 I'm pleased to introduce our speaker today, Roxana Radu, from the Stein Eye Institute, which is my home institution, that's where I did my residency many, many years ago. And Roxana and I go back a very long way. We both, and we share a common interest, we both, when we both, Roxana started out as an MD and she grew up in, or she was trained in Romania. And she came here in the mid-1990s and she was not an ophthalmologist. And just like I was not, I had no idea I was going to go into ophthalmology, but I was put into a, on a project on the visual cycle and that's exactly what she was, what happened to her when she came to Texas at UT Southwestern. And obviously the visual cycle is a great, great area to be studying and she's been very, very productive. She was at UT Southwestern working mainly with Gabriel Travis there. And when he moved to UCLA, she came in the mid-1990s in the early 2000s, she went to UCLA with him. And she, even though she didn't have, she doesn't have a PhD, she learned how to do research and learn from the ground up and eventually was publishing in very, very high-profile journals like Neuron and PNAS and has done very, very well and now has an R01 in her own independent laboratory. She's going to be talking today about complement dysregulation and the link with Stargardt disease and age-related macular degeneration and I'm very happy to have the clinicians here and also have the basic scientists who think so much of her that they actually did get up at eight o'clock in the morning to be here. So Roxana? Thank you very much, Paul, for the kind introduction and I am truly honored to be here at the Moran Eye Center and visiting the center and also have this incredible opportunity to share with you data that we are generating in our laboratory. And looking forward to meet some of you today and tomorrow. So today I'll be talking about complement dysregulation as a possible link between Stargardt disease and age-related macular degeneration. So how many of you have heard that complement system is dysregulated in Stargardt? Some. Okay. I certainly hope at the end of this talk everyone will consider this pathogenic pathway that does play a role in Stargardt. And although both AMD and Stargardt disease have multiple cellular targets that includes of course photoreceptor, retinal pigment, epigelium, brux membrane and coroid capillary, for today's talk, the majority of the study that we've implemented was to focus on pathophysiology of the retinal pigment epigelium. This is heavily studied in the past as Paul alluded because I've been implicated in evaluated multiple functions of this retinal pigment epigelium. This is the outermost layer of the retina. It contributes to formation of the blood retinal barrier by formation of those tight junctions and allows selective trafficking of all sorts of solutes, ions, glucose, vitamin A from the basal lateral to the apical side. They are polarized cells and this apical microvillage, the RPE, have a very intimate connection with the outer segment of the photoreceptor and by doing so they also participate in daily phagocytosis that allows regeneration of membranose disc of the photoreceptor which are critical, contains the visual pigment and the molecules, life-sensitive molecules that are needed for our vision. It also, RPE also secretes the growth factor necessary for both site neural retina and also colloid capillaries such as VGF and PEDF. As Paul mentioned, visual cycle is a key component in RPE because RPE expresses major enzymatic protein that are essential in regeneration of the visual chromophore 11C's retinal dehyde. Also, due to the presence of melanin or other non-visual opsin that I've been implicated on my own as a postdoc with Gabe Travis studying retinal RPE-RGR or RPE G-protein cover receptors and peroxin, they both modulate vitamin A metabolism in the RPE and that's also a function pretty critical. You could imagine that any impairment of those function of the RPE could mount complement reactivity. Compliment and I'm pretty sure everybody here is aware it's our innate immune arm. We are born with this, so pretty much everything that goes wrong in a cell, in a body, will mount and complement reactivity. It has numerous biological function, which I listed, opsinization, chemotaxis, agglutination, cell lysis, all with the goal of killing pathogen. From which cell lysis I think it's more dramatic, it's the one that has the ability to build this membrane attack complex with the intention of, you know, break the pathogen membrane and open up the cell and release its content that leads to cell death. This beautiful diagram, I'm not going to go through the steps. It's just to kind of elucidate, show you how this MAC is formed, membrane attack complex is formed, it's a cascade activation starting through various pathway and Dr. Hegemani is the one who elucidated most of them. It could be a classical pathway, alternative pathway, lacking pathway or an interesting pathway. But what they have in common is the, this stage where they form this C3 convertase, so C3 convertases and C5 convertase that all have the powerful ability of lice molecules of complement that becomes more reactive and combined with other fragments to lead to this powerful C5B9 where other complement components get together. It is on all the time as I said and it's important to provide this powerful defense mechanism but it can also be detrimental to self-sal if it's amplified over the normal level. So there is a constant