 Hello good morning everyone. We're gonna go ahead and get started with today's grand rounds. Today we have a guest speaker and what I'm gonna do is slightly a little different from what was expected. I'll introduce Brian Jones who will then introduce Dr. McCall. Thanks. Not sure how this usually works but Dr. McCall is actually gonna give two lectures today as usual for a basic science person so I'd encourage you also to attend the noon lecture where you'll get a different story. So it's my distinct pleasure actually to introduce my friend Maureen Dr. McCall and we've known each other for a while. Maureen Dr. McCall got her bachelor's in psychology at the University of Maryland and then did something very cool. Got a master's degree in psychophysics and psychophysics is not taught really much anymore which is a tragedy especially given all of the clinical therapeutics that are going in into play. It's actually one of the great shortcomings. I'll leave that editorial there. And then she got her PhD in neurobiology at Albany in New York, did a postdoc in Wisconsin and then started shortly thereafter a little while after at University of Louisville where she's been for a number of years now. So Dr. McCall thank you. So thank you for inviting me and in particular because it's snowing in Louisville and the university's closed and also thank you for having grand rounds at 8 a.m. instead of at 7 30 the way we do in Louisville. And lastly you know I talk fast and I get going and so sometimes I don't see a hand raised so please feel free to say hey slow down and explain something to me as I go through my seminar this morning. So my lab has two arms to it and this morning I'm going to talk to you about the work that we do that's more translational and related to retinal photoreceptor degeneration. And so just to give you an idea of where we're going to go with where I'll go with this lecture is I'm going to briefly touch on the retinal circuit and then the changes that occur in retinitis pigmentosa and so most of you probably are more of an expert in this than I am. And then I'll talk a little bit about the genetic nature of retinitis pigmentosa because all of this leads into why why this is a disease that can be addressed in terms of potential therapies with gene therapy which is where I will end up. And then in between I'm going to tell you about the creation and characterization of a pig model of RP because the pig is a very good model for the human disease. And then finally I'll tell you a little bit about some of the data that's coming out of our gene therapy approaches in this model. So this is just a schematic of a retinal circuit and from an electrophysiologist point of view the photoreceptors are on the top and I know that anatomical views usually put the photoreceptors down here and so I'm just going to tell you that most of the time in my slides you'll see photoreceptors on the top and the ganglion cells on the bottom but occasionally when we are collaborating with our our anatomists the slide will be flipped upside down and I'll try to remind you that that's the case. Okay so we all know that the way this works is light comes in through the transparent retina and then that light energy is transduced into a signal within the photoreceptors and then that results in a change in glutamate release from the photoreceptors which then leads to a vertical information flow through the bipolar cells to the ganglion cells and out from the ganglion cells to the rest of the of the visual system. So in in retinitis pigmentosa because of mutations that occur in proteins in the rod photoreceptors you get the first part of the first step in the sequence of neurodegeneration is that the rod photoreceptors die and they die over a certain length of time and then it because of reasons that we don't understand but are active areas of investigation the cones can't be sustained in their normal morphological and functional normally and normal morphology and function and what we end up with at end stage disease are cone photoreceptor somas that don't have their outer segments or their pentacles so these structures have have retracted but even late in in in disease in both the in both the pig model that i'll talk about in the rodent models that we know about in humans there are these cone photoreceptor nuclei that remain and so this is exciting from the point of view that if those cells are still there there's the potential that they can be rescued and remake their connections but in this in this stage when the cones now have retracted and no longer make connections with the second order neurons we get blindness and and so in in the end stage disease in humans they are legally blind and don't have any any real light perception at all so this occurs in a lot of patients and in as an autosomal dominant disease 20 to 30 percent of the of the patients that you see will have an autosomal dominant mutation that means that they only need one mutant copy and that and then they get the disease manifestation and out of those 20 to 30 percent eight and a half percent of these autosomal dominant rp arise from mutations in the rhodopsin gene which is shown here and the rhodopsin gene is this transmembrane protein here's the extracellular side and here's the cytoplasmic side and you can and there are mutations in a bunch of different places the one that i'm going to focus on today is a proline to