 All right, so I'm excited to talk to you today about a project. I've been involved with inductor and bodies lab and in collaboration with other researchers in the lab and I'm very fortunate They're allowing me to present some of their findings as well so that I can tell you about kind of a full story of How we have looked at the modulation of soluble flit one within the eye and beyond so We all kind of know the clinical impact of the angiogenesis and Just as a quick reminder it certainly does impact all aspects of ocular pathology from the cornea to the retina and has Significant implications for patients and their vision Angiogenesis on a molecular level is known to be quite an elaborate process It was really the research of angiogenesis was really pioneered by dr. Judah fulcman who was a surgeon in Boston and I love to highlight that he was a surgeon because many times people think surgeons aren't real Investigators so he really is credited with a lot of the Initial work in the field of angiogenesis and this schematic just kind of details Actually, some of the basic steps necessary for Angiogenesis to occur so clearly it is An elaborate process with lots of molecular Signals and cues, but we do know now that a final common pathway in angiogenesis is regulated by vegev so the Jeff is released in response to primarily an ischemic stimulus and Once released it then binds its receptors on endothelial cells to promote Sprouting of new blood vessels from existing blood vessels, which is the process of angiogenesis pictured here and then this process is Integral to a lot of non pathologic processes as well as pathologic One so for example endometrium growth wound healing, but also solid tumor growth And certainly a number of pathologies in the eye, which I've already highlighted so The cornea then is kind of a unique molecular environment in that it is a vascular and It needs to be certainly to have a clear medium to look through as we know, but We also now know thanks to doctor and body's work as a graduate student that this avascularity is Entirely dependent upon the expression of soluble vegev receptor one and And the way that this works is so the Flip one is also a name for the vegev receptor one so on the DNA level Once transcribed we now know that soluble flip one Is expressed due to an alternative splicing event that occurs at the level of the pre-mRNA of this Vegev receptor one so in in this way you get a soluble form Containing just the ligand binding domain and not the transmembrane portion of the receptor And you get the full-length form which includes both the soluble Or both the ligand binding domain and the transmembrane domain and so that is pictured here and so doctor and body's work was so instrumental because he essentially showed that Expression of soluble flit one is essential for the avascular nature of the cornea and that furthermore if you Take away expression of soluble flit one and he did this in a number of ways at the protein and the molecular level Then you see spontaneous vascularization of the cornea so If we take this information and add it to the schematic I presented earlier soluble flit one then acts to Bind vegev prohibiting interaction with the membrane bound receptor Which is what is necessary for angiogenesis to occur and so in this way we block angiogenesis All right, so based on what I told you Is it possible then that the presence of an expression of soluble flit one could? represent a Way to modulate angiogenesis Such that we could alter pathology for example if we increase soluble flit one so thereby inhibiting angiogenesis would this then allow us to modulate disease states such as corneal vascularization solid tumor development, etc and alternatively By decreasing soluble flit one promoting angiogenesis Could we then modulate disease states such as stroke heart disease encourage wound healing things that? would benefit from more vessel growth and Indeed this is the question and so to to address this Preliminarily some key questions needed to be addressed so number one Can we effectively alter the expression of soluble flit one in vitro or in the Tissue culture dishes as well as in vivo in mouse models and etc and then To kind of get right to the point are there molecular tools allowing direct bench to bedside Translation so can we use tools in this setting or in the mice that then could have potential to be used in humans Thereby getting to the end result more quickly Secondarily does increasing soluble flit one assuming we can do this does that then reduce or regress new blood vessel growth within multiple model systems? because we want to Apply this broadly and so we need to test our our hypothesis in multiple models to show that Ubiquitous application and then finally is the level of inhibition that we might see here is that sufficient to alter disease progression Okay, so we'll focus on this first question So there are tools called morpholinos which are Have been used for a number of years in zebrafish, but are now being used more and more in mammals mice and humans actually and this work was pioneered within the Though research for Duchenne muscular dystrophy because it's known that the dystrophin gene has mutations such that There is improper reading frame and splicing etc So people have used morpholinos which are essentially