hello everyone. My name is Noah Wise Leader, and I'd like to welcome you to the GEG NEM Symposium Speaker Series presented by the Nebraska Disease Foundation. I'll be administering today's call. My name is Noah's Leader. I'm a professor and director of Graduate Studies in Physiology and Cell Biology at the Ohio State University, and it's a great pleasure to be here today again with you in this series to talk about gene myopathy and some of the efforts involved in the study and treatment of the disease. I'd like to give a quick reminder that if you have any questions during today's seminar, we're using the online question and answer feature of Zoom that you should be able to access the Q&A tab towards the bottom of your screen, depending on how things are configured. But if you have questions, please enter them into that section, and we can, if they're urgent, let me know. Otherwise, we'll probably hold these to the end, and then during the, at the end of the

session. I will administer the Q&A portion using those questions as well as probably some other ones that come up. Uh, so, uh, I would also point out that we have a couple of other events coming up in this seminar series in the near future. I wanted to point out that, uh, Dr. Paul Martin will be presenting, uh, on December 9th, and then, uh, uh, that Dr. Francis Rossignal will be presenting as well on December 18th. So, we're excited to have those two coming up very soon, uh, and there'll, of course, be the announcements that go out about that as well. But today, uh, we are really excited to have, uh, have one of our rising stars in the G.E. myopathy field here, uh, to give our today's presentation. Uh, today, I will be Catherine Kozwara. Uh, Catherine Kozwara, she's currently a Ph.D. student in the LEK lab at the Yale School of Medicine. She received her Bachelor's of Science in Biological Sciences from the University of Notre Dame and then went

to work at Yale on developing our PhD. Her studies focus on concentrating on both computational biology and genomics and combining those two interests in order to study human genetic diseases and with a particular emphasis here, as you can see, on GNE myopathy. She's been investigating the application of CRISPR-Cas9 technology in terms of editing to correct patient mutations and gene myopathy as an example, and also has been instrumental in establishing a biobank of GNE patient cells, many of the uh many obtained through donors by the uh by the NDF, and this has become a really cornerstone resource to be able to spread the amount of GNE research that's done both in the United States and throughout the world. So, we very much appreciate your research and the efforts you put into helping to build the field, and I will turn this over to you today to present your talk on developing an AAV gene therapy for GNE myopathy. I think so for a great introduction. Um, yeah, so I'm going to be talking a bit

today about the work that I've been doing in the lab regarding developing an AAV gene therapy for genome myopathy. Um, so, uh, Dr. Julie Crudell gave a part one to some of this work at a previous seminar, so I highly encourage people to watch, uh, watch that as well. But diving right into kind of the work that I've been doing on this project, the aim of the project as a whole is to develop an AAV gene replacement therapy for genome myopathy. Kind of the work that I've been doing has really been focusing on optimizing the A part of the AAV called the transgene cassette in vitro, so in cell lines, before moving to more in vivo mouse studies. And so first, a bit of background into GNA myopathy. So, gnomyopathy is, as many people know, an autosomal recessive disorder that leads to progressive skeletal muscle atrophy and weakness in the legs and arms. It is caused by mutations in a gene called GNE, affects about one in one million people worldwide, and

characteristics of the disease are characteristic of Rim vacuoles and inclusion bodies, which you can see here. Some of the staining and the genome apathy patient. You can see these inclusion bodies and remedicules that you don't see in healthy muscle tissue. So, as I mentioned, GNE is caused by mutations in a gene called GNE, which is diagrammed here. So, there are two domains that the Gene and upper Mary's domain and a kinase domain. As you can see with some common mutations listed across the bottom, these mutations are spread all across the disease, and there are many different mutations that have been found to date to cause GNA monopathy. And lastly, um, GNE is an enzyme and a solic acid biosynthesis pathway. So, it's here diagrammed as the silic acid biosynthesis pathway, and you can see that GNE performs two of the steps in this pathway, the epimerase function and a kinase function, and eventually this will lead to the production of cyalic acid, which is then used to silate.

