 Okay, and it appears that we are officially live. So hello everybody and welcome to another session of our Sussex Vision Seminar Series, as always within the Worldwide Neuroinitiative. I'm George Caffetzis, a master's graduate from Thomas Euler's lab and currently a PhD student with Tom Badden. As your host for today, I would like to once again begin by thanking Tim Vogels and Panos Bozellos for putting forward this initiative, this ever-expanding initiative towards a greener and much more accessible seminar world. Of course, having said that, allow me to get back to the reason we all gathered here for today and introduce our guests from Louisville University, Professor Maureen McCall. Following her undergraduate studies in psychology and visual psychophysics at the University of Maryland, Maureen went on to State University of New York at Albany for her PhD, where she worked with Helmut Hirsch studying the physiological effects of monocular deprivation. During her post-doc years in different groups at the University of Wisconsin, Maureen worked on the plasticity of the visual system and the circuits, retinal or higher, underlying basic signaling, while at the same time developing genetic tools to manipulate the nervous system. In 1997, she joined the University of Louisville as an assistant professor and has been located there ever since, nowadays holding the title of professor and vice chair for research at the Department of Ophthalmology and Visual Sciences. So without any further ado from my side, please all welcome Professor McCall for her talk entitled, Mutation Targeted Gene Therapy Approaches to alter rod degeneration and retain cones. Maureen, the stage is officially all yours. So I'll share the screen now. Thanks for the introduction, a very nice introduction and thank you for the invitation to talk today and to talk about some of the work that we've been doing, applying all the basic science tools that we've developed in the lab over the last 20 years and now trying to use those tools to create animal models for gene therapy and then to assess the efficacy of these gene therapy models. And today I'm gonna talk about, primarily about one of our approaches to try to alter rod degeneration and retain cones in an autosomal dominant RP pig model. And then at the very end, I'm going to talk a little bit about some new approach that we've been working on in collaboration with another biotech company. So there are two biotech companies that we've been working with on these two approaches. One is precision biosciences and the other is wave life sciences. So we focused on the P23H mutation in the autosomal dominant retinitis pigmentosa and we did that in terms of gene therapy because it is an inherited retinal disease which is one of the things that's required in order to do gene therapy. It is not a stop codon, it's a mutation. And so we have to fix that mutation in order to be able to fix the disease as opposed to the alternative, which is if you have a null mutation, you can just replace the gene and the protein that's absent. It's, as I said, inherited. It's patients come to the clinic when they experience night blindness initially and or tunnel vision, which is the next thing that happens. And that's because primarily the defect is in the rod photoreceptors and it's only after the rod photoreceptors die that the cone photoreceptors start to die as well. So autosomal dominant forms of RP and this P23H mutation is one of those, they account for 30 to 40% of the patients. Many of these are mutations in the rhodopsin gene, which is what P23H is. And in terms of the commonality of P23H, it's the most common autosomal dominant RP in North Americans. It's a proline histidine P to H substitution and it's at position number 23 of the rhodopsin allele. And so it ends up accounting for about 10% of the autosomal dominant RP cases. The thought is that the P23H mutation creates a mutant protein that is misfolded and this misfolding causes at the endoplasmic reticulum to be stressed. And that is the, so this misfolded protein response is the reason why these cells die. As I said, there's the initial result, the insult is to the rod photoreceptors and then once the rods are gone, we know that the cone photoreceptors can't survive very well in the absence of rods. And then eventually then we get to blindness, but the first thing that happens, as I said, is congenital stationary night blindness or tunnel vision. And the P23H rhodopsin mutation has been addressed in a number of gene therapy forms, primarily in rodent models. And we did a little bit of rodent work on this, but today I will focus only on the work in the transgenic pig model. But even so, even though there's been a lot of work on this, there's still no definitive cure. There's still nothing that has actually been taken to the clinic to help to ameliorate the night blindness and or eventual blindness in these individuals. So let me introduce our model. We started out by making a transgenic mini pig model and this pig carries the human P23H, the complete rhodopsin coating sequence. And we created several different lines of these pigs. And in the line that we chose to expand and study, the animal has somewhere in the vicinity of six to nine copies of the P23H human rhodopsin gene. They are inserted on a chromosome, and I can't remember which chromosome it is right now, but they're inserted head to tail. So they're all in one place. They're all in one integration site. And this pig also has two copies of the wild type pig rhodopsin. So they emulate the human condition