University of Washington campus

[Date TBD] Seminar from Dr. Jack Parent

This seminar was originally scheduled for 2020; however, due to health and safety concerns around COVID-19, this talk has been rescheduled for 2021. Check back for updates!

Targeted Augmentation of Nuclear Gene Output (TANGO): A novel therapeutic approach to treat SCN1A-linked Dravet Syndrome

Background: Targeted augmentation of nuclear gene output (TANGO) is an antisense oligonucleotide (ASO) technology being developed by Stoke therapeutics for the treatment of severe genetic diseases. This ASO therapy targets naturally occurring, non-productive RNA splicing events to restore normal levels of the target protein. In collaboration with Stoke Therapeutics, Dr. Isom has tested this technology in a mouse model of Dravet syndrome.

You Will Hear: Dr. Isom provides an overview of TANGO and presents results from testing ASOs in a mouse model of Dravet syndrome.

Speaker Bio: This webinar was presented by Dr. Lori Isom, the Maurice H. Seevers Professor and Chair of the Department of Pharmacology, Professor of Molecular and Integrative Physiology, and Professor of Neurology at the University of Michigan Medical School.


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Audience Q&A with Dr. Isom

Is anything known about the mutation susceptibility of SCN1A?

Oh, you mean how susceptible the gene is to mutation? Very, very, very susceptible. It’s a huge hit. It’s a huge target for hits. We knew that back in the day when I was in Bill Catterall’s lab and we were purifying and cloning sodium channels and cloning the SCN1A cDNA was just nearly impossible. Everything you did made it mutate. When we saw this later on, we were not surprised at all.

Where are you measuring expression levels in whole brain tissue or are you looking at specific brain regions?

What we do is we take the whole brain and then take a slice that goes through the cortex and the hippocampus and use that to make the mRNA and protein.

Are you able to targets cell specific populations with ASO22? For example, inhibitory cells versus pyramidal neurons to see whether the therapeutic effect may increase?

This technique, as it stands, is general, so it does not discriminate between cell type. In fact, it will hit every kind of cell in the brain, including non neurons, okay? It seems to have this remarkable effect in spite of that. Now there are and encoded another biotech firm who was looking at this very similar technique but using viruses that target to PV positive interneurons. You may have seen their work at the AAS meeting last year. They have a very similar result with the SUDEP model that we have of stopping, of ceasing, of preventing the seizures and SUDEP.

There’s a question about quantification of the protein. Are you using Western blots to do that or are you doing something else?

Yeah, we’re using the Western blots. It’s more of a high throughput, so we’ve done it both ways. When you look in the paper, we’ve shown it by Western, which is more low throughput, but for all those millions of samples, we used a more high throughput protein, a high throughput modified method.

There’s a couple questions that came in about age of administration, especially since you’re talking about P2 versus P14. How did that translate to humans?

That’s a really good question. This is where mouse models have limitations. If you ask 10 developmental biologists, what a P2 mouse translates to in terms of a human, you will probably get 10 different answers whether or not that is a newborn, whether or not that’s embryonic or not, and whether or not P21, at weaning, is the equivalent of one year. I’ve read some papers where that’s more the equivalent of an older individual. It’s really difficult to predict what the mouse ages are going to translate to with humans. That’s why we wanted to push this up to the time of disease onset to see how much we could prevent. One of the limitations of this model is that there’s such a small window between the time of disease onset and the majority of the SUDEP, so we start losing animals right around P22 to P23. There’s very little window for us to do the injection and then have the full analysis before the animals start dying. I think that’s where we’ve pushed this particular mouse model to the limit. That’s why we’ve now doing the ultimate experiment, which is the clinical trial.

Have you looked at cognitive deficits in your mouse model?

We haven’t, and that’s a really good question. I get this question all the time. That’s going to be a very, very large study. At present, we’re looking at more detail at the electrophysiological details. We’re going in and looking at sodium current and firing of the inhibitory neurons and whether or not we can reverse the disinhibition that we see. Once we get that nailed down, then perhaps we could go on and do some of the behavioral deficits as well.

You did mention that no other sodium channel genes are affected by ASO22 treatment, but did you look at the entire transcript on?

We didn’t, and that’s a good question. No, but that’s something that we ought to do. Yes.

Do you think that you might need multiple injections to maintain the effect or is the single injection going to be enough?

