[Tom Zinnen, Outreach Specialist, Biotechnology Center, University of Wisconsin-Madison]

Welcome everyone, to Wednesday Nite @ the Lab. I’m Tom Zinnen, I work here at U.W.-Madison Biotechnology Center; I also work for U.W.-Extension and Cooperative Extension. And behalf of those folks and our other core organizers, Wisconsin Public Television, Wisconsin Alumni Association, and the U.W.-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 U.W.-La Crosse, where he majored in Finance and Business Administration. He has been at U.W. 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?

[Dhanansayan Shanmuganayagam, Assistant Professor, Department of Animal Sciences, University of Wisconsin-Madison]

Jaffna.

[Tom Zinnen]

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

[laughter]

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]

[Dhanansayan Shanmuganayagam]

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. And 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 team’s 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 – as the metaphor you’ll hear later. And the – our journey really began with a baby boy who had a pigmented mark –

[slide featuring a photo of Mason Konsitzke, the boy who had a pigmented mark on his back side as a baby]

– on his back side. And I’ll let Chuck start that.

[Charles Konsitzke, Associate Director, Biotechnology Center, University of Wisconsin-Madison]

Thank you.

So, 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 – Ive got attacked for helping very unique projects come to fruition. And I had one challenge ahead of me.

So, Mason was born in 2010. We had some early concerns – five pounds weight. He had G.I. issues, which required a special diet.

[slide titled, My Son Mason, featuring a baby photo of Mason next to a list of early signs of concern – low birth weight of 5 pounds, gastrointestinal issues that required a special diet, and jaundice]

He had jaundice.

[new slide titled, Neurofibromatosis Type 1 (NF1), featuring another baby picture of Mason]

And then at six months old –

[slide animates on a green arrow from the top of the slide pointing downwards and noting at 6 months old Mason has his first caf au lait spot on his rear end]

– we noticed a pigment on his rear end. And myself being who I am, I started to investigate this. And come to the horror, I – well I shouldn’t say horror –

[Charles Konsitzke]

– it was just what scared me is I noticed something called a caf au lait spot in Googling it. [chuckles] And the worst thing to do is Google anything.

[laughter]

So, at his nine-month appointment, we talked to the pediatrician –

[return to the Neurofibromatosis slide now animating on 9 months old with the bullet point regular check-up]

– and we stated, Well, 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 up. So, I immediately changed my insurance company and I transferred over to the U.W. Children’s Hospital, where at one years of age he was clinically diagnosed with NF1.

[slide titled, NF1 Progression, featuring photos of Charles holding Mason and Mason playing in the leaves at 15 months old. Additionally, there is another green arrow starting at the top of the side and pointing down with the headline – 15 years old, Plexiform Neurofibroma, and a bulleted list of symptoms – reduction in speech, difficulty hearing, gait and balance issues, and behavior changes]

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, kind of, 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. And so, we took him in and they did a hearing test on him. And they believed that –

[Charles Konsitzke]

– 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 M.R.I. prior to putting a tube 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 looked – it was spreading to the naval cavity as well because what a plexiform neurofibroma – neurofibroma is, it’s not like your standard tumor. It’s not like a benign tumor where it’s – its 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.

[slide titled, Living with NF1, with a photo of Mason curled up on a hospital bed and a bulleted list of consequences of NF1 – many M.R.I.s, medications, therapy, and a lifetime of parental woes]

So, after many M.R.I.s, cocktails of medications where we – we – 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.

[Charles Konsitzke]

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]

[laughter]

So, I started –

[slide titled, Local NF Community, with a bulleted list of things that Charles did – networking, discussing issues, avenues for fundraising including Links for Lauren, The NF Team, NF North Carolina]

– 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 –

[slide animates on the logos of all the fundraising initiatives for NF]

– because I’m priming myself for what the next step is. So, a lot of the stuff was occurring at home, very long nights. I have my file right there.

