Lecture: Gene Therapy for AMD: Are We There Yet?

During this live webinar, we will discuss the concept of retinal gene therapy and its use for rare and common diseases, and how the type of vector, viral dose, and mode of delivery dictate efficacy and safety in both preclinical and clinical studies. We will also discuss ocular immune privilege, the impact of biological barriers, and factors that determine the degree of ocular inflammation related to gene therapy – also known as gene therapy-associated uveitis (GTAU). Finally, we will review gene therapy programs in development for age-related macular degeneration (AMD), as well as early studies using CRISPR-based gene editing technology as potential treatments for AMD. (Level: Beginner and Intermediate)

Lecturer: Dr. Glenn Yiu, Ophthalmologist, University of California, Davis, USA


>> Good morning. For those of you in different time zone, good afternoon and good evening. I’m Dr. Glenn Yiu. I’m a professor at the University of California in Davis at the health system. I’m a clinician scientist. I am partially a retina specialist. I see patients with macular degeneration, diabetic retinopathy, retinal detachments among various conditions. I do surgeries on them. I spend half my time involved in research. My research program covers the spectrum from translational work. I focus a lot on nonhuman primates. Rhesus monkeys and mostly trying to develop new therapies for macular degeneration and using gene editing and gene therapy. But I also am involved in clinical trials of note we’re involved in some clinical trials that involve gene therapy in the retina space. So today, I’m very excited talk do you a little about the prospect of gene therapy for macular degeneration, are we there yet? So let me try to see if I can advance my slides. There we go. Here are my financial disclosures. I’m a consultant for several companies. The ones that are bolded are those that might be relevant to this discussion. What is gene therapy? In the simplest manner, gene therapy involves the delivering of a gene. A copy of the genetic code for a specific molecule in the eye, in the person. And you deliver the gene and that gene can express the protein or the transgene product. One of the steps in gene therapy, usually you need to identify the gene of relevance. You need a vector, something that carries the genetic material. And you need a way to deliver it to a person and in this case it’s the eye. When we talk about genes we think about the inherited retinal diseases where there is a specific gene that is mutated or dysfunctional like RPE65 et cetera. When we talk about a vector, usually they’re viruses. Characteristics that really make a good gene therapy include the viruses that make a lot of protein, high expression. Long durability. A large capacity that can encode genes of different sizes. Low immunogenicity and low risk of mutations: You don’t want anything to cause problems like cancer. A common one is lentivirus which checks off most of the boxes but it integrates into the genome, the viral vector can integrate, it can integrate into the wrong place, say a tumor suppressor gene and cause cancer. There is a small risk there. AAV does not I want grate and it’s safe but it has a smaller cargo it can encapsulate. For larger genes like ABCA4, it’s unable do that. You have to break the gene up into two different parts or use a different vector. Adenoviruses have largely been abandoned because of the risk of causing uveitis and inflammation. And synthetic polymers and nano particles are still in development. They’re exciting because they’re generally safe. They don’t integration or cause mutations but they’re not as effective as what nature has evolved to be a gene-delivering vector. Once you identify the vector, you need a way to inject it into the eye. That can be done through intravitreal route or sub retinal route. Sub retinal is nice because the viral particles get up against the cells you want to induce like photoreceptors or RPE. However the treatment effect is limited to just the area of the sub retinal bleb. The small area of the bleb. Intravitreal injections work well because the viral particles go everywhere but it has limitation of entering the retina by the internal limiting membrane. The intravitreal injections can be done in the clinic, easier to do. Sub retinal injections require a more complex surgical setting. It has to be done by a vitreal retinal surgeon. Once you have all these three items determined, then you can go ahead and design a gene therapy and that really was what led to the FDA approval of Voretigene Neparovovec, which was the first approved in the US gene therapy to treat Leber’s congenital amaurosis resulting from RPE65 deficiency or mutations. I will start with my first poll question, which of the following delivery vectors is most commonly used for ocular gene therapy. AAV, adenovirus, lentivirus, synthetic materials. I’ll give everyone about 30 seconds. Once that question is answered, we’ll see some — the majority of people answered AAV. I wanted to get a sense of what was people’s knowledge about gene therapy and by far AAV is the most common delivery vector for ocular gene therapy. Thank you for closing that. I will move on to the next slide. We’re very familiar are the fact that gene therapy can be used for inherited single gene disorders. I talked about Leber’s congenital amaurosis and others here. But gene therapy can be used to treat degenerative diseases like macular degeneration, diabetes, it’s complex. It involves an interaction between many genetic variants. There are not often