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Intro: Welcome to SCN2A Insights bringing you the latest research and clinical updates on SCN2A and genetic epilepsy from around the world.

David Cunnington: So welcome to this episode of SCN2A Insights. I’m David Cunnington.

Kris Pierce: And I’m Kris Pierce.

David Cunnington: And in this episode, we are talking about modeling. So why modeling? Well, modeling is one of the key ways of better understanding changes in gene function and also what happens if you then try and manipulate gene function to predict what might happen with treatments. And so, very important ways of developing treatments. And a lot of the community have been asking us about different models, what’s a mouse model, what are stem cell models, and we hope to be able to answer that during this episode.

Kris Pierce: In this episode, we interview Dr. Snezana Maljevic. She is a senior research fellow at the Florey Institute of Neuroscience and Mental Health, working on ion channels and human diseases. She has published many papers on SCN2A and other genetic epilepsies and we’ve had the pleasure of working with her through GETA and she has also presented at our conferences in the past. We really appreciate her involvement and contribution to the landscape of SCN2A and the development of potential treatments.

Snezana, thanks for joining us today.

Dr. Snezana Maljevic: Thank you for having me.

Kris Pierce: We are going to talk about what you do in the lab and why it’s so important for families of rare conditions. Why do you need models to research rare genetic conditions?

Dr. Snezana Maljevic: I guess not only rare genetic conditions but actually genetic conditions because one of the things that we want to understand is how do the changes we’ve just seen in genes translate into what is happening in the body and why is the disease developing? So to study disease mechanisms, we always try to actually understand what is that specific changes in a certain gene producing within a neuron or a cell that we are studying because of these neurogenetic disorders or within the brain and how does that translate to the behavioral changes, ED changes.

So can we actually, by introducing or making a model with the mutation that is identified in patient with a certain condition, can we have more insights into disease mechanisms? Will that model reproduce the things that we’ve seen in patients? And also, will we have some kind of biomarkers or disease markers that we can then screen the therapies against?

So we start with different models. There are different ways to model genetic disorders and the simplest way is to just take some cell lines because the majority of the ones that we are working on, majority of epilepsy genetics are actually ion channel disorders so you can actually record currents and we use really simple models like  Xenopus, so frog eggs where you can then search your gene of interest and the mutated genes are the ones that had a change found in patient and just compare how do they behave differently in eggs, what kind of currents do they give, are the currents larger, smaller? Are there any changes in biophysical properties that you can actually then use to explain what happens in a patient? But that’s super simple because of course frog egg is not a human brain. Still, it gives a lot of information at least where to start.

Also, one of the questions that geneticist needs to answer because once you have a mutation identified, it doesn’t exactly always mean if it’s a de novo, that that is the cause of the disease. You kind of need a bit of functional confirmation. So you need some data to cite, well yes, that specific change in a gene does change the way that a protein is acting and that can lead to disease.

So it’s kind of like that functional part is really important and I think it has become a bit easier to deal with numerous mutations that they identified now when we do some predictions, identity programs and software that you can use and proteins that you can use in the meantime to look at how conserved the protein. Was that Immunocidin an important region? But unless or until you kind of measure and see something in some cellular model, you kind of don’t have a real answer. So that’s how we start.

And then the next thing to do is of course to go to a bit more complex model so you can use some of your neuronal cultures or neuronal cells. We sometimes get them like a primary mouse brain cultures and then you put your gene of interest or your mutation and you then look at the behavior. So it’s one of the approaches. It’s probably not as informative as creating a mouse model on its own but you can of course turn and go slowly towards the more complex systems.

And then most models have been around to study disease for many years. They seem to – on the first ones, the first mouse models for genetic epilepsies appeared when the first mutations were identified. And the nice thing that was kind of becoming obvious is that in many cases, they will reproduce some of the phenotypes. So like just carrying the mutation, will the mice well have seizures and will have changes in behavior that you can study?

And then you have many chances to dissect the disease mechanisms so you can look at how the mouse behaves overall, do they have seizures, do they – are the most susceptible for developing seizures, can you see any changes in the way the networks and neurons connect in the brain, can you see changes in single neuron behavior and then movements like more complex behaviors, how you can analyze all that, and then use them to screen drugs.

Dr. David Cunnington: So how do you actually make a mouse model?

Dr. Snezana Maljevic: There are different ways to make mouse models. Some were there a long time. So basically, you are using genetic approaches to modulate the early sort of mouse genes. And different models that we use or that we can profit from can actually be, for instance, only the ones that are already expressing a certain gene. So basically, they have their old genes and you give them a bit more of other genes and look what happens. Sometimes people make it seem that you give them green fluorescent proteins so that they can have like green urines or green cells and they can study those.