histidine mutation here at position 23 but there's another there's several other models there's a there's a model out here that has a mutation at position 347 and then there are a bunch of others in between so what happens is that because this protein is mutated it doesn't traffic properly and to the outer segments of the of the photoreceptors and it becomes toxic and the photoreceptors die as a consequence of this so it's a little bit like the neurofibrillary tangles that you hear about in Alzheimer's and they gum up the works and because of that the cell goes into into stress and the cell dies in rp most of the it's a progressive disease and as you all know we're our visual system is really good at fooling us you don't see patients in a lot of cases until they're very well progressed into either rp or glaucoma any of these things because our visual system fills in and and is redundant in some respects and so these patients really don't come in until they experience night blindness is usually the first complaint when they're seen in the clinic so once they get patients have come in they've lost a lot of their rod photoreceptors already and so we don't know what the early sequences are in these patients it might become more obvious now that we have with that we're better at diagnosing because these are autosomal dominant and so that we can do genetic testing and then we could track children as they start to progress into the disease but that hasn't become one of the things that's that's that's currently done a lot and so what we've done is to turn to animal models to try to study the early steps in in the sequence and and then and in these models to try to intervene early mid and late in the disease to see what what our therapies can do so the classes of animal models there there are two one are spontaneous mutants and these are animals that are found usually by mistake we found a mouse model for congenital stationary night blindness because one of our collaborators was using mice from a particular colony and the control animals didn't have an ergb wave and so all of a sudden that opened up a whole set of ways of looking at congenital stationary night blindness other mouse models are that are common for rp are rd1 and rd10 and again these have mutations that are part of not the rhodopsin gene itself but part of the photo transduction cascade there's a rat model with a the royal college of surgeons rats that that's a mutation in the rpe is important because it fagocitizes the outer segments and if you don't fagocitize the outer segments then that becomes toxic to the photoreceptors themselves and then they die again from a slightly different point of view and then there are several inbred dog models and these arise because because of the inbreeding in in particular strains of of dogs and so then the puppies come up blind and then those puppies are usually donated to a facility at the University of Pennsylvania where they study them and then more recently we've been able to molecularly manipulate the genome of mice and a little bit in the rat and then a little bit in the bunny and most recently in the pig and so what we can do is we can create either one of two kinds of molecularly manipulated animals we can create transgenic animals and they mimic the human defect because they they can carry actually you can actually make them carry the human a mutant gene but they don't mimic the gene complement so in a in a retinitis pigmentosa patient as I said if it's autosomal dominant you'll have one copy that's a mutant copy and one copy that's a wild type copy in the transgenic animals you get two normal copies and then the trans gene that's that's inserted into the genome as an extra protein and and examples of this are the p23h redoxin mutant mouse that's not spelled right rat and then the pig that i'll tell you about today there's another mutation in redoxin at the at 334 this is a searing to a to a termination stop codon and this is a rat model and then the original pig model that was that was made in in john and uh rom petters lab was a pro lean to lucine at at at redoxin 347 this was a full-size pig the pigs i'll tell you about today are mini pigs so now more recently we've been able to knock in the mutant gene so you replace the one copy of the wild type gene with the mutant gene and part of this uh the ability to do this is because of the work of mario kopecki who i you probably all know as well as oliver smithy's who who died just recently and in here this now you can get the human defect because you can knock in the human uh the human mutant gene and and you knock out and and and they knock out one copy of the of a wild type gene at the same time so now you have the human defect that that is mimicked and the gene complement that's mimicked we have not been able to make a knock-in pig yet but but the but there's a recent recently developed p23h knock-in mouse that's been very useful made by uh chris paulchevsky as well as by ted ted wensel so these are the animal models that we have available to us and like and as i said i'm going to tell you a lot today about this p23h rhodoxin mutant pig that we developed and we did this in collaboration with the national swine research resource center uh that's located at the university of missouri and they are the they are the cutting edge of of making transgenic and knock-in uh pig models and so they have been successful in