신경근병증에 대한 AAV 유전자 치료의 발전

구델 박사는 GNE 근병증을 효과적으로 치료하기 위해 이중 프로모터 AAV 유전자 치료제를 개발하고 있습니다.

### 신경근병증에 대한 AAV 유전자 치료의 발전: 줄리 구델 박사의 인사이트
* 👋 워싱턴 대학교 조교수인 줄리 구델 박사가 GNE 유전자의 돌연변이로 인해 발생하는 질환인 **GNE 근병증에 대한 AAV 유전자 치료**에 대한 연구를 발표하는 GE nem 심포지엄 연사 시리즈에 오신 것을 환영합니다.
* 🎓 구델 박사는 **다트머스 대학에서 학위를 받고 펜실베이니아 대학에서 유전자 치료에 관한 박사 학위**를 받았으며, 현재 **듀센 근이영양증과 같은 질환의 근육 유전자 치료**에 관한 연구를 진행하고 있습니다.
* 🔬 그녀의 발표는 근육과 간 모두에서 GNE 유전자를 발현하는 이중 프로모터 개발의 중요성을 강조하며, **간 발현을 활용하여 근육 교정을 강화**하는 것을 목표로 합니다.
* 📊 실험 설계에는 탠덤 발현 카세트를 만드는 것이 포함되며, 초기 결과에 따르면 **간과 근육 조절 요소의 특정 조합이 두 조직 모두에서 발현을 크게 향상**시키는 것으로 나타났습니다.
* 💡 구델 박사는 **AAV 벡터, 특히 이전에 사용했던 것보다 훨씬 적은 용량**으로 근육에 효과적인 전달을 보여 임상 적용에서 잠재적으로 안전성을 향상시킬 수 있는 유망한 AAV myo의 선택을 강조합니다.
* 💉 이 치료법의 제안된 전달 방법은 **정맥을 통해 근육과 간 조직을 광범위하게 전달**할 수 있어 치료 효과에 매우 중요합니다.
* 🗓 구델 박사는 규제 당국의 승인과 자금 조달에 따라 일정이 달라질 수 있지만, **2~3년 내에 GNE 근병증에 대한 임상시험**을 시작할 수 있을 것으로 예상하고 있습니다.
* 🤝 근육병 퇴치 재단의 지원을 받은 이 연구의 협력적 성격은 **향후 10년 이내에 시장에 출시**될 수 있기를 바라며, **GNE 근병증에 대한 유전자 치료 솔루션**을 발전시키기 위한 공동의 노력을 강조합니다.

Hello everyone. My name is Noah Weisler. It's my pleasure to welcome you here today to the GE nem Symposium speaker series presented by the No Muscular Disease Foundation. We have a very exciting speaker here today, and we're very happy to welcome Dr. Julie Credell to our virtual podium to make her presentation. Dr. Goodell is an acting assistant professor in the Department of Neurology at the University of Washington in Seattle. She received her undergraduate degree from Dartmouth College in Biochemistry and Molecular Biology, as well as Art History, and then put on a stint teaching chemistry in high school for the Teach for America program, a great program and a great service to our communities. But then moved on to do her graduate work in AV gene therapy for hemophilia and cancer in the Cell Molecular Biology PhD program at the University of Pennsylvania School of Medicine, where she worked in the lab of Dr. Walder Arruda. After completing her work there in the gene therapies and vaccines program, a very foundational program involved

in the development of a lot of the fundamental technologies used for gene therapy and many different diseases. She moved on to work at the University of Washington, working on muscle gene therapy as well, including Duchenne muscular dystrophy, X-linked myotubular myopathy, and gene myopathy, which is what brings her here today to talk to our group about the progress that she's been making in her laboratory developing an AAV gene therapy through GNE. I'm very excited to have you here today, Dr. Goodell, and I turn the podium over to you. Thank you. So today I'm going to talk to you about AAV gene therapy for gene myopathy. I'm really going to focus on the work that I've been doing over the last year on promoter development, and then a little bit at the end, I'm going to show you some other work that our lab has been doing looking at AAV serotype screening. Before I get into today's topic, though, I actually want to give a big shout-out to

some of my colleagues. I am a seasoned gene therapist, but I'm brand new to GNE myopathy. Thanks to NDF, these people have really done an amazing job of sharing their knowledge and resources about GNE myopathy so that I could get up to speed quickly and not have to reinvent the wheel. So, a huge thank you to these individuals and to the NDF for funding me. So, GNE myopathy, just a quick overview for anybody in the audience who may not be as knowledgeable, as I'm sure a lot of people in the audience are. So, GNE myopathy is caused by mutations in the GNE gene, and therefore gene therapies should look at replacing this gene. The GNE gene encodes for the GNE protein, which is an enzyme that's vital to the ability of cells to make sialic acid. Sialic acid is a sugar that gets attached to other proteins in a cell. These proteins are called glycoproteins, and if you want a great overview on this, you should go back to the recordings if