engineered fragments of DNA which are Complementary to your target of interest And they've used these to modulate gene splicing and then therefore expression in humans And you can see here they've started clinical trials with these tools and have been quite successful so Thinking about that we wanted to apply the morpholino system To our question so this is a schematic here of then the flit one Receptor at the DNA level so we've engineered some morpholino Sequences that are complementary to regions flanking the alternative splice site necessary for The transition of soluble flit one expression versus membrane flit one expression So using the morpholinos we predicted an increase then By altering the splicing event we predicted an increase in soluble flit one expression and In fact we did find this to be true in multiple cellular backgrounds And so these are several these are two different cancer Cells that were grown in vitro and you can see that using the morpholino We do see an increase in soluble flit one expression That's on the RNA level and then on the protein level in human endothelial vein Cells we see an increase when measured both by Eliza and on the in the western blot we see an increase In the protein level of soluble flit one and so we believe that these morpholinos Do in fact modulate the expression of soluble flit one so then the question becomes in using a disease model system What does this mean in terms of vascular? The vascular nature of the disease and the disease pathology All right, so the first model we used was corneal neovascularization. Why did we pick this? It's convenient It's clear and it's very easily accessible It also has been very well studied in terms of neovascularization and just to remind About some data. I mentioned earlier. Dr. and body was able to show in this exact model system that when you Inhibit soluble flit one expression By knocking it down you get spontaneous vascularization of corneas and Conversely when you have spontaneously vascularized corneas for example in a pack six heterozygous mouse That is the way they are at baseline due to this mutation When you add soluble flit, which is low in the in the normal condition you regress those vessels So you're able to restore an avascular cornea So having this basis was important for our future studies with our morpholinos Also, you know, it's a real clinical problem as well Corneal opacity which includes that due to neovascularization represents the second most common cause of blindness worldwide and It is known that VEGF plays a central role in the pathogenesis pathogenesis of corneal neovascularization Therefore It is likely that molecular mechanisms that we understand from these experiments will be broadly applicable So here's the experimental design and how we tested our hypothesis So we used a suture model. This is a suture in an eye a mouse eye So we used a suture model of neovascularization whereby we Put a suture In the mouse eye and waited a week for the vessels to grow in and then we Injected these eyes with either the morpholinos Which would increase soluble flit PBS as a control as the vehicle or a standard morpholino just to control for the presence of the morpholino and then we looked at the the nature of the blood vessels and and Quantified this in each of the conditions and then finally to confirm that the result We were seeing in this condition was in fact attributable to expression or modulation of flit one We knocked down. Oh, this is Actually standard morpholino, but we knocked down soluble flit one To show that we could restore the phenotype Found in the absence of increased flit one. So let's go through the data So in in the first data these bright field images show What happens when we have corneal neovascularization and we introduce side increase soluble flit one levels so These two are standard conditions and you can see at day zero, which is one week after we sutured You can see the vessels growing into the cornea and these are unchanged You know a week after injection with either of our standard conditions and then There are vessels growing into the cornea day zero in our Flit one morpholino condition. However at a week after Injection with our morpholino you see that these vessels have regressed so furthermore when as I mentioned when we Co-inject with not only the morpholino first flip flip one But also an RNA I that is designed to knock down flit one So we're kind of there are two things that are acting on flit one one is to up-regulate one is to down-regulate So you see that you lose this regression of vasculature and so this helps us to ascribe This effect of vascular regression specifically to flit one And so these are the data Represented graphically what I've just shown you so you can see that you have a decrease and a regression of vasculature when you have increased flit one and that this is Reversed when you knock down flit one so in summary for these data the Morpholinos do increase flit one transcript and protein expression The morpholinos in doing so inhibit suture induced corneal neovascularization in our first round of experiments and then in the second round of experiments When we specifically block flit one we see that restoration of vasculature indicating to us that this Flit one is the Mechanism for the regression of the vessels All right, so