Rescue of SynGAP expression in SYNGAP1 Syndrome: Antisense Oligonucleotides (ASOs), small molecules, and viral genetic rescue

Here are our introductory comments: 

Sydney: Our talk today is with Dr. Richard Huganir and is titled "Rescue of SynGAP expression in SYNGAP1 Syndrome: Antisense Oligonucleotides, small molecules, and viral genetic rescue". He is a professor of neuroscience, psychological and brain science at Johns Hopkins University School of Medicine where he has been since 1988. In 2006 he also became the director of the Solomon H. Snyder department of neuroscience and we are proud to have him as the chair of the SRF Scientific Advisory Board. Dr Huganir has been interested in the science of learning and memory since his early childhood. His desire to understand the biochemical processes of learning have led to a career focused on investigating mechanisms that regulate synaptic transmission and synaptic plasticity, including the molecular mechanisms that regulate neurotransmitter receptor function. This has included research on glutamate receptors to better understand how the phosphorylation of these receptors modifies some of their functions related to learning and memory in cell models. In addition his lab studies the processes by which certain types of these receptors, AMPA receptors, are recruited to and regulated at synapses where they play an important role in the connections between synapses.

His work with the SYNGAP1 gene and its protein has evolved since 1998 when he first discovered that the SYNGAP1 gene's protein plays an important role in regulating synaptic plasticity. Since then his lab has also created mouse models which reflect human mutations in SYNGAP1 and have characterized the resulting phenotypes. Potential routes to therapeutic treatment of Syngap disorders is another focus of his lab and that is what he's here to share with us about today. Earlier this year he published a review titled "20 years of SynGAP research from synapses to cognition" where you can read more about the many advances he has contributed to in our understanding of SynGAP.

At the end of this presentation you'll have an opportunity to get your questions answered. We'd love to hear from you, so you can please write your question in the chat. For those of you just joining us, welcome to our talk today by Dr. Richard Huganir titled "Rescue of SynGAP expression in SYNGAP1 Syndrome: Antisense Oligonucleotides, small molecules, and viral genetic rescue".

Webinar Overview

Dr. Richard Huganir is a SynGAP researcher at the Johns Hopkins University School of Medicine. He begins the webinar with an overview of how to regulate protein receptors. He then explains how SynGAP is very important for human cognition; SynGAP mutations underlie 0.5-1% of non-syndromic intellectual disability and many of these mutations lead to haploinsufficiency. Additionally, all kinds of mutations, whether it is a subtle splice site mutation or a severe frameshift mutation, lead to haploinsufficiency. Therefore, regardless of the mutation, therapies need to increase expression of the SYNGAP1 gene from 50% to 100%. One way to regulate gene expression is using an ASO (antisense oligonucleotide) which binds to regions within the gene and recruits a protein to carry out a specific function. Dr. Huganir has found an antisense transcript (non-coding RNA) of SYNGAP1 which can be targeted with an ASO to recover SYNGAP1 expression. In addition to this discovery, Dr. Huganir closes the webinar with the work he is doing to rescue SynGAP plasticity in SynGAP splice site variants.

Other Relevant Publications by Dr. Huganir

Twenty Years of SynGAP Research: From Synapses to Cognition

SynGAP isoforms differentially regulate synaptic plasticity and dendritic development

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Here is the transcript:

Dr Huganir: Great, thank you Sydney. It's a real pleasure and honor to be here speaking to parents again and it's really been a pleasure to work with Mike and Ashley and SRF in, you know, helping promote the research and also to help develop therapies and engaging biotech companies and and many many other researchers to push this field forward. So I thought what I'd do today is give a little introduction to SynGAP and many of you know about it but I thought I'd give an introduction and how we got into it over 20 years ago now and then also talk about some of our approaches using mouse models to generate models of SynGAP haploinsufficiency, and then talk about some of our approaches we're taking to try to develop therapeutics, so using antisense oligo treatments and then but at the end I'd be happy to talk about other approaches, small molecule approaches or AAV type rescue approaches and I'll briefly talk about, at the end, looking at different splice forms of SynGAP. Because as many of you know, or will discover today, there are many different forms of SynGAP and they all don't work in the same way. And so to deliver a, to rescue, SynGAP expression using an AAV or viral type rescue approaches we really can only rescue one isotype at a time and so we really need to know which ones are the key isoforms to rescue. So, can you see my cursor, Sydney?

Okay, so I'll just start out with the brain, of course, the brain is a 100 billion neurons in humans and it's an obviously extremely complex organ and here's a video I'd like to show, it's an animation of signaling between neurons in the brain. So these are all neurons and you can see the action potentials and the electrical signals going between these various neurons. So there are 100 billion neurons and each of these neurons connect to about 10,000 or so other neurons at specialized areas called synapses. And here we're zooming in on the synapse right here and this is where the neurons communicate with each other through a process called synaptic transmission.
So here is one neuron touching another neuron. So if we zoom on this, into this schematic here, here is one neuron, the end of the neuron, it's called the axon terminal and this is the second neuron and this is where the synaptic transmission is going to occur. So it's a blow up of this contact here of the synapse. And what happens is an electrical signal comes down, releases neurotransmitters which then go across this gap between the two neurons and activates receptors. And this process of signaling is how the brain works. It is forming a network of neurons communicating with each other through circuits and these circuits are of course what underlies all our behaviors, all our intellect, our emotions and all brain function.