because they are expressing the human complete transgene, but they don't emulate the human condition because in the human condition, you only have one copy of the wild type human rhodopsin. So they're a good model, they're not a perfect model. And so we tracked what happens to these animals in terms of their natural history here from P0 and P3, which are shown here morphologically, and then functionally all the way out past P120, but I'll show you using the ERG, I'll talk about that in a moment. So when the piglets are born at P0, there is the outer nuclear layer is still a little thinner than the outer nuclear layer of a wild type alliterate. However, they still have inner and outer segments and there's still a large number of rods, which is a good thing because that means that there are a large number of rods available to be treated. And there's not a huge reduction in the O and L thickness between P0 and P3. However, the degeneration of the outer nuclear layer does start to really accelerate between P14 and P30. And then by P60, we have two layers of photoreceptors, one layer of the cones and one layer of rods. And then by P90, we have almost no rods left in these untreated transgenic animals. In terms of their scotopic vision, which tracks pretty well with the degeneration of, I'm sorry, doesn't track well with the degeneration of the rods. And that's because we never see any real scotopic function in the rods, even as early as P3. So the transgenic animal scotopic response, this is the rod-driven, rod-isolated response is shown here in black. And you can see that this is just basically hugs the zero amplitude of the B wave. Whereas in the wild type, you see an increase as the retina develops and then a plateau after 60 postnatal days. In contrast, the cones actually develop quite normally through P30 in terms of their visual function. And at that point, then there is a divergence of the wild type B wave amplitude. This is the photopic B wave amplitude from the transgenic animals. And then after P20, there's a very slow, but a slow but steady decline in cone function with minimal cone function out in these animals when they're about five years of age. So here's our schematic model that one of the postdocs, Archana Jolly-Gomplup put together. Here is what we envision as a rod photoreceptor with all of the rod, all of the Rodopsin shown here in normal wild type Rodopsin shown here in red. And then a P23H rod has a mixture of both the P23H Rodopsin and the wild type. And then as a consequence of this, then their outer segments start to shorten and shorten even further. And then with the shortening comes a mislocalization of Rodopsin throughout the rod cell itself. And then they lose completely lose their outer segments. They exist only as a very small nucleus with a small pedicle and maybe a little gnarly bit right here. And then eventually this goes on to cell death. So just to remind you again, this is the dominant negative allele. So only one allele is mutant and it's a dominant effect. And the phenotype results from the expression of this mutant P23H protein which is hypothesized to be toxic. So, mutational targeted gene therapy approaches, what they do is to edit the genome in some way, shape or form, either at the level of the gene or the level of the RNA. And they target the mutant allele, hopefully only the mutant allele to show specificity. The issue is can they eliminate the expression of the P23H mutant allele or protein and will that then arrest degeneration? And so, and then the last couple of questions is about specificity and safety. And that is that there's no editing of the wild type allele and no alteration of the wild type protein. And thus, if you have this normal protein now in the cell, what can it do to help these rods to recover from this insult? To remind you about the AAV virus because that's what we're going to be using in our first approach to deliver our gene therapy tool. Here's the AAV with its capsid coat. It's carrying this payload. That is the therapeutic DNA. It's taken up into the cell, injected. In our case, in here, we're doing an intravitrile inject. Sorry, a subretinal injection. It's targeted to the photoreceptors both because of its subretinal injection and also because of the capsid and the promoter, which is a GRK1 promoter. It's taken up by the cell by endocytosis, sorry. The endosome breaks down, the virus is released into the nucleus where it forms an episome. And then from that episome, the protein is translated and expressed. And this is, so the protein is our therapeutic drug, for so to speak. So we teamed up with Precision Biosciences and they have what they call a mega-nuclease, which is also known as a homing endonuclease. And they have a proprietary patent on these mega-nucleases. We called the one that targets the P23H, row 1-2. The mega-nucleases are made from iCREI, which is from the chloroplast genome from Green algae. So they have a mechanism of action that's very similar to CRISPR-Cas9, but it's different in that it's a mega-nuclease. It's small in size. It's only 930 base pairs of coding region, which makes it very good for packaging and delivery of the AAV. The mega-nucleases are known for their high specificity and they are low toxicity. So here is a schematic of row 1-2. It's a self-complementary AAV-5. The promoter is GERC-1. Then row 1-2 is the nucleus that we're looking, that we're testing the efficacy of. And this nucleus recognizes a 22 base pair target sequence that's in the vicinity of this mutation in P23H. So the idea here is that this nucleus