you haven't already seen it and watched Dr. Kelly Crow's recorded lecture for more information. So, every cell tape actually makes the genie protein, and therefore every cell makes its own cyalic acid and its own glycoproteins. But in addition to that, cells can also recycle cylic acid off of glycoproteins in the blood, and these glycoproteins in the blood are primarily made by the liver. So, we're hopefully going to use this to make our gene therapy better, and in fact, Dr. Paul Martin's lab has already shown that this can be done. He gave a talk in last year's ndf Series where he showed this data. So, at the top here is a genie mouse that was untreated, and so the muscle has very little scialic acid on the surface of the muscle cells. The liver has a bit more, but it's still hypocyalulated. So, when these mice are treated with a gene therapy directed towards muscle, you can see that there is an increase in the cyalic acid on the surface of the muscle.

fibers, but there's not a change in the liver. Whereas, if he treated the mice with a gene therapy directed towards the liver, the liver obviously had a huge amount of cyalic acid made, but so did the muscle. And so, you could even say that the muscle looked better than it had when we directly treated the muscle. And so, it's really proof of concept that liver expression of the genie protein can help correct the muscle. So, before we go any further, we need to answer the question, really, what is gene therapy? And it can be broken down into this slide. It's putting a gene cassette into some sort of vector and then delivering that vector into human cells. Of course, each of these steps requires you to make choices. So, what is your gene cassette going to be? What is your vector going to be? How are you going to deliver this vector to the patients? And we're going to go through the steps one at a time and how they relate specifically to GNE myopathy. I want to acknowledge that this figure came from

unicure's educational resource called Ingenuity Hub, and I recommend that you look it up if you want more information on AAV gene therapy. They have a lot of great brochures and videos that you can learn more there. So, the first step is really designing your gene cassette, and I'm working on that quite a bit thanks to NDF funding. So, our gene therapy cassette needs two important things: it needs a promoter and it needs a transgene. There are a few other things in the cassette as well, but today I'm just focusing on these two. So, for the transgene, as we've already mentioned, we need to replace the GNE gene. It turns out that there are a few different versions of the GNE gene, and Catherine from the LEK lab, who's speaking here in a few weeks, did some great work to show that we want to use the version called GNE1. So, our transgene is going to be GNE1. Then the promoter is what drives the expression of the gene, and this is what my NDF funding has been spent on.

developing, so we want a promoter that can express GNE in skeletal muscle, and we want a promoter that can express GNE in the liver. Because remember, we're trying to utilize that liver expression to get even better correction of the muscle, but we don't want a promoter that expresses everywhere. There are safety concerns with that, and in addition, many of those promoters get silenced over time. So, our work has been combining regulatory elements from muscle-specific promoters and liver-specific promoters to get dual expression, and we call these promoters tandem expression cassettes or texts. So, the experimental design for my study is to design a tandem expression cassette and then to test these sets of tandem expression cassettes in HepG2s, which are liver cells, or C2C12s, which are muscle cells, in dishes in the lab. And then, based on how that expression looks, go back and do another round of tandem expression cassette design and test those as well. So, for our first set of texts, this work

was done by a graduate student named Maggie Rexopage. So, for this round of text, we have our muscle regulatory element, and we didn't change anything about these. But we have this set of liver regulatory elements, and we changed size and orientation to look and see how that impacted expression. So, in muscle cells, this set of texts, when tested in muscle, there really wasn't much of a difference in expression, which is not that surprising because we didn't change anything about the muscle regulatory elements. But when we looked at liver cells, we did see some variable expression, and Tech 1B and Tech 1C really seemed to lead to the best expression. We did this series of experiments a number of times to confirm our results, and here is one other example of this experiment where we also then compared it back to the muscle promoter, just this blue part alone. And as you can see, all of our texts did lead to better expression than our muscle promoter alone in the liver. So, we are amplifying our liver expression by adding these.