sublitic deposition to this level of membrane attack complex at the cellular level but this complement it's tightly regulated to avoid this pathological level where the host membrane can be damaged and lead to cellular deaths. So you have to imagine this MAC complex, in fact it's like punching holes in the cellular membrane. All the cellular content is released and cell dies. So what is key for the retinal pigment epithelium is the fact that it expresses almost all the major cell surface, membrane bound and also fluid phase of those complement regulatory protein. And this is literally similar as liver, liver is the major organ that produce those complement regulatory protein to be available to other cellular target. But in the case of RP it does have this ability and we analyzed with express other laboratory also shows data that they are present there. And that it's a key factor of this RP cells because it can define itself without relying on the systemic circulation resource. And this is a beautiful image from an eye donor of a woman 86 years old just to show you how strong immune reactivity takes place that those cellular targets that are implicated in AMD. So in blue it's RP, it's represented in blue right here. And this draws, it's the pathological hallmark for age-related macular degeneration. This particular individual didn't have any genetic background, it was just an H.I. And no complaints basically, no opal or pathology or vision impairment unless, but it shows that this activity it's on all the time without really causing major problem. And despite this tremendous accumulation of those complement molecules within this drusen. So drusen consists of cellular debris coming from different origin. It's photoreceptor, retinal pigment, epithelium, Brux membrane, coroid capillary. So all those debris mount or trigger this complement activation. And the end product it's deposition of those fragments of complement that manifest mac and negative regulator or other regulators that also try to control this activation. And here in orange you see the mac deposit promising the central area of the drus and complement factorate which is a soluble form of soluble protein, complement regulatory protein that surround this mac. Suggesting that the RP constantly puts out this molecule to defend itself by this detrimental effect of mac. So what other knowledge we have from past study? Well 2005 was a critical year. It's the year that there was this major discovery that mutation in complement factor age which this discovery was made by Dr. Greg Hageman and in parallel three independent labs came up with the same findings made the news that there is a direct association with development of the age-related macular degeneration. And that was quintessential. Since then, numerous study has actually shown that there are other mutations, other complement genes that shows mutation and shows association with AMD. Well we also learned that star guard carriers of mutation in the gene responsible for star guard is a risk factor for AMD. So they start to sort from the genetic standpoint that is another correlation. Clinically and pathologically both this is manifested as central vision loss due to loss of cells in the macular region. There are no suitable models for AMD because AMD is not just genetics age, there are other environmental factors smoking that contributes to this build up. So it's hard to model experimentally. There are models for star guard disease. This is a monogenic disease and today I'm going to go over several models that we generated or we obtained from other places to investigate complement system in star guard disease. So I'm going to share with you some of the published and unpublished data using the star guard mouse model, the ABCA4 knockout mouse. And I'm also going to share with you part of the data that we rescued the ABCA4 phenotype because we also generated an albino mouse that in addition to biochemical phenotype that's been published initially when Dr. Travis generated this mouse with back cross on an albino background and this particular ABCA4 knockout mouse has also photoreceptor degeneration. Then I'm going to show you some data using star guard donor eyes and that's from collaborators of us in Cleveland, I institute. And then we generated a star guard in a dish model where we obtain fibroblast from star guard patients and we re-derived them in RPE. So just a brief background about star guard disease, as I mentioned, it's caused by a single gene mutation in a single gene, the ABCA4. It is the most common inherited juvenile macular degeneration. It affects about 1 in 10,000 individuals. Part of the clinical presentation, patients manifest with progressive central vision loss and delayed dark adaptation and those manifestations are similar with AMD. As pathological markers, star guard presents with accumulation of autofluorescence bis-retinate lipofusion material in the RPE. It has developing RPA trophy and photoreceptor degeneration. And this is just the topological model of ABCA4, so the gene encodes for a protein, which is an ATPase binding cassette family member, type 4. And it's a cytoplasmic, directly ATPase-dependent flipase that flips retinaldehyde, it's a form of vitamin A, also condensed with phospholipid, the phosphatidylethanolamine. So N-ray-P is the substrate for this protein, ABCA4. More studies have shown that ABCA4 has no specificity for the substrate, it could flip the eleven-sis or all-trans retinal, N-ray-kinelidine-phosphatidylethanolamine. So