MED C-LIVE Transcript

JUNE 2020 Mario Capecchi, Ph.D. NOT FOR PUBLIC DISTRIBUTION

Michael Keel My name is Michael Keel from New Jersey. And it is my honor to introduce Dr. Mario Capecchi. Dr. Capecchi is the Distinguished Professor of Human Genetics at the University of Utah School of Medicine. He is best known for his groundbreaking work in gene targeting, in mass embryo derived stem cells. He was awarded the Nobel Prize in Physiology for Medicine, for his work in finding ways to manipulate the mammalian genome by changing mammals’ genes. His research interests include analysis of neural development in mammals, gene therapy, and production of murine models of human genetic diseases, from cancer to neuropsychiatric disorders. Dr. Capecchi, welcome to the Congress of Future Medical Leaders.

Dr. Capecchi Thank you. First of all, welcome to the Medical Leaders’ Congress. It's a pleasure to be here. My name is Mario. I'm going to be talking about gene targeting. Next slide please. My laboratory has developed a technology called gene targeting that allows you to change any gene in any conceivable manner in a living creature, such as a mouse. Next slide. Why do we do gene targeting? Next slide. There are many reasons, but they boil down to two. One is basic research and the other is applied research. In basic research, you investigate the biology of an animal. For example, how do you make a limb or a heart or a brain? Those are basic research questions. The other is applied research. That is we can use gene targeting to generate mice with human diseases. Next slide please. There are over 5, 000 human diseases that cause, by single gene defects, that are caused by single gene defects. Next slide. Gene targeting allows us to generate mice, for example, with defined cancers, diabetes or neuropsychiatric disorders. Next slide. We can study disease in mice in much greater detail that is possible in humans for ethical reasons. Next slide please. The disease model can be first used to develop the how, the mechanism of disease starts from beginning to end when it shows the full pathology. Then once you understand the disease, you can use it as a platform to develop new therapies, the exact same mouse. Next slide please. In 1980, I submitted a grant to NIH proposing to develop the targeting intraembryonic cells. Next slide. The project was deemed impossible. Next slide. And the reason they gave for refusing the grant was that the newly introduced DNA that's being introduced into the living cell will never find the same sequence in the genome of a living cell. Since this is the very first step of gene targeting that said the project would not work. Next slide please. So I had two choices. One is to drop the project, or two is to continue the project using funds from another grant. Now the second possibility had enormous risk. Next slide, please. If I failed, I would be finished as a scientist. Imagine trying to get a grant after you've used their money for four years, and you have nothing to show for it. The next grant would be very difficult to ever receive.

However, I chose to go by this second route. Next slide, please. And the reason was the second option, if we were successful, the payoff would be enormous. And so we staked everything on that. Next slide, please. Four years later, we were successful. We could do gene targeting in a mirion cells. Next slide. This was fortunate because grants cover five years of work, but renewal occurs every four years. So you have to renew the grant every four years. And four years later, we had results and we could document that we could do gene targeting. Next slide, please. So I submitted a renewal to the same NIH section that refused the first grant, this time increasing the ante and proposing to do gene targeting not in mammalian cells, but in living mice. And their reply was Next slide, please. We're glad you didn't follow our advice. They have a sense of humor. And from then on, we could pursue it. Next slide, please. Now to understand the targeting, we need to appreciate the essence of DNA. Next slides. This shows you the structure. All of you are familiar with the DNA structure. This shows two DNA strands and they're bound together in a double helix. The red strand and a green strand. And what's holding them together is a ribose phosphate linkage, and then the basis, the information part, are the steps of that helix. Next slide, please. Now, the information content in DNA is the order of basis. And it's an alphabet four letters ATG and C rather than 26 letters. Next slide, please. And this shows you the essence. On the top, you have one strand of DNA, and then two base pairs, and that opposite strand. Okay, so we're looking at just two base pairs. And if there's a T on one strand, then on the other strand you automatically always have an A. Or if you have a C on one strand, on the other strand, you automatically have a G. And the two base pairs are held together by hydrogen bonds, which is shown in red. It's only this configuration of T and A or C and G, that allows the spacing to be exactly the same. And therefore you can construct a stable helix that's present in DNA. So this is everything that we ever do with DNA is dependent on this base pairing. That's the essence. Next slide, please. And this tells us right away how DNA functions to archive information. You simply have one double strand of DNA; they separate and each forms a template for making the opposite strand. And now you've duplicated with precision going from one molecule to two molecules, one mother cell to two daughter cells or one generation to the next generation. And this allows you to transfer the information from one generation to mother and father down to their siblings. Next slide please. Now, Gene tightening takes advantage of a process called homologous recombination. What is homologous recombination? Next slide. The major role of homologous recombination is to repair DNA damage. Next slide. However, at that time, when we started these experiments, that wasn't known. It only became apparent from our own experiments. And that's a separate story, which I don't have time to go into. Next slide, please. Now, in DNA, there are two major grooves, there are two grooves, a big one and a little one. The big one’s called the major groove, and the second one's called the minor groove. Next slide, please. Now, there's enough surface on, revealed on the major groove, to allow a molecular machine to identify which pair is present at any given position without having to separate the strands. So all you have to do is travel along the major groove, and you know exactly what sequence of DNA is through this machinery. Could I have the next slide please. And this homologous recombination return, it does something quite unexpected and magnificent. OFFICIAL TRANSCRIPT NATIONAL ACADEMY OF FUTURE PHYSICIANS AND MEDICAL SCIENTISTS © 2020 ALL RIGHTS RESERVED 2 JUNE 2020