So as Sydney mentioned, we've been studying synapses and particularly receptors for many years. How these receptors work (shown here) and how a modification of receptor function may be involved in dynamic regulation of synaptic strength and thereby circuits in the brain and we think this is really critical for what we call "synaptic plasticity" and processes such as learning and memory. So we've been studying this by looking at phosphorylation of receptors I've mentioned these are, I won't go into detail but these modify receptor's function and then also we've been studying... this is a reversible process...and then also we've been studying how these receptors work.

So here is a schematic of a neurotransmitter receptor, one of the most major receptors in the brain, AMPA receptors, and we study how this receptor is regulated both by adding these phosphate groups but also by how they are trafficked to synapses and how they interact with proteins that regulate this trafficking of receptors to the synapse. So over the last 25 years or so we've been studying the regulation of receptors and we characterize a variety of proteins that interact with the receptors and regulate their targeting to synapses and this is where we discovered the SynGAP in 1998. So we were identifying proteins that bound together to form this complex and we were able to identify this protein called SynGAP. At the same time Mary Kennedy also was studying synaptic proteins and she identified SynGAP by studying high abundance proteins in synapses and it turns out SynGAP is one of the highest abundance proteins at a synapse. So she was able to identify it as well.

So SynGAP is a really key for regulation of synaptic function as I'll show you and the one of the key molecular mechanisms we study is a process called synaptoplasticity and this is occurs during learning and memory and other higher brain processes and it's a process where receptors are recruited to the synapse and added to the synapse and this actually increases the synaptic strength. This increases the synaptic communication between neurons and this is really key during the learning process to form a circuit that would encode, for example, a memory. This process can also go the opposite direction so if you look at something called LTD or synaptic depression, receptors can be recruited away from the synapse and weaken synapses. So this process of weakening and strengthening synapses is really key for all brain function and in particular we've studied its role in learning and memory and other other forms of learning.

So coming back to this again: SynGAP was a key to this and so we've started to study this in learning and the way we did this back in the day and also continue to today is generate mouse models where we delete SynGAP and then ask the question: does deletion of SynGAP regulate learning, behavior, or development? And so we did this in the early 2000s and just to show you how beautiful SynGAP can be here's a picture of SynGAP in a neuron. So this is a neuron that's been filled with a red protein shown here and all these little green dots are actually SynGAP which are located at synapses. So each one of these little dots here is a synapse so you can see it's located at the tip of these sort of projections which are called synaptic spines and it's decorated by SynGAP.

So SynGAP is, the structure of SynGAP is shown here. It's a protein that has several different domains and I'll talk about how there are many different forms of SynGAP but we and Mary immediately recognized that it had a what's called a GAP domain, identifying it as a GTPase-activating protein which I'll talk about briefly in a minute. So this is my newest favorite picture of SynGAP. It's a beautiful picture taken by Yoichi Araki in my laboratory just again showing the beautiful synaptic localization of SynGAP along the dendrites. Each one of these is an individual synapse.

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So as I mentioned SynGAP is a RAS GAP and I'm not going to go into detail but these RAS GAPs have been studied for for decades and they're involved in regulation of signaling molecules and again i'm not going to go into the details. So we knew something about its function, that it probably would be regulating RAS signaling in neurons and to study this further as I mentioned we did a deletion of SynGAP gene in mice.

So interestingly when we knock out the gene for SynGAP, in the homozygote, so if you knock out both copies of the gene, the mice actually die within a few days after birth, you know, about three or four days after birth, but the heterozygotes are fine they live a normal life they breed relatively well but it turned out they had significant cognitive and behavioral deficits as well as deficits in plasticity. So this is a classic form of plasticity I mentioned earlier LTP, long-term potentiation, and what we're looking at here on the left is just the strength of synaptic transmission.

So we just start out with a baseline. So we're measuring this electrophysiologically in a slice of brain from mice and if you record synaptic responses, it's very stable until you give this sort of induction protocol which induces LTP. This long-term potentiation that's thought to be involved in learning and memory. And this is a wild-type littermate. So this is a sibling of a heterozygote knockout. So this has two copies of Syngap. You can see you get a nice strengthening of synapses about threefold. In contrast, this is the littermate which is a heterozygote. So this is a brother or sister of these these mice and you can see this potentiation is strongly inhibited.
So these mice have very strong deficit in plasticity but they also have a variety of deficits and behaviors and so just one example I'd like to show is a working memory and this is a standard assay we use to look at mice behavior. When you put a mouse in a Y maze like this it'll run back and forth for a few minutes sort of exploring the neighborhood and it will seek out and seek out novel areas. So when it comes to this choice point it will always go to the arm not the arm it came from but it will go to a novel arm and this is so it should alternate if it remembers where it's been it's going to alternate and go down on the alternate arm. And this can be seen here. Here's a wild type animal. If the mouse was just making random choices at the center of the Y maze it would be a 50% but if he was seeking out novel, forging for novel spaces he would remember where he's been and he'll take the alternative arm each time it reaches that choice point. So this is a standard response. About 70 to 80 percent of the time mice will take the alternative arm and in contrast the heterozygote basically just randomly chooses one arm or the other. Interestingly, if we observe this they actually do these repetitive behaviors. They'll go back and forth, back and forth, between two arms.