mutations. There are many proteins. There are small variations in many different kinds of genes that bind with environmental factors, dietary, age, et cetera, that leads to the disease. The other big difference between treated and inherited and degenerative disease is inherited diseases you’re usually treating children. When you do a gene therapy, the duration of benefit is for their entire lives of the child. Whereas degenerative diseases tend to occur in older individuals. Some people say it’s not worth the risk because they already, you know, you don’t get that much benefit out of it but you can argue the Vices is smaller because if there is some long-term effect 30 years from now, you don’t have to worry about it if you’re already an 85- year-old person versus if you’re a child. One thing that ties all these complex diseases is VEGF. I will talk more about gene therapies related to VEGF for my talk today. Why treat macular degeneration or wet macular degeneration using a gene therapy? We look at the top here, when you do an intravitreal injection, most of the drug is inside the vitreous cavity and small amounts diffuse into the retina. When you look at the bio distribution inside the eye, the majority of the drug is in the vitreous and a little in the aqueous and a little bit in the retina. The pharmacokinetics is called pulsatile. You get a dose and you get a super high dose, maybe more than you need and over time it dwindles quickly. You have this peaks is and troughs of the drug distribution. In contrast, with anti-VEGF gene therapy, you’re trying to transduce the retinal to produce the drug. What you do is you end up having most of the drug where you want it, which is in the retina, and less in the vitreous and the aqueous and potentially less secondary side effects and adverse effects, et cetera. Pharmacokinetics is much more sustained as well. Meaning the drug production is constant. So you don’t have the peaks and troughs of these pulsatile injections. One of the first attempts at gene therapy for wet macular degeneration involves an AAV injected into the eye that makes a soluble VEG-F receptor. Very similar to aflibercept or as it’s known in the US, Eylea. There have been studies in nonhuman primates. This is a monkey, looks similar to a human. They did a sub retinal injection. This vector glows green because it’s engineered to have green fluorescent protein. When they did a sub retinal injection of the green fluorescent protein that makes this solid VEGF, it does a laser induced CMV. It’s a model to test or simulate wet macular degeneration. They found they can suppress the CMV development suggesting a prompting attempt at using AAV to treat wet macular degeneration. However, when they did the first clinical trial, this was back in 2016, the primate end point for safety was met. It was not unsafe but there was no difference in vision or the number of rescue treatments. This was a very early trial and I won’t go into the details but there were some design flaws. Many of these patients had already been treated by many injections and it was hard to detect a benefit. Since then, there have been a few new programs to develop wet macular degeneration gene therapy, one is called the RGX-314. This is probably the most advanced program. It uses an AAV that expresses an anti-VEGF fragment. It’s similar to ranibizumab. A fragment of an anti-VEGF antibody. This is a phase, an early phase dose escalation study. They’re increasing the dose of this virus as you’re going up. And what’s interesting is that many of these patients now, they’re now entering phase 3. So they’re close to pivotal trial analysis. Many of these patients have been followed up to 4 years and what is interesting is many of these people can maintain the vision’s gain after anti-VEGF delivery. And this is very in stark contrast to real-world studies or long-term extension of, these are real-world studies on the right side and long-term extensions of the clinical trials on the left side. Every patient after 3 or 4 years start to show a decline in vision. In real-world settings it’s hard to sustain monthly or regular intervals for intravitreal injections. Due to attrition, many people start to lose vision. This gene therapy, because it’s constantly generating this drug inside your eye is not susceptible to that. This is a swimmer’s plot. Every line is an individual patient. On the left side are people’s previous injections prior to entering the trial. You can see many people require many shots. And then here after a single dose of RGX-314, you can see that almost half the patients can become injection free. And this is now gone out to 3 year long-term extension study and many of these patients remain injection free after requiring many injections the previous year. Now, that was using a more typical AAV virus. But newer generations of AAV are also being designed. If you remember earlier, AAVs have to be given subretinally because when you do it subretinally, the virus can get up against the photoreceptors. If you just inject into the vitreous membrane, it can’t get into the retina. We have use directed evolution. You can get it directly into the retina from the vitreous. You take a virus and mutate it many ways. These are small variants of an AAV and you injects it into the vitreous cavity and some of the viruses transduce the photoreceptors. Say you put in the virus something that makes the photoreceptor green. Once you transduced it, you dissociate the cells and all the green cells you do fact sorting. You sort through and identify the green cells and take the green cells and purify the library