proteins on the cell surface. So, in terms of developing and treatment for GMA myopathy, there are kind of two main ways to think about doing this. The first is by supplementation, something like salic acid or manic supplementation that some collaborators with the NIH are working on. And this aims to supplement with some of the compounds produced in the silic acid biosynthesis pathway. So, I'm not targeting GME but looking to add some of the compounds that it would normally produce that it can't in patients achieving myopathy. The second approach, and the one that I'm going to focus on, is a gene therapy or Gene replacement strategy. So, this aims to replace the mutant gene and add a healthy copy of the gene, introduce that into the cells so the healthy copy can then produce the silic acid. And so, I've mentioned a latching replacement therapy, but for those of you who don't know, I can go a bit into what that is and what that entails. So, Gene replacement therapy aims to

deliver a healthy copy of the GNE Gene to cells affected cells so the cells that don't have have a mutated Genie Gene. We want to introduce a new functional gene into the cell, and so this funk new functional Gene can work in the cell function. In this case, GNE enzymatic activity can be restored. And so the way gene therapy works is um it's an AV vectors are used to deliver healthy genes to the cells. So in this case, a gene cassette pictured here is packaged into an aav vector, and this combined Vector is introduced into the cells, which then allows the gene cassette, in this case, the DNA Gene, to be expressed. And so once that Gene is delivered to the cell, it undergoes a process in which the DNA that we deliver is transcribed to RNA, and this RNA is then produced into a protein. So we can kind of measure use this to measure how well our delivery is working by either looking at RNA or protein expression to see if we see the G and E Gene expressed.

so the first step in the work that I've been doing in the lab is first to design this Gene cassette for gnomyopathy. And so, there are a couple of key components that I wanted to optimize that would lead to the best of the highest GNE expression. The first is the promoter, so this is the thing that's driving the expression of the gene. Second is an intron that is between the promoter and the transgene. And then lastly was this trans Gene itself, so which kind of version of the GNE Gene that we want to use. And so, the way that I did this was generating a bunch of different vectors that have different combinations of promoters in trans and trans genes, introducing them to cells in Excel culture, and then harvesting the protein and looking to measure how much protein, how much GNE protein expression we can see with the variety of different vectors that we have, and see which one leads to the highest GNE expression. And so, the first thing that I wanted to look at was which promoter to use. So

since GNE is a muscle-specific disease, we wanted to use a muscle-specific promoter. So, there were two of interest that we wanted to use and test to see which one worked well. The first is called ck80, and the second is mhck7. And so, I created constructs that have either of these two vectors, introduced them into cells, and here you can see is a blot of just looking at protein expression. So, we can use an antibody for G and E to look and see how much protein was in all these samples. So, we're looking at the higher brand here. We can see in Lanes four through seven our constructs with the ck80 promoter, and then Lanes 8 through 11 have contrast with the MHC K7 setup promoter. And what I hope you can see is that in general, these Lanes, especially in Lanes four and six that have the ck80 promoter, have stronger bands meaning more GNE protein was expressed using this promoter compared to the MHC K7 promoter. So, that's the promoter that we

decided on moving forward. The second thing that I wanted to look at is the inclusion of an intron. So, you can either have an intron here, and we're using the SV40 intron, or no intron at all. And so, I created a bunch of constructs. The ones that have the SV40 intron have a red asterisk, and those that do not have a blue asterisk. And so, what I hope you can see is that in all the lanes that have the red asterisks, we have a very strong band, meaning a lot of G-E protein was expressed from the constructs with the SV40 intron present, compared to the lanes with the blue asterisks that don't have as high a strong of a band, meaning not as much protein is expressed. So, we thought it was, we concluded it was very important to have this SV40 intron included in our construct. And then the last thing that we kind of wanted to look to optimize was which transgene that we wanted to use.

so g e has a few different isoforms, and what we wanted to see is since Genius of muscle disease, we wanted to see which of these isoforms was expressed highest in the muscle tissue. So fortunately, we had a few samples from healthy donors, healthy muscle samples, and looked to see in these healthy muscle biopsies which RNA, the RNA from which isoform, was expressed higher. So we did an RTPCR, and what you can see is in these four lanes, looking at the GNE2 isoform, they're very weak or faint, meaning not a lot of GNE2 isoform was present compared to these four bands in that used was looking at the GNE1 isoform, and they're very strong, meaning that in these muscle samples, we saw a high expression of the GNE1 isoform. So that's the one that we wanted to use moving forward. So um basically in conclusion from all of that work, I'm looking to kind of create this construct that has the promoter, the intron, and the transgene.