That’s a really good question that I get asked all the time. Based on our observation that we had a single injection at postnatal day two, and we ended our experiment 90 days later, and we still did not, we saw a single seizure and a SUDEP. The question is there may be two possibilities. Well, maybe three. Okay, so two possibilities that I can think of is that in a mouse, at least, the ASO gets the brain past a critical period of development with the increase in Nav1.1, and then after that, the brain takes over and plasticity happens and that it’s normal development ensues, or this is a really long lasting ASO. We know that, that this chemistry really protects the ASO from degradation, so when you look all the way out at P90, you can see the ASO is still there. There’s still some there. It may be that you need very little over a long time, and it’s, it’s very effective, okay, or maybe mice are not small humans, and there’s a different process there, but at least in our hands, all you need is one.

Now, you notice in the clinical trial, they have two phases, right. They’re doing a single dose, and then they’re doing multiple doses to see what effect that is. If you think about the original ASOs news [inaudible 00:55:04], right, for spinal muscular atrophy, you have to do multiple doses. I think they inject quarterly, I think it is, in intrathecally in order to keep that therapeutic benefits, so that may be true.

Have the preclinical tests been performed in other mouse models? If yes, which ones?

They haven’t. I think it would be an interesting experiment to do the test in one, in a model that actually makes a protein, okay? The models, most of the models that are out there now are haplo-insufficient. This was a null, but you could also do it in a, Dr. Yamakawa has a very interesting mouse model that has a knock-in of stop code on, okay, which is a humans variant. That would be interesting to do. Now, that’s also haplo-insufficient, so we’d expect to have the same result as we have here. It would be interesting to prove the, what I proposed that if you use a variant that actually makes a protein, that it also increases that protein and can have some deleterious effects. We should do that, but the only mouse model that we’ve used so far is the null.

What proportion of patients with Dravet would you say are good candidates for this therapy?

I think at least 60%. More than half of Dravet patients have variants that cause haplo-insufficiency. And so the nice thing about this ASO is it’s a very antagnostic, right? Because so all of those variants cause nonsense-mediated decay of that other allele and this ASL upregulates the wild type allele, which is wonderful. I think at least 60% of the, and maybe more of the, maybe as close as 80% of the Dravet patients may be helped by this therapeutic.

What is known about the location and type of SCN1A mutation related to the efficacy of TANGO in increasing protein production?

Totally agnostic. It doesn’t matter. If the mutation or the variant causes a premature stop or a deletion and targets that mRNA for nonsense-mediated decay, then this ASO will work. It doesn’t matter where. On that map that I showed you that you could have a mutation at the end terminus or the C terminus and still have Dravet due to haplo-insufficiency, the ASL will work with all of those, as long as it causes haplo-insufficiency. That’s the beauty of this treatment that it doesn’t matter where the variant is.

Biological Mechanisms of SUDEP

Background:  Sudden unexpected death in epilepsy (SUDEP) is the most important epilepsy-related cause of death, occurring in at least 1:1000 people with epilepsy each year. The risk of SUDEP increases dramatically in uncontrolled epilepsy. The events leading to SUDEP are thought to be caused by a destabilization of autonomic cardiorespiratory compensatory processes. Dr. Simeone’s research has focused on determining progressive changes in cardiorespiratory function that could increase the probability of SUDEP in preclinical animal models. Identification of temporal biomarkers that can be monitored could lead to opportunities for intervention to postpone or prevent SUDEP.

You Will Learn: Dr. Simeone discusses the progressive cardiorespiratory dysfunction seen in the Kv1.1 knockout mouse model of SUDEP and the potential role of orexin as a central regulator of SUDEP.

Speaker Bio: This webinar was presented by Dr. Kristina Simeone, Associate Professor and Director of Master’s in Neuroscience Program at Creighton University’s School of Medicine.


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Audience Q&A with Dr. Simeone

Did you notice in your knockout mice, were the animals having more frequent seizures during the sleep period as they neared SUDEP, or was that not related at all, the sleeping seizures?

So, that is a fantastic question. That is a study that we haven’t done yet. So, the study that looked at the hypnograms, that looked at sleep architecture, we analyzed the data. And then after the study was done, we sacrificed the animals for histological studies. So, we didn’t let them live until they died naturally. That study was actually conducted before we started doing that with our endpoints.

So, we know that the animals that had very disrupted sleep that they were a higher risk. They’re I think probably SD70, at the age of SD70, where 70% of the knockout colony had passed away or died of sudden death. So, I think that would be a great study. Are there more seizures that are coming out of sleep? Is the sleep architecture disrupted more as within subject as they approach sudden death? We haven’t done that. That is a fantastic question.

How do you actually calculate disruption in sleep when how much sleep does a wild type mouse get and then they’re going through these bouts of sleeping, waking, the much minimum amount of disruption that’s required to increase to SUDEP risk?