When it finds two very long stretches of identical DNA sequence on homologous pairs of chromosomes. Remember, you always have two chromosomes in it for each for your information. It lines up the sequences, and then literally cuts and exchanges the strands with almost perfect precision. This process is shown in the next slide. Here, the top is a double helix, yellow, and then there's a blue one. They actually represent the exact same sequence, but I've covered one yellow, one blue, so you could distinguish them. So the sequence across that region is identical. Every base pair is identical to the other one, okay? Once they're lined up, and that's what the homologous combination does, then it literally cuts and rejoins them. And since we've made them different colors, you can see that they're now joined together with an opposite partner. But what's important is and in the process, it's done with perfection. So now the sequence is still the same from one end to the other on both strands. So it hasn't changed. It simply exchanged partners. Okay? And it's this machinery that we take advantage of, to generate, to do gene targeting. Now, the next slide, please. Okay, so this is what we're going to do. And then the next slide shows the actual mechanism. So here it is, it's very simple. On the top bar is your favorite gene. Okay. It represents, for example, an average gene would have 40, 000 letters. And in the middle of that, we've created this in a test tube. And in the middle of that we make a change and that's the asterisk, the red asterisk. Okay. And that change can be anything. A single base pair. 10 base pairs. A hundred. Thousands. And now we're working on a technology to make it many millions, so we can transfer enormous amounts of information to a single chosen spot on a chromosome. Okay, now, the asterisk is different. But on the sides of that asterisk is a sequence that is identical to what's present in the genome. So we introduce this DNA into the nucleus of a cell. And then the harmonics recombination searches the entire 3 billion base pairs. Remember, if we wrote this out, it would occupy 1000 volumes, each 1000 pages long. Except the podia Britannica has maybe what 40 volumes. This is 1000 volumes of a book that size. Huge amount of information. Okay. It searches that entire genome, finds the exact same sequence outside of that asterisk, and then mediates that breaking, rejoining at both sides of the asterisk. So two homologous recombination breaks, okay. And then the process transfers that DNA sequence that we introduced from the outside exogenous sequence, replaces the endogenous sequence, okay. And therefore, we now have changed that sequence in the genome. And we change it in any way we want, by any kind of information we want. We can destroy the gene, we could change where the gene functions and so on. How much of the chain project is made, and so on. Next slide, please. So this diagram appears simple, but it wasn't actually easy. It took over 10 years to figure out how to do it, and to finally accomplish this goal. And we did it. We did it in actually a little less than 10 years. But I had. Next slide please. Now we decided to do it in mice. The next question you should ask yourself is why the mouse? Next slide, please. Next slide. Okay, next slide. So, all mammals, doesn't matter whether it's a mouse, a human or an elephant, share the same set of genes within what. . . 99% of the genes are identical, all of those atoms. So in 1% that makes us, it distinguishes us from an elephant or a mouse. Because we're all mammals, we have a common developmental plan, physiology, nervous system, reproductive, biology and so on.