from that and make another round of mutation and again inject it into another eye. So in this way you’re doing rounds and rounds of evolution because you’re selecting, you’re selecting viral particles that preferentially transduce photoreceptors. That lead to the development of this AAV-2, 7, and 8 viral particle platform. It’s a vector capsid that allows an AAV injected intravitreally to get into the retina itself. This was done and tested in mice. It doesn’t work as well in monkeys but these have entered clinical trial as the AAV.M-022. It use the capsid and used to encapsulate aflibercept. Similar to the previous one. The previous one was a subretinal RGX-314, a subretinal AAV8 to make something that looks like ranibizumab. This is an intravitreal that uses this 7M8 and can generate something that looks like aflibercept. In this trial, many patients required multiple injections. But after a single dose of ADVM-022, many became injection free or the injection frequency has gone down. One challenge is this treatment was used both for patients with diabetic macular edema and AMD. There were two trials. In the diabetic macular edema or the infinity study, a number of patients developed inflammation. Particularly at the higher dose compared to the low dose and compared to Aflibercept. You can see a lot of red and orange dots here that correspond to severe intraocular inflammation. Now, fortunately, this seems to be focused primarily on the infinity study and not on the optic study which is the studying the testing this therapy in macular degeneration. Some of these patients if you look carefully developed hypotony. Our hypothesis is when you do an intravitreal injection, some of the viral particles get into the ciliary body which may affect the ability of aqueous production and cause the eye tons go inflammation and hypotony. Why do you get inflammation in the eye with a gene therapy? I think in order to understand that, we have to understand the principles of the immune privilege. Why is the eye what we call an immune privileged organ? First of all, like, unlike various parts of the body, many parts of the eye do not have lymphatic drain age including the anterior chamber, the vitreous cavity and the retina and sub retinal space. The conjunctiva, the sclera, the cord, the walls of the eye do have lymphatic drainage. Inside the retina has a number of barriers just like the brain has a blood brain barrier, the retina has a barrier, an inner and outer blood retinal barrier and they express molecules that are immunosuppressive like TGF beta and IL10. This is a process known as ACAID. Those involved in corneal transplants may be aware that ACAID is a Noble Prize winning concept where antigens introduced into the anterior chamber in the front. ACAID stands for anterior chamber associated immune deviation. Antigens in the chamber induces immune tolerance and activates immunosuppressive T cells. And in this way, it induces immune tolerance. That is how you can do a corneal transplant without rejection. Now, gene therapy has been done for diseases like X-linked retinoschisis. You don’t need the viral particles that are directly evolved to penetrate into the retina. Conditions like X-linked retinoschisis, the retina itself is mottled and unhealthy. You can give intravitreal injections that transduce the retina in this way. In these studies by Seving at the National Eye Institute, using increasing doses of viral vector, they saw increased levels of inflammation, especially at high doses. This is a many of anterior chamber inflammation and antibody titer. What was interesting is when they looked more carefully at the X-linked retinoschisis patients, many of the patients had higher CD 4 to CD 8 ratio even before they get gene therapy. When they got the gene therapy, that AAV — these levels go up and those with higher CD 4 to CD 8 ratio developed stronger inflammation. Essentially what this told us is that maybe patients with retinal diseases unlike healthy patients have already a baseline proinflammatory status. Their eyes are more prone to be inflammatory. When you inject the AAV, it revs them up even more. The other thing is if you notice the inflammation that was found in the ADV.M-022 program was primarily associated with an intravitreal injection. If you look across both large animal studies dogs and monkeys versus human studies, all these are different vector doses, top is higher doses. And these circles are different clinical studies and green means no inflammation. And orange and blue means eye or systemic inflammation. You can see in general higher doses of AAV have a higher tendency to lead to inflammation. What we also find is those with sub retinal injections tend to be able to tolerate higher doses without inflammation. So it seems like intravitreal AAV is more proinflammatory. So because of that, there’s another program now called the 4D-150 program. This is from a company called 4D molecular therapeutics. This cap side is designed by directed evolution but it’s done in monkeys rather than in mice. In these early monkey studies you can see that transduce the macula really well. In fact, much higher levels than the 7M8 variant. This is showing its superiority compared to a standard AAV2 for transducing the retina. They designed a program which is pretty complicated. It makes aflibercept as well and encapsulates a VEGF C interfering RNA. It can block all different pathways of VEGF signaling. This is using this special R100 capsid. It can be given intravitreally. In patients with wet macular degeneration using this 4D-150 platform, particularly at the higher