So that’s a really great question. Because, yeah, these animals, they’re rodents, so they don’t sleep for eight hours at a time, right? So, how do you measure sleep deficiency? So, that was a hard question. We analyzed it several different ways. What we ended up with is using actigraphy. So, actigraphy is a non-invasive way of looking at rest and active states. So, these animals were video monitored. They were put into an actigraphy cage, which is an infrared beam that measures activity and rest. They were in that for their entire life. So, we took all of the wild type data.

We found that at the ages we were looking at, there were no changes. It was really, really robust with how many rest EPICs they had during their rest period, during the 12-hour rest period in their light-dark cycle. So, it was constant throughout every single age. So, we took that wild type value of what is standard rest. We compared it to the epileptic animals. We found that as they got closer to death that they had, they had more…

Sorry, I know my arm was not a great line graph. If this was wild type rest, every single day, there was very little variation. If this was the younger knockouts, they look just like the wild type and they had the same amount of rest. And then as they got closer to SUDEP, the rest started to go like this. So, the rest efficiency was this difference between the average all of the wild types together and then each knockout.

If you started treating chronically with DORA starting at, say, P30, and then did you happen to see it enhance survival in very large manner, say up to P20 or P50?

So that’s the study that we still need to do is give a DORA… There’s lots of different doors out there but give a DORA early on at a much younger age and then see if it can prevent everything from happening and if it can postpone SUDEP…

[I will] expand on that just a little bit is that for research purposes, if you give it before the onset of epilepsy, that’s not clinically relevant, right? So, you want to wait until some of the problems have started before you give a drug, so that it’s more clinically relevant. But I do think both of them need to be done, both studies need to be done, where you wait for the onset of some of the pathophysiologies to occur and then you give it as a treatment, but then you can actually start a treatment maybe earlier more as a proactive measure.

Now, it’s important to know that the DORAs improved sleep. So, you don’t want to give it during the period of the day that you’re supposed to be awake, because it’ll just knock you out and put you to sleep. So, this is really restricted to things that we can only do during the sleep. You can give it at lower doses to help protect against some of these pathologies, and it won’t induce sleep, but those are again nuances that we still need to figure out.

Are there any side effects to doing this chronic treatment with DORA?

So, DORAs are really safe. So, in 2009, when we first started working on these studies, we were using Almorexant, which was just about to be approved by the FDA. It has since been pulled from the market. Suvorexant and Lemborexant are now FDA use drugs. So, now we’re using drugs similar to those. So, the benzodiazepine and benzodiazepine-like drugs, when you give them as hypnotics, as a sleep aid drug, they do improve sleep, but they also can cause daytime drowsiness the next day. It can cause other kinds of side effects as well, like cognitive problems the next day and that kind of thing. So, the DORAs are really safe. They’re very well tolerated. You don’t end up with that next day drowsiness during the day.

So, in terms of their safety profile, clinically, these are very safe drugs so far. They’re still relatively new. So, let’s wait 5 to 10 years and see what happens. There is something interesting that we just recently found out. We have a study that’s actually under review right now, I didn’t have time to share this data. But we looked at sleep architecture and EEG of the epileptic animals that had been treated with many different anti-seizure drugs, traditional anti-seizure drugs.

So, in that study, we were able to separate out effects of a drug on sleep versus effects of a drug on a seizure. Because I know a lot of basic scientists and clinicians want to know, “Do the seizures arise more out of REM or non-REM?” We’re finding that for at least in our animal model, that the seizures arise both out of non-REM and out of REM just at baseline, and then the drugs change that up a little bit. Hopefully, that paper will be coming out soon.

What’s New at the ETSP?

Background:  The NINDS Epilepsy Therapy Screening Program (ETSP) is a preclinical screening program that provides researchers the opportunity to screen potential therapeutic agents in established rodent seizure models. Since its establishment, the ETSP has played a role in the development of several FDA-approved epilepsy drugs.

You Will Learn:  This webinar broadly discusses the scope of the ETSP and new models and assays available at the University of Utah.

Speaker Bio:  This webinar was presented by Dr. Cameron Metcalf, a Research Assistant Professor in Pharmacology and Toxicology at the University of Utah. He is also a Co-Investigator and the Associate Director of the Anticonvulsant Drug Development Program.  Dr. Metcalf’s primary research interests include the evaluation and advancement of novel therapies for the treatment of epilepsy and pain.


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Audience Q&A with Dr. Metcalf

In the intrahippocampal model, what areas do you record the EEG from?

So I would refer you to our colleagues at SynapCell for greater detail; they published their pharmacology and their methodology recently. But these recordings are deaf electrodes in the hippocampus. And since I don’t have any direct involvement with the actual performance of that model, I can’t say more than that. But I can refer you to their publications.