making knock-in pig models but not for rp yet this is one of the goals that we are continuing to work with them on and randy prather is the is the lead at the national swine research center and we work with both eric walters and and and jason ross to create founders of the p23h mutant pigs so this is a mini pig uh and the reason that the pig is such a good model is that its eye size is similar its developmental program is similar to humans it's vision it has a visual streak so here's the fundus image of one of our our transgenic pigs here's the optic nerve head the blood vessel pattern is somewhat similar to to humans we have a a blood vessel free uh what what is called the visual streak it's not a phobia it's not macula but it's the next best thing given that the only other organisms that have phobia and macula are non-human primates some non-human primates um and and human primates so uh we they used somatic uh cell nuclear transfer to do this and then what we did was to create and characterize six different founder animals um and the important thing is that they carry this human p23h redoxin mutation and so any therapy that we find that is efficacious and safe uh can then be easily translated to the clinic because it's the same mutation that you'll be treating in in the in the human patients so a lot of what we do um in terms of the electrophysiological assessment relies on the erg and i know all of you are familiar with the erg here's one of our uh more uh mature uh pigs with a jet electrode on his um on his cornea this is the lkc gansfeld and we we literally shoved his place gently his head into the the the gansfeld and what you know is that the erg is a gross potential and so it it it reflects all of the processing of the retina both peripheral and central and that's going to be important uh in terms of some of the uh in some of the challenges that we face in gene therapy so the a wave as you are all probably familiar is the is the the change in the polarization of the photoreceptors after a flash of light it's a doubt and it's just a and it's followed by the b wave which is the post receptoral component primarily from the depolarizing bipolar cells and if you see these little tiny wiggles on here those are called oscillatory potentials and they reflect a lot of the circuitry here in the inner in the inter plexiform layer so we can uh assess photoreceptor function and bipolar cell function and you can and as i said if you have congenital stationary night blindness you'll see a normal uh amplitude a wave but no b wave in rp what happens is that the a wave decreases and the b wave also decreases because it's dependent on the polarization of the of the photoreceptors and we can change the lighting condition so we can dark adapt the animal and and use very dim flashes to assess rod photoreceptor function or we can put an adapting background which saturates the rod response and then and present a bright light light flashes and assess cone function so we can get both rod and cone functions separately uh in using the erg then the other thing of course you know is that the erg is a non-invasive gross potential so we can use this on a weekly or monthly basis to track what's happening in terms of the degeneration process or in terms of functional recovery after therapy so i'm going to take you through the characterization of the of the six founder pigs that we that we did uh to figure out who to use to for for the rest of our experiments and what we found electrophysiologically is that if we looked at the scotopic b wave and we started at three months we found two different classes of founders we found the ones that were severely affected and had no rod function at from three months all the way through 18 to 24 months there were some that were moderately affected so they had rod function at three months and then that declined um and then in terms of their cone function uh the the severely affected had some change in the in the cone b wave uh at even at three months and then that is pretty solidly maintained all the way out to 18 to 24 months so this is very similar in in terms of the the human function except that the humans have rod function so they're more similar to the moderately affected but when we were trying to figure out which animal to use as our founder for the rest of our experiments we had one other factor that we needed to take into account and that is the size of the animal so these are mini pigs but mini pigs are still pigs and so once you get out to about uh you know 22 months these are substantial animals they weigh in at about four 500 pounds and so we decided that from the point of view of animal husbandry uh that that a severely affected animal should give us a phenotype that we could work with in a time frame where the pigs only got to be about 150 or 200 pounds at termination and so a lot of this electrophysiological work was done by Juan Fernandez de Castro uh who was a postdoc in the lab and is now one into private practice in in Florida so here are some beautiful histological preparations from Brian Jones who helped us with looking at these founders this is an adult domestic pig so here are the photoreceptors out here in the outer nuclear layer and the and their outer segments and the rest of the lamination pattern is beautiful and normal in this adult in the two moderately affected animals at at 18 and 22 months you can see that there's still