[Charles Konsitzke]

So, then I did the pre-ample 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 U.W. 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 –

[slide titled, My Investigations, with the bulleted list of Pre-Ample – questions of conflicts of interest, Prescouter – Custom Intelligence, On-demand – offering monthly reports in depth investigation of Researchers – capabilities, strengths, weaknesses, National NF Researchers, NF Publications and Manuals]

– 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 – 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 investigations – 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 –

[Charles Konsitzke]

– national NF researchers and I was asking them, What are the bottlenecks, what are the issues you are seeing ahead of you? I started – I was reading a plethora of NF publications, manuals.

And what it came down to, was the model size. So, 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 – they are 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 – a – on a cardiovascular issue with NF. So, the – 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 – or you see this in NF research.

So, swine model, I was think – I communicated with a – a senior faculty member outside of U.W., 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 U.W. Day at The Capitol, where we meet a bunch of legislators, senators, and we’re kind of explaining –

[slide titled, Serendipity – U.W. Day at the Capitol, featuring two photos from the Capitol rotunda of U.W. Day at the Capitol]

– what we do on the Capitol, kind of promoting what – what activities happen within the -the center or on campus, and after, you know, four or five hours, you’re kind of, like, exhausted, so I was standing next to the railing almost – 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, you know, what both of us do. And then he said, Well I – I do biomedical research with swine, and I said, Huh! I have a project for you!

[laughter]

So, I’ll hand it off to Dhanu.

[Dhanansayan Shanmuganayagam]

So, animal models. Ultimate goal for why somebody is looking for an animal models is because they’re trying to find a therapy or a cure for something.

[slide titled, Finding Cures and Therapies, featuring a photo of a hand working with test tubes]

You know, they’re trying to study science, but ultimately, you’re looking for a cure or a therapy – or a therapy.

[slide titled, Drug Discovery and Development – A Long Risky Road, featuring a chart showing the progression of a new drug and new drug approval starting with basic research to drug discovery to pre-clinical (3-6yrs) to clinical trials in three phases (6-7 yrs.) to F.D.A. review (6 months to 2yrs) to post-approval and research and monitoring]

And if you look at what drug development, for any disease is like, its – 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 F.D.A. 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%.Okay.

[Dhanansayan Shanmuganayagam]

And if you look at the cost of development, right now we’re somewhere –

[slide titled, Cost to Develop One New Approved Drug, with a bar chart featuring cost in billion dollars on the y-axis and decades of development on the x-axis, showing an increase from $179 million dollars for a new drug in the 1970s to now $2.6 billion dollars for a new drug in the 2000s]

– on $2.6 billion per drug. It’s fairly unsustainable. If you look at what the – the cost is, the cost usually comes from the failure rate, okay? So, what’s figured into the cost of any successful drug, is all the other drugs that failed.

[Dhanansayan Shanmuganayagam]

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, its – there’s no turning back. Alright.

[slide titled, The Research Gap – Benchtop to Bedside, featuring the two goals of development – Scientific Discovery and Basic Research to Clinical Therapies]

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. Okay.

Most of the time we use rodent models to try to get across this gap.

[a photo of two mice animates on the slide]

And rodent models are extremely valuable. They’ve been very valuable in our understanding a lot of disease process.

[Dhanansayan Shanmuganayagam]

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 –

[return to The Research Gap slide now with a photo of a monkey next to the mice]

– they just dont – cannot be the closing of that gap.

[a question mark animates on where the monkey was in the slide]

Which then comes to us, how do you close this final gap in a way that’s translation – translational and where you can get to success with the lowest amount of expenditure of time and money?

[new slide titled, The Ideal Translational Animal Model, featuring a profile photo of a pig with its head held high wearing several pearl necklaces]

The pig.

[laughter]

[Dhanansayan Shanmuganayagam]

Pigs are quite amazingly very similar to us. In fact, I’ve – Ive studied cardiovascular disease for most of my career, and I would say they – from – from the perspective of cardiovascular disease, the pigs are far more similar than our 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.

[slide titled, We Are Similar, showing a Ven Diagram of with the outline of a human and a pig showing intersections in genetics/epigenetics, anatomy and size, and physiology and pathophysiology]

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.

[Dhanansayan Shanmuganayagam]

You could actually feed them very human-like diets. You can actually feed them McDonald’s and they’ll eat it, okay. Good luck trying to feed a Big Mac to a rodent. [chuckles]

So, however, I mean 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 is – 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.