So I'm not going to go into all the details but over 10 years of work, 15 years of work or so we came up with a model for how SynGAP is working during plasticity during LTP and so here SynGAP is tightly associated with the synapse as shown here. It's bound to this complex that's involved in interacting with receptors as well during LTP the induction of plasticity actually what happens and i'm not showing any of this data but what happens is actually SynGAP will leave the synapse and this will allow activation of downstream signaling which is really required for plasticity and what we see is that the synapse not only gets more receptors as I described earlier but also becomes larger so this normal plasticity in heterozygote does not occur and so what is shown here is that in normal neurons we have a relatively small size of synapses these little spine like modules but in the SynGAP mutations the synapses are already activated it already has a lot of receptors and the synapse is already big just to illustrate this further this is a figure from the recent review that during LTP induction you would have this structure shown here with receptors and synapses and the synaptic scaffold during LTP you get more receptors and you get the size of the synapse increases. In contrast when when the SynGAP is only fifty percent there with the haploinsufficiency the synapse is already big because the signaling is already activated because SynGAP is gone and it does not - no longer responds - to any stimulation as shown here.

So SynGAP also is involved in a variety of other properties of the synapse and I'm not going to get into this in too much detail today. It's involved in signaling but we also think it's involved in the structure of the synapse. As I mentioned, it's a very abundant protein in synapses and so you can see here that the the scaffold of the synapse is schematized here and in a paper published in 2016 in collaboration with Mingjie Zhang's laboratory we found that SynGAP is really important we think for the structure this actual physical structure of the synapse.

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Now this paper like many of us changed the way we think about SynGAP and really change the direction of the SynGAP team in my laboratory as you know this also changed Gavin Rumbaugh's research program as well to really... because it showed that SynGAP not only was important for learning and behavior in mice but was really critical for human cognition as well. So of course this is the a paper by Jacques Michaud's laboratory which discovered the first three
mutations in SynGAP that led to intellectual disability and these are the first three girls having severe mutations in SynGAP that likely lead to haploinsufficiency, destabilization of the message in one copy of the gene. So these individuals are likely just like our heterozygote, they have one good copy and then they have a mutated copy which is probably not producing protein.

So recently we've of course this then expanded tremendously and there are hundreds of children as you know and probably thousands, probably ranging into tens of thousands if not hundreds of thousands. So this has become one of the most common mutations in underlying non-syndromic intellectual disability, ranging from 0.5 to 1% and it's really a, you know, critical underlying cause of intellectual disability. Now we it's been a real honor to get associated with the families and with both SynGAP Research Fund but also Bridge the Gap family foundation. It's really been an honor to meet all of the parents and you know many if some of you have come to our laboratory and come to Kennedy Krieger Institute at Johns Hopkins. It's really been motivating for my students and and postdocs to understand SynGAP and also develop therapies for SynGAP.

So here are some of the mutations that this is a figure from our review just showing that there are many many different mutations in SynGAP across the exons shown here, exons and introns. And so many of these we think are haploinsufficient, lead to haloinsufficiency we could talk more about this later most of these are nonsense mutations but some are missense mutations, some are frame shifts, truncations, splice site variants, and so it's not clear if all of these lead to haploinsufficiency but I think that and most of them will lead to a haploinsufficiency. So we've started to make some mouse models based on these mutations and this is all unpublished work so here we honor this young girl to make her mutation and this is a really severe mutation and we predicted this would lead to to haploinsufficiency and so we generated a mouse with exactly the same mutation and bred this mouse out and again this is, we're looking at the heterozygote, and here what we're doing is we're looking at ex- in this mouse what is the level of SynGAP expression. And so here you can see this is a what we call a western blot where we're probing four different splice variants of SynGAP and you can see that indeed, just like the heterozygote knockout, this is the old knockout we made in 2000 or so, it's 50 percent of SynGAP expression and in this new mouse the mouse model of this mutation it's basically exactly the same that she has. We would predict that she had 50 percent of synthetic expression in her brain.

So looking at the behavior of this mutant versus the conventional knockout, so this is the conventional knockout I showed you earlier, the working memory is deficit. Here's the wild type littermates. Here's the mutation, the knockout. This is the new patient mutation. Very similar it's, you know, it's littermate it is fine but this mutation leads to a poor working memory. Now these mice are also hyperactive and so you can measure this in many ways but here you see an increase in activity in mutation in the patient mutation but also in the normal heterozygote knockout and we also as I mentioned there's repetitive behaviors and here we can see this repetitive behavior in the Y maze in the patient mutation but also in the heterozygote knockout. So we're characterizing this mouse further and and these mice will be useful to study, you know, mechanism of SynGAP function but also for testing therapeutics to try to as we'll talk about and shortly test methods to raise the expression of SynGAP from the 50% back up to 100 percent.

So recently we've also generated mouse model SynGAP splice site mutation and so this splice site is shown here we thought this was an interesting variant because it's actually located in an intron and would promote a cryptic splice site but it's interesting because it's really near the end of the gene it's near the end of the gene and we thought maybe because it was so close to the end of the gene that this RNA would be stable it would produce some protein and possibly this mutation would not lead to a decrease in in SynGAP expression. So we generated this mutation using CRISPR techniques in a mouse model and somewhat surprising we found that the RNA was was unstable and we see about 50% of the RNA but importantly also we saw fifty percent of SynGAP expression at the protein level maybe a little bit of more of the beta isoform which i'll talk about later but really essentially a 50% of SynGAP expression. So this argues that both the severe truncating mutation and also a more subtle splice site mutation is leading to haploinsufficiency.

So we are going to make maybe one or two more mouse models for example make a missense mutation which should produce protein in theory and we're going to test that in vivo in the mouse model, make the mutation and see if protein expression is normal. In many instead of our preliminary studies we think that a lot of these missense mutations actually destabilize the protein and so they may be haploinsufficient as well.

