 Well, it is a distinct honor for me this morning to introduce to you and to welcome back to Gustavus, Dr. Michael Blaze, the first Gustavus alumnus to be a presenter at a Nobel conference. While I was gathering some information for this introduction, I contacted some friends and some emeritus faculty and what, the one or two remaining faculty that had Mike in class while he was a student at Gustavus. And to a person, they remembered him. In fact, they remembered him quite well. So I started looking a little deeper, hoping I might find some little story or foundations for a story or two that I might share with you. And as I was talking with his daughters about this, they're kind of perked their interest a little bit, and they thought maybe he'll find something that's a little bit shady here, you know, that we can hold over old dad's head, but they were disappointed. I apparently, but I did not delve into his fraternity activities or a lot or into some of the other extracurricular activities. So maybe I talked with the right, with the wrong people. But so I really didn't, it was kind of disappointing to them, I think. I couldn't come out through with that. What I did find was unanimity, though, amongst these faculty and praising him as a student and a respected person around campus. He spent only three years at Gustavus going, attending the University of Minnesota after three years and transferring some credits back so that he graduated with the class of 1961. Although he did not create any unusual kinds of waves at Gustavus, he certainly has done so as a medical clinician consultant and research scientist. He became involved in immunological research while a medical student in the highly acclaimed immunology laboratory of Robert A. Good, the University of Minnesota. After an intern in residency program, he joined the National Cancer Institute or the National Institutes of Health in 1966, where he has worked for 26 years. Currently, he is head of the cellular immunology section of the metabolism branch and is very active. In the NIH clinical center. His work with rare immunodeficiency diseases in children had earned for him worldwide recognition. But more recently, he has integrated his clinical and research background in immunology with molecular biology and using the tools of molecular biology has been able to help develop gene replacement techniques that may open a new era in medicine where certain genetic diseases may be contained or even conquered. And in the well-known landmark experiment in 1990, Dr. Blaze and colleagues, Dr. French Anderson and Dr. Kenneth Culver, performed the first federally approved use of gene therapy on a human patient. This patient was a four-year-old girl with a denizen, deaminase, or ADA deficiency, a condition that paralyzes the immune system. And I'm sure that Dr. Blaze will discuss some of the results of this historical event. I believe that event also will be there are plans to include some of that the mementos and so on from that event in a history of American medicine being set up at the Smithsonian Institute. He continues his research interests in other immune, immune deficiency disorders, cancer, and AIDS. Dr. Blaze is the author or co-author of over 250 publications. He served as consultant, presented numerous lectures around the world, received a number of prestigious awards, and his active and other professional activities, as indicated in your program. But what is not mentioned in your program, but I think appropriate to emphasize here, is the distinguished alumni citation in medicine that is awarded to him by Gustavus Adolphus College in 1983. His concern for and willingness to contribute to the encouragement of young developing scientists is indicated by his involvement in intern programs. A number of Gustavus students, plus other undergraduate students from across the country, have had the privilege of interning in his lab while participating in the normal volunteer program at NIH, and he has maintained a contact with a number of these persons. And they remember his very warm and considerate and somewhat laid back demeanor, but also his keen interest, his high energy, his knowledge and the competency in his lab. I also would like to extend a hearty welcome to the members of his family who are here with them. This is sort of a double as a homecoming and a family gathering. His wife, Julie, is here as a Gustavus graduate of 1962, his daughter, Elise, a graduate from 1989, Christy, is a currently a student at the University of North Dakota, and his father, Robert, from Minneapolis. His daughters have assured me that dad is just a wonderful speaker. So I'd like you to join me in welcoming Dr. Mike Blaze, who will talk on the topic Gene Therapy, Medicine for the Future. It's really been a special thrill for me to be invited back to the Hill and to be invited to share this podium with the distinguished scholars and scientists that we've already heard. It's extra special for me because my family is here and especially because my dad is here. In all the years I've been in science, he's never heard me give a lecture about what my work is, so if you talk about pressure, we've really got it this morning. As I've been listening with you to the wonderful talks that we've heard yesterday by Dr. Manasaroff and American Gallo, I've been pondering about how to present my story. In these days of high-tech science, advances often take the contribution of many different individuals with a variety of different skills and talents and backgrounds. One part of this team of individuals is the physician scientist. It's our job to take the insights that have been gained from the basic scientists, so wonderfully presented in the last three lectures, and to try to figure out ways to apply these advances in knowledge to the treatment of human disease, that is to help our patients. What I've decided to do this morning is to take you on a quest with me. I'll try to share with you some of the sense of excitement and frustration, some of the advances and retreats and re-advances along the road to try to develop a new kind of therapy for human disease, that