 Down to our last speaker of the afternoon, thank you all for sticking around for this. Nico Franco is one of our first-year residents. He's going to talk to us about senior locan syndrome and I guess enlighten us about where we are in terms of gene therapy. Thanks everybody for sticking for the last presentation. So let me tell you our latest data on gene therapy on senior locan syndrome, but let me tell you first our initial work before we get there. There's really three points that I'd like you to get in here, understand what senior locan syndrome is, the human genetics, the mutations involved in it, and the animal model that we made. And then lastly, how we do gene therapy pre-clinical trials before we go to humans. So no financial disclosures. So I really just wanted to start with a case presentation highlighting senior locan syndrome. We have patients here, but this is from a published JAMA article. In this article there was a 20-year-old male that presented with increasing night blindness and difficulty with his peripheral vision on examination. He had some mid-peripheral pigmentation, vessel attenuation, as well as waxy power of the optic discs. And this led to the diagnosis of retinitis pigmentosa. What was weird about this case and interesting in this publication was there was a flame-shaped hemorrhages as well, which is not typical for retinitis pigmentosa as well as for a 20-year-old. So they suspected high blood pressure, and indeed this patient had a blood pressure of about 230 over 130. And he was diagnosed with advanced kidney failure with a creatinine of 8.2. He had subsequent workup with an ultrasound and showed smaller kidneys with increased echogenicity, and the biopsy was advanced kidney disease. So nephrinopthesis is a triad of small kidneys, cortical medullarisis, which explains the increased echogenicity as well as interstitial fibrosis, which is not specific, but you can see it in any advanced kidney disease. And retinitis pigmentosa, early retinal degeneration with nephrinopthesis defines what senior locan syndrome is. Just to finish up with this case, three years later he had a kidney transplant, which is usually the case for all senior locan syndrome patients. If not, they usually do not survive. A little bit about what senior locan syndrome, but this was first described in 1961 by two separate groups, and I've mentioned already it's both an early type retinal degeneration as well as nephrinopthesis. The visual symptoms, as most of you know, can present early in life first to second decade of life. And usually they present with polyurea and polydipsia, but it's very, very interesting to see flame shaped hemorrhages. That's why I really presented that case. This is a very rare disease, prevalence is one in a million. We actually have here in Utah, we've identified two patients with senior locan syndrome, which I won't talk about too much today. And we also know that this is autosomal recessive. So there's seven genes associated with senior locan syndrome written here, and HP stands for nephrinopthesis, and the protein it encodes are called nephrosistines. There's actually a lot more nephrosistine genes that I reviewed when I was still a PhD student, but all of this just caused a kidney disease. These seven are the only ones that cause retinal degeneration, and really why I wanted to concentrate on this. And for some reason, I really was interested in NPHP5, and because it just has all the mutations cause 100% retinal degeneration phenotype. Other reasons why I was interested in NPHP5 is not only does it cause senior locan syndrome, but it can also cause non-syndromic LCA, meaning without the kidney disease. I mapped out all the mutations known in the disease. So this is the gene, these are just the mutations. And all the mutations cause deletions, nonsense, or frameship mutations. Basically, we think it makes a non-functional protein. There's no NPHP5 in cells, and that's why it's causing disease. So to study NPHP5, we know it's non-functional in humans. We made a knockout mouse. We basically added a gene trap early in the gene, causing basically a non-functional protein. I'd like to draw your attention on this graph. This is a histology from a post-day 15 of mice. These are the blue things here are the outer nuclear layer, and then the red things is where outer segments should be. And the red thing is NPHP5 and knockout mice. We don't have NPHP5. Functionally as well, we recapitulate the absence of both scotopic and photopic function. The red line here are the knockout mice. You can see it's flat lined very early at postnatal day 14. To give you a sense, mice open their eyes at P12. So two days after opening, they're already blind. So we also know that in the knockout mice, it causes early onset retinal degeneration. So in here, in this column in here are the knockout mice. And I just want to draw your attention on the thickness of the outer nuclear layer compared to normal animals. And as you can see at one month of age, it's almost complete. This is just showing you statistically of progression of the retinal degeneration. So why does it cause retinal degeneration? We think that NPHP5 is important in this little area right here in the connecting cilium, which connects the inner segment to the outer segment of photoreceptors. And in here, what I can show you is so the green things in here is what the staining for basically this little structure of the connecting cilium. And in normal mice, we see that it elongates. But in the knockout mice, we see that it's just little two dots that never really elongates. It's really hard to see in those un-magnified views, but I did electron microscopy. And in here very early postnatal day 10, and then wild type animals, we see the nice connecting cilium in here and the outer segments. And then in the NPHP5 knockout mice, the connecting cilium just does not look, it looks abnormal. Outer segments never form. So that's the animal model that we've established for NPHP5. I just want to let you know that after this, there's two other spontaneous models that was identified, a cat that was blind or shown NPHP5, and as well as a dog, a pit bull terrier that has a spontaneous mutation. This dog is very important because we're both our mouse and this dog is the preclinical models to use for the gene therapy we're using. And there was just a talk by Gustavo Aguirre on gene therapy in Arvo recently, one of our collaborators as well. So why do we think NPHP5 or Cedralocon syndrome or this disease would be a good model for gene therapy? We think that it's good because there's a window that cells survive that we can add back the gene so that we can regain function. That was the hypothesis. So again, early on the disease, we see retinal degeneration in the knockout mice. There's still some cells remaining and we think most of these cells are cone cells. So because in this model, everything degenerates pretty fast, we did a genetic trick to make basically all the outer nuclear cells become cone cells. And with this, we show that even at two months, the cone cells remain, they never degenerate, but even if they don't degenerate, they still have a flat line in their ERG. So it's non-functional, but the cells are there. So