9UNP1682HD_Charles_Konsitzke

- Welcome everyone, Wednesday Nite @ the Lab. I'm Tom Zinnen, I work here at UW-Madison Biotechnology Center, I also work for UW-Extension and Cooperative Extension. And behalf of those folks and our other core organizers, Wisconsin Public Television, Wisconsin Alumni Association, and the UW-Madison Science Alliance. Thanks again for coming to Wednesday Nite @ the Lab. We do this every Wednesday night 50 times a year. Tonight, it's my pleasure to introduce to you Charles Konsitzke, of the Biotech Center, and Dhanu Shanmuganayagam of Animal Science. Chuck was born in La Crosse, Wisconsin, and went to high school in Tomah. Then he went to UW-La Crosse, where he majored in Finance and Business Administration. He has been at UW since 2001, and he's been here at the Biotechnology Center since 2008, and he is currently the Associate Director of the Biotech Center. That means he is my boss. 
(Laughter)
Be nice, if you can.

I can. Dhanu was born in Sri Lanka, in the city of Jaffna.

And then went to high school in Singapore. He came to UW-Madison back in 1992 and studied Bacteriology, Genetics and Biochemistry for his undergraduate degrees. Then he got a PhD in Nutritional Biochemistry here at UW-Madison. He post-doc-ed at the Primate Center working on that project that shows caloric restriction lengthens lives. That's why I will live to 61. 
(audience laughs)

He is now an assistant professor in the Department of Animal Sciences. And he's gonna talk with us tonight about turning piglets into personalized avatars for sick kids. Please join me in welcoming both Chuck and Dhanu to Wednesday Nite @ the Lab. 
(applause)
- Good evening everybody. Thanks for coming out here. I appreciate it.

We were asked to sort of talk about how we got to creating pig avatars for personalized medicine. And so we're gonna tell a story about how we got here.

But before I do, I wanted to preface by saying that most of what we've done so far is a work of a lot of people. Some of my research teams' out here, but it's actually a collective effort by multiple research teams, and it took a lot of people to get this shuttle launch, as the metaphor you'll hear later.

Our journey really began with a baby boy who had a pigmented mark on his back side. And I'll let Chuck start that.
- Thank you.

Mason is my son. He's eight years old right now.

And as Tom stated, I'm the Associate Director here at the Biotechnology Center. However I've been facilitating and administering research for about 17 years. I've got attacked for helping varying projects come to fruition. And I had one challenge ahead of me.

So, Mason was born in 2010. We had some early concerns at five pounds weight. He had GI issues, which required a special diet. He had jaundice.

And then at six months old, we noticed a pigment on his rear end. And myself being who I am, I started to investigate this. And come to the horror, well I shouldn't say horror. It was just what scared me is I noticed something called a café au lait spot in Googling it. 
(chuckles)
 And the worst thing to do is Google anything. 
(audience chuckles)
 At his nine month appointment, we talked to the pediatrician and we stated, we believe our son has neurofibromatosis one. And the pediatrician said, what's that? 
(audience groans)
 So, right there, my alarm, you know being a parent, having an infant, your alarms go off. So I immediately changed my insurance company and I transferred over to the UW Children's Hospital, where at one year of age he was clinically diagnosed with NF1.

At about a year and a half, we started seeing—Well, from a year to a year and a half we saw regression. There was a reduction in the speech. We could tell that he was having difficulty hearing. His gait and balance was off, his behavior was changing, he was hitting his head.

So we took him in and they did a hearing test on him. And they believed that he was having a hearing issue. And they also saw that he had a pretty substantial ear infection. So they went in, and they did an MRI prior to putting a tub in. And what they saw was a benign mass in that region of his left ear. About the size of a ping pong ball. And it was spreading to the naval cavity as well because what a plexiform neurofibroma is, it's not like you're standard tumor. It's not like a benign tumor where it's a solid mass. This is a type of tumor, it's your nerve cells that meshes and it grows wherever there is space. So you can imagine what my wife and I were up against.