Our lab came up with this construct that has the CKAD promoter, the inclusion of the SV40 intron, as well as using the GNE1 isoform, which we also tagged with the protein tags so we would be able to detect it later. Previous work done in the Metron and Rosa bomb lab has also developed a separate AV gene therapy that uses this construct that has a different promoter, the MCK promoter, but then the rest of the construct is very similar with the SV40 intron and the GNE1 isoform. So, we wanted to test both of these constructs, from the one that we developed as well as the work that the Adriana Rosa lab has done. And so again, we introduced these into cells and looked to see if we saw GNE protein expression. So, as you can see in lanes five and six, were our two constructs, and you can see both had very strong bands, meaning very strong GNE protein expression in several different cell lines, both in a human cell line as well as in a mouth.

Mouse Zone, and we also looked at the RNA that was produced when we introduced these constructs into cells. You could see that at the RNA level, both the ck80 construct from our lab, as well as the mck construct from Accuration Bob's lab, also had high RNA expression. So, both of these constructs showed promise in being used for an AV gene therapy. So, kind of the next step was to take these constructs that we had optimized in cells and designed in cells and moved forward to a dose in biodistribution study in mice. Um, and I mentioned, but I want worth pointing out again, is this effort is a very collaborative effort from many labs and many groups. Some, and I'm just working on a very small part of this step right here, um, but have had many collaborators that we're working with, um, and are currently, um, working on some of the mouse studies right now. And so, the way this first dose of biodistribution study worked is we take the constructs that either the Luck lab or the Medrani Rosa Bob lab have created.

we generate the AAV9 particles containing our cassette of interest. We'll dose at different doses into mice, and then we can look to see, we'll harvest RNA or protein from different tissues and look to measure the GNE expression that we're getting from our AUD. And so, I did this first looking at several different muscles at the RNA level, so looking at the exogenous GNE1 expression in the gastroc muscle. And you could see here, compared to mice that didn't have any construct, we see an increasing response of low dose for both the MCK construct as well as the CKAD construct. We see GNE expression at a lower dose, and then one that we use a higher dose dosing these mice, we get even more DNA expressions. So, we see a dose-dependent GNE1 expression in the gastric muscle. We see a very similar thing in the quadricep muscle, which is what we expect because our promoters are muscle-specific, where we don't see any expression if the mice are not treated, low expression if they're treated with a

low dose in either of the constructs and then a higher GNE expression when we use a higher dose. We also look to see in the liver because we want our therapy to be relatively muscle-specific. And while we did see a little bit of expression in the liver dose-dependent as in the muscle, when we normalize across tissues, you can see that in the liver we had very, very little expression compared to the two muscle samples we looked at, like in the gastroc and the quadricep. So, this is an encouraging result that we're seeing good RNA expression of gne1 across our two muscle tissues that we've looked at so far. We also wanted to try and look at the protein expression. So again, going from RNA to protein to see if we get strong Gene protein expression. So, we're looking for strong bands, and we hope to see, as with the RNA results, that we wouldn't see a lot of expression in the untreated samples, a little bit of expression in the samples treated with a low dose, and then higher expression um with this in the

samples treated with a high dose of these AAV gene therapies. Unfortunately, that didn't seem to be the case. We didn't see a strong pattern in either the gastric muscle or in the quadricep muscle. It was not a very strong trend. So, we're still working on doing a few things to see if we can find a better way to detect this GNE protein in these samples. And that's where we're currently at. I'm still working through a variety of more tissues from these mice, as well as looking to optimize being able to detect the GNE protein expression. And so, following this biodistribution study where we're looking across more tissue types and looking to optimize detecting our protein expression, we also are in the process of trying an even higher dose that we're looking for to see if that will improve our ability to detect protein expression. So, that is in progress. And then also, work at the NIH is currently being done to characterize and treat a mouse model of gnomyopathy. So, they're working to

characterize it now so we would be able to do a future efficacy experiment. Um, so kind of takeaways a what so far what I've been working on and where we hope to go is that we've optimized some AAV vectors in vitro and shown that the CK80 SB40 gene 1V5 construct results in high DNA expression in myoblasts. Some preliminary results of the dose and bad distribution study in mice show dose-dependent muscle-specific expression using both the construct we developed as well as the one that Dr. Sala materials and Bob developed in her lab. And sort of future and ongoing work will aim to look to just show that we're getting strong Genie protein expression and then as well as look to move into a disease Mouse model to determine the efficacy of any potential gene therapy treatments. Um, so with that, I'd like to thank everyone in my lab and especially the NDF for funding a lot of this work. It's been and giving me the opportunity to present today. Thank you and I'm happy to take any