ABCA4, it's been shown, so the gene was identified by randualic mites over 20 years ago, and Molday actually showed that the protein localizes on the rim of the membrane disc of the outer segment of photoreceptors. And our lab also recently shows that ABCA4, it's also expressing the RPE cells in addition to photoreceptor. And that's quite new, and it does change the paradigm of pathogenesis of Stargard. And this part of the studies, I'm going to present at the research seminars, so I will, hopefully if your time permits, I hope you join me because it does have a significant clinical relevance in disease pathogenesis. And what I'm showing you here, this is a section of frozen fixed eye that came from the collection of Dr. Redhageman, and it wasn't meant for RNA in situ hybridization, it would use specific probes against ABCA4, and as expected, the probe, every single dot, red dot, represent the molecule of RNA. So as expected, the mRNA, it's present in the cell body of the photoreceptors, it's not present in the outer segment because the outer segment only contains the protein of that mRNA. And it's also heavily expressing the RG monolayer, as it's shown here. Well this is an H-pigmented eye, so it's hard to see, but there is more data to show this evidence of RPE later at the seminar. So regardless, it's in the photoreceptor and RPE, the lack or mutation in ABCA4 leads to build up of bisretinoids or dimers of vitamin A. Without going through all these steps, the idea is, all we need is aldehyde, and it can be in the form of old transcript and aldehyde as shown here, bind with phosphatidylatanolamine, and this is the first reaction, which is reversible form N-red P, and in excess of aldehydes, 116-Altrans or any kind of aldehydes, condensation to this N-red P, a second molecule of aldehyde condensing with N-red P, give rise to this dimers form. So there are various forms of this dimer, depending on their composition of having or not having the fatty acid included. And upon phagocytosis, those components are released in the RPE, so RPE has to deal and further process some of them and form this A2E, and I think that's the most abundant fluorophore, because these bisretinoids also have the property of autofluorescent, and depending on their absorption spectrum, or less, you can visualize them in a living eye as well. Giving their chemical composition, they're not final compounds, because they all have this double bond along the polyane chain, and those double bonds can oxidize, and that gives rise to a series of totally more reactive, or they can fragment, and that also become more reactive. It's hard to monitor them. What I'm showing you here, every single compound, we can analyze on a normal phase liquid chromatography, but the oxidized form of this dimer, so vitamin A, could only be analyzed using a mass spec where you have a particular developed method for the individual one. And it's hard to control this level of oxidation. So consequences in a mouse model, the ABCA4, the Stargard model, ABCA4, with age they build up these components, and what I'm showing here is the A2E and the immediate major precursor of A2E. A2E stands for two molecules of ultranslating aldehyde bound with ethanolamine. What's interesting, a wild type eye also increased this dimerized form, so with ageing we have processing of this vitamin A in the RBE that are not really clear also, a wild type also accumulates those. So this is sort of the image coming from a living eye. And what I'm showing you here, from study of Fischli at all, found this outer fluorescence of a Stargard patient, top images, and geographic atrophy AMD patients at the bottom. So I mentioned that those base retinates have property of outer fluorescence. And here, this massive bright outer fluorescence granulence kind of accumulates along the edge of the atrophic lesion. Both patients turn to have mutation in ABC for genes. None of these patients have mutation in the complement genes. However, I'm going to show you later that complement is regulating such case. So how do we know those material, outer fluorescent material in a human eye contains base retinates? Well we had the opportunity to obtain some tissue from Stargard eyes and what I'm showing you are representative chromatogram of normal sample and two different Stargard patients. So we extract the base retinates and analyze by HPLC. And you could easily estimate that the normal eye has very low, lower abundance of A2E compared to the Stargard individual that the peak heart rate is much higher. And the inside actually shows the specific spectra for A2E in blue and the precursor of A2E in red. So what else we know about complement? So Dr. Sparrow labs have done incredible amount of work in evaluating the effect of the base retinates and oxidative form of base retinates on complement. And most of those study were done in vitro in stimulating different cell type loaded with those components. We also evaluated the effect of base retinates in the mouse issue and I'm going to show you some of those evidence. But we had a seminar study a while ago where we showed that the activation of the complement in bi-base retinates is direct dependent on the genotype of the RPE cells. So in other words, not only that activation was done via the alternative pathway, I'm not going to show you the data that's been published, but it's clearly that the AMD complement factor risk haplotype cannot defend itself properly when it's challenged with the outer segment