some of photoreceptors that are around these are primarily cones and and here's our one of our severely affected animals at 12 months and you can see that all they have left are these cone photoreceptor nuclei and then the inner nuclear layer and then the ganglion cell layer so uh but this is so when when we first started with these guys they wouldn't let us work with them before they were before three months of age and so we didn't know whether they had any rod function at birth and so that was something that we needed to do in terms of once we got the f1 or the offspring from whichever founder we we selected so we selected this one called 53 one and as i said we knew that he had no uh no no rod function at three months of age but we knew that his cone function was similar to wild type and we knew that there was a slow and progressive decline of cone function so because of these limitations in terms of husbandry we selected him as the founder and so then we wanted to know uh for in terms of what the model could do for us whether the this this founder transmitted the trans gene in Mendelian fashion so as an autosomal dominant mutation whether the f1s have the same phenotype as the founder and then we wanted to be able to use these guys so that we could look and see whether rods were present at birth and whether they functioned and then declined and then we wanted to know whether the cones developed normally during those first three months before that they declined and so i'll go through those data now so here are the morphological data first and so here are the wild type animals on the left and the p23h transgenic pigs on the on the right this is at e105 so you can see that they have a full complement of photoreceptors right prior to birth the gestation time in a pig is about 113 days and if you count the number of photoreceptor nuclei you can see that they're identical at p0 there is a small change in the in the outer nuclear layer that's shown here in the summary and here's p0 and p3 there's a there's a further i'm sorry this is p3 there's a further decline and then as you go past between p14 and p16-60 there's a real decline in rod photoreceptors that at p90 and all the way out at p120 you see that there's a single layer of nuclei and and they have the morphological appearance of of cone nuclei and and they and we have i'll show you we have cone function out at p120 and then these data were generated by Wayne Wong and Patrick Scott in our department there's a central to peripheral decline in rod photoreceptors that's shown here summarized on this slide in these butterfly plots so we go from p0 to p3 all the way to p120 and this this image of that's a schematic of the of the fundus shows where the most effect is which is here in temporal ventral retina so if you take a look at what's plotted here is that there's more of a decline in temporal retina early here and here temporal retina is lower than the nasal retina and then the ventral retina is more affected than the dorsal retina until you get out to about p120 which is why this dark shading is here in this area in terms of the in terms of the ultra structure these are much higher power images than i've been showing you before and you can see that at p0 the cones have very nice structure they have inner segments and outer segments and there are even some rod photoreceptors that have inner and outer segments sprinkled in here at even higher power at p0 you can see ribbon synapses in both the wild type and the p23h and you can see that the cone pedicles are normal in both of these animals at p3 and as we go through from p14 to p60 the cone outer segments retract and so do their pedicles so at greater than p90 the cone pedicles have retracted as well and even though they are they've retracted we still have some signaling through the cones in the retina and i'll show you that right now so over here are representative traces of ERG responses at p3 in gray is the wild type and in black are the transgenic offspring so we have these animals born at the University of Louisville and so we can begin to to assess them as early as p3 we find that before p3 it's a little difficult to anesthetize them and then bring them back but they're pretty hardy by p3 and at p60 this is so at the top here is the rod response so as i showed you in the in the founder animal there's there's no rod function here but unfortunately we have no rod function even though we have full complement of rods nuclei at this point but but at p3 and all the way close to p60 the cone function is is pretty good and pretty robust and the and the the summary data is shown here on the right so this is the scotopic or the rod driven responses we don't see any in the in the offspring of of 53 one just like in 53 one which is plotted here in the triangles whereas the wild type pig has a has a normal development of rod function and then that plateaus after about p60 in terms of the photopic response p3 and p14 and p30 are almost exactly the same between the wild type and the transgenic so this is nice because the cone system seems to develop normally and in many of the very aggressive forms in the rodents the the the degeneration of rods and cones occurs at the same time as the normal development so you have these issues of are you looking at a change in developmental sequence or are you looking at a change in photoreceptor degeneration and so we in terms of our