questions. Thank you very much for that update on the progress relating to both Vector development as well as the biodistribution studies, which are very exciting. It's seeing them go into mice and start to understand which one of these are going to be effective in gene therapy is going to be very helpful. So, we have a couple of very a couple of questions coming in. Let me start with the first question: Are your studies with normal myotubes? Have you tested in GNE myotubes as well? Yes, so actually we have tested them both in both normal myotubes as well as the GNE myopathy or knockout myotubes as well. And both in our studies showed that using our constructs, we were able to get strong wild-type exogenous gene expression from our constructs in both the normal myotubes as well as myotubes that are missing the GNE gene. Right, I did. I maybe as a follow-up that I could throw in there too is that, you know, now looking at Wild

type mice the buyer distribution study. Do you think that's one of the reasons kind of obscuring a clear conclusion there? Because you have the native present there as opposed to what's taking place in the, uh, taking place maybe with a knockout mouse or something like that? Yeah, exactly. So, we do have the endogenous gene expression from the healthy mouse, um, and we're trying to detect any exogenous, so any gene expression that we're introducing over that, which could be making it difficult. Um, so we did add the protein tag to our exogenously expressed gene. So, we added a V5 protein tag. So, I'm currently, I'm working on some things to help you use that to be able to distinguish between the endogenous gene of the healthy mouse compared to the exogenous that we're expressing from our construct. Kind of make sense there? Um, additionally, I wonder if there's, uh, just to follow up on that as well, they had some variation there in the protein level of expression, whereas the PCR was pretty consistent where the

look in the RNA level is pretty consistent. Do you see variation in the level of, uh, it looked like you had it just even in control background too. So, do you think that's consistent that you see kind of variation in the level from one mouse to another mouse, even in the same anatomical muscle? Mm-hmm. Yeah, hard to tell, um, given that our loading control, some of our controls were also pretty variable, um, so I'm going to repeat a couple of times and see if we can get a clear answer to that question, but inconclusive for now. Sure, sure. I mean, but you know some, you do see that with some proteins, you do see, uh, you know, variation even in a well-defined genetic model. You can see variation, you know, time of the day and lots of different things that could contribute to that, uh, anyway. Um, another set of another set of questions coming here. This is actually a two-parter, so I'll start with that first. Say thank you for a very interesting talk, and once we're ready to use patients.

with AV gene therapy, in theory, be a single treatment therapy, or would multiple rounds of gene therapy be necessary? Yeah, so um, typically AAV gene therapies, at least as they're currently used, tend to be one dose um, but as a still emerging field um, you know, lots of things are still changing, and people are making more progress with the deliberate method um, so that's kind of outside the scope of what I was working on in the study, but definitely something to me hi. I can jump in. This is um Angela. Like currently, in terms of AAV gene therapy, it's a one-dose deal because once you dose um, your body develops antibodies to the virus, which prevents them from um, transducing muscle cells when you try to do repeat dosing. But having said that, there are some emerging technologies in the field now that are um, looking to address the redosing issue. The idea is that right now anyway um, what gene therapy drugs aim to do is to um, to hit all the muscle fibers all in the one go.

um, so that um, we get as much expression of the missing gene of the healthy gene um, in the one shot um, so that it doesn't need to be repeated. But in the event that it does need to be repeated because um, because maybe the transgene has um, dropped out or has been diluted in some way, then um, uh, we're gonna need to rely on newer technologies to to lower antibodies in individuals um, in order to to permit redosing. I hope that answers the question. Yeah, I think that that addressed that. Actually, there was a follow-up question that I'm sure they can follow up too if they have a follow-up question. But uh, the second part of that question was somewhat related to what you were just saying, but sort of the other side of that coin. Would there be any risk of immune reactivity that would be taken into account when designing a final therapeutic approach? Immune reactivity? Yeah, so currently in the fields um, there's definitely immune responses to to AAV. Some people handle it well.