additional liver regulatory elements. So we did an additional round of tech design. So our last text were tech one a through one e, and now we're on to numbering one through six. So looking at the muscle, we're comparing our expression to the liver-specific promoter alone and the muscle-specific promoter alone. So the liver-specific promoter alone, you really don't get expression in the muscle. For the muscle-specific promoter, we get a decent amount of expression. But then when we combine these elements, we actually increase our muscle expression, which is great because that is the target tissue for correction to treat the genome myopathy. So text one through four really do lead to, well, maybe not two, but text one, three, four point eight, and four point ten do lead to an increase in expression in the muscle. Tech 5 and Tech six don't look great in the muscle. Type 2 also doesn't really look great in the muscle. Then when we look at the liver again, we have the liver-specific promoter, and it has excellent expression in the liver. The muscle-specific

promoter doesn't leak a little bit into the liver, so there's a little bit of expression. But then our combination text can dramatically increase how much expression we get in the liver over this just Baseline muscle-specific promoter. And here really all of them fared better than our muscle-specific promoter alone. And then remember we really are trying to limit expression just to these two tissues, so we also looked in a Cell line of kidney cells, and our liver-specific promoter is very tightly regulated in the kidneys, so there's almost no expression. The muscle-specific promoter does leak into the kidneys, so there's a little bit of expression there, and our combined promoter texts um some of them have some decreased expression compared to our muscle-specific promoter. So if we were to combine all of this data into a single graph and normalize it to our muscle-specific promoter because this is really our starting point, if we were to go in with the gene therapy today, we would use this muscle-specific promoter. So it's our Baseline comparison, and we look in the muscle, which is the

purple bars. Our Tech 1B, 3, 4.8, and 4.10 all lead to increased expression in muscle, which is great and sort of a benefit we weren't really necessarily thinking we were going to improve muscle expression. Her goal here was to get expression in the liver, so this is a nice added bonus. We look at the liver expression again. Tech 1B, 2, this time Tech 3, Tech 4.8, and 4.10 all lead to increased expression in the liver. Tech 5 and 6 look great in the liver, but they have basically no muscle expression, so we've discounted them. And then looking at the kidney, we see a little bit of a reduction in expression in Tech 1B. Tech 4.8 and 3 are about the same, uh, in Tech 4.10 is a little bit reduced. So based on this data, we've decided to move forward with Tech 4.8. We have about equal expression in the muscle or an equal increase in expression in the

muscle and liver without having a bunch of extra expression and kidney. So, we've established what our Gene cassette is going to be. It's going to be Tech 4.8 expressing gne1. So, now we need to decide what our Vector is going to be, and our Vector is going to be an adeno-associated viral vector. These vectors, um, they're really popular right now because they are non-pathogenic viruses. So, 90% of adults have been infected with some version of aav and not even known that they had it. It does not make you sick, so it has a safer profile than a virus that would make you ill. But there are a bunch of different versions of aav, and so we need to decide which one of those we're going to use. But first, I want to go over what the difference between a virus and a vector is. So, a virus uses their genetic material to replicate. So, you have a viral capsid, proteins that make up this shell, and inside is the Genome of that virus.

that virus then infects cells, and that DNA gets spit into the nucleus, and then that DNA is used to make more virus, and that virus then bursts out of those cells, goes on to infect even more cells in the body. So, those are replicating activity. That's different than a vector. So, for a vector such as AAV, we're still using that viral capsid, still the same proteins making up this particle, but inside, instead of there being a viral genome, we've taken all of that out for AAV, and we've replaced it with our therapeutic transgene cassette. So, there's nothing in here encoding anything viral. So, when a vector then enters the cell, we don't call this an infection; we call it transduction because it's not replicating, but a lot of the steps are the same. The virus, or sorry, the vector enters the cell, spits its genome, which is now our therapeutic genome, into the nucleus, and then that genome is used to make proteins, but instead of making viral

proteins. Now, it's making therapeutic proteins. So, for us, that's Genie. Um, and then if it depends on whether that's an intracellular protein, which for genomopathy it is, or if it's created. But basically, there's no additional production of new vector, and it's not replicating, so that makes it safer. And we don't have to worry about a lot of the issues of a viral infection when we use a vector. So, vector choice is incredibly important. We want to pick an AV that transduces skeletal muscle. Historically, that has been AV6 or AV-9, or perhaps even AV8. Those tend to be the stereotypes that are used to treat skeletal muscle diseases. We also want an AV that transduces the liver. That's not so much a worry because most AAV ends up in the liver anyway. The liver's job is to clean stuff out of your blood. So, if we put AV in your blood, a bunch of it goes to the liver. So, we're really focusing on picking an AV.