containing the base retinates. So that was a seminar study that told us these cells over time being on this predisposing background cannot really mount the proper regulatory reaction. So here we move towards what evidence do we have for complement disregulation in star dark mouse model? So this was also done several years ago and it started very simple. We learned about the complement system in 2005, we learned about this clinical similarity in star dark and AMD and I was like, okay, if we know that this base retinates stimulates complement then we ought to find evidence by looking at complement specific protein. And what I'm showing you here, these are confocal images of suction of retina where we use an antibody against C3 fragments, actually those were specific for the fragments, but we use antibody that can visualize the whole lens of the protein. The fact that the whole thickness of the RPE link up suggested that all this activation of the complement that normally takes place on the plasma membrane are internalized, further contributing to pile up or debris that are not really normal. And they do co-localize with autofluorescence, which autofluorescence is sort of a marker for base retinate lipofusions. In the wild type, however, the level of the complement fragment internalization needs more. It wasn't surprising the internalization because I have to remind you, RPE is a phagocytic solid. It's not only phagocytos outer segment, but it also internalized fragments from the basolateral and that is one of the defensive mechanisms of the cells. So we further test for MAC production in this and what I'm showing you are representative confocal images of the RPE flat mounds of the wild type on the left and ABC for knockout on the right. And here, I hope you could appreciate a significant more acquisition of this MAC on the RPE cells of the knockout. And surprisingly, despite this massive complement activation and deposition of MAC, the negative regulatory protein in the mouse, the starter mouse, are downregulated. So that was puzzling to us because we would have expected that the RPE is going to express higher level to counteract this aberrant activation of complement pathway. And the only explanation we had at that time was, well, maybe that, it's a reflection of RPE declining and providing the necessary protein to defend itself. So over time, this is just contributing to the pathogenesis of changes that we've seen over time in this particular cell. And of course, we confirmed by protein amount that those are truly lower in knockout mouse. And mouse has a duplication in the gene DAF1, it's a homologue of CDD 45, and CRRY, it's actually a homologue of human complementary sector 1. So with this data, we decided to take, ask the question, is this complement dysregulation in the starter mouse pathogenic? So in other words, is that an independent pathway that takes place in the RPE and the side of the gene defect, this just contributes further to damage of the cell? And one way to address that was, can we upregulate negative regulatory protein in the RPE and rescue the photoreceptor degeneration in this mouse, and perhaps, you know, create a much healthier or promote healthiness of the RPE, I would say. So how did we do that? So we choose the CRRY, the CR1 homologue, human homologue, because it acts much early in the complement cascade, so not really building up all this sort of full cascade of mountain map. And we also tagged the protein with me, because we wanted to distinguish between the endogenals, protein, and visualized by immunofluorescence. And here I'm showing you on top section the experiments with the antibody against meek and the AAV injected mice. This was a single injection at four weeks, and we monitored the mice after one year. And we showed that the expression of meek tag protein is specifically in the RPE, and when we use an antibody against C3, and this is full line, and it also can identify fragments of C3 protein, it's significantly lower. But this was qualitative images. We did quantitative immunoblood and showed that there is a significant reduction in the deposition of those C3 in the RB. And here it's not biased, the injection was sub-regularly. We usually, in our hands, transduce about anywhere between 40 to 65 percent, but there is also diffusion. And as I said, this is biased, we use the whole eye to homogenize and subject to this type of analysis. So surprisingly, when we extracted this retinoids and analyzed by HPLC, we also noticed significant reduction in this retinoids in the CRRY injected mice compared to the null injected. So that was the appropriate control for that. And the interpretation we had, okay, so that means the fact that we reduced the burden of RB with the complement fragments, the RB may recycle or clear out those retinoids. So that speaks towards healthiness and much more robust activity of the RB functions. And that's consistent with quantification of the lipofusion granules, and they are pointed by the yellow arrows. And also some rescue of the photoreceptor cell. And this quantification of the photoreceptor cells were done in the region that we knew the RB cells were transduced. And in that region, the number of photoreceptor actually matched the number of wild-eyed mouths. So that was quite important finding that not addressing the gene defect, but just dealing with the complement, this regulation in the RB and lower the burden of complement fragments and activation can partially rescue the phenotype