And finally, now that we can manipulate the mouse genome, we can change anything we want in any conceivable manner. Could I have the next slide, please. So look. So whatever we learn about the function of the gene in the mouse, it's likely to also be the same function in a human. And I'll give you an example of that in just a minute. Okay. Next slide, please. So how do we go from cells, from changing a gene in a cell to making a mouse from it? We use very special cells. So these are embryo derived stem cells. And here we're showing you those cells are growing on a feeder layer and the feeder layer is another set of cells, which then produce factors which keep the cells happy. What are embryonic stem cells? They are cells that are pluripotent. They're sitting in limbo, haven't received instructions, whether to become muscle, or skin, or teeth, or hair, any type of cell in your body, these cells are capable of doing. So they haven't chosen anything. We can grow them forever. We can freeze them, put them back out of the freezer, keep growing them. And most importantly, we can use them to make mice. So these are the cells that we then genetically manipulate. Embryonic stem cells, and specifically mouse embryonic stem cells. Next slide, please. Okay, and here is how we actually make the mouse. This represents a needle that's holding a blastocyst. That's the circle. Okay. A sphere. And it is a pre, a very early embryo. It hasn't attached to the uterus yet. Okay. Represents just two cell types. There's the outer cells, which are going to be called the placenta, and the inner cells at the bottom, the inner cell mass, which are going to give rise to the embryo proper. In the needle, we have the embryonic stem cells, so we change a single gene. And we inject that right in. You can see it going into the embryo right there and depositing those cells that we've genetically manipulated right into that embryo. And then they mix with the cells in the inner mass, and then start making a mouse. Next slide, please. And here's how we make the mouse. So in the top diagram, on your left, we have essentially the blastocyst and the needle going in and putting the egg cells in. Once we've introduced the egg cells, they mix with the cells in the inner mass. Then we take that embryo, make a small slit in a mouse, a female mouse and deposit it right in the oviduct, which then travels down to the uterus, implants, and then starts making an embryo. A mouse embryo. Okay, and here we introduce a trick. The source of the embryo that we're injecting into the blastocyst, it's from, for example, black mice. The ESL, a source of the egg cells, were from a brown mouse in terms of its coat color. Okay. So then the first progeny that we make are both black and brown. And you can see that there, okay. And then that says, “The experiment worked, or both cells contributed to making the mouse. ” But most importantly, they not only contributed to making the mouse, but also sperm and eggs. So whatever change we create in that mouse, it can then reproduce and make as many copies of that as possible through breeding. And then by breeding, we can get mice that are entirely generated from the cells that we injected. The embryonic stem cells, where we changed a single gene. And therefore that single mutation now is in every cell of that body, of that mouse, but also in the germline so that we can breed males and females together and get as many progeny. And literally a mouse will have 10 to 12 progeny, every, all the time. Okay, so it's working. Could I have the next slide please. I'm going to give you an example. So, we have a gene that we're interested in, it's called hoxsey 13, that's at the top. Okay. And what we can do with that gene is introduce another gene. OFFICIAL TRANSCRIPT NATIONAL ACADEMY OF FUTURE PHYSICIANS AND MEDICAL SCIENTISTS © 2020 ALL RIGHTS RESERVED 4 JUNE 2020

And in this case, we actually introduced two other genes. One derived from bacteria that makes cells blue, and another one that is from jellyfish, and makes cells fluorescently green, called green fluorescent protein. So, by introducing these other genes in the middle of hoxsey 13, we destroy hoxsey 13 function. But still the gene that’s there, either blue or green fluorescent, will show the same pattern, the same cells will be functioned, will have those gene products as that gene is normally functioning in. Okay? So you'll have blue cells, or you'll have green fluorescent cells. And what you can see in this experiment, that in the embryo, the vibrissae, the whiskers of a mouse, are either blue or fluorescently green. Okay, that says that hoxsey 13 functions in those cells. So that experiments worked. We know where it functions. Then the next question is, what is it actually doing in those cells? Okay. So for that, we have to destroy both genes. Remember, we receive two copies of every gene, one from your father, one from your mother. If you destroy one, then you essentially still have a function because the other can do it. But if we destroy both then all of a sudden you see what happens. Could I have the next slide please. And here we see blue nails. Next slide we see that this gene also functions in the next slide. Fluorescent. Next slide. Fluorescently green nails there it is. Okay, next slide. The mice themselves. Here's plus plus, perfectly normal mouse. Plus minus, still a normal mouse. But minus minus, now we destroyed both copies. And now what do we see? No hair. So that tells us that this change is required for making hair. Next slide. This shows that it’s the same genes required to make nails because you have defective nails when you have minus minus. Those genes destroyed hoxsey 13, and therefore that shows what the function of that gene is. Now we can go to humans and ask, “Are there people that are bald and have dysfunctional nails. A) There are, we look at their DNA, and they have two defective copies in hoxsey 13. That tells us whatever we're learning in mice is also applicable in humans. Next slide, please. I'm going to. . Next. I'm going to go through the next 10 slides very quickly. Okay, keep going. Next, next, next next. This is my mom. Here's my mom living way up in the mountains. And she is a poet. And she went to Serbo. She got her training there. And there became very political, making a plack from Hitler Mousolini, saying that these guys are really horrible. You shouldn't follow them. That became very dangerous because it became very popular throughout Europe.