The more classical method is kind of based and use of embryonic stem cells and that it will be then injected and modified and then put back into kind of foster mom and then developed into mice. That method is actually pretty long so that’s why they were not that many models for a long time available.

With CRISPR-Cas which I presumed many people know what it is. CRISPR-Cas9 was then introduced to the kind of do that modification in a quicker way without the need to screen for many offsprings of mice that are generated in different – in the other way, in a previous way. So that has accelerated the whole process. And so we are now able to get mice within a few month, earlier than could have been more done a year.

So basically, you just want to change some of the starting cells in development of mice and then let them develop in the organisms then grow them in foster moms or do the embryo transfer and let the mice be born and then screened if they have the mutation. That’s how it starts.

Dr. David Cunnington: And that’s for one particular gene mutation. As we know, a lot of genetic epilepsies, there are variances within a particular gene mutation. Do you need a mouse colony or a line for every one of those variances or can you use a model that uses the large mutation?

Dr. Snezana Maljevic: Some years ago, we were happy to just start with knockout of that gene to see what kind just generate amongst a peace, you don’t target specific part of the gene you just remove or somehow break it down and then look at whether the consequences of removing that specific gene. That did give some information. It was useful and we kind of thought that it is helpful in those cases when you cited a mutation is or variance is causing the loss of function because that should sort of correspond.

We have learned in the meantime that it’s not always the case. Most models have that ability or like as a system, they compensate in different ways so they will not always develop exactly in the same way as the disease will develop in humans. And we realized that in most cases, the genetic disorders are one, at least mutated, the other one is so preserved. And this is enough to result in disease.

In a mouse model, you often need more than one mutated allele to kind of – we often study what we call homozygous, which means both are alleles carry mutants, and that gives the green peck.

Why do we need to study every single mutation in a mouse model? It’s probably not feasible. But there are some ways to select for the ones that we are studying. And that is like in cases of SCN2A, if you know that there are those that are early on certain light on the study there are some specifics about phenotypes. You can go for those.

You can also look which region, which part of the gene is affected. So – or which part of the protein in the end. And is that some functional important part and would that help us understand maybe the behavior of the channel on its own? Like will we learn more if we find a mutation in some region that is important for localization let’s say in the neuron. Like if you would make that and that is broken in a patient, how can you then modulate that later to kind of produce a contrary effect and maybe find a therapy?

So I think like it’s kind of we are trying to understand or separate different mechanisms, just not also to make mutation for hundred something variance and mouse models. I mean yeah, with many labs and of course funds. But there is sometimes a rationale behind how you select and sometimes there is not. You just – you start studying and you’re kind of, “Well, let’s see what the mouse model would do,” and you just go for it.

Kris Pierce: We have a lot of genetic groups on Facebook and across social media and we see lots of families or family groups raising funds to try and develop these mouse models. What’s your thought on that and how can that move the science forward?

Dr. Snezana Maljevic: You can’t make a mouse model for every single variance. If there are enough groups that are involved – if you all have enough funds and enough people working, of course, the more models we make, the better we will understand the disorder. That’s the hope.

The thing is that we don’t always work in the same way. We don’t always do the studies exactly the same way. But I think this is kind of improving and people are sharing and there are bigger collaborations and it is just sometime it is like a good approach to have more modules for a disorder and we understand that and it is, of course, the interest of parents and the interest of groups to know like what is actually happening. It is more that it’s not feasible from our time. So our group cannot handle more than we have, and we have already quite a few.

Dr. Snezana Maljevic: And it is not a cheap thing to do.

Kris Pierce: Yeah. What does it cost to …?

Dr. Snezana Maljevic: The cost of generating a variable so you can find always – we are actually outsourcing for now but it’s really making mouse models anymore in-house, we are not, because there are companies that can do that for you within a few months. They are just like focusing on that. So, you can practically order and buy a mouse.

The analysis and the cost already are high because you’re kind of keeping the mouse colony for a long time. You are paying the cost per cage per week. You are then doing all the studies and the analysis. So it is expensive. Just breeding costs per week that we are paying for our lab are huge.

Dr. David Cunnington: Yeah. It is in the tens of thousands rather than the thousands?

Dr. Snezana Maljevic: Yes, tens of thousands would be, yes. Yeah.