of the RB. So the data are clearly slow, C3 activation, it's protective against complement attack and preservation of the photoreceptor it's shown, so overall the RB, it's healthier. So this complement is regulated also seen in patients with stargardt, and here we had about, we had four donor eyes from stargardt patients, they've been clinically diagnosed as stargardt, and some of them were genotype for ABCA4, some could not be genotype because the tissue was fixed, so pretty much all we can do is evaluate the complement system using biochemical approaches. So this is the first eye we had, and it was fully characterized by our collaborator, Dr. Bonilla, and we specifically asked from her to provide tissue from two regions, the perimacular region and peripheral region, because to assess the complement reactivity in the RB, we needed to have RB cells, if we go to a trophic region it doesn't tell us anything, and I'm going to show you some experiments from both regions, and to do quantification, all the quantification was done from the peripheral area, and we also confirmed, so we got the blocks for electron microscope, and we did section from the peripheral area to ensure that that regions have still an intact, not intact, obviously change morphological changes, significant morphological changes in term of height or thickness and border of the RP, but the RP is present. So this side section from one of the stargardt eye, where we used the top section are just controlled, so there is no primary in this section because we wanted to know what's the background, and the bottom section comes from a control eye on the left, and stargardt eye on the right, where we used an antibody against MAC protein, and as it's shown on the control, MAC primarily deposits on the curate endotelium, and it's pretty well deposited around those endotelium. In the stargardt eye, and this is a perimacular region, and clearly there is a lot of disruption, the RP cells are a little wider, in some cases with totally missing some of the cells. MAC deposits sort of immediately in the base of lateral of the RP. Brook's membrane is pretty thick, thick compared to the control, also probably some of the layers are dispersed and contains this MAC proteins, but what's interesting, some of the RP also points out to have internalized some of this MAC. In the periferous area, where we do maintain this monolayer in both control and the stargardt, we've been able to quantify that, and the diagram, the graph bar shows about 1.5 fold increase in this MAC, and this comes from three independent stargardt eyes, so measurements that we've done from all of them, and we average. So C3 activity, we measure by looking at using an antibody against C3 protein. Again, top section come from the premium, so secondary antibody, and the second sections are stained with an antibody against C3, and the control eye shows very minimal deposition of C3 fragments internalizing the RP, and on the right, and this is a peri macular region, we can see a small drusen in this particular eye, but you could clearly observe significant more deposition of C3 fragments as we shown in the stargardt model. In the perifer area, the C3 seems to be higher, there is a lot of background, there is a lot of pigment, it's not easy to deal with eyes that weren't quite processed for proper monohistocanistry, they had different fixative that we normally do, but none of the less we've been able to quantify this as well. Compliment factor H is mention here, it's one of the major fluid phases, it's secreted by the RP, and here we show that in this particular section of the stargardt eyes, although the cells are present, they don't really secrete or stain for complement factor H that much, whereas other cells do have, so there is a lot of heterogeneity, depending on what stage the cell is found at that given time point. Compliment factor H was also quantified in the perifer area from only two stargardt eyes, so we are still in process of acquiring data for this, and it's shown to be significantly increased. So to summarize this part of the top, using the stargardt eye donors, similar to AMD we've seen increase C3, C3 fragments deposited or internalized by the RP, and map deposit on the RP cells, and this suggests that complement is regulation, it's also an important etiologic factor in stargardt pathogenesis. So, so far we kind of correlated the eye donor study with what we've seen in the mouse, but to get a closer look at the real human RP dynamic of complement activity, we took the step to generate an IPS derived RP cells from stargardt patients, and here we had much more control over selecting patients that we knew they have mutations in ABC4 genes, and they've been also controlled for the complement-related genes. So we wanted to first study a patient that doesn't have any mutation in the complement to estimate the contribution of this gene mutation in the RP to trigger complemented dysregulation, and this is basically the team that contributed to that. Our collaborator, Dr. Michael Gorin, collected the fibroblast, we have the Eli Broad stem cells at UCLA, Dr. Karumbaya, it's induced the fibroblast to create the pluripotent stem cells. We had three lines for independent patients, we generated three clones from each of the line, and they've been screened to the ground in my lab to make sure that the development and of the RP cells is normal, with the exception of ABC4, so we can estimate what's the effect, downstream effect and complement