cones we can say that the cones develop normally and a lot of this erg data was done by a graduate student in my lab who's finished and heard whose name is jennifer noel so to summarize what we know about the offspring is that they have no evidence of rod function from birth onward but their cone function is normal and then it declines so in terms of cone function they're very similar to what happens in the human patients in terms of rod function they are they are not as good a model so and the other thing is that they all what i didn't tell you is that they inherit in the Mendelian fashion so about 50 percent of the animals that are born in every one litter are transgenic or animal wild type and so they all have the same phenotype as the founder so they're in in terms of the genetics they're a good model for for rp so this slide shows what happens in terms of the redoxin expression and now the transgenic is over here on on the left and the wild type is here on the right and this is a anisostein to remind me to tell you that this is from our one of our anatomical studies and so it's upside down so the photoreceptor inner and outer segments are shown here with and and then in the transgenic the redoxin is actually expressed all the way through the cell bodies and into their terminals and so this misolocalization is probably part of the reason why these rod photoreceptors are dying in terms of the expression pattern the wild type now is shown up here between p 30 and p 120 and we can see redoxin expression in the transgenic at p 30 and just a smathering at p 60 so even out to p 60 there's some redoxin that's still being expressed and so the hope is that if we can do something in terms of eliminating the mutant protein that we might be able to rescue some function and i'll show you some data about that later in the talk okay so let me stop here for a moment and summarize so what we see in the in this p 23 h transgenic pig model is what is the way we interpret this as three stages of retinal degeneration in terms of retinal structure and function the first stage is is the is the is a loss of rod driven function okay that's that's happening here p 0 to p 14 and then we have a mid stage p 30 to p 60 where the the world we've lost the rod function completely and the cone function starts to decline and then we have this idea of end stage disease which is larger than p 90 and so depending on the stage the the the therapy is going to have to be tailored to the stage of disease and i'll talk about those therapies in a second but what i want to do is also tell you one other way that we assess visual function in these animals so as i said we when we use the erg it's got the advantage that we can use it successively over time and we can actually assess weekly if we want to but as i showed you in that earlier slide it gives you the the view of what the entire retina is doing and i also showed you that the cone function has a central to peripheral gradient and so at any one point in time with the erg we are looking at how good the peripheral retina is whereas the central retina has probably lost function but what we'd really like to know is what happens it in particular locations in the retina and so you can use the multifocal erg but i'm not a big fan of it um but what my lab also can do is actually take portions of the retina and place them on a multi electrode array and put them into a dish and then use visual stimulation to activate the retina in vitro and we can record from the retinal ganglion cells and ask questions about visual processing using different light flashes or using different kinds of visual stimuli and we can do this either here in the central retina or we can take a we can dissect a piece of retina out from peripheral retina and and if this is where we place our treatment then we can ask whether treatment has affected this very particular localization in the retina versus in an intra eye control or in the control fellow eye which may not be which we may not have treated and what we end up doing is recording the action potentials of these ganglion cells on these electrodes and the nice part of this approach is that we have 60 electrodes we can usually get about 90 different ganglion cells on our on each recording and so therefore we get a nice survey of what the ganglion cells are doing in a particular area versus in another area and so we did that with these p23h animals and here's the here's a here are schematic not schematic these are what we call post stimulus time peristimulus time histograms and at the top each of these little ticks is an action potential and so then we can look at the average over many trials we usually do 10 to 20 trials and we use different brightnesses of of light stimuli and what you can see is that you get this nice peak firing and then sometimes a maintained firing for the two seconds that the light is on and that sustained component usually increases with the brightness of the light and sometimes we see a decline in the peak but this has to do with light adaptation when we use the same visual stimuli in transgenic animals when when we're in rod isolated conditions we see no response so the rods are not driving the ganglion cells at this low level but you can see that once the the we reach cone function that you can see these beautiful responses among the remaining cones and so here's the summary of this kind of an experiment