is gene therapy. To begin with, if we're going to talk about gene therapy, we should discuss a little bit about genes. Genes are the set of instructions or the blueprints of life. They're the set of instructions used for the assembly and function of all living things. So people have genes, mice, insects, pine trees, wheat seeds, bacterium viruses. All of the instructions that make up the development of each of these different organisms are related to genes, and all of the genes use the same fundamental coding apparatus. In human cells, the genes are located within a cell in the nucleus, and they're arrayed along structures known as the chromosomes, which are made up of long chains of DNA. It's the DNA that contains then the genes that we're going to be talking about. Let's start off with a definition so we can at least begin at the same point about what we're going to say that gene therapy should be. And we're going to call gene therapy the insertion of a gene or genes into human cells to get a new set of instructions to those cells. And you can think of at least two different ways to apply this technology. One would be to repair or correct an abnormal gene that has led to a disease. There are over 4,000 genetic diseases of man. You've all heard of sickle cell disease, juvenile diabetes, cystic fibrosis, and some of the diseases I'll be presenting later on are much rarer than these. But this is potentially a very powerful technology to use this coding information to treat other disorders. As you can also think of providing a new or an enhanced function to a cell. That is, if we could figure it out, we might be able to add resistance to T cells so they can't be infected with the HIV virus. Or we may program a cell to be more effective in attacking and killing cancer. So this is the kind of context that we're talking about for gene therapy. I'm going to pose some questions for you and I'm not going to give you answers to these questions, but I'd like you to think about the implications. Let's assume for a moment that you're all physicians and that you're dedicated to helping your patient. And now we're going to have several situations to think about. Let's assume that you're caring for a child with a disfiguring, extremely painful, and untreatable genetic disease. We may have another situation where the parents of a child with terminal cancer plead with you to help their child. You may have a couple of already had one child die from a disease that was inherited from them and they come to you and ask you to fix them. So that they won't pass that trade along to other children or to their grandchildren. You may have parents come to you with their newborn infant requesting that you give the child a treatment to increase for IQ to 250. You could have a couple come to you and ask that you treat them so their children and their grandchildren will have IQs of 250. A professional mining engineer might come to you and ask that you treat him so that he will be able to work in an environment heavily contaminated with asbestos so that he does not develop cancer. But you could also have a company come to you and ask that you develop a treatment for its employees so that they can work in an environment filled with carcinogens without incurring the liability for that company of inducing cancer. Interesting questions. There are many ideas about where to draw the line for this kind of technology. I think we could probably all agree or most of us will agree that the application of gene therapy for the treatment of a desperately ill child that has no alternative therapy is an appropriate use of this technology. Many of us would feel very uncomfortable with the idea that as a condition of employment you would have to accept gene therapy to make it resistant to something that might be in the environment of your workplace. Well, where do we draw the line and who decides? And all we can say at the moment is that line has not been drawn definitively. But there are some organizations, some structure in place to help us decide. And the principal one in the United States is the Recombinant DNA Advisory Committee at the NIH. And this is a committee that's made up of physicians and scientists. But it's also made up of attorneys and ethicists and lay people, clergy who get together and as a scientist when I want to do gene therapy I have to make a formal presentation before this body. And they discuss the ramifications both medical, the possibility that we might be successful, but they also discuss the ethical implications and it's this body that must give us permission. So there's someone out there looking out for your interests. There's someone asking these crazy scientists the hard questions and making us justify that what we want to do is within the bounds of at least what society believes is appropriate today. The Food and Drug Administration is also involved in giving approval for these different proposals so that you know that the materials that are being used are appropriate for human use. Well, one of the questions that was raised on that list of questions that I gave you was the issue that we'll talk about saying somatic cell versus germline gene therapy. What does this really mean? We talk about somatic cell gene therapy as the insertion of genes into the diseased tissues of the patient only. So that you try to correct the disease cells but you specifically avoid introducing these exogenous genes into the reproductive tissues of the patient so there's no possibility that they could transmit these genetic alterations to their offspring. That latter technique would be called germline gene therapy. So if you want it to be fixed so your children don't get a disease and you don't transmit it to your grandchildren, the only way we could really do that would be to affect your reproductive tissues and to remove a defective gene or alternatively add something to it. Now it's been the decision of the Recombinant DNA Advisory Committee in this country and in fact in all countries in the world that are considering these kinds of technologies that will restrict all of our efforts at least at the moment to