After many MRIs, cocktails of medications where we don't know the past of what medications, therapy, however the therapy couldn't commence until he was diagnosed with autism first. Then the therapies could commence. So NF1, as itself, wouldn't allow the doors to open to therapy. We had to get him diagnosed with autism, which is a byproduct of NF1, to open those doors to therapy.

It is a lifetime worry for us, because mortality can occur at any moment.

So, I don't step down, I grew up in a military family. I fight forward. And I told everybody I knew this disease picked the wrong person. 
(chuckles)
 
(audience chuckles)
 So I started networking with other individuals with NF. It's a very small community.

Started discussing issues with NF.

I started looking at avenues of fundraising for NF because I'm priming myself for what the next step is. A lot of the stuff was occurring at home, very long nights. I have my file right there.

So then I did the preamble processes because of my position, I needed to make sure that I'm not conflicted. So I spoke with leading faculty members on campus, I spoke with UW Legal, I explained the situation, I explained how I'm proceeding, and I explained how this isn't interfering my position.

I got the green light, so I started moving forward.

A company by the name of Prescouter, which focuses on custom intelligence on demand, what they do really is they look at all the academic research that's happening in the nation. And they look for companies that would want to acquire it.

So the companies actually hire Prescouter to seek out these kind of interesting types of research. Well, they gave me one of their teams of post docs for a year to investigate the bottle necks and weaknesses in NF1 research. This occurred in 2013 and 2014.

So I was receiving monthly reports. I was receiving in depth reports on researchers, internationally as well as nationally. I was looking at their capabilities, their strengths and weaknesses. And you can see the chart on the right there, that's from one report, just one month's report. 13 academic institutions, three others, one technology, looking at everything in depth. I started reaching out to national NF researchers and I was asking them what are the bottlenecks, what are the issues you are seeing ahead of you?

I was reading a plethora of NF publications, manuals.

And what it came down to, was the model size. NF is very unique, and we'll get into that a little bit later but, what was occurring with the model sizes with mouse and rat, which are exceptional models, don't get me wrong. They're definitely needed in research. But, when a mouse would have possibly a neurofibroma tumor, as my son does, it could grow to a mass where the mouse then dies and the research can't continue because of the death of the mouse.

Cardiovascular issues, you couldn't perform some of the research. This was a post doc out of Canada. She couldn't move forward with her research on a cardiovascular issue with NF. So what the outcome with that is she completely went off of NF altogether. She went down the cardiovascular route, which was devastating. And you see this a lot in NF research.

So swine model, I communicated with a senior faculty member outside of UW, and I'd asked him, I said, have you ever thought of a swine model? And he said, no, but that's a great idea. So right there I had my goal. Because we're in Wisconsin. I worked on a farm. 
(chuckles)
 So, one day I'm up at the UW Day at The Capitol, where we meet a bunch of legislators, senators, and we're kind of explaining what we do on the Capitol, kind of promoting what activities happen within the center or on campus, and after four or five hours, you're kind of exhausted, so I was standing next to the railing, and you can see my table's over there, but I was standing on the railing right there. Dhanu's table is right here. And Dhanu and I started just chatting.

And we're talking about what both of us do. And then he said, well I do biomedical research with swine, and I said, huh! I have a project for you! 
(audience chuckles)
 So I'll hand it off to Dhanu.- So animal models.

Ultimate goal for why somebody is looking for animal models is because they're trying to find a therapy or a cure for something. They're trying to study science, but ultimately you're looking for a cure or a therapy. If you look at what drug development, for any disease is like, you sort of wonder why it even happens because you often start out with about five to ten thousand different compounds, and you move across. A drug discovery phase, you go through preclinical testing, and three phases of clinical testing, and finally FDA approval.

From these 10 thousand compounds, you're lucky if you get one approved.

This is a very long period of time that this goes through. And drugs that reach the clinical, by the time they get to Phase Two of clinical trials, the failure rate is 95%.

And if you look at the cost of development, right now we're somewhere on $2.6 billion per drug.

It's fairly unsustainable. If you look at what the cost is, the cost usually comes from the failure rate.

What's figured into the cost of any successful drug, is all the other drugs that failed. And the primary cause of failure is that they didn't have the right model earlier on in the development phase. Because if you fail fast, fail early you would save a lot of money. If you let it go all the way 'til the last minute, to the clinical trials, you've already spent a lot of money, a lot of time invested at that point.

