David Williams: Okay. Well, thank you so much for the opportunity to tell you about the AGI that we've been involved in at the University of Rochester. It's been a wonderful five years for us. We've developed new collaborations that we're very excited about. And we're eager to tell you about our plans for continuing them. But let's start with the things that we have done. Here's the cast of characters. I'll be telling you more about these individuals as we go through the presentation. Alright, so here's the basic premise for all the work that we've been doing over the last five years. Our goal is to accelerate various methods, two method different methods for vision restoration, with a non human primate model. Very important to have a non human primate model, we feel because we, we believe that that the similarity between a monkey and a human is is so great that there are many, many things that we can do in that model that we could never do in another species, allowing us to have a make, to generalize to what we expect to happen in the human case, much more easily. So that's been a very fundamental aspect of everything that we've done and everything I'll tell you about today. The second part of, our second premise for our project, is the use of adaptive optics. This is a high resolution imaging method that allows us to, in the living monkey eye and the intact animal, to observe single cells directly. And not only can we look at the anatomical structure of those cells, but we've developed methods to record the function of the cells that in several ways. But for example, we can record the light response of single neurons in the living monkey eye, especially the retinal ganglion cells, the output neurons of the retina. So those two things together have advantages for accelerating vision restoration, because what we are able to do now is track the efficacy of various approaches, any approach really, to vision restoration in a living animal over time without the need to sacrifice the animal to find out what happened. And without the need to, under in this case, to undertake psychophysical experiments ultimately that's what you want to do, you'd like to know how well people see when you have restored their vision, obviously. And that's a very important final step. But we believe that the process of studying vision restoration methods can be greatly accelerated by having a direct view of the retinal circuits we're trying to restore and adaptive optics provides us with the opportunity to do that. Now, one of the first things we needed for the monkey was a good model of retinal degeneration that was workable for us. And as you may know, such models are very difficult to come by. So we developed our own using light to destroy the photoreceptors. And not just ordinary light, but we used an ultra fast laser delivering femtosecond light pulses, which allows us to confine the damage that we produce to the photoreceptor layer. And we use that in conjunction with adaptive optics to achieve this goal of localizing our damage and you can see that in an adaptive optics image on the left here. And you can see it in an OCT image on your right. You can see that this area here is, is where is where the damage is localized. We've destroyed the photoreceptors and we have other measurements. I don't have time to tell you about that confirm that the inner retina, in particular, has been spared by this highly selective light exposure method. Now in order to record the ganglion cells in a living monkey eye, what we do is we do a single intravitreal injection of a viral vector that's been loaded up with a payload that allows once the cells, the ganglion cells have been infected by this virus, allows the expression of the viral vector content so that flourophores can be expressed in the in the cell. So the cells become fluorescent and, more importantly, they're ganglion cells that allow the flourescent report allows calcium imaging, so we can do functional imaging in the living eye. Now here you can see, for example, of the photoreceptor mosaic image with adaptive optics in a monkey eye, in a living monkey eye. Once we've got the expression of this for fluorophore GCaMP6s in the eye that's shown here in the green ring around the center of the fovea. It's a ring, because the ganglion cells are displaced in the fovea from the very center that's a natural part of the topography of a primate foveas. And what we can do then is we can deliver stimuli to the cone mosiac here and we can image the ganglion cell responses over to the side because these ganglion cells are responsible for handling stimuli delivered to the cones to the very center of the fovea. That's a very convenient part of the architecture of the fovea that we capitalize to keep our stimulation and imaging separate. It took us a long time to develop this method. And one of the things that Bill Merigan, in particular, demonstrated was that we could get much better and long term stable expression of this calcium indicator through the use of immune suppression and you can see that here where well without immune suppression over time, in this case 13 weeks, the ganglion cells died that were expressing these the fluorescence protein that we need. But with suppression, we get much better stability and we're able now to record for periods of up to two years or more in single animals, which is, which was wonderful, from the point of view looking at the long term viability of vision