Here, what happened was that I knew things were going to go wrong. She actually gave money to a farming family. And here's the farm that I was going to live in when she was picked up. The Gestapo came, took her to Dachao. And I lived on this farm for about a year and a half. And then money ran out, and I was put out on the streets. And for five years I've traveled throughout Italy, getting my own food by stealing it, and living in shelters, which mean bombed out houses. And then five years later here, the next slide simply shows you where my mother had it even worse. She was at Dachau. And this is a picture from Dachau. Next slide, please. And after the war in 1945, Dachau was liberated. She actually survived. And then she spent two years looking for me. She finally found me. Next slide, please. And I was in the hospital. Next slide. Next slide. She found me in the hospital, with essentially I have typhoid fever and malnutrition. She recognized me, and then bought some clothes for me. We went to Italy.

We arrived Sunday night in New York after the long two week book trip, and looked at the Statue of Liberty all night long. Next morning, went throughout this island, there my aunt and uncle were waiting for me. And they took me to Pennsylvania. They lived in a commune in Pennsylvania, which was completely different from living in the streets. It was having now 160 parents instead of none. And they nurtured me and that provided opportunity for me to become a scientist. Next slide, please. This is myself just after arriving in the United States in 1946. Next slide. And this is my uncle. He was a famous physicist. He actually developed, first made, the first electron microscope and here he is doing something he didn't enjoy. Working on TV as a developer of both color and black and white TV. Next slide, please. From there, I went to Antia. Next slide, please. Next slide, please. And there I ended up at Harvard for graduate school. Next slide is my mentor. And this is Jim Watson, who actually worked out the structure of DNA with Francis Crick, won the Nobel Prize in 1963. Next slide, please. And then I went to Utah. And that's where I am now. And here we're looking all of this is Utah. In spring. Next slide. Summer, next slide and fall, winter. Next slide. So now I'm going to take you a very fast trip through Stockholm. Thank you. Next slide. And that's a trip to Stockholm. Beautiful city. Lots of, all the ocean, and this. All the cities, everywhere you look, your boats. Next slide. Beautiful hotels. We stayed at this particular one. Next slide. Here are the winners in 2007, Martin Evans, who developed TS cells which were crucial to the experiment. And Oliver Smithies. Next slide. Here he’s sitting waiting for the Nobel Prize. The Nobel Prize winners are in the front, and all of the 50 people that are involved in making that decision are in the back. Next slide. Here's the Kenyan queen, the king and queen. The next queen, this is over here on your right, and the, a younger sister, and the ceremony is beautiful. And all you have to do is follow the king. If he sits down, you sit down. If he gets up, he gets up. So you know exactly what to do throughout the ceremony. Next slide please. This simply shows a funny picture. Next slide please. And this shows receiving a Nobel Prize. Next slide please. And here is an intimate dinner of seven thousand people. This is the. . . Next. The queen and the person who won the physics prize from France, and then myself on the other side. Next slide, please. I should point out that the Nobel Prize ceremonies last 12 days. You get up at 630 in the morning, go to bed 330 in the morning, because every night there's a dance. So they keep you busy. You have to dance with many, many people, and then finally get to go to bed. Three hours later you'd have to get up and start the day over. 12 consecutive days, exhausting, but also exhilarating. Thank you.