somatic cell gene therapy. But I'm sure in the future there are going to be discussions and that we as informed citizens are going to have to keep aware of these discussions about the potential uses and potential abuses of germline therapy as this becomes technically possible. Well how do we achieve gene transfer without exposing the germline to these exogenous genes? And so I've listed a couple of things here called ex vivo and in vivo treatment. In vivo treatment would mean that we would take the genes and we would infuse them into the patient. Now there are many genetic diseases where you might have to do this procedure and we can talk about it later on but it's a real problem because obviously if you infuse genes they have the opportunity to reach the sperm and the ova. That is they have the opportunity to reach the germ tissues of the individual. So at the moment we're not going to consider doing in vivo therapy. What's the alternative? Well the alternative is something called ex vivo treatment where you remove the diseased tissue from the patient, take it to the laboratory and attest to, insert the corrective genes then in the laboratory and then re-infuse the corrected cells back into the patient. So we're going to focus at least initially on this ex vivo treatment and there are some real constraints now about the kinds of diseases that we can think about treating. What cells can you remove from the body and effectively put back? Well we know that you can do bone marrow transplantation so you can take the bone marrow from someone. Theoretically you can remove skin or some of the cells lining the blood vessels or the immune system cells that they'll emphasize that we've heard about in this conference. It might be possible to remove liver cells, possibly some kidney cells but there are lots of cells that you can't effectively remove. We can't take the brain out of someone that has mental retardation, treat it in the laboratory and give it back. So we're very restricted. You can't remove all the skeletal muscles from a patient that has muscular dystrophy, fix them and give them back. So this constraint of doing ex vivo therapy severely limits what we can do at this stage in the development of this kind of therapy. Alright now as physicians we're going to think about what are the candidates that are going to meet these requirements that we've talked about. First of all we certainly want to pick a disease that's a very serious and life threatening disorder. This is a new technology, it's not trivial and we don't want to try it on something that really doesn't need extreme kinds of treatment. We obviously have to have a disease where we've isolated the gene. As I said there are more than 4,000 genetic diseases but the number of genetic diseases where we actually have isolated the gene that's responsible for that is a relative handful. It's growing every day and part of this human genome project that you've heard about in the press is to try to map the entire human genome so we have the genes available so we can study them, understand how they work and potentially apply them for treatment. We want a disease where the treatment is not adequate. If you have a perfectly satisfactory treatment there's no sense in taking any potential risks of this kind of new treatment. Some technical issues we want a gene with simple regulation. What does that mean? Well let's say that you were going to try to do gene therapy for diabetes. That you would want to give an insulin gene. You know that the amount of insulin in the body changes drastically with time. It depends on how much carbohydrate you have in your meals. So that would require a gene system that would allow insulin production to respond to those physiologic signals. That's really a tough problem and it's too hard for us to approach just yet. So we want something that we call a housekeeping gene. A gene that's simply been turned on and works at a certain level and doesn't have to be modulated in its level of expression. We want a gene for a single-chain protein. What does that mean? Well a lot of the proteins in our body are actually made up of multiple chains of protein that are coded for by different genes. One example is for instance hemoglobin, the red pigment in your blood cells. Hemoglobin is made up of two different chains. But during development from the time of the fetus to adult there are actually four different hemoglobin molecules that are expressed at different points in differentiation. Well to do gene therapy for a hemoglobin disease you'd have to put your corrective gene in and then figure out how to get to express at the appropriate time in development so that it only came up when it was needed. Now that is also a very difficult problem so we're going to be restricting ourselves to situations where we only have a single chain that we have to worry about. So related to that is we want a disease where the defective gene does not produce the pathologic gene product. What does that mean? Well all of you have heard about sickle cell disease which is a hemoglobin disease predominantly of blacks where the hemoglobin molecule becomes rigid and so the red cells cannot squeeze their way through the capillaries. Now that's a defective gene that produces a defective gene product. So even if I knew how to put the correct hemoglobin gene into those patients I'm going to have to figure out how to get rid of the bad one. So I have two problems and again that's a difficult problem that we're just not going to be ready to handle. And finally as we've already discussed we need a tissue that permits exfivo treatment. And the disease that fit those criteria are best illustrated by Chelsea here one of our patients with severe combined immune deficiency. Severe combined immune deficiency was popularized I think in the press if you remember back a number of years to David the bubble boy who was put in a plastic bubble from the time of birth in Houston where he lived. He was kept in that isolated sterile environment of a plastic bubble until he died at about the age of 12. He was put in that sterile environment because he had no defense