where we're plotting the light intensity on this on the x-axis versus the percent of visually responsive ganglion cells wild type again is in gray and transgenic is in black so you can see at under rod only conditions we have no ganglion cell ganglion cells being driven in the transgenic animal but as soon as we get up to someplace where the cones can become sensitive we start to pick up on the visual responses and the cones then saturate in a hundred percent of the cells of the cones that are there have have you know have really good responses and this is out at p120 so this plots the percent of visually responsive cells as a function of age and that's stable in the wild type animal but in the transgenic animal you can see that uh that there's a decline in in cone function all the way out to p90 but the cones that remain have very good and robust responses so we we're losing drive to the cones but the cones that have responses are being maintained in a in a in a very stable fashion and this actually also is similar to what happens in in humans until they start to lose cone function all right so this takes us back to that schematic that i showed you earlier about the three stages of transgenic photoreceptor degeneration when we're here the question that we want to address is can we restore rod function and prevent cone degeneration by getting rid of the mutant uh rhodopsin and then in the mid stage the question is can we prevent further cone dysfunction so can we do something to support the cones in some way shape or form so that they they are arrested from the time of treatment onward and then in end stage disease there are a lot of treatments that other people are trying and i'm not going to talk about this but they include prosthetic devices that will drive the circuit optogenetics that will replace the the drive of the photoreceptors and stem cell therapies and and other and other transplantation strategies so what i'd like to do now is just to talk about what what these two what what you might want to do with gene therapy in in these two stages of the disease so as i said what we'd like to know is whether we can restore rod function by manipulating and in our case the p23h rhodopsin gene expression and then here we want to know whether we can just delay cone dysfunction and degeneration and the idea here is to in in our hands is to provide some kind of trophic support or some neuro protection strategy so just for everybody in the audience who might not know about gene therapy this is a brief summary of how we deliver the virus and how the the viral delivery of the genes and and the protein expression so we use adeno associated virus because it's usually well tolerated and we use these and and we we pull pull out the guts of the of the viral genome and replace it with whatever genes we want to use in terms of our in terms of our functional strategy so we can manipulate the AAV vector coat and this is what some of my collaborators do and and in manipulating that coat to the protein then it will recognize certain receptors on the surface of some of some cells but not on others so this helps to create a selectivity of targeting of the virus to particular cells and in our case what we want is is to either target broad photoreceptors or cone photoreceptors so the coat may change depending on which of the cell classes we want to target and then the delivery method so there are two ways of doing this you can use a sub retinal injection and that's good if you want to target photoreceptors because now they're very proximal to the injection volume or there's the idea of using intravitrile which is from us from a therapy approach in humans is probably more palatable because it's more easily tolerated you don't create a retinal detachment that has to settle down and then the promoter is also used to enhance the selectivity of of the of the gene is being delivered so for example we want to target the broad photoreceptors the redoxin promoter is a really good thing to do that and the down here is summarizing either sub retinal or intravitrile injections so the red is a really great place to try approaches gene therapy approaches and part of it is because of the transparency of the ocular media this allows this allows us to see really well when we're doing both intravitrile and sub retinal surgeries like a lot of you do in in patients the limited access of the sub retinal space and even the eye eliminates systemic side effects so inflammatory in general systemic inflammatory responses there's a certain amount of immune privilege that occurs in the intraocular environment so that's also helps with any inflammatory responses that might occur in some gene therapies you know for example those that are targeted at liver because it's a bilateral disease we always have an untreated or a control treated eye as the as the control for the treated eye and so that's a nice intra-animal control and then we can track changes in noninvasively with oct so here's another fundus image off of our oct equipment and here is a one of the one of the one of the animals oct where the photoreceptors now here or at the bottom and the ganglion cell layer is here at the top so we use oct as well as the erg is a noninvasive way of tracking what's happening in terms of degeneration and also therapeutic all right so what I'd like to do now is to talk about how we're tailoring gene therapy to the mutation