[slide titled, Requirements for Biomedical Swine Research, and a bullet of availability of swine models]

One is, you gotta have availability of swine models. Okay. But with having swine models –
[the Requirements for Swine Research slide animates on Expertise in Swine Models including swine genetics, anatomy, physiology, breeding, husbandry, and research/surgical techniques]

– you gotta have expertise in swine. And that’s kind of also a dying thing, especially with agriculture becoming less and less popular, agricultural research then, those who use to have a lot of knowledge about swine, have sort of 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.

[the Requirements for Swine Research slide animates on the bullet – Space, Facilities and Supporting Resources]

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.

[the Requirements for Swine Research slide animates on the bullet – Integration and Medical Research – Preclinical to Clinical]

And then you generally wanna have close integration with medical research. Because if you’re gonna use these models, you’re gonna need imaging technology, tech – techniques, various imaging modality, M.R.I., C.T., whatever it might be. So, the closer you are to a medical facility that has all those, makes those studies feasible. And luckily at U.W., we have – have it all. Okay.

[the Requirements for Biomedical Swine Research animates on the U.W. shield and green check marks next to all the bullet points]

And in fact, it’s been sort of the very basis why I have the career I have.

[new slide titled, Our Previous Successes – Wisconsin Miniature Swine, Swine Model of Cardiovascular Disease, featuring a photo of a swine heart]

So, I wanted to talk about – so, when Chuck approached me with this –

[Dhanansayan Shanmuganayagam]

– 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 sort of the miniature swine breed.

[slide titled, Wisconsin Miniature Swine, featuring a photo of this new breed of swine]

This is a – a miniature swine that actually stays human size, allows us to a lot of research that you wouldn’t be able to do with sort of the conventional meat swine in the meat industry because those animals –

[Dhanansayan Shanmuganayagam]

– tend to get to be 200, 400 pounds, and they are not – they don’t quite any more replicate the physiology of the human anymore. Okay, they’ve been selected for agriculture lifestyle breeding. So, so, one of these – of – 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 –

[slide titled, Plasticity of Body Composition, featuring an X-ray and M.R.I. of three miniature swine next to a graph of percentage of body fat by age for the swine]

– has very plastic sort of body composition. Which is that, if you were to feed the miniature swine more calories, they’ll get obese.

[new slide titled, Diet-Inducible Metabolic Syndrome Features, two bar graphs one of insulin sensitivity and one of glucose effectiveness]

And they will also start to develop sort of obesity associated sort of phenotypes, which is metabolic syndrome and so on and so forth. And we’ve been able to study those in our – in our models. So, we -we believe that it sort of mirrors sort of the complexity of the human physiology.

[new slide titled, Swine Model of Atherosclerosis (FH Swine), featuring a graph of various L.D.L. receptors showing with a circle the mutation in the L.D.L. receptors that leads to Atherosclerosis]

One line of this miniature swine that we have used fairly extensively is one – one we call the FH Swine. This particular line of the miniature swine has a naturally occurring mutation in its –

[Dhanansayan Shanmuganayagam]

– L.D.L. receptor in the liver. Basically, this receptor is responsible for clearing cholesterol out of your circular – or regulating your – your cholesterol in circulation. Okay. So, it has a faulty receptor, like many people do. Those who have high cholesterol levels, a fair number of them have a faulty L.D.L. receptor. So, this swine essentially over – over its age –

[return to the Swine Model of Atherosclerosis, now animating on a Serum Cholesterol graph by age next to the L.D.L. receptor graph that shows an increase in serum cholesterol as this swine ages]

– gets high cholesterol.