system. He had no T cells or B cells and so his immunity basically did not develop and he would have died of overwhelming infection with just a matter of weeks if he was removed from his plastic bubble. Well Chelsea here has a similar problem but we know the gene that causes Chelsea's problem and it's a gene for a protein enzyme called adenosine deaminase or ADA. We know that Chelsea's disease can be cured by bone marrow transplantation and that's very important because now if we can cure her by giving a brother or sister's bone marrow we can also potentially remove her own bone marrow insert a corrective gene into that bone marrow and then give it back to Chelsea. So one of the requirements she would fit this ex vivo treatment model. We also know that the ADA corrected cells in this disease should have a what we call a survival advantage. What that basically means is that the cured cells will ultimately take over from the defective cells in her body. We don't have to do something special to kill off the remaining defective cells. We don't have to add any extra insults to her like total body irradiation or chemotherapy that's often has to be used with bone marrow transplantation. Now this slide is going to show us the pathway for the chemists in the audience. This is deoxyadenosine which is a toxic substrate that accumulates in the body of these children because they're missing this enzyme adenosine deaminase. And what ADA does is remove this amino group and convert deoxyadenosine to deoxyenosine which can then be excreted in the urine or removed by other catabolic pathways. Now in the absence of ADA this compound which is ordinarily not found in cells accumulates to very high levels. And unfortunately as it continues to accumulate it gets forced along one metabolic pathway in the immune cell specifically. And it becomes phosphorylated to make a compound called deoxy ATP which is a poison and it kills the immune system cells in these children. So basically these kids end up suiciding their own immune system because they can't get rid of this toxic substrate. Now as I mentioned the strategy here is to try to put the corrective gene in the bone marrow stem cell. This slide looks at the development of our immune system and our blood system. So down here in the bottom are all the mature cells that are found circulating in your blood. They have the red cells and the different granulocytes and monocytes and the T and B cells from the immune system. And we know that all of these cell types originate from a particular specific grandmother cell that's found in the bone marrow that we call the bone marrow stem cell. And as you need more lymphocytes the stem cell will differentiate to different progenitor cells which will ultimately give you a mature T cell or B cell and so forth. Now for gene therapy what we would like to do is put the gene up here. That would be the ideal place because if this cell is genetically corrected then it's called upon to give off daughter cells to make red cells or T cells. All of those cells will be genetically corrected. So one treatment should theoretically cure the patient's disease. And so that's what we're going to be focusing on to try to do stem cell gene therapy. Now how are we going to get the gene in? Well there are lots of ways in the laboratory that we have for tuning genes but nature has been working on this problem for several hundred million years. And we might as well try to take advantage of what nature has been doing and nature in fact has infectious genes. Those are the viruses. And so what we're going to try to do is develop a strategy for taking infectious genes from nature and changing them so that they do our business for us without causing disease. And we're going to focus on the retroviruses, something that you heard about extensively from Dr. Gallo yesterday. Now Dr. Gallo showed us a slide similar to this. Let me explain why a retrovirus is particularly interesting for our purposes. If you remember back to that slide that had the reverse tree structure going from a stem cell on down, you have to recognize that each time a cell divides you have the possibility of losing an inserted gene. If you put DNA in there it can be diluted out as a cell goes through multiple steps of division. So what you really want is a gene that will go into the nucleus and stick itself into the chromosome. So as the cell divides and the chromosomes duplicate themselves the new gene will go along with it. So we're looking for a gene insertion process that would integrate. Remember Dr. Gallo told us that retroviruses integrate into the genome. What does that mean? Well here's a retrovirus that binds to the cell surface receptor. And it inserts its RNA into the cell along with some little dots here called reverse transcriptase which convert the RNA molecule into DNA which can then go into the nucleus and it inserts itself randomly into the chromosome of a cell. Then that gene is just like any other gene along that chromosome. It can be transcribed and translated into proteins. In this case the proteins are going to be the virus protein so that this virus can make more viruses. But this is a very attractive feature. This is the ability to integrate because it would suggest that if we can figure out how to use this system for gene transfer we'll be able to integrate the genes into the chromosomes to be able to handle something like the immune system which is actively dividing. Now you heard about retroviruses and they sound nasty things aren't they? I mean HIV is a retrovirus. So what we should do is to try to use the properties of the virus that integrate but get rid of the bad parts. And this is a very simple series of slides that are oversimplified but if you remember Dr. Gallo said that the retroviruses, at least this is a mouse retrovirus called the Maloney-Muring leukemia virus and it only has three genes. It's the simplest of viruses. And it has these things out on the ends that are called the long terminal repeats which Dr. Gallo pointed out are the places where this virus will integrate into the human genome. So we'd like to keep these parts