So coming to therapeutics, so if most of these mutations are haploinsufficiencies they have 50% of the protein, the most straightforward and the safest way to rescue and try to develop a therapy is to increase the expression of the SynGAP up to 100% and so there as many of you know there are um there's been recent success using uh anti-sense oligonucleotides or ASOs to to do exactly this to modify the expression of genes in many different diseases so I'm not going to go through this in too much detail but I want to point out there are many different ways you can use an ASO.

ASO is a small oligonucleotide shown here and it's used to bind to regions within the gene so for example in in this schematic shown here an ASO would be a synthesized that would bind to the the RNA of the gene, Syngap for example, and this ASO would be designed so it would recruit a protein called rnase h and this would chew up the RNA so it would destroy the RNA and lead to degradation of the protein. So this is not what we want to do because this would actually decrease the SynGAP expression further but we have a little twist on this which I'll talk about in a minute. We're using this approach in sort of a novel way. There are other ways to regulate expression using ASOs. You can decrease the expression by binding to the start site and inhibiting expression but one of note is as shown here (this is called splicing modulation) and in this case what you do is you use ASOs to either increase or decrease with the efficient splicing alternative splicing and this will either increase or decrease the production of the protein and this technique is what's been working so well in Spinal Muscular Atrophy and has shown success in several other disorders as well.
In the Syngap case this is the approach that Stoke Therapeutics at least is taking in a pre-clinical level to try to use these ASOs to increase SynGAP expression.
So the twist we took is that we were interested at looking at the Syngap gene. Here's the Syngap gene shown on chromosome six. You can see all the exons shown here and what was unique about this is that it turned out there was an antisense strand. So there's actually a gene that is read in the opposite direction. The RNase starts here and it's read in this direction but it overlaps with Syngap. It has a very similar sequence it's called an antisense transcript and this gene is not encoding a protein but it's actually just encoding a non-coding RNA, a small RNA. Now why this is interesting is that this antisenses usually regulate the expression of this homologous gene so this RNA could in theory regulate the expression of SynGAP. So we looked at this in more detail and interestingly this antisense transcript is only found in a human brain and not in mouse and rat and this is a problem of course for our studies if we want to study in mouse and rat systems but we are, you know, starting to look at this in the human cells currently.

Now this is looking zooming in on this structure so here is the normal the end of the Syngap gene. We have three exons shown here this makes - these combine to make the SynGAP protein but we found that there are two antisense forms that antisense non-coding RNA and remember they're going the opposite direction and they overlap with exon 17 and exon 16. So this antisense will bind to the sense strand and as I mentioned in many cases these antisense when they bind, they lead to degradation of the RNA.

So to test this we actually just, since we couldn't do this in mice, we tested it in human cells. These are not neurons but we - called human embryonic kidney cells and what we could do is sort of reconstitute a system to see the impact of this antisense strand on SynGAP. So we we you can put in Syngap gene shown here and you can see the SynGAP is highly expressed (this is the protein) and if we titrate in this antisense strand shown here you see that the SynGAP expression goes down.
Using two different antibodies shown here. So this confirms what we we hypothesized: that this anti-sense strand actually inhibits SynGAP function, SynGAP expression. So the twist we we decided to do is to take anti-sense oligos and not target them to SynGAP because that would decrease SynGAP, but target them to the anti-sense strand. So if the anti-sense oligos, the ASOs would down regulate SynGAP the anti-sense strands bringing this level of the antisense down, we could increase SynGAP expression as shown here.

So we designed a variety of antisense oligos against the antisense strand. I know this is sort of a double negative that's hard to say, let alone understand but using these antisense oligos we try to target this antisense strand to decrease its expression and that would therefore increase the expressions of normal SynGAP.
So we screened a variety of these and using first shRNA techniques but then making synthetic ASOs the small antisense oligos and you can see a few of these will target the SynGAP antisense strand. So this is the non-coding Syngap antisense here you can see the expression is very high. If you add in oligo number five it decreases the expression of SynGAP antisense "syngap2" and here again if we use a the small oligonucleotide based on the sequence of number five you can see it again it decreases the expression of "syngap2".

Now the the proof in the pudding though is will this regulate SynGAP protein level? And so here what we've done again is reconstitute everything. We put in a Syngap gene, Syngap itself and we add also a Syngap antisense and then and so we get a very low level of expression because the antisense strand is knocking down SynGAP but when we start adding our ASOs you can see that we rescue the expression of the SynGAP protein. So this is saying that knocking down the SynGAP antisense construct is regulating the sense strand the SynGAP expression from the sense RNA.

So we're continuing that work and trying to look at that in human iPS cells and studying which ones will work best in human system and and that that work is ongoing and I can hopefully give you an update next year. So I want to turn to isoforms of SynGAP and so here is the gene structure of Syngap. This is sort of the most common form that's been studied over the years called the alpha-1 form. There's different splicing here and I'm not going to go into it in detail but some of these exons are for example, not expressed in brain.

This is exon 11 and this you may know this is so-called "poison exon". Stoke is also using ASOs against this region to try to increase expression of SynGAP and we can talk about that at the end if you want. But there are also other isoforms as I'll show you in a second. Some other exons are only found in brains so such as this alpha-1 form and some are suppressed expression in brain tissue.