cuts the DNA in this 22 base pair region for the P23H allele. That creates a double-strand DNA break, which is then repaired by non-homologous and joining. This forms indels, many of which create a premature stop codon, and so the protein is inactivated and as a consequence degraded. This is the experimental design and timeline for our first set of experiments. So we've developed the ability to do sub-retinal injections in these neonatal piglets. And we do this in the vicinity of three to seven days post-birth. This timeline is a couple of days apart because if the animal is born at the end of the week, the surgeon isn't available until the following week and nor is the surgical suite. So we're always in this area. We do a baseline, so when the piglets are born, we do a PCR to genotype them. We confirm that genotype with a baseline fulfilled ERG. So we know that our animals have no scotopic, rod-isolated ERG, and so that confirms our PCR. At that point, we do the sub-retinal injection of row 1.2, which is mixed with the GFP. So the row 1-2 does not contain the GFP itself, but we have a GERC-1 GFP AAV-5 that's mixed in with the row 1-2. We do both clinical exams and fundus imaging, both pre and post injection to make sure that we don't see any signs of inflammatory responses. We rarely see inflammatory responses with this row 1-2. And then we follow up the development of the expression of row 1-2 and GFP over time. And so what you can see is we have this fluorescence fundus image that shows the GFP related area. So this is a proxy for where row 1-2 is expressed as well. We begin to see the GFP at about four weeks post injection, four to five weeks post injection. And then we continue to follow this. The GFP area does not change relative to the blood vessel pattern. And we've taken some of these animals all the way out to 42 weeks post injection. We also look at the OCT along the way. So this is an injected wild type animal. Here is the retinotomy site. These are blood vessels that smaller and larger blood vessels. And then the outer nuclear layer is this nice wide dark hypo reflective where this is the external limiting membrane, the myoid and outer segments, the RPE, the coroid, and then the rest of the retinolamination for the outer retina. This GFP area we've calculated is about 12% of the entire pig retina. So here are some of our pigs. They're in a little pig pile. They like to sleep like this. The study design was a dose escalation study. And we went from two times 10 to the nine all the way up to six times 10 to the 10. You can see the number of animal eyes that we have treated with each of those. And we have untreated eyes as a control in either the same animal or in a litter mate. We also did these injections in the wild type animal to make sure that row one dash two was safe and non-toxic. So this is a representative example of what happens in one of our treated animals. This is an animal that was treated with two times 10 to the 10 viral genomes. And so here's the animal's baseline response to the rod-isolated flash. So these animals are dark adapted for about 30 minutes. And then the flash is 0.01 candela second per meter squared. And as I said, these animals don't have a rod-isolated ERG response at baseline. And at six weeks post-injection, we begin to see this beautiful little B wave in the injected eye and then not in the, this eye was a control that was injected with PBS. That B wave amplitude grows at nine weeks. And then again at 15 to 16 weeks, whereas the response in the control eye is still is negative. So that's a representative example of what we see in animals that are given a dose of two times 10 to the 10 or six times 10 to the 10. Here, I'm depicting what if the family of ERGs at P120 as a function of dose. So untreated animals here in gray with the dashed line. And then with increasing doses, we see that there is an increase in dose. We see that there is an increase in the B wave that results at P120 with six times 10 to the 10 and two times 10 to the 10 overlapping each other. And then two and six times 10 to the nine smaller than that. There's also a dose response correlation in that, as I said, every one of the animals that was injected with six times 10 to the 10 or two times 10 to the 10 showed a B wave in the eyes that would not necessarily have one. And then in the other two doses, we saw fewer animals that had an induced B wave as a function of both six times 10 to the 10 and two times 10 to the nine. So there's two types of dose responses that we're seeing. And this shows you how those doses arise as a function of postnatal age. Here is the baseline for the uninjected animals, and then the dose response for these. And when you do the statistics on these data with an analysis of variants, both the two and the six times 10 to the 10 are significantly different from the baseline at 120 and then out greater than 140. So what this does is it sets up a minimal effective dose, which is two times 10 to the 10. So we know that six times 10 to the nine doesn't give us a nice consistent response across injected eyes and two times 10 to the 10 does. So this slide summarizes what happens in terms of this family showing the two times 10 to the 10 animals. The solid line is the mean and the standard error of the plus and minus one standard error of the mean is shown by the shading. So you can see here that at P30, which is just about three to four weeks post-injection where we begin to see what my friend Neil Peachey calls a little wiggle in the B wave, in the scotopic B wave. And then that