but we want to get rid of the bad genes from the virus. So we're just going to cut them out and throw them away and then glue in the place a gene of interest. Now it's not quite that simple but you got the idea. Now this particular gene is a gene that we've taken from a bacteria. This is a gene that actually encodes for resistance to an antibiotic called Neomycin. Now you can put, so now we have a virus that we're putting a bacterial gene in and let's make it more complicated, let's put another gene in there. We're going to put in the human ADA gene. So now we've made this complex structure which still has the long terminal repeats so it should be able to integrate but there are no viral genes left in there. So it can't produce more viruses and it can't cause disease. So we're going to try to take advantage of this hybrid guy now to insert the ADA gene into some T cells from patients with this disease. Now this is a data slide and let me explain this a little bit. We've taken T cells from our patients and T cells will grow very actively in tissue culture and that's what this shows. So you get more growth as you go up. And now we're going to challenge those growing T cells with that toxic substrate, the deoxyadenosine. And the red line here, I'm sorry these are not drawn very well but this line here is what happens to normal T cells. And you have to get out to a very high concentration of deoxyadenosine before you inhibit the growth of the normal T cell. You have to get out to about a thousand units of deoxyadenosine. This is a T cell line made from Jimmy Fox who was my first patient with ADA deficiency and you can see that Jimmy's T cell line is inhibited by only about 3% as much deoxyadenosine as the normal. And that's an accurate reflection then of the deficiency of this enzyme that they just, the cells die when they're exposed to this toxic substrate. And we're going to take this population of cells and we're going to put that ADA virus into it. And lo and behold, here's what happens when you put the ADA gene in using the retrovirus. You can metabolically cure these cells using this technique. Now this was very exciting data that we had back now, seven years ago, but in 1985 and we were wondering, gee, we can cure these cells and we start treating these patients. But we thought before we went into patients that we really ought to step back and think about it a little bit and to try to show that this technology would work in an animal model and particularly in a primate. Now there's a problem here in that we don't have any ADA deficient animals. So we have to figure out some way of testing it in a system that will tell us whether it potentially could work. And since these techniques have never been used in man, we went to non-human primates that would be as close to man as we could do. And we did a bone marrow transplant with this gene therapy and let me work you through this. So we took rhesus or cinemologous monkeys and we isolated their bone marrow and we mixed it with this retrovirus vector to insert the gene into the bone marrow cells. While this was going on in the lab, the monkeys received total body irradiation to kill off any bone marrow that was still there so that when we gave them the bone marrow transplant they would have to get their marrow from the infusion. So that hopefully if the gene went into these cells when these animals recovered they should now be producing human adenosine deaminase. And this is the result from two of our monkeys Bonnie and Clyde. You get a prize in my lab if you come up with good names for the animals. But here's the effect on the white blood cell count of these animals that got total body irradiation. And this is what we see, the white cells go away. And then the bone marrow transplant starts to work and you can see the white count came up to normal. And we got very excited out here at about two months when we started to detect the human ADA protein in the blood cells of this monkey. And we got even more excited when the value started taking off and then our emotions went the other way as we saw this very drastic fall off and finally the disappearance of the human protein in these monkeys. Now this isn't going to work. This is the best we can do for gene therapy. There's no sense in going ahead. And it was a very bad time for us because we couldn't figure out what was going on why it wasn't working for us. And I'd like to, I mean what happened is it didn't work and where do we go? I don't know but let's think about where we might go. We can think about what was the problem and the problem comes back to this initial diagram that I showed you. And we're trying to get the gene in up here in this toticotin bone marrow stem cell. Well one of the things that Dr. Gallo mentioned yesterday about retroviruses is they only insert their genes into cells that are actively dividing. A cell has got to be synthesizing DNA for a retrovirus to insert its gene. And unfortunately this cell is usually just sitting there. It's not actively dividing so it's not susceptible to the gene transfer system that we're using. And what happened in Bonnie and Clyde is that we got the gene way down in here. These cells are very actively dividing because they're actually providing the peripheral cells with the mature blood cells and lymphocytes that we look at. Eventually these cells will die off. They'll synapse and be replaced by a more immature cell that never got the gene. So we see this burst of expression of our introduced gene but it disappears. Well we're really frustrated now because we couldn't seem to get the gene in the cell we thought we had to get it into. And then it occurred to me as I was writing a chapter for a textbook and reviewing some of the literature that there might be a way for us to actually go at this cell. I've already showed you that we could cure that cell in the test tube. And so we're going to look to see whether a T cell can be used for gene therapy. Lymphocytes have a number of properties that make them sort of interesting. As you know they're the cells in the immune system. They're present in the peripheral blood so they're