we're moving on in terms of the broad applicability of these tools We wanted to also look In for example a cancer model system as we know angiogenesis to be very important for solid tumor growth so I chose to look at breast cancer and Why breast cancer well? In general tumor vasculature is a really important therapeutic target Due to fulcman who I highlighted earlier was the first to really show that a tumor cannot grow beyond about 2 millimeters a solid tumor without inducing Neovascularization so it's absolutely essential for solid tumor growth and then breast adenoma Carcinoma is known to demonstrate marked dependence on VEGF and Neovascularization for growth even among solid tumor malignancies and these data are from some of the Sentinel work and done in late 2007 That led the FDA to approve a vast in for treatment In breast cancer and then finally VEGF inhibition specifically has been shown to reduce tumor growth In both the experimental as well as the clinical setting so in both mouse models and in patients so Breast cancer also plays a role in ophthalmology So dr. Shields is a well-known ocular Oncologist and has published some data on on this topic It breasts adenocarcinoma is the leading source of primary malignancy in women with ocular and or orbital tumor metastases as you can see here and Then this is just one example of a caroidal metastasis found here Also seen on ultrasound here With some sub foveal. It's it's not Obviously directly beneath the fovea, but causing some swelling. So these are things that we could see clinically So to test our hypothesis regarding soluble-flit one in this system. I use breast cancer cell line that is widely used in the field to grow xenograft tumors in immunocompromise mice pictured here and Then after two weeks of growing the tumors I started to do intratumoral injections of the morpholino on a bi-weekly basis And this was for two reasons number one we wanted to show regression of tumor growth because that really is the strongest data available in the field and We also did it on a bi-weekly basis after a number of trials of Understanding how long the morpholino is present after injection. So we wanted there to be essentially a steady state Okay, and then our outcome measures Were three-fold we looked at tumor size using digital calipers measured bi-weekly And tracked over a period of a month, which was our treatment time We also looked at Expression of soluble-flit after we harvested these tumors we Harvested the RNA and looked at transcript level of soluble-flit and finally we looked at the degree of vessel growth In the tumors again after completion of the experiment So prior to starting our morpholino injections the tumors this is at a two-week time point They were approximately The same size although you do see a degree of variation and I will point out that the larger tumors are in the treatment group So I didn't place them to skew towards standard Okay, and then these are our data so you can see this graph represents change in volume So from the time we started injection with the morpholino to the endpoint How did the volume of each specific tumor change and these represent our standard tumors? And you can see to some degree or another they all increased in their volume with standard morpholino treatment and then these represent the The morpholino treated tumors and you can see that they all did Regress in terms in terms of their overall size And then this is a graphical Average representation of the trajectory of the entire treatment groups standard versus morpholino treated And so when we looked at specifically soluble-flit one expression again at the RNA level We found that there was increased soluble-flit one Versa in the treatment group versus the standard group so confirming that Our morpholino was acting in our tumors and then finally when we looked at vascular density Between the treatment and the standard groups you can see using isolectin staining, which is specific for endothelium You can see that there's decreased staining in the treatment group as compared with our standard So presumably the increased soluble-flit one was is leading to a Decrease in overall vasculature and this decrease in vasculature makes it Makes the tumor unable to grow and in fact it does regress so as an aside Certainly, this is still at the bench level and there are other studies that we need to do to Show definitively that soluble-flit one is in fact responsible for the findings However, there is a timely need for innovative treatment I guess there probably always is in any field, but the FDA is likely to pull its approval of a vast and based on two studies which were actually Put out by Genentech so their Company studies to follow up the original study showing efficacy and these studies Apparently are unavailable to most people, but I've read other accounts saying that they show essentially no overall survival benefit When a vast and is added to the standard chemotherapeutic regimen and given that there are side effects of a vast and It's likely that this will be pulled Their recommendation will be pulled so getting back to our original questions that we set out to answer Regarding soluble-flit one expression. So can we alter? Expression of soluble-flit one with these more falleno tools. Yes, we can Does increasing soluble-flit one expression