In the testing scheme which you showed at the beginning of your presentation, is it a step-wise process to go from the identification to the differentiation step? Or, how do you determine when a compound can move into the differentiation?

Sure. Thank you for the question. So one thing that I’ll say is that the decisions to advance compounds from one part of the testing scheme to another … we’re not always privy to those, and so I would defer to my colleagues at NINDS for specific examples.

What I can say generally is that the scheme is designed for it to be step-wise, meaning that as a compound advances through the identification phase, it would be a candidate to move toward the differentiation phase. What you might also envision is that while there are many compounds that could initially be tested in identification, not all those compounds have been advanced.

And so, there are some benchmarks and some go/ no-go decisions if you will, that help the ETSP in advancing compounds. There’s a lot of different factors that can come into play, and not all participants enter the program in equivalent ways. Some have to enter and exit for various reasons. Others may be limited in what their desires are to obtain information from the program. So I think it really varies, but in a very broad sense, it is intended to be step-wise.

Which amygdala nucleus is injected with kainic acid?

The model was designed around injection of the basolateral amygdala with kainic acid. And I should mention, this is also a good opportunity to highlight that we do have an upcoming manuscript that’s being prepared that will describe some of our methodologies.

Do you have any preliminary results in the intra-amygdala kainic acid model in mice, in terms of the seizure frequency and progression of the spontaneous seizures?

Yeah, great question, and again I would refer you to a paper that we will have forthcoming. We’ve also presented these at AES in the past. What I can say very broadly is that we do see variable seizure frequency similar to what I described for the rat kainate model. In a very general sense, we see on average about one seizure per day, but that really can vary quite dramatically. We can have animals that have a very high seizure burden, and we can have other animals that don’t have as many seizures, or seizures at all. And so we’re trying to find the best way to optimize our use of this model, and that’s going to be something that we’re going to continue to look at going forward.

When you do the tolerability toxicity assays, are you only working with models to look at motor activity, or are there other models that you look at for other types of toxicity?

Yeah, great question. This is something that we’ve thought a lot about recently. Historically, the models that we used to look at tolerability were really geared toward motor activity, and so that would include the rotarod assay in mice. We used a modified open field observational assay in rat. And we also used an open field automated locomotor open field assay.

However, in recent years, we’ve also brought on board a modified Irwin test. And while this isn’t as comprehensive as many you may be familiar with, it continues to be largely geared toward motor activity, but it also does allow us opportunity to look at other potential means of toxicity, such as autonomic, audio-visual and others. And so, this is helping us, but for those of you that work with rodent behavior, there’s only so much we can do, I think, to look at tolerability and be predictive of what happens in the clinical setting. But we have recently expanded our abilities to look at tolerability, particularly in rats.

What mice strain do you use for the acute seizure, the 6 Hz instead of epilepticus, because different strains can have drastic changes with regards to the severity of this?

Wonderful question, thank you, and this is also something we think a lot about. It does vary by model, so for 6 Hz and MES we use the CF-1 mouse strain from Charles River. For the corneal kindling assay, we have used the CF-1 animal in the past, however recently we’ve gone to the Charles River C57 black 6 model of mouse. For the TMEV assay, and for intra-amygdala kainate, we use C57s from Jackson. And so it does vary, but most directly to answer your question, we use the CF-1 mouse for our acute seizure assays.

But I do take the point that there are notable differences, not only in seizures but also in seizure pharmacology.

Status Epilepticus: 2019 Update

This talk will:

  • Define Status Epilepticus, its clinical stages, and treatment
  • Discuss the Ictal-Interictal Continuum
  • Apply new monitoring techniques

Stay tuned – additional details are coming soon.

Astrocyte Dysfunction Contributes to the Development of TBI-Induced Epilepsy

For years, scientists have focused on neuronal damage as the cause of neurological disorders, such as epilepsy. A growing body of evidence shows that impaired astrocyte function happens before seizures manifest.

Traumatic brain injury (TBI) is a primary cause of epilepsy, which is characterized by scar formation that seals off injured areas from healthy brain tissue. These scars are formed by astrocytes. Scars inhibit normal function and have been associated with seizure genesis.

Researchers modeled TBI in mice, using a weight drop injury paradigm, in order to determine whether astrocyte scar formation is involved in TBI-induced epilepsy. While animals developed epilepsy, no scars formed, but astrocytes behaved atypically, with decreased protein expression and decoupling with brain vessels. There were also more atypical astrocytes in epileptic animals. These data support the hypothesis that altered astrocyte function contributes to the development of TBI-induced epilepsy.