serotype that is good at transducing muscle, with the assumption that then a bunch will just end up in the liver on its own, for AV gene therapy clinical trials that are going on right now. And for the one muscle disease that has improved therapy, which is zolgensma for SMA, those treatments use greater than 1 times 10 to the 14 Vector genomes per kilogram to treat patients. So, this is more than 1 times 10 to the 14 Vector particles that have our therapeutic genome in them per patient kilogram. So, doses are very, very, very large. Unfortunately, these doses have been associated with a lot of severe Adverse Events, and in three different diseases, they've also had deaths. So, we are really aware that we need to reduce the doses of AAV to increase the safety profile of these treatments. While the deaths have been exceedingly rare, thousands of patients have been treated, and there've been seven deaths. It's still obviously tragic; every death is tragic, and

anything we can do to make the treatment safer is going to make it a better treatment. It's also going to make it a more used treatment and a treatment that you can feel confident using earlier in life. So, we're gonna reduce these doses by carefully choosing both our promoter and our vector. So, we've already shown that by making differences to the promoter, you can increase expression, but you can also have a profound impact on expression levels by choosing your vector carefully. So, as I mentioned, the AAV 6, 8, and 9 have historically been used, but in the last couple of years, there have been two really groundbreaking papers looking at AAV serotype design and finding or designing AAVs that are excellent at transducing muscle. This is one of them, and it's the one that we are testing in our lab. We have a collaborating company that has licensed this product, so that's why we're focused on this one. It's called AUB mile, and it was identified by the Dirk Dirk

Grimm's lab. So, we've tested AV Mile in our lab. This work was done by postdoc and Jeff Chamberlain's lab. Hashem tasked out, and he tested doses of 1 times 10 to the 13 vector genomes per kilogram of ab9 or AV myo. So, this is 10 times less than what is currently being used for muscle diseases. It's also in the range of what's used for a lot of liver diseases, which have seen no deaths. So, it seems to be a much safer dose. And Hashem tested this in our dystrophic mouse model using the transgene expressing micro dystrophin. But the results still apply because we're just looking at expression levels. What the transgene is here doesn't really matter. So, looking at the histology at this pretty low dose of 1 times 10 to the 13 vector genomes per kilogram, you still have a lot of pathology of the tissue, and we don't see much expression of micro dystrophin.

which would be on the cell surface, and you can see a little bit, but not much. The same exact dose using AV myo, though we have correction of the tissue pathology, and we see great expression of our transgene. He also looked at the Western blot. He did Western blots to quantify how much expression he was getting in the muscle of each group of mice. And so, this is an MDX mass that is untreated. You can see there's no dystrophin expressed at all. When he gave mice 1 times 10 to the 13 Vector genomes per kilogram av9, no detectable expression by Western blot. But when he treated with the same dose of aav myo, he got great expression of the micro dystrophin. And in fact, it was at even higher levels than just normal wild-type mice express dystrophin. And when we looked at the strength in the limb muscles of these mice, these are normal mice versus dystrophic mice, and he treated the dystrophic mice with ab9 or AV myo, and he saw a

correction of the muscle weakness in these mice with this more moderate dose of 1 times 10 to the 13 Vector genomes per kilogram. So, my lab's really focusing on using AUB Mayo moving forward. And then the last question, um, is how would we deliver this to patients? And the answer is we would deliver it intravenously. We want to get transduction of the muscles throughout the body. We also want to transduce the liver, and the best way to do that is to inject the vector directly into the bloodstream and allow the blood to disseminate the vector through the body. So, our key takeaways are we have designed a promoter that has dual liver and muscle expression to be used for AV gene therapy for genome myopathy. We've confirmed that AV Mio can be used at 10 times lower doses than av9 to treat body-wide muscle disease, and we're now testing the AV myo Tech 4.8 Gene one in mice that have mutant Genie to look at expression and

silhoulation and see if we can restore silation in these animals. I'd like to thank everyone in my lab and Dr. Chamberlain's lab, who I'm a part of. Dr. Hauske's lab, also at the University of Washington, who falls under the Chamberlain lab umbrella, has been helpful in giving me advice on designing promoters. And then, as I mentioned, the LEK lab has been really great at sharing resources and providing the data to support the use of GNE1 as our transgene. That's it. It's a faster talk than I realized it was going to be, but I'm happy to answer any questions. I don't think anyone will argue with brevity when it comes to a talk, so there we go. No, thank you for that. Very interesting talk, and we actually have some questions in already. I'm gonna... There's a couple... There's a hand raised, but I'm also going to ask a question that's in the question and answer box right now. And if anybody else wants to raise their hand and ask a