dose, people who required many different kinds of intravitreal injection after a single dose of 4D-150, almost all are injection free at the high dose. And those with the lower doses, many can become injection free. There are lower rates of intraocular information in these patients because we’re using lower doses. One thing to note, this is a fresh hot off the press result. They said there is no inflammation but most of the patients have only reached about the 28-week time point. They used a 20-week prof laxative steroid taper. The majority of this period of time, they were using eye drops to prevent inflammation. So whether this will remain as they come off of the taper is unknown. So my next poll question is which of the following treatments are not used for wet macular degeneration? I talked about the 4D-150 platform, the AAV8-RS1. The ADV.M-022 and the RGX-314. It’s now the ABBV-RGX-314 because it was purchased by Abbvie. I know this is a challenging one because it’s the alphabet soup of many different letters and numbers. We’ll see how you guys do. Good. It’s a bit of the mix. The answer is the AAV8-RS1. The RS1 is retinoschisis. The RGX314 were for gene therapy for macular degeneration. All right. So we can move on. I’ll talk next about a novel mode of delivery. Most of this is being designed from our lab. So we all know that when you do an ultrasound, if someone has a posterior scleritis, they have the T sign. You can see the actual hypoechoic space between the cord and the optic nerve. That’s a space that you can see on OCT imaging as well. Back in 2014, I described when you use an OCT imaging and enhanced depth imaging you can see this hypo-reflective or dark band underneath the choroid. That corresponded to the super-choroidal space. This super choroidal space can be accessed with a micro needle. A micro needle is a very short needle that can penetrate the wall of the eye without perforating into the vitreous cavity and you can deliver a drug just into the space that can better transduce the retinal. There is also something called micro catheterization. You make a small incision in the wall of the eye and pass the catheter through this space. This is a picture of a sub retinal needle designed by a company called orbit. It’s accessing the supra choroidal space. What is interesting about the supra choroidal space, when you inject the intravitreal drug, it goes everywhere and you don’t see it in the wall of the eye. Whereas with the other, you can see it concentrating in the wall of the eye where the retina is. The other interesting thing about supra colloidal delivery, it works better with larger particles. The larger the particle, the longer it can stay inside the supra choroidal space. The choroid is a high flow structure and small molecules get washed out quickly. This works well for viral particles because viral particles are large and can be sustained in the supra choroidal space. In the clinical trials where they did supra choroidal injection of steroids, like a suprachoroidal triamcinolone injection, you can see the suprachoroidal space. And we have done work where if you look in this space, this is from the tanzanite study. A study they injected patients with RVO, and inject steroids into the suprachoroidal space. The edema went away because it’s a steroid. But if you look at the thickness of the suprachoroidal space, there is a statistically significant expansion of the space. You can inject into the suprachoroidal space out to three months. I just want to get a sense of your familiarity. Out of curiosity, I wanted to see if you guys, if the audience had experience, are you very familiar and have given many of these suprachoroidal injections. You’ve done a few. Never done of it but heard of it. Or never heard of it to begin with. I will give a few seconds for people to click here. Very good. Seems like there is one person that has done many suprachoroidal injections but many of you have heard of it or not heard of it but have not given these injections. What can you do, how does suprachoroidal delivery work for transducing the retina. The first attempt was made in a rabbit back in 2011. This is many years ago. The pictures weren’t very clear but they showed you can transduce many of the layers of the retina. There is work from the University of Iowa where they did rat injections. They’re saying you can stick the needle into the suprachoroidal space in rats and do a sub retinal injection or a suprachoroidal injection but it’s hard to see where the suprachoroidal bleb is. There is work from the Wilmer Eye Institute where they said they injected some pigs and monkeys and showed diffuse transduction of parts of the retina with cells at different layers. Many of these are using just regular needles. It’s hard to use a regular needle and perfectly thread it into the right place. What we did at the California national pry mat research center at UC Davis is use these micro needles that are FDA approved in the United States for use in humans and there is a shorter version that is 700 microns long and we use it to inject viruses into monkeys and test at a week, a month, and three months and do imaging. For this one we only use green fluorescent protein. We want to know what cells are transduced and how far it lasts. After a month, you can see stark differences. This is using the same batch of virus. When you do an intravitreal injection, not mump of the AV gets into the retina. With a sub retinal injection, you can create a bleb where all the AAV is transduced and some ganglion cells can project back to the nerve. When you do suprachoroidal, you