some people don't, and that adds to the risk of delivering AV gene therapy. Currently, the field is trying to work together to figure out who is at risk because it seems like some people just don't take it well and are at risk of death. We still have a lot to figure out in terms of the safety of AAV gene therapy, but currently, it is the most efficient way of delivering a healthy gene into a human that we have to work with. Yeah, I think maybe they were also one aspect of that, not just the AAV itself, but the expression of GNE in the specific context. There, I think it would be less concerning about the immunogenicity of the GNA protein exogenously expressed since there's the endogenous protein. Most patients are still expressing the endogenous protein, usually with a small mutation in it. So, do you think that

would affect the likelihood of immune response to the actual expression of the transgene. Yeah, I think that, um, given that, um, most GNE patients have a mutant form of GNE, as opposed to not expressing anything, that's sort of desensitizes the immune system and sort of, uh, when we try, when we go to introduce the healthy copy, the body would have sort of seen the protein before, um, as opposed to not having seen the protein at all. I think there's usually very little difference in the structure of um, uh, one, uh, a mutant GNE protein with one missense mutation versus like a healthy copy of the GNE gene. Sorry to um, budget, but Catherine, if you have anything to add, um, sorry that I had jumped in there. No, it's great, great to hear your inputs, great, uh, and always, of course, feel free to put any follow-up questions in the Q&A box. Another question is the aim to recreate normal levels of GNE expression in muscle cells or to exceed that normal level. Well, obviously, normal would be

ideal, but um, we'll see what the mouse tells us. You know what, what levels maybe we need to outcompete it, the mutant, the mutant genie, in order for the wild type healthy copy to play the more dominant role. So, this is something we also need to figure out, but um, the good news is that um, studies by you know, previous groups such as Stella Rosenbaum have shown that over-expressing genie is actually not, does not have overtly toxic effects. So, we don't really have to worry about that because of, I think, the negative feedback inhibition of um, of the genie, the genie gene. Um, we were pretty safe in that we're not going to make, you know, too much cyalic acid if um, if we were to introduce a lot of the genie gene. There are sort of molecular feedback loops that prevent um, the pathway from um, from being on overdrive. Yeah, as most metabolic pathways have multiple levels of regulation. So, that is, I think, a reasonable expectation. Um, Catherine, anything you want to add?

there yeah another question. Is there any consideration as to how one might ensure an efficient balanced situation of aav to skeletal muscles in patients? Yeah, so that um. I've talked today about basically the transgene cassette that's packaged into the aav. So the actual distribution to skeletal muscle is controlled by The Stereotype of a be used. Um, so not in our lab, but there are body Labs out there that are working on improving and finding different and optimizing stereotypes of a b. So in this case, these aav9 that would address that question, but that's something that we're currently working on doing great. Another question. Uh robust discussion here, that's great. Please keep the questions coming. Do you have any data on the amount of Genie protein are required for a therapeutic purpose? Yeah, so that would be um kind of the next future work in hopefully a disease Mass model would be the point of that efficacy study would be aimed to determine exactly that. How much healthy Genie expression you would need.

to have a dose that was effective in storing it sure, and when where you you label them as low in high doses uh on your figure, you know, trying to keep it accessible to uh you know everybody's level. But I think really uh the what you're listing there is the high dose kind of Falls in a medium dose for what's used in Mouse models for neuromuscular diseases sometimes, right? So in theory, you can Envision going to a even higher dosing level potentially if necessary, isn't that the case? Yeah, so the ones that we did currently um for people were 2013 Vector genomes per kilogram and 5e13 Vector genes per kilogram, which are both relatively low doses in terms of how high you can go um. And so in progress, we've also um dosed some mice at 3 e 14 after genes for kilogram, so a very high dose um, and are waiting for it's nice to age, so hopefully that can we'll see the country effective. I need a higher dose as well.

right, right. But that'll certainly give you a better idea how much you'll certainly need for that therapeutic purpose for sure, or at least set it up to do it in another mouse model where there's a phenotype to correct. Excellent. Is there any possibility that different forms of g e have greater expression in different muscle types? I guess that's if you're talking about the different isoforms, which I see a couple of questions about. Yeah, so that's something that people have looked at. I believe Mario's group or Stella's group have done and looked at that and looked at the different Genie isoform expression and concluded on using gne1 in general for muscle therapies. But there are several different types of forms that other labs have looked into to see which is expressed across different tissues, right? And so you went with the g e one just based on that literature before, on the previous literature for that. Yeah, both previous literature, some