question in person. Feel free or um feel free to put in the question and answer box. So the first question is, is there a schedule for human clinical trials for this? And once started, how long does it generally take to become a viable solution to treat GNE patients? Sure. So I want to acknowledge that the Luck Lab is also working on developing a GNE myopathy um with the backing of NDF, and they are a little bit further along than they're quite a bit further along than I am. And so this should be thought of as like we're taking multiple shots on goal um. It's a lot of IND supporting data is being collected, has been collected, will be collected. I believe the NDF's goal here is to try to get to clinical trial in two to three years. That's a very ambitious timeline. I'm not saying it won't happen, but it is very ambitious. My guess is though that within five years, I think that we can be in a clinic in clinical trial as to how long it takes them to

get approval really depends. Uh, it depends on a lot of things. It depends on how well the trial goes, depends on how quickly enrollment can be met. Um, if there are absolutely no problems and the treatment is amazing and very clearly works really fast, it would still take quite a few years to get to market. So, probably would take at least five additional years to get to market. But in the meantime, there would be these clinical trials that interested patients could enroll in. Uh, so I would hope that within 10 years we could have something on market, but a lot of things can go wrong. Um, if you look at other gene therapies, the Zolgensma, which is on the market, probably took about seven years between the start of the clinical trial and reaching the market. But then that, like the hemophilia clinical trial, has been going on for 15 years and they're still not tonight. It depends, right? Thank you, thank you for that. Uh, I see Dr. Well, since we just talked about the lab, let's see Dr. Angela likes.

her hand raised. Did you have a question there, Dr. Leck? I believe you're still muted. Ah, yeah, no good talk. Um, Julie, no, I just wanted to clarify that it's not the leg Club per se that's pursuing the genie therapy-like development, but we're contributing to a bigger Consortium involved. So, it's an NDF-led effort, and it involves many scientists. So, it's not specifically just a lab that's pushing the effort along. It's several labs that are involved. So, uh, yeah, we just often the construct to a full for the project, but um, they will work because uh, in multiple labs. Yes, thank you for clarifying. Also, uh, would you like to clarify the timeline that I just hypothesized? Yeah, yeah, um, to be honest, that it's really unpredictable. I mean, it all, it's a conversation with the FDA, the regulatory bodies, to see what further experiments they want us to do. Um, and uh, I mean, it's really hard to

say I, as a scientist, I've always hesitated to put an exact number on, you know, when people say when it's, it's, we're kind of, we're not, we're not close, and it's when you're close, then I think you can say a number, but right now, we're not, we're not in a position where we can say, um, yeah, the earliest would be two years, but nothing sooner, I don't think, yeah, but that's also being very optimistic, as you said, yeah, and, and the fact is too that we are behold holding to the FDA, and FDA changes what they want based on other trials, and so it's a constantly shifting goal post, correct. Sometimes, you know, it'll depend on whether they want to see safety studies in a large animal model, for example, in which case that would severely extend the timeline of the development process, or whether they're happy with, you know, just Mouse studies, so it's, it's really, it's almost impossible to put a timeline to the project, yeah, I agree.

and I would also mention that, uh, on October 30th, uh, we'll have Catherine Kakazawa from the, uh, from the Black Lab, who's been an important part of that Consortium project, giving another talk about GNE gene therapy. So, I'm sure we can discuss that in more detail then as well. Um, great. I know it's a very active area with many groups working on it, so it's really great. I mean, um, and everyone is pursuing a slightly different strategy, so it's really good that we're not sort of putting all our eggs in one basket, so to speak, like different promoters, different stereotypes, and yeah, definitely. Yeah, the biggest issue, of course, is, um, in order to get into clinical trials, we need, there needs to be a lot of funding in the order of millions, um, and whether we can interest a biotech to make that investment or whether NDF can raise those funds. If you've got grassroots fundraising, that's um, you know, we could, the scientists, we can

have the therapy, but at the end of the day, there needs to be funding to take it into clinical trials, and that, to us, is the biggest hurdle. Do you agree with that, Julie and Noah? I do, and they're very, very expensive trials. I mean, just making the AEV alone is hundreds of thousands of dollars, and then you have to contract with the locations, and this is a slightly tough sell to Industry, to biotech. So, I do think that NDF is working hard on this correctly. I think that they need to be prepared to have to fund this themselves, so definitely fundraising everywhere possible. It's going to be really important. Yeah, it's a tough sell, as you say, because it's an adult onset disorder, and it requires much more vector, and it's slowly progressing. All those things make it less attractive to a biotech company than a disease that's more rapidly progressing and affects children. So, there's more to it than just the science.