So this is where it gets complicated (more complicated) and this is the sort of core structure of SynGAP. This is the core domain this is found in all of the SynGAP isoforms but there are two areas of variation. One is in the n-terminus region and this is because there are alternative start sites so the gene starts at three different places but importantly down here there's a lot of alternative splicing on the c terminal region. There are at least four isoforms: alpha-1, alpha-2, beta, and gamma and they have distinct structures. They have distinct structures that they see terminus that lead to distinct functions and I can go into this if people are interested but recently we and others have started characterizing this. Gavin and I have been working in parallel complementary methods to look at the function of these different isoforms and we wanted to know which one of these isoforms is really important for a SynGAP function in the brain and specifically in SynGAP function in that synaptic plasticity that I talked about earlier. So this is a recent paper (I'm not going to go into it in detail) we had characterizing the different isoforms. Where are these isoforms expressed and when do they get expressed? Where do they get targeted? And what we found is that the alpha-1 isoform is the most critical we think for synaptic plasticity and is the most synaptically targeted isoform.

So SYNGAP1 alpha-1 goes to synapses very efficiently and is really key for plasticity. And if you just focus down here what I'll just go through this simply is that remember I told you that during LTP induction during plasticity the synapse gets larger. So here you can see this is just in a wild type neuron in culture that's such as a wild type mouse. If you induce LTP you see the the increase in volume. Now if you knock down SynGAP, so if you decrease the level of SynGAP using shRNA techniques, you can see this volume goes up. As I mentioned earlier the size of the spine is already large and in fact when you try to induce plasticity nothing happens. Now what we're going to do is we're going to do what's called a molecular rescue and so we can actually transfect into these neurons a normal SynGAP the normal alpha-1 SynGAP, and you can see you rescue sort of the behavior of the wild-type mouse. So here you're getting expression of the alpha-1. This spine size gets smaller again and then it responds to LTP induction. So with alpha one when we rescue the knockdown of SynGAP without the one we get a nice response, however when we rescue with the other isoforms alpha 2, beta and gamma we don't rescue as well this plasticity. So you see the volume stays, the spine sizes stays up, and again you don't get much of a response during plasticity. So this is, we think, an important result because it certainly says that a key form of SynGAP, potentially for AAV rescue, is the alpha one isoform and I think this agrees with recent unpublished data from from Gavin's lab as well that the alpha-1 isoform is really key for plasticity and behavior in mice.

So I'm going to stop there. I know it's been a mouthful and hopefully I can clarify things if you have questions but I just want to acknowledge the people involved. So this all started back with Jee-Hae Kim who was a graduate student in the laboratory in 1998. She cloned, as part of her thesis, SynGAP. She now works at Regeneron.
Hey Kyoung Lee did the physiology on the knockout back in the early 2000s. She's actually now a faculty in my department, Johns Hopkins she was a postdoctoral fellow. Kogo Takamiya made the knockouts, knocked out in the mouse back in the 2000's he's now a professor in Japan. Gavin Rumbaugh, as many of you know, was a postdoc in my laboratory and worked on the SynGAP as well as other things in the laboratory and of course now he's become a major contributor to the field and I'm very proud of him and he's working in the Scripps institute and he and I and Jimmy Holder constantly are interacting and collaborating and sharing information and reagents etcetera. Sarah Ju actually was an undergraduate student she's now at Oxford university. And this is the SynGAP team shown here in yellow. We have several different people doing different aspects of SynGAP and this is the rest of the lab who are doing other interesting work at least i think so. And again i want to call out Minji Zhang's laboratory who we worked on this together for collaboration for the the structural work and then finally funding NINDS NINH and NIA and I also want to thank SynGAP Research Fund for funding our work as well it's really you know, research takes a lot of time a lot of effort these students and postdocs work night and day and it you know it's not free. It takes generating these mice and even maintaining the mice is an incredibly expensive endeavor and so I just wanna now I'm ready to to sort of have a discussion about potential therapies and be happy to start a discussion with talking about ASOs versus small molecules and viral rescue but I think we can probably take down... stop sharing and we can just begin a discussion.

Mike: So Rick thanks for that. It was a tour de force and I'm sure parents will watch that a lot and email both of us lots of questions. In the Q&A Dr Hans Schlecht who is a Syngap parent and leads a team of Syngap parents who are MDs and PhDs has been hitting you with four different questions and generally with these webinars we have a few we have a good number of parents join and then we have a lot of parents watch the videos later there's also a lot of scientists on here who I think are going to come in swinging with great questions and they're welcome and we we love seeing scientists interested in Syngap. But I just want to hit you with the hardest question first: you've got some very promising data on your ASO for syngap2 and at the end of the day parents are blown away with the science and glad for the decades of work you've done and they just want to know when that's going to be in the clinic. And as I think about it the question behind that question is like
how fast could that be in the clinic are you partnering these I'm gonna they're it's already coming to my inbox how fast it's gonna be the clinic? Are you already working with the drug company? What's the game plan? I just want to get that one out of the way before we let people get now...

