 good morning to try to summarize four years of research in 40 minutes please interrupt at any point so as most of you know our lab focuses on angiogenesis in the eye today's presentation the compass is really about AMD and in the lab we have three broad areas vascular demarcations intracellular therapy and drug novel drug delivery using nanoparticles to focus on this condition now as you all know I'm a cornea guy and we started off 10 years ago in the lab focusing on what keeps the cornea clear and we found that a molecule known as soluble flit or soluble VEGF receptor 1 which is the high affinity receptor for VEGF was the main mediator of corneal avascularity and that when he deleted it in the mouse obtained a spontaneous corneal neovascularization and the second strand of our work over the last 10 years has been trying to use that insight of what keeps the cornea a vascular physiologically as a novel anti angiogenic by focusing on intracellular pathways so today we're going to talk about some novel models of AMD and targeted not targeted targeted intracellular therapeutics this audience is well aware of what happens in AMD with subruntinal neovascularization and hemorrhage and the clinical impact in America it's the leading cause of blindness in worldwide it's the second leading cause of actually third at behind corneal disease leading cause of blindness simply because there's a lot more corneal injury and glaucoma in the African population in the United States there's 10 million patients with AMD it more patients with AMD than cancer not just breast cancer or prostate cancer but all cancers put together there's more patients with AMD than all cancers put together and worldwide there's 40 million patients blind bilaterally from macular degeneration and when you consider the state of Utah we're talking eight times the state of Utah's population right actually no more than that we're 15 times the state of Utah's population that are worldwide just from macular degeneration so it truly is an epidemic so what happens here we have corneal neovascularization growth of abnormal blood vessels from the retinal space a subretinal space into destroying vision and what we wanted to achieve in the last several years is to first understand what normally protects vascular zoning of those photoreceptors when you think about it it's quite remarkable that the a vascular photoreceptor layer sits adjacent to the highest flow of vascular bed in the body so what controls that vascular zoning is a very important research question now mice share a lot of things with humans but they don't have a macula they do have very similar VEGF and VEGF receptors they do have very similar genome the retinal architecture is quite similar except for the absence of a macular because in the mouse the optic nerve comes right out of the center of the retina now I mentioned to you previously S-flit one S-flit is the soluble form of VEGF receptor one because it's soluble it's not membrane bound it's floating around in the extracellular space it thereby acts as a decoy for VEGF binding it in the extracellular space essentially suppressing VEGF in tissues where it's present so our hypothesis in this big question of what controls vascular zoning underneath the retina was if we knock down S-flit one will we increase free VEGF and induce corridor angiogenesis that was our initial starting hypothesis now would that lead to a new model of AMD which could be better than the traditional laser model or some of the genetic knockout models that exist was our secondary question okay so that's our purpose first is S-flit present in the retina these are in C2 hybridization images so basically looking at mRNA and interestingly enough S-flit is present in the RPE in the inner segment a little bit in the outer nuclear layer and the outer plexiform layer at an mRNA level you don't see it on the sense control as you don't expect it to is it decreased in AMD patients relative to normal so we don't have that many human specimens we only have about 14 AMD specimens compared to about 10 normals it's a lot easier to get corneal tissue than retinal tissue but we find suggestive findings that in normal patients or normal retinas there's significant S-flit in the RPE and in the photoreceptor layer but it's decreased in AMD both by fluorescent staining and by histologic staining okay so we examined using three different approaches knocking down S-flit in the mouse at a protein level a transcriptional level and a genomic level so strategy one was injecting S-flit antibody or anti S-flit antibody with a sub-retinal injection and first we want to check distribution so if you inject GFP sub-retinally in the mouse you do get a lot of GFP expression in the RPE and in the photoreceptor layer now doing a sub-retinal injection in a mouse is probably one of the technically the hardest things you can do I think from an animal perspective and I have to give a lot of credit to my postdoctoral fellow Dr. Ling Luo who is a retina surgeon and apparently a mouse retina surgeon as well when you do that what happens you inject anti S-flit antibody you get significant