restoration methods. Here you can see an example of recording where we're drifting a grating over the photoreceptor mosaic on the left and on the right you can see the cells changing their fluorescence, the amount of light they generate based on the delivery of the stimulus. With the red cells, behaving in a different way and the opposite way from from the green cells, labeled with the green arrows that is, indicating that we may be looking at on and off center cells. Now just to show you very quickly the value of this technique. We can do some very basic neuroscience experiments with this method. Here are the three most common ganglion cell types in the primate retina. The Parasol, Midget, and Bistratified cells. If you're ever going to be doing vision restoration through direct stimulation of ganglion cells, you'd like to know how these cells are distributed, what are their response, what are their stimulus requirements that to get them to respond well so that you can drive these cells in an optimal way. So using this calcium imaging to examine cells is an important and classify cells that are important capability. So here, Tyler Godat has been developing stimuli like this, a flickering light presented to the center of the monkey fovea. That's a luminance direction. A red green direction and a blue yellow direction. These three directions are chosen to select for each of those three cell classes I showed you in the previous slide, and indeed to be very brief here, you can see clusters of cells emerging from this kind of analysis where we can see what we are putative parasol cells here in the middle, putative small bistratified cells here that have S cone input, and also midget cells that have L/M-cone input on the left and the right in the plot. So how do we apply this recording method in various approaches to vision restoration? Well, let me tell you the progress we're making working with Dave Gamm and his colleagues at the University of Wisconsin doing cell replacement therapy in monkeys and Juliet McGregor and Bill Merigan at Rochester are leading this effort. So basically what we can do with adaptive optics is we can do a subretinal injection of precursor photoreceptor cells, human photoreceptor cells, into the monkey eye and observe over time, because we've labeled them with a fluorescent dye, tdTomato, we can watch them over time in the living animal. And here are examples of what's happening with these stem cells over time. These are individual cells injected into the subretinal space. And what we've discovered is based on both of our adaptive optics in vivo observations and follow on ex vivo histology observations we can see a number of exciting things about what happens with these photoreceptors when they are implanted into the beneath the retina. First of all, they migrate into the outer plexiform layer. They definitely can survive under the right conditions. We can see expression of opsin, and I'm not showing the data for all these things that we have specialized markers that show us that this is happening. So we do get the expression of opsin they form neurites. So they're growing processes and in some of those neurites that reach toward the bipolar cells, we see the expression of presynaptic proteins, which is especially exciting because, of course, what we ultimately want to do is demonstrate connectivity. between the photoreceptors that we've implanted the donor photoreceptors and the host retina. We haven't demonstrated that connectivity yet and that's the next step in the project. Now another project that that we're engaged in is in collaboration with Connie Cepko's laboratory at Harvard. And this case, the goal is to increase stem cell survival by creating the right microenvironment to allow these transplanted cells to survive. And the way we're assessing that microenvironment is through a technology that Jennifer Hunter has been pioneering which is adaptive optics flourescence lifetime Ophthalmoscopy. Now, I won't. I don't have time to go into fluorescence lifetime in great detail, but basically, what you're doing is looking at the rate with which fluorescence decays in very short timescales, in order to assess what's happening to to the microenvironment in which the cells have been transplanted. Jennifer's able to look at individual cells now and see see what's happening inside of them in a living mouse eye, and in particular, she's using, thanks to Connie and Connie's lab, they've developed a glucose sensor using an AAV8 vector that allows the measurement of glucose uptake in individual cells in vivo. And so here you can see the instrument that Jennifer has built at Rochester. Thank you Alf Dubra for your help in designing this instrument that was a great collaboration across partners in the AGI. And now we're getting, Jennifer's getting the first results with this rebuilt instrument using Connie Cepko's vector. And here you can see a pattern of RPE imaged with with this glucose vector, which was conveniently labeled called Sweetie. And so Sweetie produces a pattern of glucose uptake that you can see here when you've got the Sweetie in place. But when you don't have the Sweetie you see a much more uniformed pattern, providing some preliminary evidence that this is going to tell us about glucose use in the retina and allow us to assess whether the death of photoreceptors and retinal degeneration is related to glucose uptake in cells. Finally, we're also using this technology, the