very easy to obtain. You don't have to chop out some of these liver to get the appropriate tissue as an example. They're very simple to grow in tissue culture. We know a lot about these cells. They're probably the most widely studied cells in biology. T lymphocytes can actually be very long-lived in the body. One of the examples I often use is that I had my last tetanus shot over 30 years ago. But I'm still immune to tetanus. And it's conceivable that the cells that saw that tetanus antigen 30 years ago are still around. So if I had put a gene in those cells 30 years ago, it might still be in my body and functioning. Finally, T lymphocytes have antigen specificity and they can actually go to deposits of antigen in the body which might be important if we were trying to deliver a gene to a specific problem like a deposit of cancer. So we started looking at lymphocytes and again we've been to animal models. And this is just to remind me to tell you about these studies briefly. We took T cells from a normal mouse. We put the gene for human ADA in that normal mouse. And then we gave the cells to this guy that you've also seen. This is a nude mouse, a mouse that does not have a thymus. So it doesn't have any T cells of its own. So we could follow how long the T cells survive because they should have come from the original donor. And in fact, after a lot of studies in mice and then in monkeys, we showed that in fact T lymphocytes would work. And we can get expression for a long period of time if we put these foreign genes in this cell type. So we were ready to go. Let me read this for you though. One of the questions, if you're talking about using retroviruses in man, you know the prince here is saying it wasn't black magic, it was mead contaminated with somebody's recombinant retrovirus. Okay? We're concerned about the implications of using these modified viruses. They'd never been used in man. And so we started on an odyssey of getting approval for this process. And I just say that this is sort of a flip inside, but we spent seven years of very extensive studies looking at the safety of these retroviruses in a whole variety of animals including about 50 different primates to demonstrate that in fact this procedure was safe. But then we had to convince these guys and this is a review, a list of the public reviews that we went through for our first gene insertion starting in 1987 and finally 18 months later we were authorized to go ahead. Now the first use of gene transfer in man was not to treat ADA deficiency. I really didn't think we'd ever get permission to go into a child. And so we looked for a different population of patients and the first study was done in some patients with cancer and I'll come back and describe that later on. But what it showed us when we did the initial studies in cancer patients is that this procedure was safe and it was effective and so it allowed us to go ahead then and try to treat ADA deficiency. Now, we have another problem. You always have to consider the alternative treatments that are available to you before you decide whether you should take the risks associated with introducing something new. And for ADA deficiency there are a number of important alternative treatments. This is an incredibly rare disease and I was asked last night how we could justify spending the money to treat such a rare disorder when there are so many people with poverty. And I gave the response that studying rare disease is often very important and let me just say how important this has been. Because these children are so desperate and they're dying within a few months of life, there have been a lot of innovative new treatments introduced for the treatment of severe combining inefficiency. Bone marrow transplantation which was first used to treat SCID as you now know is used to treat all sorts of cancers and other inherited disorders. It's used to treat thousands of people a year. That treatment was developed to treat this disease. Enzyme replacement which is a very important concept in therapy of actually giving the missing enzyme to a patient which has actually been used in ADA deficiency. The point was also proven in this disease that you could give exogenous enzyme to treat a disease. And now as you'll hear gene therapy is also being applied to this disease. So this disease that affects five or six patients in the United States a year has had an enormous impact on the development of new and innovative kinds of therapy. Well the purpose of this slide is just to talk about these alternative therapies and what we can do. The treatment of choice for this disease is bone marrow transplantation if the patient is lucky enough to have a matched sibling donor. Unfortunately because family size is so small in North America, less than a quarter of our patients now will have a brother or sister that match exactly. So we have to look for some other kind of treatment. Those other treatments involve using donors that are unrelated. You've all heard the advertisements for trying to have you come in and have your blood type to be a potential bone marrow donor. There are now several hundred thousand people in North America who have been tissue typed and we're now doing more and more bone marrow transplants from unrelated individuals that happen to match for these tissue antigens. It's still not perfect though and about half of the children that get these other kinds of transplants still die. Enzyme replacement I'm not going to talk about but it was another way of trying to treat this disease and it works very nicely for some children and not so well for others and we don't understand it. But what we decided to do was to take children that don't have a donor and who are not doing very well in this enzyme replacement and try to do gene therapy. Now bone marrow stem cell correction I've already told you we can't figure out how to do it so why do I think that we could go for T cell correction? And I'll explain it on this slide and basically the observation came from those children that were cured. I think we have a slide to drop here. The children that were cured by bone marrow