[new slide titled, Atherosclerotic Lesions in FH Swine, featuring a graph of the prevalence of different coronary artery lesion types in the FH swine as well as two photographs of the lesions in the swine arteries]

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 is – you can’t get this type of complexity in any other human – any other animal model. In fact, if you were – when we have taken a vessel and sent it for a pathology, the human pathologist –

[Dhanansayan Shanmuganayagam]

– they often can’t tell that we gave them a human – sorry, 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 just highlight a few. We’ve used it to develop and advance –

[slide titled, Intravascular Ultrasound and Optical Coherence Tomography Imaging of In-Stent Restenosis for Novel Stent Development, featuring a series of photos showing the differences in FH Swine from Conventional Swine and the use of a vascular stent in a FH Swine]

– new stents, new drug alluding stents –

[new slide titled, MRI for Atherosclerosis Screening, showing three photos of M.R.I.s of the FH Swines heart measuring the pulse wave velocity]

– we’ve even used it to develop new imaging modalities. This is a M.R.I. imaging technique. We were interested in whether we could use M.R.I. 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 heart disease. So, we’ve used this model in many different contexts.

[new slide titled, Unique Resources at U.W. featuring a photo of the concrete U.W. shield on the side of the Kohl Center]

The reason we could do all these studies –

[Dhanansayan Shanmuganayagam]

– isn’t just the model, but the unique resources we have at U.W. And I mentioned that earlier. And this one aspect of is the facilities, and I just want to show – a lot of people are amazed, even those who have been at U.W. for a long time. In fact, Chuck, when I first told him, he was amazed that it even existed. He’s been – hes been here a long time; he knows the research infrastructure. We have facilities.

[slide titled, Swine Research and Teaching Center (SRTC), featuring a photo of the research facility as well as the blueprint diagram for the facility next to a bulleted list – the facility is off campus; it is a S.P.F. shower-in facility; it is A.A.L.A.C. accredited; it is a large facility – 40,00 square feet, 1,500 swine capacity, a breeding facility, and a surgical site with pre and post-op recovery rooms]

One of our – our breeding and sort of housing facilities houses up – up to 1500 animals. Okay. Its – it is officially designed by actually 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 really – it doesn’t get as big as you’d think. If you do it right.

It’s also a – what we call an S.P.F. 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.

[Dhanansayan Shanmuganayagam]

And it’s an icebreaker when youre –

[laughter]

– 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. And you know, its – its – 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 – on campus also houses a fair number of swine.

[slide titled, Translational Research Facility (TRF), featuring exterior and interior photos of the facility next to a bulleted list – it is on-campus; it is A.A.L.A.C. accredited, it is a medium-sized facility that is 30,000 square feet, has a 100-200 swine capacity, and has a surgical suite with pre- and post-op recovery]

I’m gonna give you some comparison. Even some of the top-notch universities – research universities, Johns Hopkins, for example, generally they can only handle anywhere between three to 10 animals at a time, okay? And – and from a cost perspective, what we call per diems, you know, we are at about $8 a day per head, per – per animal. Whereas most of these universities who CAN do –

[Dhanansayan Shanmuganayagam]

– swine research, are at about $45 to $50 a day, per animal. So, you can see how that works out. And so, we have fully equipped surgical suites, and so on so we can do all this research.

So, this sort of allows us to do a lot of research.

[slide titled, Biomedical Large Animal Transport, featuring a photo of a truck hauling the large animal transport facilities next to a bulleted list – it is custom designed and fabricated; it exceeds current biomedical requirements; it has environmental controls – three 1,500 BTU air conditioning units, two 20,000 BTU heaters, and its own H.V.A.C. system powered by a 10,000 watt diesel generator; it has its own monitoring systems including closed-circuit television for driver to monitor animals, and internal and external temperature telemetry]

In addition, we also have our own luxury, biomedical large animal transport. This is actually designed by Jamie Reichert, who is actually one our facility managers. And it – it surpasses all the standards of – 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.

[Dhanansayan Shanmuganayagam]

Because we wanted to have animals that we 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.

So, I mentioned that we have all the requirements for doing swine work.

[return to the Requirements for Biomedical Swine Research slide with all the requirements ticked off with green check marks]

But one thing – there was one more thing that we didn’t have.

[the bullet point – Genetic Engineering of Swine animates onto the bottom of the list on the slide]

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.

[Dhanansayan Shanmuganayagam]

And what we realized really soon was that genetic engineering of swine is sort of like launching a shuttle.

[slide titled, Genetic Engineering of Swine is like Launching a Shuttle, featuring a photo of a space shuttle blasting off]

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 –

[Dhanansayan Shanmuganayagam]

– and they have to come together with window of launching, literally for – 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.