information on g-text, and then also our own study. I'm looking at healthy muscle samples, saw that we saw more gne1 expression compared to genie2 and some control samples that we had in the lab. Great, uh, what cell lines were the AVS tested on? Xvivo, yeah, so it was a mild blast line, so we tested both in healthy mild blast line as well as in a mild blast line that was deficient in GNE. So, oh, a human one, which is some of those cell lines that were from the g e bank, I would imagine. Yes, we also might do that in the future, yeah, but for now just a mild last line. Okay, with my last line. Oh, okay, so a modified another human myoblast line was modified. Yes, great, excellent. Uh, let's see another thank you for your talk. Great, um, and then we've already talked a little bit about gne2 and Genie one expression levels in muscle. Uh, is there any evidence that expression of the aberrant Gene is what causes damage to cells as opposed to lack of

expression of normal GNE, I guess, sort of is it is it the my interpretation that would be is there is there a uh is it the mutant form doing something different than what you would expect the uh the the wild type healthy version of that to do you know is it causing damage actively as opposed to just the absence of it um so as far as I I'm aware now and other labs have looked into studying that um and we see that in the patients it is the decrease of enzymatic activity that a lot of the mutations um show so we don't expect that they'll be any um negative effects of continuing to have the aberrant um or mutated form of Genie In addition to the normals you need but that is also something that when we look to do the efficacy study in a disease pass model we can look to answer that question yeah certainly I'd say there's it's still uh not totally clear exactly what the pathogenic mechanisms involved in G and Em are so you know you could you

could envision there certainly some effects that could be related to these specific mutations that cause genomyopathy, causing some other effect taking place. However, you know, I think we'll have a lot more knowledge about that based on some of the studies that are currently being funded by the NDF, developing tissue-specific knockout models in mice, and I think that will help to resolve that exact question. One of the reasons that the NDF is sponsoring those studies as well is to produce models for clinical studies, so definitely can I just add, yes, the prevailing model is the loss of function of the gene. The mutations cause the gene protein to function less efficiently rather than the mutation causing a gain of function behavior and conferring a function that it's not supposed to do, and that's the current theory. Because if mutant gene were to confer gain of function, then I think the patients, individuals who are carriers, meaning

that they have one healthy coffee and one mutant copy would um show a phenotype, but they don't um. The carriers of a single GNE mutation are typically healthy. Does that make sense? Yeah, well, yes, but you know there's certainly plenty of examples where there's hypomorphic mutations where expression level is much more dependent, and you get precise penetrance of it. Yeah, but I mean not to get too far off the reservation on molecular genetics in this sort of format, but no, I mean I think you're exactly right. I mean that's the that is certainly the prevailing hypothesis, but I think that at least in my opinion, we don't fully understand everything that Genie might do outside of the actual uh salary cap, the synthesis pathway, and I'm sure there are most proteins in the body do more than one thing, and I think we're still at early stages of understanding other potential functions of G and E. Another question, is there any oh I'm sorry, here we go down though, that's

Thanks for more of the information. That's great. Um, why is AAV the best option to use for gene therapy versus other possible viral carriers? It's not just for gene therapy um. It's currently known as the most efficient um. Gene delivery vehicle for the neuromuscular field. Actually, not even the neuromuscular field, for gene therapy um. For the entire gene therapy world um. There are certainly um. Other non-viral vectors that are being developed, such as lipid nanoparticles and just antibody-based technologies, but those are not very efficient um. When you compare them to AAV gene delivery side by side um. And moreover, they penetrate muscles less um. Less efficiently than AAV. And also, yeah, it's just I feel like um. In terms of AAV, it's a virus that nature has sort of um. Evolved over millions of years to be really effective at infecting cells. And we're just sort of hijacking that mechanism to make them deliver the gene that we want um. And it seems that we can't uh.