get more diffuse and broad expression. This is circumferential, 360 degrees. It’s hard to steer the height of the camera and you run into the monkey’s nose because their snout is so large. This is a magnified view of that. When you look at the suprachoroidal injection, it looks like a lot of green is everywhere. That is great compared to a sub retinal or intravitreal where you see a little where the needle went in and a bit near the nerve. When you zoom in, the only area of transduction is in the peripheral retina here. And the rest of the green and the eyeball the sclera and not the retina itself. In fact, when you do histology, you cut the retina and focus on the green cells, you can see with sub retinal injections you have good transduction of the photoreceptors and the RPE. The intravitreal injections doesn’t touch most of the vitreous material which is blue out here and RPE which is red. With suprachoroidal you get transduction of the RPE and we saw a lot of photoreceptor degeneration in these animals in the area of transduction. The other thing we found was that if you follow these suprachoroidal injections out to 3 months the transduction disappears and this can be seen up close here. Why is that? One thing we noticed is when you do a suprachoroidal AAV transduction, we’ll notice sometimes occasionally, some of the patients develop dots in the retina. And also vascular, perivascular sheathing along the vessels. On OCT, you see hyperreflective foci. These little dots, suggesting some mild vitritis. This is subclinical. When I looked in the eye, it looked healthy. If you look carefully, there are punctate coral retinitis. And this went away after a short course of a week of oral steroids. It’s definitely responsive to steroids. Let’s talk about, remember we talked about immune privilege and how intravitreal injections of AAV is proinflammatory whereas, subretinal AAV tends to be more immune privileged and you can get away with higher doses as long as you keep it under the retina. When we think autoimmune response, there is B cell response, humeral response, and T cell, the cellular response. There is immune responses to the viral vector, the AAV that you’re delivering. Or it can also be to your transgene. We’re testing GFP which is green fluorescent protein but in human trials we wouldn’t use that. We would use aflibercept. This is a busy slide. But to summarize, the suprachoroidal AAV led to a host of immune responses mostly to the transgene rather than the vector which is what intravitreal injections lead a lot more to. The other thing that we found, was that when you do, this is a few of these monkeys, you can see one animal got intravitreal injections in both eyes. A couple of animals got suprachoroidal in one eye and intravitreal in the other. The animal that had the most virus leaking into the systemic circulation getting to the spleen was the intravitreal. Giving an intravitreal AAV is almost the same as giving someone an IV injection. The virus particles can get out into the spleen and cause an immune response. When you do a sub retinal or suprachoroidal, with sub retinal the viral particles are confined to this space. Little gets into the systemic circulation. The GFP circulation is within the blood retinal barrier. When you do the suprachoroidal injection, a lot of the green is in the wall of the eye, the sclera. A lot of this GFP expression is outside of the blood retinal barrier. You can get more immune responses to the GFP transgene of concern. Now, fortunately, in human clinical trials, that is no longer the case. We’re not, that is not the case, we don’t use GFP in people. GFP is a foreign protein from a jelly fish. So the Regenix bio program has extended the RGX-314. They have extended it to deliver suprachoroidal. RGX- 314 was done subretinally. That is entering phase 3 trial. They want to inject suprachoroidal because a suprachoroidal injection like an intravitreal injection can be done in the clinic. You don’t need to go to surgery like a sub retinal injection. In these trials it did show there is up to 70 to 80 percent reduction in annualized injection rate. This is a swimmer lane plot. Many patients that required intravitreal injections previously, after a single dose of the ABBV-RGX-314 became injection free. Many can have fewer injections compared to those who did not get the gene therapy. We have to talk about inflammation because it’s a whole thing about the ADVM02 program. You can see as long as you give some kind of topical steroids and that seems to be most effective. There are zero cases of ocular inflammation noted in that course. When you do a suprachoroidal AAV, a lot of the AAV is in the wall of the eye. Maybe it’s important to pay attention to this episcleritis and hyperemia. A third of the patients developed some level of epi scleritis, it’s easier to treat. Much less harmful to the interior of the eye. That program is continuing to advance right now. So I ask the next question, which of the following is an important advantage of suprachoroidal AAV delivery. It can be given in the office, does not trigger immune responses, good expression in photoreceptors, the highest level of expression or the least amount of inflammation. Again, this is suprachoroidal. That’s what we just talked about using the micro needles. I will give you a second to answer that question. Let’s see. About a quarter of you got it correct. It can be given in the office. I wouldn’t say that generates the least amount of information which is the second answer, that’s a sub retinal. Sub retinal is underneath the retina, that has the least amount