CNV at the injection site confirmed on both FA and OCT and we know this is not due to interactions with the membrane flip receptor this is directly a result of taking out the soluble receptor because when you inject the antibody in a flip tyrosine kinase knockout animal where the membrane receptor is non-functional because the kinase is not functioning you get the same phenotype you get CNV at the site of injection we know it's not due to interactions with PLGF which is another potential target of flip antibody because PLGF doesn't change so that's at the protein level strategy 2 was knocking down S-flit at a transcriptional level taking out the mRNA expression of it by a virus an adeno-associated virus that expresses a SH RNA short hairpin RNA so sure all of you have heard of RNA interference where if you express double-stranded RNA inside cells you will selectively delete the target that is cognate to that double-stranded RNA because in people mRNA is single-stranded double-stranded RNA is generally viral and cells recognize double-stranded RNA is foreign and destroy any genes that look like that double-stranded RNA so this is a way of selectively taking out mRNA transcripts by delivering double-stranded RNAs technical terms SH RNAs into cells and when you do that and the appropriate controls are non-specific SH RNA GFP expressed by AAV and PBS you induce sub-rattinal angiogenesis so first we show that we can knock down S-flit by this approach and not by the controls and when you inject the test SH RNA you get very vigorous CNV and minimal reaction with the control further not only do you see CNV but you see increase in free VEGF with the SH RNA knockdown of S-flit1 as you would expect compared to the laser model so let me start human CNV patient you see the classic CNV lesion here the laser model basically is doing a laser to burn a hole in the in the retina in Brooks membrane and then CNV goes through Brooks membrane at the edges and it's very different architecturally than the human condition however the virus induced a CNV when we knock down S-flit much more closely resembles the human process further laser CNV lasts for about four weeks and starts to regress the SH RNA knockdown of S-flit causes progression of CNV and secondary lesions far from the injection site and this is confirmed histologically compared to the normal mouse in addition you do see pigment epithelial detachments again unlike with the laser model in human patients PEDs are fairly common where you have sub-RPE fluid and you can observe that in the mouse OCT here and here and you can see FA negative areas where you have light up with ICG confirming the presence of a PED in the mouse mentioned to you that we increased free VEGF and you can see that here where the free VEGF level was highly elevated by knockdown of S-flit 1 and if you try to do this in a mouse that lacks VEGF in the retina and how do you achieve that you achieve that by giving enzyme called Cree in a VEGF locked animal VEGF locks means the VEGF gene has locks P flanking sites which the Cree enzyme can remove in a tissue of interest and so what we're showing here is that when you inject P Cree the Cree enzyme into a VEGF locked animal you're preventing VEGF from going up and you're preventing C and V from occurring but if you inject the null plasmid no Cree you observe the C and V and VEGF elevation so when you take out VEGF trying to take out S-flit doesn't do anything that's what you'd expect by mechanistically we're saying knockdown of S-flit releases VEGF causing C and V and where we also show in the TLR3 knockout animal that you observe the same phenotype of C and V and VEGF and so this SHRNA effect of inducing C and V by knocking down S-flit is not due to TLR3 effects but it is due to VEGF desequestration. The third strategy in this zone or this question of zoning is genomic knockdown of S-flit now you can why not make a genetic knockout you might ask if you make a genetic knockout of a flit it's embryonic lethal the mouse doesn't survive so you can you have to make a tissue selective knockout and you can do that by doing P-Cree injection that same Cree enzyme in a flit-loxed animal and as you'd expect S-flit goes down and C and V does occur and VEGF does go up or you can do some transgenic models now this is probably my favorite slide in the presentation so let me just spend a few seconds on it you can avoid the whole trauma of sub-retinal injection by cross-breeding animals that will express Cree just in certain areas so there's a VMD Cree promoter VMD as most of you recall by teleformed macular dystrophy so VM the VMD promoter is specific to RPE cells and you can put a Cree enzyme driven on the VMD promoter which when expressed in the Rosa red mice normally the Rosa red mice all of the cells are red fluorescent when Cree is expressed it shows up as green in this Rosa red the red is converted to green by the Cree and you're confirming that the Cree is expressed just in the RPE an alternative promoter that we used was iCree 75 which is specific to photo receptors and so similarly we're showing Cree expression just in the photoreceptor layer