um it's hard to find a replacement of sort of like a mechanism that works just as well. um that that's that's man-made. So does that does that sort of make sense um yeah. There are other viruses are also being used like lentivirus but they each have their pros and cons. So for example plenty viruses tend to integrate into uh the genome meaning that that that's uh potentially risky side effect in that it could insert the entire Vector could insert into a gene that um May then cause cause um that Gene to be inactivated. A gene that's normally functional and human may be inactivated if an antivirus were to insert into that region in the genome. So that's that's a problem um in terms of AEV. It is definitely the immune response that's currently um The Hot Topic in the issue and something that the field is trying to deal with and and trying to figure out the best cocktail of immunosuppression drugs in order to allow individuals well in order to manage the immune response in in the

first, uh, a few weeks, uh, post-therapy. Definitely, yeah. I mean, that, that's the, there is a reason that the field, that multiple fields have coalesced around AAV as sort of the, the Vanguard delivery mechanism for what is going to be for these sort of gene therapy trials. While there's a lot more technologies that are in development, there's, uh, it's at this point, the general consensus is it's the best, uh, vehicle available. All right, uh, let me see another question here. Could induced pluripotent stem cells generally from skin cells of a potential recipient differentiated muscle cells be used as an X Vivo model to test the safety and therapy before introducing the virus into the patient proper? What would be possible, realistic, achieved in a reasonable time frame from this approach? Yes, it's actually a project, um, that the NDF has also funded that I've been working on extensively for the past few years, is to generate kind of a biobank of patients' Alliance that, um, were from skin biopsies. And so, we actually don't

need to differentiate them into Glory stem cells. We can actually go right from the fibroblasts, from the skin cells, into a more muscle-like cell. So yeah, I've been working on that for quite a while to get a bank of a bunch of these different cell lines that not just us in our lab for the purposes of AAV testing, but also many other labs that want to study genomopathy, to learn more about the disease or test therapies, can use as a model in culture rather than moving to a mass model. Great. And also, we've shared our skin cells that we've collected from the patients with other labs internationally, including Marian Hoising from NIH, who have converted them into iPSC cells. And you're right, of course, you can convert them into myogenic cells, but I believe the question was in light of testing, was that the immune response, was that right, Noah? I can't remember. Well, no, not so much. Mostly, I think it was in the

context of the immune response. Whether that would be another way to test, uh, test the impact. It's hard because, um, uh, the immune response is something that's systemic, and it, it having like a cell model is very different to, to having like a, a systemic immune response. Yeah, I think the cell models could potentially be used to test sort of efficacy if they could restore a phenotype, but it would be hard to have them test the safety in regards to an immune response at a particular dose. Yes, I would agree with that, but I mean that these are all model, you know, all these are model organisms that are used to test these sort of approaches. It's good to have multiple models, be able to be more, have more confidence in any sort of, uh, product that you're going to move forward into the clinics. So that's, uh, an important element of what's taken place in some, as, as Catherine's mentioning, some of those NDF-sponsored research has really helped to develop more of these models. That appears to be all the questions.

that are present in the box so that, uh, is addressed well. There's one other question. It's more of a, uh, housekeeping question, so I'll answer that now. But if there's any more, uh, questions on the presentation, please let me know. Uh, the question that's present now is, How do we access the first talk? Uh, that you can access the pre-vet part one of, uh, gene therapy update, uh, either on the NDF website. We'll have the directions for that, or also the, that's within the, uh, channel for the NDF in YouTube. So you can see it there directly as well. So I'd encourage you to go back if you have not seen that first part. Also very useful and tells you a lot about the different sorts of approaches, the multiple shots on goal, as it were, that the NDF is helping to work with to try to find an effective therapeutic for GNE-M. Uh, any additional questions? If not, let's thank our speaker today for an excellent presentation. Greatly appreciated. Thank you for taking the

time, and we appreciate that very much. Uh, also in the chat box, you'll see the link for the, uh, on the NDF website to be able to access that. Um, with that being said, I think we will wrap up for today, seeing no more questions. Uh, so, uh, again, we greatly appreciate Catherine, you're taking the time to give the talk today. That's an excellent talk and obviously a very robust discussion afterwards, so obviously that shows some of the interest here, and I'm sure there will be a great deal of interest as well for our upcoming speaker on December 9th, Dr. Paul Martin from Nationwide Children's Hospital, where he'll talk about the development of a potent CSA and disease models for GNA myopathy. So, talk about some additional ones of those disease models we just alluded to and the importance for these sort of therapeutic development programs. So, thank you all for today, uh, for being here today and for all the questions, and please enjoy the, uh, the rest of your day and see you next month for another

portion of the GE Symposium speaker series

2022 NDF GNEM Symposium with Katherine Koczwara: Developing An AAV Gene Therapy For GNEM: Part ll

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