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.