Dr Huganir: So I think we'll talk about the different approaches. I think people are very excited the ASOs because they're pretty, you know, they've been successful in other diseases. They're, you know, the path is relatively clear and straightforward but the key is to find the right antisense oligos that's going to regulate expression and so you know I think there are many biotech companies looking at this. Certainly Stoke is the most you know publicly involved in developing antisense oligos and so I think that they are in the lead I would guess. They, you know, they've taken an approach to increase the expression of of SynGAP by increasing the efficiency of splicing of Syngap in general and also this recent, they had a recent paper looking at this poison exon, exon 11, what we called exon 11. Targeting that to to try to increase SynGAP expression. So we were working on that as well and there are several other labs across the country working both on that that exon and other antisense oligo approaches so I think, you know, the ASOs are probably going to be the first approach you know I'm not Stoke may be the first one to you know get to the point for clinical trials. So you have to, you know, identify the antisense oligo that works in in vitro, you know, in tissue culture or human iPS cells then, you know, in some cases you can show that that rescues in a mouse model for example but then you've got to do the human studies and you know obviously I'm not going to be doing that I have to partner with with pharma or or biotech and so I'm working with a couple biotechs to think about this various approaches and you know I think they're, as as you know, there are lots of biotechs that are thinking about Syngap. Syngap is a very attractive, you know, the syndrome is very attractive to I think pharma because it's a really, you know, one of the most abundant causes of intellectual disability and so if we could rescue the expression of SynGAP it I think would be one of the first cases where we're rescuing a sort of a cognitive disorder you know with Spinal Muscular Atrophy it's really a neurodegeneration you know it's a neuromuscular disease it doesn't affect so much cognition but Syngap would be a great example where you can impact cognitive function and behavior. So I think ASOs are probably gonna be the fastest approach and as you know that takes three to five years once you get a good antisense oligo and you know it's not clear to me where any of these biotech companies are, you know, how close Stoke is to generating those ASOs that they think are clinically important but also whether you know they've taken it on as a major... their next major project. Obviously they're very involved in Dravet Syndrome right now. Okay so we don't know if the Syngap the anti-sense to Syngap2 is gonna work yet and but that's something we're continuing to do.

Mike: Yeah I got it. There's a been there's a few questions piling in Hans has some scientific ones. James asked about small molecule versus ASOs. I want to just ask you one more I want to just abuse my position to ask you one more question that has come up a few times among different patient groups. Some people have come to us and said why aren't we talking more about AAVs and I am over my skis using any of these terms but basically what I've said to them parroting what I've heard from another rare disease group is as a haploinsufficiency we have a bad a good copy and a bad copy. We're very fortunate to have a good copy so let's use it more and that's basically what ASOs are doing right, taking down the take down you're up regularly. Whereas with an AAV you're going in, it's one it's one shot on goal it's riskier, we're not there yet, and so when people ask me why aren't we pushing harder on that i say i later we're not there yet. Please correct or amend me Professor I mean how do I respond to that question better?

Dr Huganir: So the, you know, the good thing about ASOs is the it's pretty the path forward is relatively straightforward you know. As you know, the bad part is it's not a cure, right? It's pretty invasive. You inject, you know, into the spinal cord (the ASO) and it distributes it through the spinal cord into the brain and that has to be done on a relatively regular basis. It's also very expensive, of course. So AAVs are a different approach and I think that there's going to be a revolution in AAVs. There are whole biotech companies that are only working on AAVs, developing AAVs that will target the brain well and so I think there's going to be progress both in academics and in the biotech world. So one problem with AAVs currently you can only package a certain size of a gene in there that they have a limit on the size of the gene but I think that there's progress in developing better load, you know, loads for the for AAVs. We think we can probably package Syngap in the AAV because we've been developing some AAVs that can handle something as large as Syngap. But I think the key here is which isoforms? So you can't express the whole gene. The gene is humongous. You really have to express just one of the isoforms and so if you can only deliver one of the isoforms, which one? And will just one isoform rescue the, you know, behaviors? So our bet would be to try alpha-1 because we think that's the most important. I think Gavin is taking some approaches to see at the mouse level which isoforms are the most important for for behavior or rescuing the haploinsufficiency. So I think that's that's the key problem is to which one to package. And then also AAV, you know, this techniques for therapies is not quite there yet. There's been a couple approved as you know, SMN has been there is an AAV that's been approved and so it'll be really interesting to see how those clinical trials go.

Mike: Great, thank you. So you can read probably 10 times as fast as I can. Do you want to just tear through the Q&A and knock some of these out? What do you want?
Dr Huganir: yeah so Hans, hi Hans, so immune effects ASOs for example plasma based pam triggering I'm not sure I don't know enough about that. One thing about antisense oligos in this case will be injecting them into the into the brain right, so they would be either in the spinal cord into the ventricular system and so they might be protected against antibody responses but I'm not sure more about that.

Dr Hans Schlecht: Do you think meaningful insights into the kids can be made from studying C. elegans and get ortholog? Is C. elegans used as a model organism for any similar diseases? Thanks so much.

Dr Huganir: Yeah see it I mean C. elegans has been a, you know, an amazing system for years and, you know, many genes found in lots of diseases were initially studied and identified in C. elegans so definitely I think that C. elegans can be a very useful system. It's a lot of Cryo-EM work on GLUT and AMPA receptors lately is there work being done to look at the fine structure of SynGAP, including the missense variants in an effort to do structural function studies. We're not doing Cryo-EM on on SynGAP we are, Hopkins has just recruited a young Cryo-EM person so maybe we might be able to do that. You know, the missense variants, I really think that they may be unstable so it'd be hard to pick which one to study, which one really gets expressed in kids and is not degraded by, you know, protein instability.

Okay the HGNC but I need more information about that Hans, I don't know what you're referring to. Transportation system to deliver and have a good distribution of ASOs any progress? I think people are there's lots of progress lots of work going on in that area both using modifying different ASOs a variety of different ways. There's delivering them with AAVs for example, there's one possibility.