transplantation from a brother or sister. Maybe we won't have a slide drop. Okay if we had children that were treated with this procedure and were cured and now we study those children four or five years after they're cured of bone marrow transplant. What we found was that their red cells and their granulocytes and their platelets and even their B lymphocytes still had the defective gene in them. They were still 88 efficient but the only cell type that took from the donor was the donor T cell. Now if you can cure these kids by giving somebody else's T cells then maybe if you take their own T cells out and put the gene in you can also cure them. So that's the strategy that we've developed for treatment. Let me go through this protocol with you. We take children with this terrible immune deficiency that have been receiving the enzyme treatment and that helps some. We take their lymphocytes and their peripheral blood by a process called leukophoresis and basically that just allows us to collect lymphocytes but we give the red blood cells back so the kids don't get anemic. We take those to the laboratory and we start them to divide by stimulating their T cell receptors with interleukin 2 which is the T cell growth factor and a monoclonal antibody. Once these cells start to divide we can use a retrovirus vector to put the ADA gene in and that's exactly what we do and then we grow the cells up in tissue culture for about ten days and give re-infusions of these now gene corrected cells to the patients. And this is the results on our first old girl that we started treating back in September of 1990. Let me go through this with you. Here we're looking at the number of lymphocytes in her blood and the normal value is up here. So you can see when she was initially diagnosed she had essentially no T lymphocytes in her blood and she had experienced numerous recurrent serious infection by this time. They initially started to treat her with enzyme and you can see that her T cell count came up very nicely and then during the second year of treatment this improvement deteriorated and she started having more infections again and they tried to increase the dose of enzyme and it didn't help and she basically was getting worse. We started looking at her at this time point and we evaluated her immune system very carefully and found that she had severe immune deficiency and we began gene therapy on this date and you can see that within three months of starting treatment that her T cells came back up to normal. Now what about this inserted gene? Is it actually working? This is the level of ADA in the blood of this child actually being made by her own cells. You can see that prior to gene therapy she had essentially no ADA and then each of these arrows represent times of treatment. She comes in for an outpatient treatment and you can see we had the steady increase in ADA until we stopped treating her and then it stayed steady. We've given her a couple of treatments in the last year and her ADA level has stayed in this range. Now to give you a reference this is the value of her father. So now she's gone from having essentially nothing to having a value that's about half as much as her dad. What's it's doing to her immune system? We're going to look at this panel down here. This panel just shows her lymphocytes after we started treatment and we had some bouncing around and finally it's normal. This looks at antibody production to something called an isoglutinin which is, as you know, if you're blood group O you make antibodies to A and B red cells. And we can use those antibodies as a way of determining whether you can make an antibody response normally. This little girl was blood group B so she made antibodies to A type red cells. Before treatment she had essentially none and within three or four months she's making a normal response. Now we started treating a second little girl in January of 1991 and you can see she also had a very nice increase in her lymphocyte count and one from making no isoglutinins to normal response. So this treatment is resulting in improvement of their immune system. The other side of the immune system is the T cell side. We've looked at antibody production and she's getting better. This is a representation of a skin test. All of you have had tuberculin tests put in your arms where you look for a red bump as an indication that you've been exposed to tuberculosis. Well that kind of immunity is very important in protecting us against a number of infections. And if we skin test with things that she should be immune to things like tetanus or diphtheria or some yeast in our environment what we found is that prior to treatment the children could not give positive responses to anything but after we instituted gene therapy they both acquired normal cellular immune responses. If you can drop that slide so let's summarize what we've seen in these children then. We initially wanted to put the gene in this stem cell and the idea was if you could get it in the stem cell then the B cells and the T cells would both be corrected. We couldn't figure it out so we had to step back and look at an alternative treatment and that was putting the gene out here in the mature T cells and it looks like the strategy is working because now their immune system is coming back their T cells are working and they're making antibodies. Now the most important story is how are the kids doing? You know we started with two children and we've treated and the two children are right here our first little girl the four year old and a nine year old and the reason I show this slide is that this four year old only got out of the house to go visit the doctor before we started this treatment. The parents didn't take her in public because they were afraid of exposing her clearly she's being exposed to the public here this is a party we had for the patients and these are my co-workers in this study and their families but the two children were at the NIH for this party the parents enrolled Ashi in kindergarten the following year and last year she went to school with the rest of the kids with snotty noses for the entire year and