of inflammation. The real advantage and the reason people are trying to move to suprachoroidal is it can be given in the office. So we’ll move on from there. The last part of this talk I’m going to talk about CRISPR technology. What is CRISPR technology? It’s called gene editing not gene therapy. In fact, it’s probably already in the world around you. Scientists have already used gene editing to modify things like apples, mushrooms, potatoes by cutting out the gene that is involved in iron oxidation so they don’t turn brown as easily. That is used to modify meats and produce. CRISPR is a technology that can work at the DNA level. Currently most drugs that we give like antibodies, block VEGF protein. Even the gene therapy I talked about today like the ADVM-022, RGX-314, these are AAVs that make drugs like aflibercept or something that looks like ranibizumab and even the newer technology like the 4150, that’s the other product I talked about that makes an anti-VEGF agent, they’re working at the protein and the RNA level. CRISPR technology makes modification at the DNA level. You will probably hear about a lot of different generations and prime editing and base editing. The original design of CRISPR is a nuclease. It’s part of a bacterial adaptive immune system. Bacteria have to make enzymes that cut foreign DNA. Say it’s infected by a virus itself, if we can exploit this enzyme that the bacteria use, like the CAS9 enzyme, you can program it using a segment of the RNA using a guide RNA. The programming guide. You can direct it to a specific gene in the genome and create double stranded breaks. Bauds the body has natural repair mechanisms that are error prone, it tends to make mistakes, it will cause insertion and deletion mutations that causes a frame shift and knocks out the gene function. These newer generations of gene editing are under development. I’m going to focus more on gene ablation. That is all we need to do is knock out the VEGF gene rather than block the VEGF protein. I will talk about our work starting from mice all the way to monkeys. When we first started, we were doing it in a tissue cultured plate. In vitro and we designed target RNA to target VEGF A. We got good editing and we can suppress the VEGF protein using three different guide RNAs. We can suppress tube formation, an in vitro method of measuring angiogenesis. Next we went into a mouse and injected the virus into a mouse to see if we can test to make sure it can edit out the VEGF gene and suppress the VEGF protein. In some animals we did this laser CMV. That is using a laser to create a break in the membrane and create a CMV as a model for wet AMD. First of all, we compared different types of CAS9. We looked at the enzyme from Strep pyogenes versus Staph aureus. The step pyogenes is bigger, because it’s bigger the AAV can only encapsulate a certain size. We have to break it up into two vectors. One with the enzyme and one with the guide RNA. Versus smaller CA SA9. This dual vector system works better for gene editing, better for VEGF protein suppression and better for VEGF gene suppression done in a mouse. Why cut the gene once when you can cut the gene twice? You double your chances of suppressing the VEGF protein. Also, if you want to go into a human in the future, you need to design guides that can target across different species, like two different positions. Our next experiment, we compared can we cut the gene twice or once and check. As you can see, when you do in vitro and do two different guide RNAs you’re cutting the gene twice, you get mutations at the two sites but you can cause truncation, the whole segment can be truncated out or inversions. However, when you go in vivo, we didn’t get that much better editing or knock out of the gene. This was similar to when we did the laser CMV model. We didn’t see that adding a second guide RNA worked well in vivo. In fact, when we started doing unbiased off target analysis, meaning we’re looking to see if these gene editing tools are causing unexpected mutations, we saw that there were some mutations coming from the second guide RNA location. We found the best balance between efficacy and safety would be to use the SPCAS9 and a single guide RNA and test this in a monkey. So we advanced from mouse now to monkeys and we followed them out for 3 weeks. This is what we find. It was a little unexpected. First of all, we didn’t want to do any suprachoroidal because of the inflammation. We did an injection in the macula. This is unpublished data. We see this weird fingerprint pattern where there is regularly interspaced thinning of the outer nuclear layer and a deposit of sub reflective material in the center. What is interesting is that this, first of all, continued through week 12 even after we did the laser CMV. It occurs both in animals that got the CRISPR enzyme with the active guide RNA to ablate the VEGF gene as well as just CRISPR itself without any guide RNA. Suggesting this toxicity we’re seeing may be specific to CRISPR enzyme. So we did the laser CMV anyway and we saw some suppression of the VEGF protein and some suppression of the CMV lesions. But we also wanted to know, what is in the sub retinal hyperreflective material. When you look carefully at the blob of stuff under the retina, many of the cells are transduced with both GFP as well as the HA tag, the Sp-CAS9. There was a lot of collagen in there. Most of this is RPE cells that got transduced and a lot of fibrotic materials like collagen and extracellular matrix material. My last question for this talk is how excited are you with regard to the prospect of using CRISPR gene