reactivity in those cells. So we've analyzed and I'm not going to show data from the characterization, I'll show some of those data in the research seminar, just because that's very key for the role of ABC4 in the RP. So here what I'm showing, it's build up of autofluorescence in a dish. So this is a key pathological marker for Stargardt model in the human eye and also Stargardt mouse models, and this is generated in a dish. There is no particular things, the cells are grown on an optimized condition, they have minimal retinal extract in some case or no retinal extract till three months and the retinal extract was provided for later on an age-dependent study. So this is key. So this happened in a cell that locks or have this mutation of ABC4 and we know the protein is very low abundant or non-functional. So this autofluorescence builds up over time, so we had this culture up to study up to 12 months, so it initiates of three months, but by 12 months it's significantly higher in Stargardt. Compliment C3, as I'm showing in these confocal images, it's strongly deposited on those cells, Stargardt cells, compared to the control. And complement regulatory protein CD46, which is acting early in the complement cascade and it's homologous, that's C-R-R-Y. It's strongly, it's significantly decreased and we quantify those complement protein, we quantify CD46 and we quantify the C3 deposition and you can see the inverse correlate. So more activation of the complement, it's confirmed by less controlling of this activation by having reduced level of the CD46. So how about MAC? These are flat amounts of our culture cells and we use antibody against MAC and it's depicted in red and without any quantification it's clear that this reactivity is stronger in the Stargardt cell control, compared to control. We did quantify its evidence of three months old and it's much more evidence at 12 months in culture. So what happens having this ongoing activation of the complement? Well, cellular integrity, it's lost and here we use a marker for follow-leading, DAPI stands for nuclei. The Stargardt cell starts to lose their nicely arrangements, it's cobblestone, no more hexagonal appearance and there is soldats and that impacts and we show that we measure the loss of cellular integrity, of course, creates loss in trans-resistance epithelium, trans-epithelial resistance, that means the integrity of the tight junction are no longer in place, the transport of the solutes from basal apical, it's completely lost and sold. That happens because we quantify the sol number in a dish and we show that Stargardt culture has lower number. So that's the summary for our disease in a dish. It's much clear this model red capitulate key phenotypic features that we've seen in human disease, we've seen increased autofluorescence, we've seen complemented regulation and age-dependent cell loss and I certainly advocate for this model to be used for, you know, understanding mechanism and also developing some therapeutic approaches because they truly red capitulate key features of the human disease. What is the clinical significance and concluding remark? Well, as I said at the end of this talk, I hope I convince all of you that a complement system is dysregulated in Stargardt, like we knew in AMD and I will, a second point that I'm making here, Stargardt is a true RP cell autonomous disease, we think this is the initial cell target and RP decline in function, lots of RP cells will eventually lead to photoreceptor sold out. It may, may not be true for all the AMD cases but certainly AMD cases that do manifest with mutation in ABCA4 genes and some in the complement genes may be also an RP autonomous disease. Therapeutic approaches for Stargardt and other ABCA4 mutated retinopathy I think should definitely gear towards RP cells along with the photoreceptors and giving the preclinical testing of the complement modulatory pathway in our mouse system with the AAV mutated therapy Suggest that this particular approach may be beneficial for Stargardt and AMD. So, would like to acknowledge my students both dark stuff and I highlighted the people who actually generated the data that I've shown you know, Tamara Lenis was a graduate student in my lab, she's also an ISAC resident, she's finalizing her residency this year and move on to her new stage of career. She worked on the AV gene therapy approach in the mouse. Narmind Kadi, postdoctoral in the lab, she's actually the one who performed all the car optimization of the IPS derived RP cells. Zichong Zhang, it's my staff research associate of course Jane Hu who's an amazing research associate that developed those tissue culture in Fidel humanity, tissue culture in the involved lab and my collaborator for this part of study that I show you today and of course I have to acknowledge my former mentors in Bok and Travis for introducing me to all those exciting areas of studies that I'm pushing forward now independently and thank you very much for your attention and I'll be happy to take any questions. Thank you, we had some questions here. Yes, that was a really impressive talk. Thank you. So I was wondering what you think the next step would be to try to develop a therapy for Stargardt's using focusing on the complement system, you know what would be the next experiments to do or things to try? That's a good question, I think it's still so the way I look now, having this amount of data on complement, Stargardt is not longer a simple disease so I truly view this more like AMD, it