increases as a function of time. And that is shown here in the summary graph with the statistics on it. So even though we're a little bit higher at P30, we're not significantly different from our untreated eyes. But then once we get to P60 and out to P120 and greater than 140, we are statistically different from the B waves in our untreated animals. So, all right. So that ends the summary of what we see in terms of the function of these. So what we're doing is taking a retina where there is no rod function from birth on and we are rejuvenating or inducing a B wave by using this row one dash two and the elimination of the P23H human redoxin protein. So then we started to look at the natural history of the progression using IHC before we had just used NISL. This is the published data from Fernandez to Castro at all. But we wanted to see what we could see in terms of the inner and outer segments and also in terms of the somas in the outer nuclear layer. So here's an H-matched wild type animal that's been reacted with an antibody to redoxin and then DAPI to show the somas of the cells. At P60, an H-matched untreated transgenic, you can see that they have mislocalized redoxin in the soma itself and these gnarly little inner outer segments where, and the redoxin as I said is all the way through the cell. If we go out to P160, we hardly ever see any labeled rod and if we do, it's one little isolated one that again has this really gnarly, the word is morphology. So this is the natural progression in terms of the immunohistochemistry. This is as I said before, the published data using NISL and plastic sections. So now what we wanna do is we wanna take a look at these retinas and we wanna look at the transition zone and then within the transition zone and outside the transition zone in one particular treated retina. So here's our scale bar, this is 500 microns. So we usually take a piece of pig retina that's somewhere in the vicinity of nine to 10 millimeters long. This is an animal that we euthanized at 39 weeks post injection and this animal was unusual. We had one animal that we gave one times 10 to the 10. The GFP is being driven by that AAV5 GERC1 and then redoxin is shown in red and the DAPI is in the turquoise. And so I think you can easily appreciate that we're outside of the injection area here on the right. And then there's a really very distinct transition zone very similar to the fundus image that I showed you. And you can see that there's very few, even at this low power, you can see that there's very few redoxin expression. Here's one little rod here. And then as we start to go into this transition zone then the redoxin is much more prominent. I'll show you a high power of that now. So again, here's the wild type that I showed you from previously and now here is our treated transgenic animal. Here is the transition zone. This is that one little red profile that I showed you up here in the untreated region of this retina. And I think you can appreciate that we have a lot of GFP positive rods that have beautiful inner and then outer segments and that the redoxin in these outer segments is localized just like it is in the wild type out on the tips of the outer segments. Again, here is our untreated animal and again, we rarely if ever see any redoxin in these animals. Now just to remind you that we are using the GFP as a proxy. We also have an antibody to row one dash two and we know that the expression of row one dash two overlaps the GFP expression in these sections. And I have that slide, if we have time later I can show that to you. But we have more cells in the outer nuclear layer in the infected region than outside the infected region and in the untreated animal. And we certainly don't have as many as we have in the wild type but there's certainly enough to support that rod function that I showed you. So we do something similar with our OCT images. So we overlay our fluorescence fundus image using some computer software and we overlay that based on the blood vessel pattern. This is the fundus image from the OCT and then we can define the area of the GFP expression on the OCT and then we can place that as a box on one of the B scans. So here's the untreated animal here and it's B scan and I'll show you these in higher power in a minute. And then you can see that we have overlaid the GFP and then this green line represents the B scan that you see at the bottom and that's always the way that this happens. And then we have software that allows us to measure each of the individual hyper and hypo reflective layers. So here I promised a higher power image of this. So here's that one of those B scans and I'm pointing out the damage that's caused by the retinotomy because when we do these sub-retinal injections we go through the retina and deposit the row one dash two in the sub-retinal space. You can see however that the retinal separation that occurs at the time of the injection has resolved and this almost always resolves within the first three to four weeks after the injection. And so I showed by green box here and now I'm gonna show you a blow up of the GFP positive area versus the GFP negative area and then no injection. And I think what you can appreciate and what I'll show you in the next slide quantitatively is that the outer nuclear layer of this animal and this animal was given two times 10 to the 10 viral genomes and we're looking at this animal at 19 weeks post injection. So the outer nuclear layer here is thicker than it is in the animal in the eye that had no injection and also thicker than the eye in the