I mean we can show scientists can show restoration of gene expression and in musk in Mouse muscle we can have the exact therapy. But how do we get that to the clinic? The framework needs to be put in place and the funding needs to be there. So there's lots of lots of hurdles there. Yeah, I agree. Excellent. And I believe Dr. Bhattacharya had some questions too. Um, did you want to unmute and give your, I know she has your hand raised, but I can also read them off maybe. Uh, while we're sorting that out, I'll start with, there was a question about what was the size of the tech cassette. Thanks for giving me a chance. Okay, uh, Julie, I really enjoy your talk. Um, it's very encouraging to see so many things happening. I had a few technical questions, uh, like what is the size of the tech cassette which you finally selected. Uh, you know, the concern is that it should uh, not occupy too much space for the AV vector. So yeah, so it's a, it's a thousand, it's a

thousand base pairs, and so it's there. It's quite large in terms of, you know, the promoters that are out there that are used for AV. But the genie gene is actually a very manageable size. So, even with our largest tech, using the genie one gene with an intron and Paulie and the ITRs, the whole thing still comes in under 4.7 KB, which is the normal wild-type genome. A baby, okay, so it's 1 KB that is kind of quite okay. Yeah, okay, then my other question was that you've shown expression in kidney cells, but have you also shown in lymphocytes in the sense that, you know, the immune system should not express in immune cells, right? I agree. So, we are going to do biodistribution studies in the animals. The cell work only gets you so far, and so we're gonna do a careful study in mice to see where exactly we're getting an expression. I'm hopeful that we won't see anything in immune cells because neither

the muscle-specific promoter nor the liver-specific promoter that we're starting with expressing immune cells, but obviously, it is a thing we need to think about, and we're definitely going to look okay. And did you compare your expression level with the CMV promoter? I mean, how does it compare with CNB? I did, and the CMV is a very strong promoter. So, in liver, CMV is stronger than artex. In muscle, um, it's a little bit stronger, but it's more comparable. The problem is that CMV is known to be silenced in the liver, so after gene therapy, you lose expression over time if you use the CMV promoter because it's a viral promoter, uh, and the body responds by methylating it and silencing it. So, we don't want to use CMV. All right, so you say it's too strong a promoter. It's not that it's too strong; it's that the body shuts down expression off of CMV over time, so independent of how strong it is.

um, it has these viral motifs in it that are targeted by methylation to silence the promoter. And so, while it looks great to start with over a year or two, you actually can completely lose expression from CMV. Okay, that's really interesting. Uh, and then you've tried only the luciferase reporter, or have you also tried the gene itself? So, the gene is what we're taking into the mice. [Music] So, in mice, you've tried the gene. Yeah, those experiments are happening now. Yeah, okay, okay. And lastly, I would like to ask you whether you're also looking at the translational level, or you're only focusing on transcription. We are looking at translation, right? So, in the cell studies, it was all protein expression, um, because it's the luciferase enzymes. We look at the luminescence. I didn't make my question clear. Please, I'm sorry. Uh, what I meant was whether you want to enhance the translational efficiency. Oh, I see. So, we, um, I have another grant. I have a K award.

looking at GNE myopathy gene therapy as well, and one of the aims at that grant is to do codon optimization to look at increasing translational efficiency. That's not part of the NDF-funded study; there just wasn't enough money to do that as well, but yes, I am doing that. Okay, so that is by codon optimization or any other. Okay, uh, just codon optimization for now. Okay, thank you so much, Julie. That was very informative. Thank you. Yeah, thank you. We've got a couple more questions in the Q&A here. Uh, the next one is, uh, will complete recovery be possible with gene therapy? Realistically, probably not. Uh, the good news about GNE myopathy is that it's not incredibly degenerative like Duchenne Muscular Dystrophy, and we know from other diseases that are non-degenerative, like X-linked biotubular myopathy, that you can get a lot of the lost function back, but you're probably not going to get to a hundred percent. So, I think in an ideal world

we would treat patients early, um, preferably before they really see loss of function. Now, loss of function is usually what predicates diagnosis, but the earlier you treat for these muscle diseases, the better off you are. But having said that, this disease does lend itself to recovery. We're just probably not going to get back to 100 percent. And a similar question, uh, will it be a one-time fix or reoccurring Gene fix would be necessary. At least to start, it's going to be a one-time thing. So, this is a question in the field is how long are these muscle-directed gene therapies going to last. Again, looking at other diseases, we have a canine model of Excellence bio tubular myopathy that was treated nine years ago and has shown absolutely no loss of function in those nine years. For hemophilia, we have patients that are more than 10 years out and still have great expression in the liver. So, it's probably going to last a decade or more in most patients. There will possibly be patients who need another treatment.