Even if you could redesign the drug by a little bit, you've already committed so much effort, there's no turning back.

So, essentially the paradigm of drug development is you're trying to get from scientific discovery, to clinical therapy where there's a clinical impact. And the gap in between is the critical part.

Most of the time we use rodent models to try to get across this gap. Rodent models are extremely valuable. They've been very valuable in our understanding a lot of disease process. However, they do have their limitation. All models have limitations, and rodent models particularly have limitations when it comes to translatability. And in the past, we've tried to sort of close the rest of the gap with primate models, but for many different reasons, including ethical reasons, practicality, and the number of drugs being developed, they just cannot be the closing of that gap. Which then comes to us, how do you close this final gap in a way that's translational and where you can get to success with the lowest amount of expenditure of time and money?

The pig. 
(audience murmurs)
 Pigs are quite amazingly very similar to us. In fact, I've studied cardiovascular disease for most of my career, and I would say from the perspective of cardiovascular disease, the pigs are far more similar than non-human primates. In fact, non-human primates rarely get cardiovascular disease. They don't die of cardiovascular disease. Whereas you can easily get pigs to do that.

That's because they're similar in genetics, anatomy, size, a lot of physiology and pathophysiology. But even beyond this, you can actually put them into sort of lifestyle, you can sort of model lifestyle that mimic humans. You could actually feed them very human-like diets. You can actually feed them McDonald's and they'll eat it. Good luck trying to feed a Big Mac to a rodent. 
(chuckles)

The value of swine models is fairly recognized. It's been recognized since the earliest of time. In fact, most of what we know about anatomy was modeled by swine, the study of swine. So, why aren't they popular? Well, because swine research has a certain amount of requirements that isn't easy to get, especially nowadays. One is, you gotta have availability of swine models. But with having swine models, you gotta have expertise in swine.

And that's kind of also a dying thing, especially with agriculture becoming less and less popular, and the cost of research then, those who use to have a lot of knowledge about swine, have gone away. And there aren't new investigators coming on board with that. So you need to have understanding of swine genetics, anatomy, physiology, breeding, husbandry, behavior research, surgery techniques, so on and so forth. You also have to have the space, facilities and supporting resources.

Compared to a mouse, swine takes a lot of space.

Not just physical space, but they need space in other aspects as well.

And then you generally wanna have close integration with medical research. Because if you're gonna use models, you're gonna need imaging technology, techniques, rays imaging modality, MRI, CT, whatever it might be. So the closer you are to a medical facility that has all those, makes those studies feasible.

And luckily at UW, we have it all.

In fact, it's been the very basis why I have the career I have.

I wanted to talk about, when Chuck approached me with this, this is complete switch. But, my confidence of why we might be able to tackles this comes from the fact that we've had successes with swine models. One, my colleagues and I have had created the miniature swine breed. This is a miniature swine that actually stays human size, allows us to a lot of research that you wouldn't be able to do with the conventional meat swine in the meat industry because those animals tend to get to be 200, 400 pounds, and they don't quite any more replicate the physiology of the human any more. They've been selected for agriculture lifestyle breeding.

Of all the many different advantages of the breed, one of the advantages, especially when you're studying chronic diseases in the Western culture, is that it has very plastic body composition. Which is that, if you were to feed the miniature swine more calories, they'll get obese. And they will also start to develop obesity associated phenotypes, which is metabolic syndrome and so on and so forth. And we've been able to study those in our models. So we believe that it mirrors the complexity of the human physiology.

One line of this miniature swine that we have used fairly extensively is one we call the FH Swine. This particular line of the miniature swine has a naturally occurring mutation in its LDL receptor in the liver. Basically, this receptor is responsible for clearing cholesterol out of your, or regulating your cholesterol in circulation.

So it has a faulty receptor, like many people do. Those who have high cholesterol levels, a fair number of them have a faulty LDL receptor. So this swine essentially over its age, gets high cholesterol.