same eye that is outside the GFP treated zone. So here's our comparison of our IHC measurements. So we measure the outer nuclear layer in these animals and we also measure the OSIS length and then we will compare that with the OCT in a moment. So what you can see is that we're going from 200 to minus 200 to 200 microns and we center this line at that transition zone between the GFP plus and GFP minus. And so what you can see is that there's a very small area of transition and that within about 200 microns we are seeing a change in the outer nuclear layer thickness in the GFP area versus in the GFP minus area and the black line is the untreated, the same area in the untreated eye. And the thing that was really amazing to us was that we observed that when we had sacrificed a cohort of these animals at 20 weeks post injection and we had sacrificed another cohort at 40 weeks post injection that the OSIS length actually continued to grow in that 20 week time span. So our assumption here is that the humming and the nuclease, row one dash two, is continuing to cut and that the wild type pigredopsin is continuing to work and build these inner and outer segments. And we don't know if this increases because we never took it out past 42 weeks. The pigs get kind of big at that point. So here's a quantification of the OCT and now we've lined up four B scans that are a couple hundred microns apart. And you can see the same sharp transition between the GFP positive and the GFP negative areas. And then again, the same area of the retina in the opposite eye. And this is a heat map of what we see in terms of the O&L thickness. And that summary was all for animals that were injected at two times 10 to the 10. Okay, so that's great. If we do this treatment really early, even though the rods, while the rods are there and even if they don't have any function, we can get a very long duration efficacy and an induction of a B wave and a rescue of the photoreceptor morphology. And also the Rudopsin localization. But if we wanna be able to treat more people, we wanna be able to see how far out we can go and still have efficacy with this treatment. And so we turned to, and these are new data that Dr. Jalee Gampala will be talking about at this ARVO meeting. And so we decided that what we would do is pick P18 as the next later time point to treat the animals. Again, we used two times 10 to the 10. We did a baseline ERG to make sure that our genotyping was correct. We did a subretinal injection of row one dash two plus GFP. And then we started the same timeline and we took these animals out to 20 to 27 weeks post injection. Similarly, we get about 12% of the retina infected. Okay, and so now I've shown you these data, these are the summary data for animals injected at P3 to P7, but here's the summary data for our animals that were injected at P18. And this is based on five eyes versus 13 in the neonately treated animals. But what you can appreciate is that we still see this a very nice and significant increase in the B wave and that that is time dependent. And at 20 to 27 weeks post injection, we're still seeing a slight increment, but these guys never have as much efficacy in terms of the amplitude of the B wave. And that's because we don't have as many rods to treat. But the good news is that even though these rods are another two weeks, two and a half weeks down the degeneration trail, we're still seeing some efficacy. So this is great in terms of the patient population that we know that you can even be further along in degeneration and still be able to save some of the rods and some of the rod vision. So for more details, please go to Archana's talk, which is May the 2nd at 1017 Eastern, a middle, what is it? Mountain time. So even, and so here is just a brief overview of what we see in terms of the structural outcome. So now we were interested in, well, what's happening to the cones? So here's a low power image very inside the treated area. This is an animal that was treated at P18 with a two times 10 to the 10. The GNAT labels the cones, the GFP labels the rods and M-Opson, which can be found here at the very tips of the terminals. So not only are we having a positive effect on the rods because these are the ones that are in green here, but we're also having a positive effect on the cones, which is consistent with what we think that you need rods around in order for cones to survive. This is a image of the same area of the uninjected eye of this same animal. And you can appreciate that the cone morphology is pretty unusual, it's very bulbous and it's got this very dark area here that actually is P and A positive. So we haven't figured out what that means, but that's the case. I don't have that data here. So here's my summary of what I've shown you. The row one dash two mega nuclease cuts human P23H redoxin. It doesn't seem to cut the wild type pig redoxin. This genome editing reduces and or eliminates expression of the mutant P23H redoxin. That allows the pig redoxin to rebuild inner and outer segments and localize redoxin appropriately. Then the restoration depends on the time of treatment. P3 is better than P18. And we're still now going to look and see if we can go to P30 and greater than P30 to see what is the final time point where we can't do any more restoration of either structure or function. So the other questions, now that we've shown how efficacious this treatment is, the other questions that we need to answer, and we are in the process of starting to do this, is to ask whether row one dash two is safe. And what I mean by safe