editing. All of what I talked about earlier is gene therapy where the AAV makes a drug. Whereas here you’re going into the genome and editing out the VEGF gene using a bacterial CRISPR enzyme. So I’ll give you a few more seconds. Let’s see what the results show. We have a good number of people who are still very excited which is good and some are very concerned because of the toxicity we showed. Of course, this is only our first attempt at doing this in a monkey. Obviously, there is a lot more evidence in the mouse and smaller animals. So I think obviously more research has to be done. So in summary, gene therapy can be used for complex diseases like AMD by targeting angiogenic factors like VEGF. There are also complements that I didn’t talk about but I’m happy to discuss more for dry AMV. Intravitreal and sub retinal and suprachoroidal delivery of AAV to express anti-VEGF agents show signs-over long-term efficacy. And many patients have reduced number of injections after a single treatment of this gene therapy. Ocular information however is a concern. It seems to vary in that higher doses causes more inflammation and certain routes of administration like intravitreal AAV in particular seems to be proinflammatory. Suprachoroidal is a little less, slightly different types of inflammation. And sub retinal is the least amount of inflammation. Finally, CRISPR-based gene editing enables VEGF and CMV suppression in mouse studies. But in NHP studies showed unexpected disruption. I want to conclude and thank all the people in my lab. My primate research team. People that helped make the viruses and all my funding agencies. I’m happy to take any questions. I’m going to click open my, see, the chat. Q&A. Okay. One question is asking, please, if you have any questions, please feel free to submit it into the Q&A box. What is the meaning of AAV2? AAV stands for adeno associated virus. Throughout the talk you heard a lot about AAV. It’s essentially a virus that is completely benign virus. Many of us live with a little AAV in us. The AAV has not been known to cause any pathologic conditions like any diseases. And in gene therapy, we don’t use live AAV virus. We take the capsid that’s the vehicle and take out all the infectious component from the inside and replace it with the gene that we want to deliver. So in a way we’re not delivering the virus, but we’re using the capsid or the envelope of the virus as a delivering vector. Another question, have any human clinical trials started yet? Just to be clear, all of the studies that I talked about for ADVM220 and RGX4, the main clinical trials were all human trials. And some have advanced to phase three which means hundreds of patients have been treated. And the results, patients who needed many injections and after a single dose of gene therapy no longer needed injections, those are all human trials. The only one that has not entered the human trial is the CRISPR gene editing. That has only been done in monkeys so far for targeting VEGF. There is a human trial using CRISPR in the eye, that is through the edit 101 program. That program was led by a company called Editaus and that was to target the CE290 gene. That’s only been done in non-AMD disease. Gene editing has been done. That trial has been abandoned largely because of lack of efficacy. We don’t know if they have toxicity issues. After my talk, there might be questions about that. That has never been reported in humans yet. Third general, will gene editing have secondary complications other than gene applications I presume. The question is can gene editing cause any issues outside of gene editing cause issues outside of the delivery of the gene. That’s the main question, I think. If you remember, gene therapy means that the virus just makes drug. These drugs are generally drugs that we believe have been proven to be largely safe like aflibercept. There are probably future gene therapies that can make higher doses of the drugs. But gene editing is not just making a drug, it’s cutting and making an edit in the gene. When you make a gene edit, you can cause, you can potentially cut or edit the wrong gene. You can also edit the gene ineffectively. You can cut it and it just heals itself and it didn’t knock out the gene product. Also, in humans or every animal, there are two chromosomes which means every human has two copies of every gene. It takes two copies and it’s not easy to do gene ablation using CRISPR technology. There are concerns about off target gene mutations, ineffective gene editing. Those are all limitations we need to work on. At what stage of AMD will gene therapy be used if approved for humans. What we discussed throughout is that we’re primarily looking at wet macular degeneration. That’s the current stage. If you’re someone that needed a lot of injections for wet macular degeneration, there are gene therapies that can make the drug for a long time. There are some work using complement inhibitors. Like there is one from a company called Gyroscope that had an AAV that expresses complement factor I. It’s supposed to be an inhibitor complement for treatment of geographic atrophy. That trial kind of started but it has also been abandoned. We don’t know if there are other programs that are going to be starting up soon. What is the patient subjective response? Do they get better vision? Do they have comfort? Better vision, probably no. Most patients we’re not trying to improve the vision but sustain the vision gains from anti-VEGF. We don’t give gene therapy unless they got some intravitreal injection of the same drug. Say Ranobizmub. Now you give them gene