Dr. David Cunnington: And mouse models are one model. So Will, our son, is participating in a study where he had some skin biopsy and some stem cells harvested. What sort of model does that allow you to develop?

Dr. Snezana Maljevic: With stem cell technology is kind of were started being introduced in modeling diseases. That became a new approach and probably preferred at the moment because it’s the kind of most straightforward in the sense that you are – by getting a skin biopsy, you are actually having a direct patient background and a starting mutation in that setting. It is of course also not cheap because the whole stem cell workup and just establishing the laboratory and having reagents and having skilled people that also requires some effort.

But the nice and the advantage of that is like first, as I said, you are having exact patient background. What we then do is fix the mutations. So we kind of turn it back to what it should be. And then you have that perfect control so you have only removed the cause of disease and then can look in what is the behavior of cells that you derived from stem cells.

So the whole process is getting skin biopsy, we get fibroblast out of that. We are having coloration with the stem cell core that will then turn those cells into – or reprogram them into pluripotent stem cells. It means they now forget that they were a skin cell and can become any type of cell again.

And those are then stored. It’s kind of an easy way to bio then, do a bit of quality control and see there’s nothing else happening during the process. And then you can keep them and you can grow different types of tissue though to there if you want but we, of course, want to study what happens in epilepsy so we are differentiating those cells into neuronal cultures. And those can be again either in what we call 2D, 2-dimensional or 3-dimensional cultures. And the 3-dimensional ones are more – are well-known as organoids, brainoids or mini brains.

There are two I think major advantages and the first is that we are working with a human biology background, which is of course not what we have in mouse and we know that some genes behave differently. And that also is important for the development of novel molecular gene therapies because sequence like gene sequence is different between mouse and human so you can lead a model that also carries actually human gene so that you can see what is the effect of a therapy that you are applying and that is specific only for human sequence.

And the second advantage is of course the patient background. So we have all the other modifiers and things that could have influenced how the mutation expresses. And then of course, you have the cells available to reproduce and repeat your experiments.

They have of course disadvantages. And that is, the methodology is still developing so there is a heritability even within the same laboratory if two people are doing the same work, you’re still getting a large variation. But it is improving. We have analyzed a few variants so far. It’s getting more stable. We are learning as we are doing.

The advantage of making 3-dimensional, so mini brain cultures, is that the protocols that are currently in use, they are actually enabling to look a bit into early developmental process. So we are not only looking at ready-made brain cells, but we also look at how actually does cells develop, so what happens in the early stages of development.

You can then – you can follow neuronal precursors, look if they are reaching the position you then expected to reach within the brain. So they kind of reproduce a bit of early brain development in a very primal way of a very rudimentary way. But they are getting some process you can analyze. Plus, you can then look at the activity and see whether there are some pathological changes or some changes between groups of neurons, their localizations. It kind of gives a new tweak to the story. So it gives it bit a more chance to look into early development.

Dr. David Cunnington: Does it help with safety if you were trying to develop a protein for example to modify gene function? I would imagine without an organoid, you might need to actually test it in a live human rather than being able to test it in an organoid and get a sense of does it work? Is it safe?

Dr. Snezana Maljevic: Yes. I think it is going in that direction. I think that stem cells at the moment – you can’t exclude mouse models as a pre-clinical development because they still provide that insight into how the whole system will be affected. But there are more and more studies including actually stem cell developed neurons as pre-clinical models because of that, yeah, because we learned especially in epilepsy that using just mouse models or rat models or rodent models to develop drugs was not the most productive way. So many drugs failed later when translated into humans.

So we obviously need a different type of model. So whether it’s the stem cells or at that level without really perfect predictors of the behavior, well, we don’t know. We are looking into that. So – but I mean, they have a good chance because we do see some phenotypes that we kind of expect from what we see in other models. So if we expect more firing, it should be in epilepsy. We do see that in stem cell models or less – you can already see that that is happening. There are not that many studies on epilepsy in organoids at the moment but I know that there are many happening. So it is more of just to understand what is the readout, which is the good readout that we can actually use to see what is the phenotype.

Dr. David Cunnington: So we’ve got the frog eggs.

Dr. Snezana Maljevic: Frog eggs.

Dr. David Cunnington: Mouse models, organoids, mathematical modeling and prediction. What’s next? What other models are coming in the next five years or so?

Dr. Snezana Maljevic: I’m pretty convinced that we are going to probably have some kind of a merging of technology and stem cell modeling so that – because one of the problems we have with organoids is that they get necrotic tissues. So they are inside, because you just like grow them without blood vessels so they can only feed the cells that are in the surface. So there are people trying to grow them together with some kind of micro blood vessels.