is, so it's a different difference than specificity. So specificity indicates that it cuts only at the P23H allele and not at the wild type allele, but safety has to do with whether it cuts any place else in the genome that is unexpected. And we're gonna do some of this work in tissue culture. We're gonna do some of this work in mice. And we're gonna do some of this work in collaboration with David Gam on human retinoids. So stay tuned for those kinds of data that will come out in the next couple of years. So then the question is, what if we could go earlier? So we can't go earlier in this pig model, but if we could generate another pig model where the photoreceptor degeneration started later, then that will allow us to try to treat the retina prior to any sign of degeneration. And then can we keep the retinas from degenerating, the rods and the retinas from degenerating completely? Then that would be important because if you have a treatment that is efficacious early, then you can use some kind of a genetic screen to determine whether a child in a family carries the P23H mutation and you can try to treat them previous. And that also expands the therapeutic window. And so we're in the process of working with the National Swine Research Resource Center to develop a new transgenic pig model. And I hope that I'll be able to talk about that model next year. All right, so now I'm gonna switch gears to talk about this new editing approach that we have been looking at with a different company called WaveLife Sciences. And what Wave has done is to have a proprietary invention in terms of these antisense oligonucleotides, they call them stereopure. And the reason that they call them stereopure is that they have a proprietary thing that I have this little circle over that keeps the stoichiometry of all of the alleles, all of the oligos in the same orientation. And so they figure out what the best orientation is for this oligonucleotide and everybody's got that same orientation as opposed to other oligonucleotides where these things change their chirality. And so you can have only a few that have the appropriate stoichiometry for optimal cutting amid all of these others. So we call this Wave1. It's been designed to target, again, the P23H human rhodopsin allele and to maintain the wild type allele. And then what the mechanism of action is shown here. So these ASOs are injected intravitrally. They make their way through the retina to the photoreceptors where the ASOs are endocytosed. And the ASO goes here into the retina where it attaches to the mRNA. And through an RNA H, it cuts the mRNA for the P23H rhodopsin eliminating thus the protein expression of the protein expression of P23H mutant rhodopsin. So as I said, they have a proprietary backbone and this increases their stability and protects the configuration. It's very different from other oligos that are random. It also gives them a neutral charge so they're endocytosed more easily. And I said that administration is intravitrial and there's no virus or other transfection protocol which just makes it a much more palatable thing. It's like injecting VEG-F into the eyes of people that have macular degeneration. The downsides are it's only effective when the ASOs are present in the cells nucleus and they don't stay around forever. So that means that it's gonna require re-administration. So from a point of view of a drug company, that's great because they'll just keep selling it as opposed to the row one dash two where it seems to be a one-time deal. Experimental design here, we're still doing P3 to P7. We do this intravitrally injection. We look at ocular exams. We don't see any inflammatory response with the intravitrally injections. And again, we do OCT imaging. And these data will be presented at the Arvo meeting by another post-doc in the lab, Jennifer Null. So here's a wild type of H-matched animal scotopic B-wave, again an untreated transgenic. And here's one animal that we injected intravitrally in both eyes. And this is after eight weeks post-injection. And then here's the same animal at 16 weeks post-injection. And these are the quantification for the summary. We don't have a lot of animals in this preliminary experiment, but we're hoping to get more money to be able to do more work because this really looks promising. The earliest that we see a really... The earliest that we've looked for an effect is four weeks post-injection and the B-wave is larger than the B-wave at four weeks with the homing endonuclease. And that's probably because the AAV takes longer to ramp up than the oligonucleotides. And the data from these two animals are plotted in these two in here and here, where the one eye doesn't change very much but the other one eye starts to decline. And so we think that there may be a decline and of course the interesting thing is once we decline, can we re-administer? And if we re-administer, do we get equal efficacy? And here's just a taste of what the Maradmore phylogy looks like at eight weeks. Here's a wild type. Here's an ASO at eight weeks and an ASO treated. And you can see that this has really maintained the outer nuclear layer, which is incredibly exciting. And this is an aid-matched, untreated animal just to show by comparison what it should look like without treatment. Okay. So to summarize, wave one, which is a stereopure ASO at its human P23 tridopsin mRNA and it doesn't appear to affect the pig row RNA. Editing is faster, as I said. And so the questions that we've already addressed is the efficacy of it. We haven't looked at the specificity yet