And, like humans do, it also gets atherosclerosis, cardiovascular disease. Here's a coronary artery of the swine. It should be all open here. What you're seeing is blockage. This is plaque that's grown in here.

And this is a histology of that plaque. And it has all the hallmarks of human plaque, which you can't get this type of complexity in any other animal model. In fact, when we have taken a vessel and sent it for pathology, the human pathologist, they often can't tell that we gave them a swine sample, because it's indistinguishable in disease. And we've used this model now to do a lot of different studies. I'm just going to highlight a few. We've used it to develop and advance new stents, new drug alluding stents, we've even used it to develop new imaging modalities. This is a MRI imaging technique. We were interested in whether we could use MRI imaging to detect cardiovascular plaques developing before they actually do. Can we tell before it happens? Mostly we were interested in predicting this before somebody actually develops hard disease. So we've used this model in many different contexts.

The reason we could do all these studies isn't just the model, but the unique resources we have at UW. And I mentioned that earlier. This one aspect of facilities, a lot of people are amazed, even those who have been at UW for a long time. In fact, Chuck, when I first told him, he was amazed that it even existed. He's been here a long time, he knows the research infrastructure, we have facilities. One of our breeding and housing facilities houses up to 15 hundred animals.

It is officially designed by the Chair of my department, he's been here a long time and he designed this facility. It's so efficiently designed that it's actually run by two to three individuals. So if you think about cost of research, it doesn't get as big as you'd think.

If you do it right. It's also what we call an SPF Facility, or Specific Pathogen Free Facility, which means you have to shower in and shower out. It's always interesting taking collaborators in, because we usually tell them ahead, you're gonna have to strip down. And, it's an icebreaker when 
(audience laughs)
the first thing you do with a collaborator is you're stripping down, and then you go through the showers, and then you get shared undergarments on the other side. But these are very unique facilities that allow us to do the kind of research we do. One of the other facilities that I manage on campus also houses a fair number of swine. I'm gonna give you some comparison. Even some of the top notch research universities, Johns Hopkins, for example, generally they can only handle anywhere between three to 10 animals at a time, okay? And from a cost perspective, what we call per diem, we're at about $8 a day per head, per animal. Whereas most of these universities who can do swine research, are at about $45 to $50 a day, per animal. So, you can see how that works out. And we have fully equipped surgical suites, and so on so we can do all this research. This allows us to do a lot of research. In addition, we also have our own luxury, biomedical large animal transport. This is actually designed by Jamie Reichert, who is one our facility managers. And it surpasses all the standards of transporting animals. It's got everything from remote telemetry, to monitoring the animals by video so the driver can actually see the animals at all times. They can monitor temperature and everything like that. Because we wanted to make sure these animals were in best condition they were in their normal physiological state. Because we wanted to have animals that if developed, they can be transported to any collaborators across the country. So they can be used by others, because we want to share what we create.

I mentioned that we have all the requirements for doing swine work. But there was one more thing that we didn't have.

That was genetic engineering of swine. So when Chuck approached me, we hadn't done any genetic engineering work. And so that was an undertaking. And what we realized really soon was that genetic engineering of swine is sort of like launching a shuttle.

It takes teams of people working on everything equivalent to O-rings and bolts that have to get assembled, and it has to come together, and they have to come together with window of launching, literally for gene editing of swine, it's months of work, everything has to be perfectly timed. We have about a four hour window to make it work. If it doesn't happen or something goes wrong, then you're back to months and months of work.

But, in addition to genetic engineering of swine, NF1 is a very complex disease. So we have the complexity of genetic engineering, and then we have the complexity of the disease that we're gonna study. Let me tell you a little bit about NF1. NF1 is one of the most common monogenic disorders. But you won't really hear that. If you haven't heard that of NF1 before, that's rather strange because if you think about it, cystic fibrosis, muscular dystrophy, Huntington's disease, Tay Sachs combined, NF1 is actually more prevalent than those. All combined. So it's not a rare disease, although most would treat it as a rare disease. It's caused by a mutation of the neurofibromin gene. It's a fairly large gene. One of the largest genes in the body. It affects about one in 3,000 births.

That's based on how we clinically diagnose it.

Our recent research seems to indicate that maybe there are a lot more people with NF1 then, perhaps are diagnosed.