[slide titled, NF1 is a Complex Disease, featuring an illustration of D.N.A. strands and other genetic illustrations]

So, we have the complexity of genetic engineering, and then we have the complexity of the disease that we’re gonna study.

[new slide titled, Neurofibromatosis Type 1 (N.F.1)]

Let me tell you a little bit about N.F.1. N.F.1 –

[slide animates on the bullet point that NF1 is one of the most common monogenetic disorders, more prevalent than Cystic Fibrosis, hereditary Muscular Dystrophy, Huntingtons Disease and Tay Sachs combined]

– 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, everything – 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.

[slide animates on the bullet point that NF1 is caused by mutations of the neurofibromin gene]

It’s caused by a mutation of the neurofibromin gene. It’s a fairly large gene. One of the largest genes in the body.

[slide animates on that NF1 affects about 1 in 3,000 births]

It affects about one in 3,000 births. Okay. That’s based on how we clinically diagnose it. Our – actually our recent research seems to indicate that maybe there are a lot more people with NF1 than, perhaps are diagnosed.

[slide animates on the bullet point that individuals with NF1 are predisposed to – starting a new list starting with benign and malignant peripheral and central nervous system tumors]

Individuals with NF1 are predisposed to both benign and malignant peripheral and central nervous system tumors.

[slide animates on – under Individuals with NF1 are predisposed to – the bullet points – cognitive impairments including A.D.H.D. and autism spectrum disorders; bone disorders; and cardiovascular disorders]

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 is – is 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 actually is malignant, it will spread. And in those individuals, it spreads, the prognosis is very poor.

[Dhanansayan Shanmuganayagam]

Absolute – 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 – some individuals are born with bone – bone deformities, or fragile bones. All sorts of 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 hit – get hit with cardiovascular disorders that come in at – at that point.

And the complexity of this disorder is that there –

[return to the Neurofibromatosis Type 1 slide with the bullet – over 3,000 unique NF1 mutations have been clinically identified – animating on]

– have been over 3,000 unique N.F.1 mutations that have been clinically identified. Okay. And no one child is like another, because of that. In fact, these phenotypes aren’t very consistent. Every child –

[Dhanansayan Shanmuganayagam]

– has different phenotypes, different severity, depends on the mutation. Probably interaction between mutation and other factors like diet, and – 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 sort of the schematic of how this works.

[slide titled, Genetic Engineering of NF1 Swine, featuring a schematic of the research featuring a timeline for the swine embryo donor and the swine surrogate and the CRISPR editing of the NF1 gene]

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 going to – we are trying to model. And then, and 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.

And 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 other will not carry – carry over to term. So, a lot of kudos to actually – to Jen Might, whos in – 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 – put this on her, and she – it took her about two months to try to piece these things together. And there were a lot of other peoples 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.

[slide titled, Genetic Engineering of NF1 Swine, featuring a 3 by 3 photo grid of photos taken during the genetic engineering process]

[new slide titled, Genetically Engineered NF1 Piglets, featuring a photo of six of the genetically engineered piglets hanging out together in the lab]

And we were happy to have the first litter of N.F.-edited or piglets carrying the mutation in the N.F.1 gene. And one of them grew up –

[new slide titled, First Genetically Engineered NF1 Boar (Tank), featuring a photo of Tank with one of the swine researchers]

– to be Tank, he was the first genetically engineered by C.R.I.S.P.R. on campus, and he was also the first genetically engineered N.F.1 boar. This is actually when he’s fairly small. He’s grew up to be quite the Tank.

[laughter]

Actually, his name was actually 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 –

[slide titled, Central Mission – Share What We Create, featuring an illustration of five multicolored hands reaching in to touch one another]

– share the models we create. Because we want to get it in the hands of people who have ideas for therapies –

[Dhanansayan Shanmuganayagam]

– 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. And so, we’re also looking at mechanistic stuff. Here is some indication of that –

[slide titled, Mechanistic Studies, featuring three photos of slides A, B and C, one with wild-type swine neurospheres (A), one with NF +/- swine neurospheres (B), and one with NF -/- swine neurospheres, and noting that the homozygosity od the mutation resulted in 5 fold lower expression of neurofibromin mR.N.A. and abnormal cell phenotype and increased cell cohesion]

– 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 – the differentiation process. So, we’re trying to understand now –

[Dhanansayan Shanmuganayagam]

– 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?