But one of the approaches that you can use is to make some kind of scaffolds. So like use materials to grow the cells and then sort of enable that they develop in a certain way so that you can analyze the connections between neurons, their positioning. So I’m kind of thinking that might be one of the ways we are going to go into the analysis because once you have a scaffold, you can actually also let it detect and sense what is happening or stimulate. So it’s kind of probably one of the ways to look into that.

Of course, you would like to – I mean, with the whole genetic and we know that there are so many variance and different genes. We still don’t know what each of these variants does. And in many cases, it is actually necessary to do that really fast. So I’m kind of also thinking maybe some kind of a platform where you could maybe use blood cell and just develop quickly some stem cell or neuron-like cell and do a quick screen.

It would probably be one of the nice places we always kind of imagined. That would be the part of, you could do that as part of diagnostics and say, “Okay, we predict this.” And then like they do in cancer. You then put on some drugs and predict what would be the behavior. That would be probably the next approach.

And yeah, I think cellular models that we are currently using are – they could still be tweaked like combined with computer modeling or maybe just have the fast way of producing variations and then studying their effects like faster readouts maybe, not always one student but quite machine, two months of work. But like having a bit of an automated workflow, a more automated workflow would probably be helpful.

Kris Pierce: Has having contact with families and patients with genetic epilepsy changed how you approach your work?

Dr. Snezana Maljevic: I’ve been in field for 20 years. But it’s only since we started working with really severe epilepsies that we kind of get that human component and touch because earlier was epilepsy for me, I don’t have cases. And family, I’ve seen a few seizures and heard about them but it’s not like I wanted to study. It was – it’s brain and how it works and what goes wrong that pushed me in that direction.

But then when we started getting – like finding out that there are severe epilepsies with people, I got really involved and personal and you get calls from parents and you meet really sick kids. You kind of start to think, “Oh my God! This is really – this is real life.”

Kris Pierce: Yeah.

Dr. Snezana Maljevic: This is happening. And I think this has changed the perspective of how we do science a lot. So like the understanding in our laboratory with many people, they all these are real people we are working with. They have seen the pictures of patients they work on. So kind of whose mutations they analyzing. So it has all become very real. I’m really doing good interactions with parents and families and also because we can learn so much, there are little details that you sort of miss when you’re just like reading or hearing from a clinician. So that kind of interaction I think does move and help and move things forward.

Kris Pierce: So Dave, what were the pertinent points that you got from Snezana’s chat with us?

Dr. David Cunnington: So I really found it interesting how Snezana was able to outline just the roles of all the different models that they are able to use. So simple models like frog eggs or a simple cell line, whole animal model like a mouse model then a min organ model in the human-like the organoids. And then using mathematical modeling. So it’s not actually any of the dish model but using mathematics and how in the future that might actually be able to combine those and mathematical predictions and organoids to really predict what may be able to happen.

It really also drives home the importance of modeling because even if you can actually model what’s going to happen with a treatment or what’s going to happen to that abnormal gene function before you take something to human trials. You are going to be more confident about what you’re taking to hum trials both in terms of its effectiveness but also in terms of its safety.

Kris Pierce: Today, we talked about all those different models, how does one model lead to another?

Dr. David Cunnington: So there is an actual progression with modeling. So you might start with a simple model like a cell line for example. And some of the work at the Florey in genetic epilepsy and particularly, SCN2A, might have been around 5 years ago starting with that simple type of model. But then as you start to understand what happens at a cellular level and then you want to take that into a more complex model, so a whole animal model like a mouse or an organoid human model like a stem cell organoid, that’s a number of years. So that’s 5 years down the track from a cell line.

And so, it does take quite a long time as you work through these gradually progressively move complex models. And so, just starting with a simple cell line isn’t the end of the process. There’s a lot more modeling to do after that.

Kris Pierce: That’s great that the Florey is moving along that process and as are other labs around the world. Keep up up-to-date with the latest genetic epilepsy and developmental epileptic encephalopathies by following this podcast.

Dr. David Cunnington: Or get regular update on SCN2A through SCN2A Australia’s Facebook or Twitter @SCN2AAustralia. And thanks a lot.

Kris Pierce: Thank you for joining us.

Outro: This podcast is not intended as a substitute for your own independent health professional’s advice, diagnosis, or treatment. Always seek the advice of your physician or other qualified health provider within your country or place of residency with any questions you may have regarding a medical condition.