Okay go down to Neil...Is the assumption that synaptic plasticity could also reflect learning improvement plasticity? Yes. So that's what we would hope and this comes to the you know the burning issue is when do you treat? When you do you treat early? And if you treat late can you still rescue some phenotypes? And if you saw Gavin's talk you know he is clear that you can rescue some phenotypes in a real, in an adult mouse and so that gives hope that treatments can have impacts even later in life but of course we really want to treat as early as possible and so in theory you know we know Syngap's involved in learning memory but those mechanisms are also involved in development of the nervous system so, you know, there are windows of time when SynGAP is critical and the reversibility of that is not clear so so during wiring of the brain SynGAP is important. If you rescue after that will you be able to rescue the behaviors? It's not clear.

Dr Huganir: Is the research assumption that synaptic plasticity could reflect learning plasticity? Yes.

Dr Huganir: What are the most promising areas for small molecules? So this is what I'm most excited about... hey there Tony...  is, you know, there are now small molecules that affect splicing. So for SMN there's a small molecule that was isolated through high throughput screening and so you don't have to use an ASO anymore you could use a small molecule to do the same thing that ASO was doing to increase the expression of the protein and so I think doing high through throughput screens to try to regulate splicing of Syngap with small molecules is really a productive way to go. Yes, in the best of all worlds they would be orally available, you would take a pill and you would have to be on on those pills for life but, you know, hopefully with not serious off-target effects. I think there's great potential for small molecules. Now we didn't talk about this too much but you know there are many ways to regulate SynGAP expression and I didn't go into to all of them but, you know, with Gavin he went his talk he discussed some of these, you know, you can regulate expression, you can regulate degradation of the protein. There are many different ways and so we understood more about the sort of the metabolism the life path of SynGAP, you know, what regulates its expression, stability of the protein and the RNA, and also the degradation, we could target those pathways with small molecules as well. The other approach that we initially took, some of you may know this, is to try to inhibit or regulate downstream signaling from Syngap using small molecules and this could be effective but we found at least in our studies we were having trouble getting that to work in vivo and intact animals. I won't go into detail about that but but again those are not cures there you know it would just be therapeutic would take every day and many cases they might have side effects.

Yeah i think small molecules would be better than ASOs if we for example the small molecule that we're regularly splicing would be better as long as the side effects that is less intrusive.

Dr Huganir: All right so Olga says which are the more severe Syngap mutations? Are they identified based on type missense, nonsense versus strictly based on patient phenotype So if you just look at the Syngap mutations the the most severe are the ones that you would predict disrupt SynGAP expression or function and so the nonsense mutations in many cases lead to nonsense mediated decay of the RNA so basically the RNA is totally gone and you get 50% of SynGAP expression. So those are severe. Some of those are more severe than others they have frame shifts and then truncations. Missense mutations as you may know is it's just a change of a single amino acid so it just changes an amino acid from one to another in some cases that can be very mild that can be have no effect on SynGAP function and that's those are commonly found in many genes in the sense mutations that produce variations in protein sequence and have no impact but the ones that lead to disease that where there's you know very clear genetics that that missense mutation is causing intellectual disability we really frankly don't know why you know and I think Gavin talked about this as well it could be that protein is expressed but it's not working well or it's as I said it's the protein to express but it's very unstable and so it gets degraded very rapidly the question of phenotype genotype so looking at the genotype and and how that impacts the phenotype the behaviors of the severity of the behaviors with your children is really not clear yet and I think that's something that all the geneticists are trying to look at now is there a correlation between phenotype and genotype you know as many of you know there are many other genes although they're they're not causal for the Syngap Syndrome but they may impact the severity of the syndrome so so the background genetics of the individual may make them more or less susceptible to Syngap mutations okay i don't know if i can answer all of these mics if you want to

Mike: i'm sorry i'm sorry I realized i'm putting on a spot there there were only a few and then people have piled on so jess duggan's question though is interesting right I may have missed this but increasing expression of syngap rescue have any impact on neurons structure itself or is it SynGAP rescued?

Dr Huganir: Yeah, so we and other people have seen this, Gavin's seen this, that there is some effect on in the knockouts of structure, the dendritic structure in particular there's a delay in, sort of, the development of the dendrites and we find that's actually affected we think more by the beta isoform than the alpha-1 isoform and so this is one complication of, you know, the AAV. If we rescue with the alpha-1 and not with the beta will that, you know, not rescue the plasticity but it won't rescue dendritic development. So this is where it gets more complicated but definitely yeah there are effects of SynGAP on sort of the development of and the elaboration of dendrites as well as the size of synapses as i mentioned as well.

Mike: So Jess has another good question but i want to just follow up on that. So if you go with an AAV you have to pick an isoform and so you go with the alpha and you have the good news but you don't have the beta but if you go with an ASO and you take down you go take down syngap2, you take off a regulatory mechanism you're then not this is a question but i'm stating it as a fact you can correct me you're then not wading into isoforms right so the you you're leaving the existing mechanisms to determine which isoform to create is that is that then arguably...?

Dr Huganir: Right so there's two ways, you know, there's there's splice switching oligonucleotides and then there's antisense oligonucleotides and so one approach is... the safest approach is to regulate the expression of all isoforms, right. So to upregulate all isoforms and then the normal regulatory mechanisms will produce the normal ratio of all isoforms. Another... but you can develop antisense oligos or splice switching oligos that will for say example increases the levels of splicing of the alpha-1 isoform to produce more of that relative to the other isoforms and that may have a therapeutic effect, right, but
if the beta form for example is very critical that may not get you where you want to be. So I think these are things we have to study and these things these can be studied in mice and that's what you know Gavin is doing.