Individuals with NF1 are predisposed to both benign and malignant peripheral and central nervous system tumors. And this isn't just one tumor, this is lifetime of tumor risks. You can combat one, you just never know when the next one's coming around. Most tend to be benign, and the complexity of that is that the tumor size tends to impair function. If it's in the brain, obviously it's pressing against things, it's gonna complicate things. But about 10% of those, where the tumor is actually malignant, will spread. And in those individuals it spreads, the prognosis is very poor. Pretty much, there isn't much you can do.

It almost always comes with some level of cognitive impairment. Everything from Attention Deficit Hyperactivity Disorders, to Autism Spectrum disorders, learning disabilities, so on so forth. It also comes with bone disorders. Some individuals are born with bone deformities, or fragile bones. All sorts of those sort of things. And if you are lucky enough to make it past a certain amount of age, into your teens, past your teens, and so on so forth, then you get hit with cardiovascular disorders that come in at that point.

And the complexity of this disorder is that there have been over 3,000 unique NF1 mutations that have been clinically identified.

And no one child is like another, because of that. In fact, these phenotypes aren't very consistent. Every child has different phenotypes, different severity, depends on the mutation. Probably interaction between mutation and other factors like diet, and what else that we don't understand.

So, we embarked on trying to create a model.

We set out to create a single model.

This is the schematic of how this works. But essentially, we have these embryo donors, pigs that are gonna donate embryos that we genetically engineer. And a lot of months of work goes to figuring out exactly where to cut and snip the gene, how we're going to recreate a particular mutation that we are trying to model. And then, the edited embryo is then implanted back to a recipient. We do this by surgical means, a recipient swine that's gonna take it and carry it to term.

Like I said, we have to reproductively synchronize all these animals to these where they're 24 hours apart in their reproductive cycles. They have to be exactly that otherwise, the egg from one another will not carry over to term. So, a lot of kudos to Jen Might, whose in the crowd here, she's my lab director-- and that's her son who is pointing at her-- who really worked a lot of this methodology out. And let me tell you, her background is in botany, so when I put this on her, it took her about two months to piece these things together. And there were a lot of other people then who actually helped carry this whole thing out. So this was a team effort. We set out to create a pig model and in addition, I think we ended up creating much more. We created a team, we created a vision, and we created actually a community around this project.

There's a lot of work that goes through this process.

And we were happy to have the first litter of NF-edited piglets carrying the mutation in the NF1 gene. And one of them grew up to be Tank, he was the first genetically engineered by Chris Berg on campus, and he was also the first genetically engineered NF1 boar.

This is actually when he's fairly small. He's grew up to be quite the Tank. 
(audience chuckles)
Actually, his name was quite sentimentally, named by-- Mason actually named him. And maybe Chuck will, if you approach him, might tell you the story behind how they came up with that name.

Our central mission really is to share the models we create.

Because we want to get it in the hands of people who have ideas for therapies, who need this to understand various aspects of the disease. But that doesn't mean that we aren't doing out own work. Dominick, one of my graduate students, is in the crowd here. He's in back there. So, we're also looking at mechanistic stuff. Here is some indication of that understanding the disease. These are stem cells that we've taken from our pigs that have the mutation versus those that don't. Those either have a single copy of the mutation, or a double copy of the mutation. And we were looking at how do those stem cells transform to become various brain cells. And we can already see differences in how they behave. If they have the mutation, they seem to sort of speed up through the differentiation process. So we're trying to understand now, is that affecting the phenotype? Is that causing those cancers? Is that causing the cognition? In fact, recently we were funded by the Department of Defense to develop a gene therapy. We have an idea for perhaps sort of interfering with these processes. At least in part of the patient population.

So how do we get from a single model to patient specific models?

Well our First Generation of pigs, we really focused on phenotype. We were trying to pick mutations that were associated with certain phenotypes. Cognition phenotypes, or tumorigenic, or cancer phenotypes.

Then we advanced to Second Generation of models where we were picking more types of mutation. The huge deletion of the gene, are there a mutation that completely disrupts the entire gene? Or somethings that changed the wording.