cause reduction or regression of vasculature? and yes, we've shown that it does in in two model systems and then is the level of inhibition significant or Sufficient excuse me to alter disease progression and yes That was the case in both the cornea and the tumor and so this is a good place to start We have more work to go from here, but we're very pleased with the current state of this project so I think the broader question is What what do these data add to how we currently treat? patients and So right now we're using a lot of the antibody based VEGF inhibition Which is certainly efficacious, but the time frame for action is limited and requires repeated injection Etc. And so a thought would be that new strategies of Manipulating the subtle molecular male you Over a would provide the potential for more long-term treatment With fewer side effects for the patient and hopefully ultimately better efficacy And so for example now laser treatments Used in the retina for example destroy presumably ischemic retinal tissue to decrease overall VEGF Production, this is just one example, but instead of for example Using the laser treatments if we might be able to keep what cells are alive intact and then Just alter their expression profile of soluble flip one might this be another way to kind of get at this difficult clinical problem So with that I'd like to thank all the people who've worked on this project certainly doctor and body and then doctor Quasi and doctor. I don't know how hero Who've been instrumental in the murine data? And dr. Alana well who is up at the Huntsman and was gracious enough to provide me with the cells that the breast cancer cells and Then Alicia and Elaine who are always very supportive And I'll take any questions Okay, I Think it's unknown in humans certainly how they compare in terms of the murine data It's a I have not seen a head-to-head So I think that would be really that would answer that question directly I think when you look at the data independent of one another understanding different groups have done the studies Etc the data are pretty much analogous in a mouse model. I think the question is How can the The morpholino tools maybe be used in a different way with different delivery than for example that antibody-based tool and Once we understand native regulation of VEGF Thoroughly, which is kind of something Derek's working on as well in the lab in addition to using our morpholino tools Can we manipulate the? Molecular environment in a more long-term way thinking about gene therapy and things like that so I think At least at the mouse level You know in the animal models. It's looking equivalent. So the questions are more Towards human application It's injected. Yes, so I Don't believe We well, we haven't tried it topically. I think that we would only be able to answer that in doing so So these morpholinos are engineered. They're called in vivo. They're engineered with this with eight arginine Sidechains on the five prime end and that has been shown to aid in cellular uptake and so a Lot of that work was pioneered by groups studying muscular dystrophy So we do know from our data that at least with the injection etc. And the way that the morpholinos are designed that this is They have the ability to Access the nucleus, but whether that can be done in a topical manner I would assume that yes that can it can be done But the magnitude of change in the vasculature and the overall effect I'm not sure because we do like it to be Diffused so what we do is we do the the suture and once the vasculature grows It's an injection that actually accesses more of the stroma but then we do kind of a massage to to get the injection to have a Kind of a wider area that is accessible for the morpholinos to access the intracellular compartment So I think that might be a limitation of topical Application, but I think it's something that because of the ease potentially I think it's something we should really look at Yeah, that's a really good point Well the soluble flit it's mostly the epithelium But the keratinocytes also produce it to it to a small degree and so Dr. And body in his original paper kind of looked at all the different cells and those are the two primary producers of soluble flit in terms of keeping the cornea a vascular and then VEGF is thought to be Produced to some extent by the epithelium, but also the surrounding endothelial cells so with loss of S-flit then you get Some VEGF in the epithelium and then once vasculature grows in I mean it's just you get a ton from those endothelial cells And when you're Yes That's a very good question, and I Don't know We haven't looked at that so that's one thing we have to look at we also now that you bring it up We have to look at What S-flit does with the morpholino so pre and post morpholino That's something we really want to assess so I assessed it to some extent in the tumor model system to show that in fact We are increasing soluble flit expression Because to ascribe our data to increase soluble flit you really should show increase soluble flit so So that's one thing we need to do Certainly Sorry, but there another So I did I we used both we used PBS as the vehicle to control for the solvent and we also used a standard morpholino Which is a non-targeting it targets them In Toronto beta-globin. Thank you very much