therapy that can sustain the delivery. So you don’t need to get 10 injections a year. They can get away with maybe none to one or two if they really have severe disease. The idea is to reduce injection burden and not improve vision. Who will absorb the cost of treatment and do you recruit patients? The cost is a big question. In the United States, there are pairs that will cover gene therapy. Gene therapies are quite expensive but at the same time we can imagine a cost differential where you’re comparing to a life-long treatment of an anti-VEGF agent. In the U.S. currently, the cost of ranibizumab and aflibercept is close to $2,000. If they need 10 of those over the next five to ten years, you’re talking about 100 injections of $2,000, that is $200,000. And you talk about how many times they have to visit, the doctor fee, the exam fee, the burden of the patient transporting back and forth. So I think there are a lot of potential secondary cost save, related to gene therapy just by reducing the number of injections they need coming in. Is there any study done on race based analysis of effectiveness of gene therapy for AMD? Robust genes specifically not prone to easy editing. That is a good question. Are there race-based differences. Many of these studies in the phase two trials even are less than 100 patients. The most advanced is the RGX14 program. That is in phase three. I hope there is some subgroup analysis of race differences. Macular degeneration has a preferential effect or usually patients who have it are Caucasians or light colored eye patients. We may not see as much heterogeneity in different races as we would like. What route of administration gives you the best corrective visual acuity? That’s a hard question to answer. Most of these injections are maintaining good visual acuity. Now I think the sub retinal route is most promising. You inject the drug off to the peripheral retinal and although every gene therapy I think causes inflammation, just a tiny bit, even for sub retinal. The effect is limited to the peripheral retina. The drug is effective in the whole eye. I think you get the benefit without the risk of the central vision and the less risk for ocular inflammation. That route is probably the least inflammation but technically the most challenging to do, you need a vitreoretinal surgeon to do it sub retinal injections can be done, you hold a 41 or a 39 gauge cannula hooked up to a person on a plunger, a syringe pushing the drug or hooked up to the viscus fluid injection on a vitrectomy machine. I use an Alcon constellation device. I have done it both ways. In human clinical trials, especially in the phase 3 trial, we’ve been doing it with the silicone oil injection setting. Mainly because it’s most controlled and also that is how you get all the different studies, all the different sites to push the virus at the same speed. The reason why this is critical is there is some studies where if you inject too quickly, obviously, when you inject too quickly you can potentially cause a retinal break or something traumatic. There are some thoughts that the speed of correcting the sub retinal bleb can induce damage like shear forces against the RPE or the photoreceptors and cause more damage that way. I don’t know if that’s really clear yet. But I think the silicone oil injection by using a specific setting can give you more consistency between injector rather than having someone push the plunger. There is one question in Spanish. When will this genetic treatment be available to market? Thank for the translation from Cybersight. When will it be available to market? Now, we’re still in phase 3, which is pretty close. If it becomes successful and the results read out, assuming FDA approval and so on, process, I’m looking at potentially 2026 as the earliest that any of these treatments will become available commercially. That’s not far from now. I’m pretty excited if these products become available to our patients. Is there any contraindication to people with autoimmune disorders? That is an interesting concept. I don’t know currently, I’m presuming things like rheumatoid arthritis or lupus or sarcoidosis. I don’t know currently whether those are explicit contraindications. I do think when you do sub retinal injections because you’re injecting into the sub retinal space which is an immune privileged space, it’s probably not as high risk. So I don’t think that would be a contraindication but it could be a problem for those getting intravitreal injections or suprachoroidal injections. Commonly asked by patients, how soon can we see this in the clinical setting. I think probably 2026 at the earliest. When will this treatment be available in Russia. I would say the earliest for any product will be 2026 will be the first one. The main question from the beginning of the talk is are we there yet — gene therapy. I think we’re getting very, very close. I think we have three exciting products in development. Some of them are by sub retinal injections and some intravitreal, some are suprachoroidal. I think it will be exciting times. The idea of a one and done permanent treatment for wet macular degeneration with a single injection versus our monthly or every other month injection is a very, very exciting prospect. So we’re right on the dot on the hour. So if there are no other questions, let me just click on one — okay. I think that’s the webinar chat. Okay. I’m all set. So I just want to thank everybody for attending this talk. And then good luck can everything and I’m happy to chat at future conferences.

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