when you cross iCree 75 cross Rosa so basically we're proving that we can express Cree enzyme selectively either in the RPE layer or in the photoreceptor layer does that make sense when you do the VMD Cree cross footlocked animal so expressing Cree just in RPE cells mating those with the footlocked P animal you see significant areas of C and V scattered throughout the retina confirmed histologically confirmed knockdown of a flit expression in in the hybrid animal compared to the normal and confirms C and V on transmission electron microscopy and when you do the iCree 75 cross footlocked P animal again you knockdown as flit in the photoreceptors compared to the control confirmed on both in C2 and in immunohistochemistry and here interestingly enough you don't see classic CNV but what we believe is happening this is just from a few weeks ago this is very preliminary data what we believe is happening is a model of not CNV but RAP retinal angiomatous proliferation where you get the retinal vessels from the inner nuclear layer going down into the photoreceptor layer so if you recall at the outset I showed S-flit expression not just underneath the photoreceptors but overlying the photoreceptors so with the previous knockout with the VMD Cree cross-flit locks where you knockdown S-flit in the RPE vessels grow up with this knockdown what we're observing is the vessels go down into the photoreceptor layer from the retinal circulation and so this we believe recapitulates to some degree what's observed in RAP retinal angiomatous proliferation which is a variant of AMD okay so summing up that first sector of our work we can induce CNV targeting RPE and photoreceptors by knocking down S-flit which elevates free VEGF and by doing this in a variety of different methods we can correlate the mouse phenotype very nicely to the human phenotype. Our second sector of work is working on intracellular therapeutics the the current paradigm of treating macular degeneration is Avast and Lucentis both of which are extracellular agents but many vascular endothelial cells have their own VEGF and their own VEGF receptors and you can imagine if a cell has its own VEGF and its own receptors it's much like a junkie who's a dealer and you have to interrupt that intracellular supply to get it that. So we start off about eight years ago looking at an interesting for amino acid peptide called KDEL which is an endoplasmic reticulum retention signal. We took a page out of our friends in the AIDS world who found that if you couple stromal derived factor with KDEL you can actually knock down CXCR4 and thereby prevent the receptor that binds HIV blocking HIV entry into macrophages and T cells. So they called that an intracellular chemokine or intracine we wanted to do the reverse express an intracellular receptor or intraceptor to knock down VEGF expression. So conceptually VEGF will bind surface receptors and induce various signal transduction events and what we're proposing to do is attach KDEL to an intracellular receptor bind VEGF within the endoplasmic reticulum keep it from ever leaving the endoplasmic reticulum. In the cornea we were able to show several years ago that we could regress corneal neovascularization induced by alkali injury and I know I'm speeding through the slides but in the interest of time but essentially what we did here was inject a plasma that expressed a subunitive flit with KDEL to sequester VEGF after alkali injury and successfully regressed it and all of this is building up to Paul Bernstein's problem where I believe on Monday afternoons he has an assembly line of patients that is quite tragic. This is the current standard of care in our retina colleagues practice where we line up patients month after month giving them an injection into their eyeball and it's the best we can do and it's those of you in residency don't recall 10 years ago or 15 years ago and for OCAPS you have to read about the macular photoclugulation study and destroying the retina so this is better but it's not good and this needs it needs to be different. So to address that we're working on a novel drug delivery system that takes us into the final sector of our talk looking at whether a nanoparticle that encapsulates those interceptor expressing plasmids that is coated with RGD which homes to neovascular tissue so RGD is a peptide that's selective for alpha V beta 3 integral which is present on abnormal new vessels but not on normal vessels so this is a way of this is a guidance system it's basically taking an anti-angiogenic therapy and putting a guiding guidance targeting system on it. Working with our collaborators Dr. Kompela in Denver who makes the nanoparticles our combined group showed a few years ago in rats that laser induced CNV can be suppressed by RGD targeted nanoparticles but not by the control nanoparticles and we wanted to see whether we could observe similar effects in mice and monkeys so we'll have three species I'm going to skip over the methods. One of the things we've developed in the lab and is and hope to work with photographers going forward is measuring volume calculations of CNV so right now my