so the what many of you may have read about is is the is the gene therapy for the null mutation in labor's congenital amaurosis and this is probably the poster child for gene ocular gene therapy and in in labors that you that you lack a particular the expression of a gene so what you have is a null mutation the mutation creates no protein and so it's a relatively simple disease to treat can you replace the absence of this mutant protein with a normal protein and in doing that can you rescue function and there's a and I think the jury is still out on whether that is working perfectly in these in these patients with labors there's there are some reports of a very good application and of the of gene therapy and there are some reports where the the therapy is not doing as as good a job and that so as I said the jury is still out on that but the idea is can the the idea initially that we had was can we use gene augmentation to try to ameliorate this dominant mutation so the idea is can you express more wild type rhodopsin and therefore reduce the ratio of the wild type reduce the mutant to wild type ratio and if you do that can that have a therapeutic effect we started to do that with Al Luen and Bill Houseworth at the University of Florida but the first thing that we needed to do was a control experiment to ask whether the the viral vector that we were using was going to give a specific infection of the photoreceptors the rod photoreceptors themselves so what we used was a green fluorescent protein marker that was driven by the rhodopsin promoter and here's an image a fluorescence image of the fundus that shows the infection area of in this particular animal so the optic nerve head is down here and so you can see that the area that we get of infection that we get is pretty broad it's not the whole eye but it's it's a it's several disc diameters in area and if you look at the sections of the animals so the blue is a dappy stain to show the nuclei of the of the of the various cells in the layers of the retina and so you can see in the transgenic we get some we get very nice infection with with the AAV and if you look there's a little row that where there where there's no fluorescence in both this animal and this animal and that's represents where the cones are so the rods are nice are are nicely affected probably not every rod but many of the rods are infected in this in this application so we then switched vectors not not not the external coat vector but now we used at the same AAV driving radopsin in this gene augmentation and then this is the experimental design so we inject we do these sub-retinal injections at p3 when we know that we have rods around and then we start to do fundus exams erg and oct at four weeks and carry it out as long as possible so this is the the the transgenic uh and wild type data untreated data that I showed you before and then we use controls that include the gfp so sometimes we mix a little gfp in with the in with the therapeutic vector to try to show where the um where the infected area is and as I said we did this work with Al Luan and a postdoc in my lab erin rising and one of our fellows uh niloforpeery to do these experiments so um unfortunately we didn't see rod rot radopsin augmentation didn't work in our hands in in this model so there's no difference between baseline and and the treated and untreated animals and either four weeks uh eight weeks or 12 weeks uh in terms of the rod isolated response so these are these are representative ergs and the shaded area is is the uh is the standard error of the mean of the responses but we did see an impact on the combs so these are the summary data from individual animals and the dashed line is is a untreated transgenic control this animal just got the gfp whereas our transgenic animal got the aav row plus a little gfp and then the wild type is plotted in black so what you can see is that at uh and this is time across here so at most of the ages past two weeks which is what we expect we expect to see that the trans gene will start to be expressed between two and four weeks and so you can see that it's helping to keep the cone function more robust uh in the in closer to the wild type than the untreated treated animal and that also occurred in transgenic animal number three but in this animal which did not receive treatment the the transgenic the the the two eyes were exactly the same and these are the individual cone responses from those animals that we've summarized here and here so red ops and augmentation is limited in its efficacy it is safe but it's but it's limited in its efficacy it doesn't help with rod photo receptor function and it probably delays the the decline of cone function but probably isn't isn't going to be something that we want to pursue in the future so the next strategy that i'd like to tell you about what that's also been done in collaboration with albulin is an idea of knocking down the rhodopsin expression and and reducing it now what we'd really like to do is be able to knock down only the p23h mutant rhodopsin and leave the wild type copies in intact but the shRNA approach isn't that specific and so what we are doing is instead knocking down and then using a replacement so we have an shRNA that's specific for rhodopsin and then a second a second expression cassette that will express wild type rhodopsin at the same time and these experiments were really exciting