in quantitatively and we haven't looked at the safety of it yet. And so we need more animals because we don't have a large N and we need to take them out a little longer than 16 weeks to see how quickly the efficacy drops. And then we need to intervene and do a re-administration probably at 16 weeks post-injection. Make sure that it's safe and that it doesn't cause an inflammatory response and that it maintains this B wave. So, and then like the other, how late in degeneration can we use this? How long will the treatment last? And a real important question is can the retina be retreated? And if so, how often can it be retreated? So a lot of people really have contributed significantly to the work. Archana Jolly-Gomplah is the postdoc in the lab that has been driving the precision work and the people at precision are listed here. Dave Morris is currently the project manager. Christy Viles was our previous program manager. She's the one that was the real enthusiast and really drove this project forward. Jed Chatterton is the person that initiated it. And then Jennifer Nall has been driving the wave project and that's been done in collaboration with WaveLife Sciences in particular, Mike Byrne. Our other important colleagues are Wei Wong who does all of our sub-retinal surgeries. Erica Toller who does all of the clinical ocular exams along with Chris Langlow. And then a lot of people from the lab, Sherri Willer, Stephen Nash, Alec Bradley, Nozarel Hassan, Joe Prestigiacomo, Olivia Jacobs, and then previous members of the lab, Nila Four, Piri, Gobinda Panjini, Buban Nassahu, James Franzen and Mahaj Bahr. And we wouldn't have had these animals nor would we have even had the idea if it hadn't been for the previous chair of ophthalmology, Hank Kaplan. So I'm always indebted to Hank for that. And the lab has been very, very happy to be supported by the NEI, Precision Biosciences, WaveLife Sciences. It's also, the work is also supported by my endowed chair from the Kentucky Lions Eye Research and all of those beautiful confocal images are made possible by the Roundsible Family Foundation. So I would be happy to take questions at this point. Great, thank you very much, Maureen, for this wonderful talk and for giving us the rare opportunity to hear in our series about different editing approaches and like about pig models as well. I have a couple of questions myself and I will seize the opportunity because there are no questions appearing in the chat already. I would like to remind to our audience that they can join us in the Zoom room that we are currently sitting at by following the Zoom room link that I just posted. And my first question is like starting from something really generic. How do these pigs behave naturally? And have you considered doing any behavioral experiments with them? That's a great question. Yes, so the pigs are kept like humans in this urban environment. They're kept under relatively good light adapted conditions. And so they don't behave particularly abnormally but we did do some mobility assays on these guys. So we had access for a little while to a large empty room and that empty room allowed us to set up a maze that was about 40 feet long and about 30 feet wide. And we did run these pigs through the maze both binocularly and monocularly at scotopic and photopic levels. And we did this using, the downside was we only had a single infrared camera but the animals do better with the injected eye than they do with their uninjected eye. And in terms of they can navigate the maze more quickly and they bump into things less frequently under scotopic conditions. And these are truly scotopic conditions because in my experience with helping to run these animals in this maze, you can't discriminate color under the light levels that we're using. So it's because we only had a single camera it's not particularly quantitative but qualitatively I can tell you and we do have videos. I just don't, I'm not particularly comfortable with showing something that's so qualitative but thank you, it's a great question. And we are trying to get together a screen task that will allow us to do both light, dark discrimination as well as acuity. Right, thank you very much. I would like to let you know that because you don't have the YouTube tab open that people both greeted you at the beginning and are thanking and congratulating you for your talk. And before I move on to the questions that have just been posted like by Marla, Feller and Tom and people are already joining us maybe like given that both Tom and Marla are here maybe after my second question they can ask the question directly if they prefer. So my second question is we know that some animals have retinal waves, right? Like that help the retina mature. And we know that this is the case for mice, for example given that in your pig models the rod photoreceptors are already not functional like at P zero. Do you know if like how accurate is this model of what happens in humans, let's say given that the dead generation part is already interfering quite a lot with the developmental part if pigs have retinal waves. Yeah, so we don't know whether they have waves it's a really good idea and it's a really good question. And so I can't tell you what the answer is but I think that we will definitely think about setting that up and doing that for some of our assays. Yeah, thank you. No, thank you very much. So given that both Marla and Tom is here Marla, would you like to ask your question? If not, then I can read it from the chat and convey it. She's trying to connect to audio. So