the question is, can we wake them up for gene therapy? And so the latest results I'd like to show you. So we have an AAV8 virus that has a flag tag. The tag is just, I'll show you pictures, but we call it red with a human version of the NPHP5. So basically, we're trying to introduce back the human form, the NPHP5 in these mice. And what we did is a very technical experiment of doing subretinal injections in these mice at postnatal day 15 and harvesting them two months later. And I just want to remind you that two months, there's basically just two or so cells left. And these are our most latest data. So in the untreated eye, the flag tag or NPHP5, it's not there, but in the treated eye, we can see more red in here in the connecting psyllium or outer segment area. There's also some expression in the RPE, but the AAV8 virus usually targets both photoreceptors and some bleed through as well through the RPE cells. So that was encouraging that we can express it again. And most importantly, we can see rescue of function. So from a flat line in here of the knockout mice after we give back NPHP5, we can see some rescue effect, which is very, very promising. This is just quantification of showing you that in knockout, there's some substantial increase. This is a very, very promising for gene therapy. So also, we've started as well looking at the proteins that mislocalize. So this is one of the cone ops in mice, but in the untreated, they are usually mislocalized in here. And we think that the outer segments are forming more and they're mislocalized less. But we're still finishing up the study looking at other proteins as well as doing electron microscopy to see if there's any more rescue of the outer segments. But with that, I just wanted to say, so with our group as well as Dr. Aguirre's group at UPenn with a dog model, these two models will be invaluable before we go to human trials. They do have two candidates already, two people with NPHP5 mutations that have surviving cone cells that we're thinking. So these data are going to be valuable before we go to the human experiments. So in summary, NPHP5 mutations cause a senior look in syndrome and non-syndromic LCA or RP. We know that in the NPHP5 knockout mice, there's early retinal degeneration and that cone cells survive longer, allowing for gene base replacement therapy. And we have shown functional rescue of retinal function and NPHP5 knockout mice after some retinal injections. It'll be interesting to see whether they really form new outer segments. I'd like to thank my mentors, Wolfgang Behr, Dr. Bernstein, Kristen Hankey, who did the sub-retinal injections, very technical experiments, as well as other members of the BEHR Lab. I'd like to just finish off with just a quality improvement project before I get to questions. But the QI project that I'm working on is a multidisciplinary approach for patients with ocular cicatricial panphagoid. I really credit the past resident Zachary Jews for starting this and getting me on board, as well as Dr. Patel. So the problem is management of patients with OCP requires a multidisciplinary team approach, including oculoplastics, cornea, and dermatology. There's currently no system to coordinate fast access of these patients to these multiple specialties, which can really delay in the diagnosis as well as treatment for OCP. And the delay of treatment could be very devastating that could result in permanent blindness. The goals for this project in the first year, what we wanted to do is to create a website for easy access to the multidisciplinary team, both in-house as well as for referring physicians, and as well as to identify faculty members from the different sub-specialties to help coordinate the care of these patients. So just for these two goals, we have already have a website that we're just finishing up, and it should be live, hopefully in the next several weeks. There's a lot of process, and I've learned a lot through this process as well. It's data-driven, for example, just to let you know how detailed the website people are. So I've learned that most people, or 80% of people that go to the Moran website, don't go from the Moran, but they Google it, actually. And so if you type in OCP in Moranite or Utah, this would be one of the first hits that we'll get. So that's very, very interesting. People really don't use these columns up here to find services. So I've learned a lot in this. We've also identified several faculty members that will be heading up or that will be leading the charge in dermatology. It's going to be, of course, the chair. Dr. Zone, as well as Dr. Clark, has stepped up in our group. It's going to be Dr. Lin and Corna, and, of course, Dr. Patel, as well as Dr. Crum in our oculoplastics clinic. The future goals really in year two and year three is going to be very, very interesting. And I hope there's some dermatology residents interested at this already, and hopefully we can guard more interest for the interns. But we'd like to develop pre-existing standardized assessments at each patient visit to consider, like, epic templates to study outcomes in the future, generate standard diagnostic algorithms, including biopsy approach, specific exams, et cetera. And, you know, to have really this group to be talking to each other, to discuss treatment algorithms. And finally, really in the future, to identify faculty from other departments, GI, ENT, for patients that have non-ocular extracutaneous disease. Also, I'd like to use this model for other faculty or for other residents interested in a multidisciplinary approach for other diseases to have the kind of a template of how to do this. For this project, I'd like to thank Dr. Patel, Dr. Joe's, Linda Bult, Elizabeth Neff in our communications department, as well as Alana Schrader. With that, I will take questions. Thank you. So, Niko, congratulations. I mean, you've taken on orphan disease, and Wolfgang's described it to the detail we've never seen before, and now it will be eventually the treatment. This is really, really promising. The big problem we see with these orphan diseases is that at your rate, you're talking about, there's, what, 350 people in the United States. So there's no pharmacological company that will step up to do that. And hopefully, though, this is an avenue and it is a gene vector and a procedure that the process will be able to utilize for other areas. Otherwise, these kinds of things, even though we can do them, the cost of going to the FDA will be a barrier in which it's unlikely to happen. You know, absolutely, you know, and I always hear that comment. Definitely, it's going to be, it's going to be difficult. But hopefully, you know, senior law consider, there's just one part of the big other class of diseases of ciliopathy that could benefit and personalize medicine if we have specific mutations and if we have a window of opportunity, then we can tailor it for patients. But, you know, it's not as straightforward as you said, but. I think the FD80s take a different approach. You've got a group of them, you have one, and it doesn't need the whole expense and cost of going through the whole process again for a variant in that class. As of now, it kind of makes you do the whole thing. Thank you.