is complex because we identify this two independent pathway, one it involves this recycling of retinates that builds off dimers of vitamin A and one involves this complement, this regulation. So I envision that therapeutics will benefit from having combination intervention, it can be one or the other, each of them will provide some rescue but at what level and how it is hard to say because each individual will come with a particular genotype and each genotype will impact the level of this disruption of the RPE functionality. So we certainly like to evaluate some of those complement modulators in the human IPS cells, we're not quite there yet because we also found that there are other protein aggregates that we've done some proteomics, those are still preliminary data but those may be also mounting this complement activation, maybe this retinates just part of the conventional pathway but maybe something else other disruption along this endolyzosomal processing of anything that comes whether from the apical side or basolateral, they all have to be processed by RPE and endolyzosomal organelles, it's where we focus right now. So the new graduate student actually it's evaluating some of the ketepsine de-mediated processes and we're going to have some preliminary data at ARVO so that's something new. And the other aspect because I mean ever since we had that profile of negative regulatory complement in an early stage of the mouse eye where they are very low abundant, I was like how could that happen? Is this because RPE just cannot really synthesize those as a consequence of the lack of or mutated ABCA4 in retrospective, no thinking because we have the the news of ABC for being expressing the RPE or is consequence of build-up and consequence of complement activity internalized fragments in the RPE that eventually decline this ability of RPE to synthesize it. So we don't know that and that's somehow we're keen to understand early processes rather than go to the end point where the cell is filled with stuff okay that you cannot really dissect out anything because they definitely internalized and that ought to impact across the board all those functions that I mentioned at the beginning. Greg. A wonderful presentation. Thank you Greg. We've given a lot of thought lately to this concept that in immaculate districts whether they're monogenic or something like stargarts but a lot of interest in how do AMD chromosome 1 and chromosome 10 risk alleles influence these monogenic diseases like stargarts. Are you aware of any data out there? I am not. I am not. I wish I do. I know that's a big question now and I will be interested. I thought you might be able to enlighten me a bit about those. Well you would guess that somebody that's homozygous risk it's a chromatic underage focus and carry stargarts mutation you would assume that the pathology would be. Definitely and that was you know that was part of a very old application that I tried to put out there like looking at in you know select but we don't have the resources that was the biggest issue is like having a cohort of people who will have mutation in ABCA4 and have a normal or protective haplotype for AMD and have a cohort of people that have mutation of AMD mutation of ABCA4 but on a risk haplotype and I'm still interested in that. We do have some of those risk haplotype and protective haplotype from Dean Collection the fetal human RPE but if you do have such eyes with this type of genotype in your collection I'll be more than happy to to evaluate some of those markers that we we did with complement biochemical markers that but the more than fetal human RP haven't been genotype for ABCA4 and we genotype some of those that we knew the haplotype for AMD risk and protective and as expected so we did six of them six of each haplotype for AMD and out of six four didn't have any mutations in ABCA4 as expected and two did have some mutation but are not really one it's possible disease-causing mutations so they weren't quite appealing to us to push forward of looking at the scorer lane if that answer your question but it's very interesting question it's definitely something that I would be interested to the IPS derived cell that I show you they are on protective haplotypes so they don't have the YY they don't have the 406 and 62 yes yeah yeah definitely yeah so with the Stargard patient we did full genotype actually Dr. Goren has done this for all the AMD genes and I requested for Stargard because I was interested to know exactly what not just being clinically diagnosed with Stargard I wanted to know what is the mutation because as I mentioned this particular cell culture we use it for two reasons one to again look for expression of ABCA4 like another system that is devoid of contamination of the outer segment to look for the expression of ABCA4 and test the function of ABCA4 in the RP okay so that was key for us to know what mutation it has and what's the protein expression there and how does it work and this is the part that I'm going to talk at the noon seminar so it was a pretty good model for us and then of course complement was the other pathway that we thought it's worth looking all right hopefully you can come to the after the noon seminar I know everyone needs to come up and thank you very much it's very exciting and this is translational we are involved in one clinical study now looking at complement inhibitor C5 inhibitor intravitrally with Stargard disease so her work is already being translated thank you