because the first treatment didn't go super well. It is also possible that 30 years down the line, we might need to give an additional treatment. At this point in time, there is no way to give a second treatment in humans, but a lot of people are working on that. I'm actually working on that for my tubular myopathy, so hopefully when we get 10 years down the line, 20 years down the line, and some patients are neat looking like they might need a second dose, we will have mechanisms in place to be able to safely do that. Excellent. I did want to, I don't see any other active questions, but if anybody has another one, please feel free to put it in the Q&A box. The uh, if not, I'll take a prerogative task one additional question, more of a I guess technical question, but uh, it's exciting work you had there with the AAV mile. It's nice to see those confirmations in somebody else's hands too. So um, you know that that 10 times

greater uh efficacy is a great thing to see. Have you seen anything that would indicate any other issues with working with AAV Mayo, perhaps a production of the capsid is different or anything like that? I'm just kind of curious uh what you've seen in your hands. Yeah, we haven't had any problems with production, so it's basically AAV9 um with I think just six amino acids, very small, pretty small difference, yeah, very small difference. So um, all the production methods are the same, and sometimes when you screw around with the vector capsid proteins, you can reduce the titers of product that get produced, and we really haven't seen that um, and especially since the GE transgene is so small um, we're not going to have those struggles for that reason. So I'm pretty confident that we can get high titer. I mean, I know that we've gotten high titer vector using uh the micro dystrophin in AAV Mayo, so I'm confident that we'll have the same high titers if

not better with the genie transgenes, right? Yeah, no, that's good to hear. Great. In your thoughts, um, in your opinion, do you envision FDA looking at myo AV or AV Mayo as completely new AV stereotype and requiring um, a longer sort of toxicity and safety study, presumably in primates, or will they accept the argument that oh, it's just a variant of aav9 and so much safety studies have been done on aav9, so we'll just go off that? So, I do think that they will have the general opinion that it's a variant today, not av-9. Uh, I will say even 89 has variance, like what one company is calling av-9 and what another company is calling calling av-9 could already have six amino acid differences between them. But given all of the adverse events that are happening in gene therapy, independent of whether we were using av-9 or AV mile, I think there's a really good chance that we're going to have to do primate studies.

Okay, uh, Noah, you're muted. I am muted. I should be. I should be better at that by now. All right, um, there is one more question. I'm gonna see if we can get this. So, can AI protein synthesis based on the gene be of any help? Is that something that's within your team's scope? So, that's not within my team's scope. Um, I believe there was a talk a couple of months ago about, like, looking at post-translational modifications and groups that are really digging into the enzyme activity and how the protein is, uh, handled after it's produced and modified and that sort of stuff. Those groups might benefit from looking at sort of these AI modeling systems for protein folding and stuff in that you. There are examples of proteins that can be made to be hyperactive with mutations. For example, factor nine. There's a variant called factor nine Padua, and it's eight times more, um, has eight times as much activity as normal factor nine. The one thing I will say is that we do need to be careful.

with GNE because overexpression of GNA shouldn't be a risk here because GNE has a negative feedback loop, and so we're not at risk of making too much cyalic acid if we leave the genie protein in its normal wild type version. But if we were just somehow to block that ability to get this negative feedback in cells that end up with a lot of vector, you could end up with a bunch of extracilic acid made, and that can be toxic. So, while I agree that it's a really interesting idea and those studies should be done, they need to be done carefully. Agreed, uh, it's a great, and I'll just, uh, emphasize that that was a great talk that we had on post-translational modification of g e that was on July 31st. And if you want to go back and see that, it's available, uh, through the ndf if you'd like to see that as well. But seeing no more questions, I'd like to thank Dr. Goodell for a great talk today.

uh, and I wrote, and everyone, for a robust discussion afterwards. That was a great discussion and good to see it take place with both patients and scientists participating, so we appreciate that. uh, finally, I'd like to, as I mentioned earlier, emphasize that we have another scientist spot coming up in our speaker series. That's on October 30th. That'll be on developing an AV gene therapy for gnem, part two. So, uh, we've got SQL coming up, and that'll be uh, Catherine Kakazara from the uh, from Yale talking about the work that she's been doing developing a gene therapy approach as well. So, very excited to have that uh, these two bookends uh, before we start to move towards the end of this year. And thank you, Dr. Cordell, for a great talk today, and I hope to see you all again soon at the next NDF seminar series.

2022 NDF GNEM Speaker Series with Dr  Julie Crudele Developing An AAV Gene Therapy For GNEM

Neuromuscular Disease Foundation

신경근병증에 대한 AAV 유전자 요법

워싱턴 대학교의 조교수인 줄리 구델 박사는 프로모터 개발과 AAV 혈청형 스크리닝에 초점을 맞춘 GNE 근병증에 대한 AAV 유전자 치료법을 연구하고 있습니다.

