chronic expression with AAV and one of the challenges again is one of the sort of the fascinating things about how the feel that works is that is that the science and the sort of you know feeling on the street just don't line up and one of our challenges all of us and again Stanford of course is one of the great incubators of translational science is to convince and explain to the lay public why their impressions are not scientifically based and much more than a science communicating fault false miss properly communicating people away from false impressions that I'd know is bad are one of the greatest challenges we face much more so than just the science so yeah we are using ad know we think it's the perfect vector for this utilization and interestingly when we went to FDA and NIH and said we want to use ad now there's no issue there completely supportive they actually not even asking us to do all the safety studies that we did in our original phase one studies and again for those who actually they knew we had the tape right very interesting paradox that goes beyond what you do in the laboratory one last question whenever gene therapy comes up I always ask what the status of for cardiac gene therapy what the status or theoretical potential of coxsackievirus would be as a vector you stumped me can't answer good are you gonna tell me no I just think I think of the natural disease process in the relationship of Coxsackie to myocarditis I mean it's a vector that's in nature designed to get into cardiomyocytes and no one has really harnessed it and it's just something that's maybe it's too dangerous to do again anyway thank you very much for that wonderful talk and I would invite everyone to join us in the photo Stanford 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