AO technology, to develop optogenetic approaches to vision restoration. In optogentics, the goal is to, also with a single intravitreal injection, get ganglion cells to express channels like data channels that we can control with light, so that if the photoreceptors are gone, the idea is to directly stimulate the ganglion cells so that the retinal image can get conveyed to the brain via that route which presumably survives the death of photoreceptors. So it's possible that with this method, you could restore some some visual performance in blind eyes with a single intravitreal injection, which was a big advantage over other methods that that might use, might require a more sophisticated surgery where an opto electronic device, for example, was implanted into the eye. So we can with a single injection both put GCaMP6s into gangliion cells. So we can record from them as I described before. And we can also express ChrimsonR, which is the optogenetic agent that allows us to directly excite cells. So we now have the ability to have a two way conversation if you will with retinal ganglion cells in a living monkey eye, on an intact eye, where we can both record their activity and modify their activity with light. So here's an example where we stimulate, first we ablate some photoreceptors of this near the center of the fovea here. And you can see the effect of that ablation, because these ganglion cells in this crescent shape in the ring are dimmer because they're no longer connected to viable photoreceptors. Then we can we can go in and stimulate that region where the ganglion cells are untethered, as they normally would be tethered to their cones and this region stimulation is shown in red here and here's the outcome of that experiment like that where you can see that area now glowing because we're successfully getting the GCaMP calcium indicator is telling us that were successfully changing the responses of ganglion cells by direct exposure to light. So future direction of that, which we're very excited about is this, this has been done so far with many cells simultaneously. But there's no reason we can't target individual ganglion cells and hopefully understand what the visual impact is of individual ganglion cells on perception, something that we've never been able to do before. And that might provide a lot of new insight into how the retina codes the retinal image and communicates with the brain. So the major accomplishments have been a new model of retinal generation based on photoreceptor ablation. We can classify ganglion cells with our calcium imaging method. We are imaging pluripotent stem cells in the retina, in the living retina to track their fate as they as they migrate and differentiate into photoreceptors. And we have made substantial progress toward functional integration of donor photoreceptors in primate retina. We've demonstrated fluorescence lifetime imaging thanks to Jennifer Hunter's work, and we've got an in vivo glucose sensor working at a cellular spatial scale. in that instrument. And finally, we have a demonstration of two way communication link with a primate retina, which could be an interesting avenue for optogenetic restoration of visual function in the blind. Just to give you a little sense in closing about our future directions and how we plan to disseminate this technology going forward. You know it's a bit like in astronomy, you know, you don't disseminate telescopes and it's difficult for us to disseminate our instruments, because they're like telescopes, they're large sophisticated devices that require special training to operate. So what we can do is like a telescope is make those devices available to people and we now have, you know, Harvard, using our technology for their work on glucose in single cells in the retina. We have Wisconsin using our fluorescence adaptive optics scanning laser ophthalmoscope technology to track the fate of stem cells and cell replacement therapy. And there's a pathway there toward working with this company, Opsis Therapeutics, to eventually commercialize this approach when we get it working. We're not ready to do that yet, but I'm excited that Juliette McGregor, just today, in fact, is submitting a new Audacious Goals Initiative grant to continue this collaboration toward ultimately hopefully clinical application. Other examples of interesting ways of disseminating I want to call out Sara Patterson who is a recent new postdoc, she's joined join my laboratory, and she's a forging ahead with the classification work I told you about. And we're working very closely Now with EJ Chichilnisky at Stanford who has a completely different approach to our optical calcium imaging approach using microelectrodes and we're sharing uur technology with him and vice versa, to, to move forward more quickly. And then finally, Bill Merigan is been championing the optogenetics work very much along with Juliette, and at the very outset of the Audacious Goals Initiative project Botond Roska was a partner with us. He is a principal in the company Gensight, which is now doing human clinical trials with optogenetics and Botond is a big fan and understands the value because he does a lot of this work himself of value of using a primate model to understand and learn things that we can then apply in the human case. So lots of I think opportunities have moved of getting our technology out there and and moving it toward eventual application in humans. Thank you very much.