so shown here are 18 the some the average of of the b-wave response in 18 wild type animals at p3 in 22 transgenic animals at p3 and in this particular animal 145-12 at p3 at the time when this animal received the shRNA plus the row of replacement 12 weeks later the untreated wild type looks again like this this is the same 18 animals the untreated transgenics as i showed you before have no rod function but in this particular animal both and both eyes received the treatment we saw a very significant b-wave so first thing i thought was we had misgenotype this animal and that the first p3 recordings weren't any good and so we re-genotype this animal three times we re-genotype the animal at 12 weeks which is shown here we took the animal out to 16 weeks we saw the same kind of b-wave response and then at termination we also took tissue and re-genotype this animal and this animal was a transgenic animal every time so why don't i have a whole litter to show you this is the most unfortunate litter of animals that we ever had and they and all of its litter mates developed a respiratory infection shortly after they had the treatment and they all died with the exception of this guy so i only have this animal to show you for right now but last week we did a whole litter of 14 with this this shRNA row and so hopefully in four weeks we'll be seeing something that's significant and i'll have more to say this is the OCT from that animal so here's an untreated transgenic here you can see that we're losing a lot of the definition in the outer retina shown down here at the bottom here's your untreated wild type animal which shows all this beautiful definition for what happens in the in the photoreceptors in the in the outer outer part of the retina and here's this 145-12 which shows some thinning of the ONL which is what we'd expect because treatment is at p3 and the gene's probably not ramping up until about four three to four weeks of age so there's going to be some degeneration in the middle but at 12 weeks this animal had a had a significant outer nuclear layer that was not present in any of the untreated animals that we've seen and these and these these experiments have been ongoing with uh buban sahu govinda pangini and genel adenir adeniron uh in who who who were just doing these experiments to replicate last week so we're really excited about this idea of the efficacy of shRNA uh knocked down with replacement and then the safety is shown here on this slide so as as as expected from everything else i've told you there is no uh the the cone function in in all these animals treated and untreated transgenics is normal at p3 and it remains normal uh at p12 so the shRNA isn't with the replacement isn't doing any harm to the to the retinas so that's also one very important aspect of any gene therapy or any therapy trial you have to both show efficacy and safety okay so the last thing that i'll tell you about is a neuro protective strategy so what was found by um joseph the hall and his colleagues uh uh was that there is a factor a secreted factor that the rods produce that appears to keep the cones healthier and happier and it's called rod-derived viability factor and john flannery and lea bern who was a postdoc in john's lab but now is a assistant professor at the university of pittsburgh they had shown in a rodent model that rdcvf could actually delay uh cone cone driven uh cone driven declines and so we tried this particular rdcvf in the in the p23h transgenic animals and as expected uh we don't see a change in rod driven function these are animals out at 12 weeks post injection uh the red is the rdcvf treated i and the black is uh is the bss treated i and in terms of cone function we are seeing perhaps a little bit of a of us of a rescue of cone decline uh in the transgenic versus the bss treated animals but these are experiments that are now still ongoing uh and we're doing a lot of more analyses to convince ourselves of whether this is uh how the prevalence of this so not all animals respond equally and that's going to be the case with with patients as well and so we're so the statistics and the analyses are pretty involved when you start to come down to trying to compare one individual animal to a large number of transgenic animals that are in our database and so if anything what we're seeing again is a is a subtle effect on cone function and as i said our analyses here are a new way one last thing that i want to point out about this is that these we use intravitorial injections and it's been a completely different viral vector for for these treatments so uh i need to before i i close and take questions i need to acknowledge Hank Kaplan uh because it was Hank's idea to create this model and i wouldn't be talking to you today and i wouldn't have this translational component of research in my lab if he had not supported this idea and funded this idea in many ways throughout the last seven to eight years um Doug Emory and Doug Dean are both people that helped with the characterization of the of the animals that i didn't tell you that i told you that i didn't acknowledge previously and then we've had some very good support from both the NIH and several of the of the foundations that are associated with the University of Louisville as well as the foundation for fighting blindness and research to prevent blindness so thanks for your attention and i'm happy to take any questions