Mike: Right I want to ask one more. There was a question about do we know how much SynGAP you'd have to add back? So this I'm going to ask you about SynGAP level. I'm going to nest three questions here. One question was how much SynGAP you have to have back to to stop seizures? I can't wait to hear your answer to that one. The other question is and following on to that like some mosaic children don't have seizures and there's this idea that's starting to circulate where because we have some mosaic patients right and we were we had a family meeting the other night and someone's like well does that mean if we treat our kids with an ASO and we upregulate SynGAP but not all the way... are the mosaic kids in our community today what our kids could look like after that ASO and I thought that was an interesting quote. I want to take it a step further: what happens if we, and you haven't touched on this, what happens if we go over 100% are we closer to understanding that question? And that brings us to titration as one of the merits of ASOs because we can babystep right so they just threw four things at you.

Dr Huganir: So we've been trying to do that. Yoichi who you know, has been trying to titrate up and see where it gets. What level it gets rescued. I don't think we have to go too forward. I don't think we'd have to go to 100%, at least in our model systems, you know, I think it's more close to you know 150% or 50% you know, back up to 25% up to 75% of wild type. So I think, you know, we might get away with less. Now the mosaics are very different though, those are, you know, those are different cells, right. So there you have different, you know, population of cells that are missing SynGAP, I would presume, and then with the ASO treatment you're up regulating all the SynGAP, you know, in theory in all cells and so it's a very different, you know, end point. In one case you have a mixture of wild type and mutant cells and the other you're just you're increasing the, you know, the levels of SynGAP in all cells.

Mike: Great. So I think we actually have gotten through most of the questions. I just i'm going to read James Clements last because I think it speaks to rescue which of course all of us who have kids are obsessed with right? Time is brain. So you mentioned we don't know what the critical periods are and I think if memory serves, Gavin's written a paper saying "this is the developmental period" and then he wrote another much happier paper like "hey, we rescued something!" So James's question is: do we know whether ASOs leads to restoring rewiring of neuronal connections to wild type level particularly after critical period? My question is,
I'll let you answer that one then I'll give you a following.

Dr Huganir: Yeah, we don't know. I think there are several people doing this now in mouse models. I haven't seen any talks on it. So what you would do is, you know, do the ASO treatments and in mouse models like it's been done with SMN and other, you know, in the early pre-clinical studies and you could do it at different time points, for example. The key here is you need to have ASOs that will target the mouse sequences, not the human sequences and do it in that direction and so for example with the antisense construct which is not in mice we can't test our ASOs in mice. So James I don't know. Somebody may know the answer to that. I don't think anybody's that far yet. I know stoke is probably trying this probably as we speak, I would guess but that's, you know, a big question is you know, if we treat later and certain periods of development have already, you know, passed, whether it's, you know, three, four, five, six, seven or ten years old, you know, are we gonna be able to rescue the important critical developmental periods, developmental plasticity that occurs during those times? You know, Gavin's results suggest that several of the phenotypes including the seizures can be rescued later in life so I think there's hope.

Mike: Wonderful. Thank you. I don't see any other -
JJ. JJ started typing now, look out. If you go to the - and I'll let have the last word and then I'll thank you so much for this incredible presentation but if you go to the bottom, JJ has two questions: What are the ripple effects of the abnormal structure of the synapse?

Dr Huganir: So the synapse, and this changes during development, you know, Gavin has looked at this but having these large, very active synapses means that they're very excitable and so that might underlie the epilepsy and the seizures. We don't know that but certainly that would make sense that these large synapses make the neurons more excitable and that leads to seizures. But also they're sort of locked in and they're sort of reached their largest size they can get so there's no plasticity they can't, you know, they can't rewire during learning. So when you learn something, as I mentioned, this plasticity takes over, you make new connections like the synapses get larger and that, you know, encodes the memory, for example. If they're already large you can't modify them and so you can't... that sort of... you lose that ability to become a plastic synapse.
Mike: And what about artificial transcription factors to upregulate the gene?

Dr Huganir: So a lot of, there's a lot of interest in regulating SynGAP function expression studying transcription factors promoters, you know, we heard a great talk about epigenetics, studying epigenetic regulation of SynGAP expression. There's not a lot known about, you know, the transcription factors involved. We can guess which ones may be involved. The problem with artificial transcription factors is that transcription factors in general regulate many different genes so so putting transcription factors in there to regulate SynGAP will probably regulate many other other genes as well and that would produce off-target effects that could be detrimental.

Mike: Okay she's kept going: so if we can correct SynGAP protein to wild type can this abnormal architecture be
corrected? It will correct the abnormal architecture in the cell, right. But the question is whether the circuit that was formed during development, whether you can you can correct that. So that's the big question and, you know, most of who would say probably not and that's, you know, the major concern in treating later than rather than earlier.

Mike: Right. Amazing. Well, thank you.

Dr Huganir: There's a lot of work we still have to do so... yeah looking forward to... With COVID, you know, I can only have 50% of the people in the lab at a time but they're working night and day. We have two to three shifts and, you know, they're working through the through the night to get the work done, so.

Mike: Appreciate it. Thank you very much Rick. This has been an epic webinar and we appreciate an hour and a half your time. All right. It's been an honor pleasure I'll see you again somewhere soon I'm sure and maybe not in person but yeah, yeah on a screen for sure in person... God only knows. Not telling. All right, thank you Rick. Thank you very much.