I will give her a second or two. Okay, so I get on to the YouTube thing. You don't need to because like I will be shortly terminating the broadcast so if people want to follow us they can join us here. Hello Marla, would you like to ask a question directly? Good morning. Hi Maureen, beautiful talk. Amazing progress. Yeah, my question was about in humans who have this disease, how early does it get diagnosed? You know, so like at what stage of the progression of the disease, are humans to be aware of it? That's a great question. So usually, so there are some case studies where if a parent has RP, children are diagnosed in the vicinity of seven to nine and they do show some defects in their scotopic vision. They don't show defects. I don't think anybody looked at the OCTs yet. The majority of these people come when they start to have night blindness and that is in their late teens and early 20s when there aren't very many rods left and that's why we're so interested in how late can you go because it would be great if you, even if you had only one layer of rod photoreceptors that could be treated, if we could rescue those rods and regrow those outer segments, then they'd have some night vision and they wouldn't lose their cones, right? Yeah, and that's where, that's really the crux of the matter in terms of the patient's quality of life is not losing their cones. You know, everybody can, you know, people with CSNB drive during the day, and we have so much light in the atmosphere in urban settings that they can do kind of okay. You wouldn't want them driving a car at night, but they can navigate, but to save the cones is huge. Thank you. Thank you. So a question that just appeared from Henrique von Gerstorf in the chat. Are you planning any multi-electrode array measurements? Yes. Simple as that. We've done some array measurements and published them in the untreated animals, but we are planning on doing this in the next couple of iterations in these animals. Yeah. Before I convey Tom's question, given that you mentioned treated undone, treated animal, my question is, because you mentioned that inflammation happens, but doesn't persist after some time, have you tried to do like a mock injection to see if the inflammation, like even the transient inflammation has an effect in what you observed? So we don't see, first of all, we don't see very much inflammation generally. It's sporadic. We do pre-treat these animals with prednisone. So there's a prophylactic, I forgot to mention that. So three days before injection and 30 days after. And it is, we have seen, when we have seen inflammation, it has only been at very high viral titers. So we saw inflammation when we were above one times 10 to the 11 in these animals. And that was pretty awful. And that was a first pass through trying to figure out what an effective dose was. And we've never seen any inflammatory response in any of the animals treated with the wave. And so, yes, we do sham injections either. So we do some with just GFP. We also do some just with the PBS solution that the virus is suspended in. And they don't usually cause much damage. There can be surgical damage. And sometimes that resolves and sometimes that doesn't. Not all surgeries are perfect. And pigs very more difficult than human. Thank you very much for that. And going to Tom's question, have you looked at retinal function in downstream circuits beyond what the ERG picks up? Not yet. But of course, Tom would know that I really wanna know. But yeah, but the bandwidth that we have so far is to be able to characterize the efficacy at this point. And then once we're there, then the plan is to either, is to use the multi-electrode array and look at the ganglion cell function. Right. And before I terminate the broadcast, like just a follow up on what Tom just asked and what you replied, we know from mice that concurrently with the death of the photoreceptors, we also have like alterations at the circuit level. So like rewiring. Do you know if this is the situation also in the pig models that you have? Like, have you looked at? We haven't looked downstream yet, except in the untreated animals and they have much lower response proper responses in terms of photopic versus scotopic response. The thing that always strikes me about these, the pigs, even in the transgenic animals that are untreated is that the outer plexiform layer doesn't collapse the way the outer plexiform layer in the mouse collapses. And so, yeah, I'm really excited to take a look at what the bipolar cells look like and whether they're in the horizontal cells and whether there's any alteration there or whether the pig for some reason because there are so many cones, for example, that they can maintain that outer plexiform layer and maybe there's some rewiring of the rod bipolar cells to the cones. And it's one of those things on my list of important things to do. It's just that we haven't gotten there yet. Yeah, and rightly so, I guess. So at this point, I would like to thank you once again, Maureen, for this fantastic talk with your pig models and the different editing approaches. I would like to thank everyone that attended once again, another seminar of ours. And if you want to keep track or participate in this post-talk informal gathering, by all means click on the link that you see in the chat and you will be dropping in immediately. I will be letting you in the room. So thank you very much once again. Thank you and thanks to you and to Tom for the invitation to speak. I really appreciated it, bye. Okay, so now we are officially off air.