GNE 근병증은 세포에서 시알산 생성에 중요한 역할을 하는 GNE 유전자의 돌연변이로 인해 발생합니다.

이 유전자 치료법은 GNE 유전자를 대체하는 것을 목표로 하며, 마우스 모델에서 GNE 단백질의 간 발현이 근육 결핍을 교정하는 데 도움이 될 수 있음을 입증함으로써 유망한 결과를 보여주었습니다.

GNE 유전자 교체를 위한 유전자 치료 카세트 설계

유전자 치료 카세트에는 프로모터와 형질 전환 유전자가 필요하며, GNE1은 GNE 유전자 대체를 위해 선택된 형질 전환 유전자입니다.

이 연구는 골격근과 간에서 GNE의 이중 발현을 위한 탠덤 발현 카세트를 만들어 안전성 문제와 프로모터 침묵을 해결하는 것을 목표로 합니다.

실험 결과 근육과 간 조절 요소의 조합이 두 조직 모두에서 발현을 증가시키는 것으로 나타났으며, 이 데이터를 사용하여 효과적인 유전자 치료법을 설계하는 것이 목표입니다.

유전자 치료: 벡터 선택 및 근육 발현

10은 근육과 간 모두에서 발현을 증가시키며, 기술 4.

8은 균형 잡힌 발현으로 인해 유전자 카세트로 선택되었습니다.

아데노 관련 바이러스(AAV) 벡터는 비병원성과 근육 및 간 세포를 전달할 수 있는 능력으로 인해 선택되었습니다.

그러나 임상시험에 사용된 고용량의 AAV는 심각한 부작용 및 드물게 사망과 관련이 있어 안전성 향상을 위해 용량을 줄여야 할 필요성이 강조되고 있습니다.

근육 질환에 대한 보다 안전한 유전자 치료법

용량을 줄이고 프로모터와 벡터를 신중하게 선택함으로써 치료의 안전성을 개선하고 있습니다.

새로 설계된 AAV 혈청형인 AAV Myo는 실험실에서 테스트 중이며 현재 근육 질환에 사용되는 것보다 10배 낮은 용량으로도 효과가 있는 것으로 나타났습니다.

이 치료제는 근육과 간을 전신으로 전달하기 위해 정맥으로 투여할 계획입니다.

신경근병증 임상시험 타임라인

럭 연구소는 2~3년 안에 임상시험을 시작한다는 야심찬 목표를 가지고 GNE 근병증 치료제를 개발하기 위한 노력을 주도하고 있으며, 이 일정은 야심찬 계획입니다.

임상시험에서 시판 승인까지의 과정은 5년이 더 걸릴 수 있으며, 10년 이내에 치료제를 출시하는 것을 목표로 하고 있습니다.

여러 연구소가 컨소시엄에 참여하고 있으며, 임상시험을 진행하기 위해서는 자금이 매우 중요합니다.

임상시험을 위한 자금 조달은 수십만 달러에 달하는 비용이 드는 큰 장애물입니다.

성인에서 발병하고 진행이 느리기 때문에 생명공학 기업에게는 매력적이지 않습니다.

기술 카세트의 크기는 1,000개의 염기쌍이며, 전체 유전자 치료 구조는 4.

7KB 미만이며, 면역 세포에서의 발현을 평가하기 위해 생쥐를 대상으로 생체 분포 연구를 계획하고 있습니다.

신경근병증에 대한 유전자 치료: 인사이트와 과제

코돈 최적화는 NDF 지원 연구의 일부가 아니지만, GNE 근병증 유전자 치료의 번역 효율성을 높이기 위해 추구되고 있습니다.

유전자 치료로 완전히 회복될 가능성은 낮지만 조기 치료는 상당한 기능 회복으로 이어질 수 있습니다.

유전자 치료는 일회성 치료로 효과가 10년 이상 지속될 것으로 예상되며, 향후 안전한 재치료를 위한 메커니즘을 확립하기 위한 노력이 진행되고 있습니다.

Myo AV 및 AV Mayo에 대한 FDA의 관점

FDA는 Myo AV와 AV Mayo를 AAV9의 변종으로 간주할 가능성이 높지만, 최근 유전자 치료의 부작용으로 인해 영장류를 대상으로 안전성에 대한 연구가 필요할 수 있습니다.

AI 단백질 합성과 단백질 폴딩 모델링은 발표자 팀의 연구 범위에는 포함되지 않지만 다른 그룹에서 이러한 분야를 연구하고 있습니다.

GNE 과발현을 연구할 때는 부정적인 피드백을 차단하면 독성 수준의 과산으로 이어질 수 있으므로 신중한 고려가 필요합니다.