understanding is in clinic central macular thickness is used to assess CNV response to therapy but we've actually developed a method where we can determine the full volume of a CNV lesion using the spectralis to stack the different slices of the CNV in vivo. Traditionally volumetry in the mouse is done ex vivo after mouse sacrifice and we can observe a very good correlation with our in vivo volumetric assessment which will allow us to trend over time response to therapy without sacrificing too many mice. So in that AAV induced model that I mentioned previously when you inject first AAV subretinally you get the CNV lesion this is about a month after AAV injection and then with a single tail vein injection so not introvitrile but a tail vein injection in a mouse of these targeted nanoparticles you can make that CNV lesion go away confirmed on both FA and OCT as opposed to control nanoparticle injections where the lesions progress and increase. Yeah so the nanoparticles we've shown express interceptors for about eight weeks and when when you assess all the different groups we could you can actually show close to 50% regression with the targeted nanoparticles compared to that which is statistically different from the controls. Yeah there are AVI. I was gonna show videos but take my word this shows a very large CNV lesion on 3D and it gets flattened out with treatment. Not with the AAV induced model the laser induced model you do get spontaneous regression. We've confirmed that the target alpha V beta 3 integrant is present in all different models but not in the normal eye so this is showing a specificity of the RGD nanoparticle and that the RGD nanoparticles do home to the CNV lesion but not to the normal retina. Skip this slide on time. Now the nice thing at Moran is that we have great research laboratories with a lot of different skill sets. Dave Kreisai on the third floor has what's called an optometer which can actually assess mirroring visual acuity. How do you assess visual acuity in a mouse? You don't use an eye chart but you can use the optokinetic nystagmus reflex and see at what grading of stripes the mouse actually responds to those gradings and that will tell you the spatial resolution that the mouse is able to observe. Now a normal mouse starts out at about point three eight after CNV induction and this is in the AOV SHH RNA model. What we observe is a knockdown of visual acuity very far down but this is restored by the RGD nanoparticle treatment so in white is post injection in black is pre-injection you observe a knockdown and a lot of that visual function is restored by the targeted nanoparticles but not by the controls and the buffer treated animals the visual acuity actually gets worse. Histologically we can show that the CNV lesions get smaller and that our treatment with a tail vein injection is not inferior to use Barbara's favorite term compared to intravitral antibody injection. So we're not using a vaston but a mouse anti-vegeta antibody injection a vaston doesn't is it supposed to work in a mouse but we actually believe our systemic therapy is at least equal to our an intravitral injection of anti-vegeta antibody. Now the most important stuff I'm going to show you is the monkey work. You can induce CNV monkeys using laser and monkeys do have Immacula and when you do laser spots in a monkey and you wait a month generally they'll progress and in our control nanoparticles they progress too but in the RGD treated nanoparticles in the monkey street with RGD nanoparticles the CNV lesions for the most part regressed and this is confirmed histologically and the effects were statistically significant and this is also confirmed on confocal microscopy. In both mice and monkeys we did tox evaluations looking at the kidney lungs skin and liver after the nanoparticle injections those are the high blood flow tissues of the body so any systemic drug I think it would be advisable to look at those organs and we did not observe any changes histopathologically in these tissues so we don't believe that these nanoparticles are toxic. They're made out of PLGA so they're degrade to lactic and glycolic acid which are degraded by the Krebs cycle. So with a single intravenous injection we can suppress CNV in mice and monkeys. We have out-to-four-week data of CNV suppression. The nanoparticles themselves deliver interceptors for eight weeks. We regressed fibrosis, improved vision, no toxicity was observed. So with that I know I've covered a lot of things but I think I've managed to fit it all in in the time. There was supposed to be audio with that and I've just taken credit for the work of a lot of different people and I want to highlight some of the main people in my laboratory. Ling Liu as I mentioned is the retina surgeon who does sub-retinal injections. Jackie and Bonnie are critical players in the laboratory. Hiro developed the AAV, SHR, and ASFLIT. Our collaborators across several different institutions and of course our funding agencies. And thank you very much. Be happy to take any questions. Just get those files earlier. I think we can make them work. That's what I'm thinking.