For those who are not familiar with some of the genetics--

Your genome is like a book.

Every chapter in that book, think of it as chromosomes. And every chapter there are passages. Those are your genes. NF1 is one of the biggest passages in this book. And we don't really understand the message of this passage because we really don't understand the protein that encodes the neurofibroma, we don't really understand all of its function yet.

All we know is that sometimes all you just change is a single word, and it changes the whole message of the passage. In some kids, they're missing a whole sentence. That completely changes the passage.

Others, it's some disruption that makes you read the passage differently.

The end of the story is different, and the way the story changes, sometimes changes to have a whole lifetime of cognition problems, tumor problems, bone problems, and we don't know how to predict that quite yet. So, as we wanted to set out this create a model of NF1, we realized, a single model isn't just gonna do it.

Because every child is different, and so what if you have a single model. We're not sure which child that will help.

So we came up with the idea that we had to create patient specific mutations.

And so model, something for a child. And we started this process, we identify the clinical population to start this work with. We're waiting for IRB approval process. And that's when we realized this is somewhat a new area, because we weren't sure how to get IRB approval.

And we weren't sure what we are asking approval for. But as we were thinking through that, we realized the dilemmas and the ethical conundrums that we had to work through before we proceeded too far. Which is, for example, if we were to create a pig model that models a particular mutation of a child, and if the pig dies, what do we do? What is our obligation? Do we inform the parents? Maybe the pig dies, but maybe the child wouldn't. So all of this had to be, we had to work it out. And we hadn't worked all of it out. We had worked some of it out, and we're trying to move forward. But our long-term goal is that we'll have a pig for every child.

And what that does really for NF1 is that right now, the way a drug for a child is figured out is you try a random set of drugs. There is no schematic for it. So that means every child has to go through the side effects of all these drugs. And usually they're on a cocktail of drugs. One for cognition, one for this or that. And the side effects of that. And if they're not lucky enough to find the one that works right away, there's time ticking away. And we're talking about tumors that are growing. And they're losing time in terms of cognition issues, and they're getting delayed in learning and so on and so forth. So, what we're hoping is that when we do this, we will use the pig as a way to screen one, the drugs. One is to also start to use the different pigs with different mutations as a way to try to dissect out, “Why is a mutation here causing this phenotype?”, or so on and so forth. Is it interacting with diet? Because we can put diets in pigs. We can put them on a high fat diet. We can put them on a high carbohydrate diet and see whether that influences it. Because we don't understand the dietary influence on this disease. In fact, it's interesting, if you talk to clinicians, they might say, well, there's no data on this. If you talk to parents, they'll say, oh, you know, my kid responds to-- I don't give my kid Skittles, or this or that because something about that makes the symptoms worse. They act up. But we don't know, because we haven't really studied, we don't have the models to study. And we're hoping this will allow us to study it. As we got this idea, we realized we could use this paradigm for many other things. Including cardiovascular disease, which is also fairly complex. Perhaps not as complex as neurofibromatosis, but it is one of those diseases with multiple things acting on, and everybody is different. This idea of precision medicine.

That led us to gene editing ideas. And in fact, we have an initiative going to create pig organs that are tailored and compatible with a particular patient requiring an organ transplant. That's a whole different area, but I can tell you that, again, we are really hopeful about that too. Because right now there's about a five to eight year wait for an organ. It's about five years in Wisconsin, about eight years elsewhere, for like say, a kidney, and about a fair number of that population will not make it because there aren't enough organs to go around. So, in fact, if you can custom tailor organs and pig organs being very close to humans, with just the right enough things to fool the immune system, we believe that's gonna open the door. We are very lucky. I feel very lucky to be at UW.

To have the resources, having worked with animal models because, the right animal model is a big game changer when you talk about diseases. And in fact, historically, animal models have made all the difference. If you're lucky enough to have a relevant model, and if you have the resources to do pig models, for example, then you can have a great impact. And so far in a lot of the other diseases that we've studied, I've been very lucky to see a lot of things that we've worked on go to clinical trials, going into clinic and be used in clinic. And we're really hoping that we can do the same for NF1. Thank you. 
(audience applause)