[slide titled, From Single Model to Patient-Specific Models, with a bulleted list animating on the title, First Generation, featuring the previous piglet photo and the words NF1 exon 30 delegation (cognition)]

Well, our First Generation of pigs –

[slide animates on below First Generation additional bullet points of NF1 23kb deletion (cognition) and NF1 and p53 mutation (tumorigenic)]

– we really focused on phenotype. We were trying to pick mutations that were associated with certain phenotypes. Cognition phenotypes, or tumorigenic, or cancer phenotypes.

[slide animates on the words, Second Generation and the bullet points NF1 micro deletions, early missense/ early stop codon, and single base-pair mutations]

Then we sort of advanced to Second Generational models where we were sort of – we were picking more types of mutation. Are there huge deletion of the – of the gene? Are there just a – a – a mutation that completely sort of disrupts the entire gene? Or somethings that sort of changed the wording.

So, for just – for those who are not familiar with some of the genetics –

[Dhanansayan Shanmuganayagam]

– its like – your genome is like a book. Every chapter in that book, is sort of think of it as your chromosomes. And every chapter there are passages. Those are your genes. And N.F.1 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 – the protein that encodes the neurofibroma, we don’t really understand all of its function yet. All we know is that sometimes all you do is just change 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 were – we wanted to set out this create a model of N.F.1, 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 have started this process, we have identified the clinical population to start this work with. We’re waiting for I.R.B. approval process. And that’s when we sort of realized this is somewhat a – a new sort of area, because there isnt – we weren’t sure how to get I.R.B. approval. And because we weren’t sure what we are we asking approval for.

But as we were thinking through that, we sort of realized the dilemmas and – and the ethical sort of conundrums that we had to work through before we proceeded too far. Which is – what, for example, if we were to create a – 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? But maybe the pig dies, but maybe the child wouldn’t. But – so, all of this had to be, sort of 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 – a pig for every child.

[slide titled, NF Child-Specific Pig Models, featuring a photo of a cute young girl holding a NF piglet]

And what that does really for N.F.1 is that right now, the way a – a drug for a child is figured out is you try a random set of drugs.

[Dhanansayan Shanmuganayagam]

There is no schematic for it. So, that means every child sort of has to go through all the – 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 dont – if they’re not lucky enough to find the one that sort of works right away, you know, there’s time ticking away. And we’re talking about tumors that are growing. And they’re losing time in terms of cognition issues, I mean, and they’re getting delayed in – in learning and so on and so forth. So, what we’re hoping is that when we do this, it will – 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 sort of try to sort of dissect out, Why is a mutation here causing this phenotype?, or – 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 would – might say, Well, there’s no data on this. If you talk to parents, they’ll say, Oh, you know, my – my kid responds to – I – 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 – it is one of those diseases with multiple things acting on, and everybody is different. This idea of precision medicine. We also – that led us to gene editing ideas. And in fact, we have an initiative going to create organs that are – pig organs that are tailored and compatible with a particular patient requiring organ transplant. That’s a whole different area, but I can tell you that, again, we are – 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 – 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 – pig 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 – I feel very lucky to be at U.W. To have the resources, having worked with animal models because, the right animal model is a big game changer when you talk about diseases.

[slide with a poster of a young girl in a hospital bed holding stuffed animals with the slogan – Its the animals you dont see that really helped her recover. Also noted is that a surgical technique perfected in animals was used to remove a malignant tumor from a little girls brain]

And in fact, historically, animal models have made all the difference. And if you’re lucky enough to have a relevant model, and if you have the resources to do like pig models, for example, then you – you can have a great impact. And so far, – so far, in a lot of the other diseases that we’ve studied, I’ve been very lucky to see a lot of things –

[Dhanansayan Shanmuganayagam]

– that we’ve worked on go to clinic – clinical trials, going into clinic and be used in clinic. And we’re really hoping that we can do the same for N.F.1.

Thank you.

[applause]