did wonderfully. So both of our children clinically have done exceptionally well and we couldn't be more pleased. Now I'd like to change gears for just a minute and talk about other applications when I listed on that initial slide for gene therapy we talked about correction of a genetic disease and also the insertion of a gene to provide a new function or property to a cell and the reason why that's important is that this is a very powerful technology and we should be able to use it for treating other diseases and specifically since I'm in the cancer institute we're going to think about treating cancer and I'm just going to show you a couple of examples this is a chest x-ray on the left here of a lady with various serious metastatic disease and these two big balls are big balls of cancer from a kind of skin cancer in her lung Dr. Steve Rosenberg, a colleague of mine at the NIH has been working on ways of trying to make the immune system work better at killing cancer and he treated this lady with immune T-cells that he actually grew from one of these tumor lumps and you can see six weeks later most of those tumors are gone so the immune system can be a very powerful approach to treating cancer unfortunately it doesn't work very often so what we're going to try to do is spend some time figuring out if we can make it work better and I'm just going to give you a couple of examples this is the lung of a mouse that has a cancer in his lung and all this pink stuff is tumor the white areas are air you can see there's not very much air left in this lung there's a lot of cancer there what we're going to do is insert a gene for a cytokine you heard about cytokines yesterday these are molecules that the immune system uses the cells in the immune system talk to each other by secreting these molecules called cytokines and we're going to put a cytokine into this tumor and then give it to another mouse and this is at the same time point what the lung of that mouse looks like first of all you can see all of this air there's not so much tumor but I think you can see even from the back of the hall all of this black stuff or this dark blue stuff around each of these nodules of tumor what that represents are lymphocytes immune T cells are being recruited to the cancer now because the cancer has been modified to produce a factor which activates the immune system and it's our hope that using this approach we're going to be able to figure out how to improve the body's defenses against cancer so that we can use some of these properties of the immune system to help us in fighting off this invader now here's another mouse with two lumps of cancer this is a what we call a wild type or the regular cancer and this is the same tumor but this time we've genetically modified this tumor and let me help you think through this you know penicillin is a great drug because it kills strap and it doesn't kill us I mean it's not like taking chemotherapy for cancer it's very effective against microorganisms and it doesn't hurt us and wouldn't it be nice if we could treat tumors with penicillin well the bacteria are killed by penicillin or antibiotics because of their genes encode for certain things that make them susceptible to antibiotics so if we could put into this tumor the gene that makes it susceptible to an antibiotic we might be able to treat cancer with antibiotics and that's exactly what we've done we've taken a gene from a fungus for an enzyme called cytosine deaminase which actually turns on an antibiotic to kill the bug but in this case it kills the cancer so one can potentially use gene insertion of very unusual things now we're putting a bacterial gene in to a human cell to allow us to treat it with a new kind of pharmaceutical approach now how could we really apply this to cancer treatment in man and here's one way that's going to be starting this fall patients with brain tumor are often desperately ill and one of the problems with brain cancer is often the cancer is in a location that surgically you can't get at you simply can't cut through critical tissues in the brain but you could theoretically put a long needle into the brain area and so we're developing techniques for actually inserting genes for these susceptibility factors into brain tumors so that the gene will be taken up by the dividing brain tumor cell and then we're going to treat with an antibiotic or in this particular model we've taken a gene from a virus called herpes we've put the herpes thymidine kinase gene into tumor cells and this is a rat that's had this treatment done he has a big tumor in this part of his brain and he had the herpes gene put in but the animal was just treated with saline and you can see this huge tumor that's grown out from the rat's brain and we're going to take exactly the same animal but we're going to treat it with the drug the antiviral drug called Ganscyclovir and you can see this litter made animal now all of that tumor has been destroyed both macroscopically and microscopically we've seen no evidence of persistent tumor so now we've been able to develop new ways of inserting genes to help us treat cancer and we really hope that in the next four or five decades that this new kind of treatment is going to revolutionize the practice of medicine and I'd just like to close with acknowledging many of the people that are involved I told you that this took a lot of people to be involved but my fellows in particular Ken Culver and Don Cohen were very important in developing these strategies Steve Rosenberg and Ed Oldfield are the surgical colleagues that I work with for both the neurosurgery and my partner in these studies for the last eight years and I'd like to close with this cartoon that I stole from the Washington Post to try to get some feeling for where this is going there's been a lot of press about gene therapy you have to recognize there have only been two or three patients in the world actually have received this treatment so far but it's such a powerful approach that I'm really sure that it's going to change the way healthcare is delivered around the world within the next few decades thank you