Cloning Dolly, How and Why?
Transcript Part 4: Practical Applications
So what are the applications of this? In my opinion, the first major applications will be by pharmaceuticals--the production of human proteins in the milk, or the urine, or the blood of farm animals, for treating human genetic disorders.
Xenotransplantation, the use of organs from farm animals for transplantation to humans, or nutroceuticals, which is a term given to the modification of animal products that may have health benefits, but they are not particularly medical benefits. It may produce low fat milk for example, coming straight from the cow.
Just a few of the projects that we were involved in when I worked for PCL Therapeutics-- alpha 1 antitrypsin, which is actually now in phase III clinical trials [and] may help treat cystic fibrosis and emphysema. Factor 9 for treating hemophilia...so there is a whole range of them. We have animals that produced all of these.
What are the problems of xenotransplantation? People have [determined] that the pig is probably the animal of choice, and that the organs would be about the same size and would probably function. If we transplanted a pig [organ] into one of us, it would last about 20 minutes and we would get rejection. However, by using nuclear transfer, we could take pig cells, and possibly genetically modify them so that they wouldn't be rejected after transplantation. We could make the embryos, make the transgenetic pigs, or take the genes away and then do the transplant, and hopefully get rid of some of the organ rejection. I know there are lots of other problems with xenotransplantation, but this is what people are working on--trying to modify the immune response to pig cells.
There are lots of other applications--in studying human disease, in studying development in aging, in studying this cell differentiation process that I was talking about in the first place, in studying reproduction. In agriculture we may be able to make transgenic animals resistant to specific diseases, for instance, improving the quality of life of farm animals. We may be able to use it for genetic preservation. Everyone says if you clone everything you get rid of genetic diversity, but I would argue that we are losing genetic diversity anyway. By freezing cells we have one way of preserving genetic diversity. And in all the news at the moment is this stem cell business, which I will come to in a minute.
Genetic preservation--these are clones of this animal, here. The sheep is still alive. These were made in New Zealand. They are all black and white and they are all calves, but the black and the white bits are all in different places, even though they are cloned or supposedly cloned..
I have just talked about all this genetic modification using cloning and using fewer animals. Can we do any of it? In order to do it, the cells have to be able to sit in the dish, go through all these manipulations, and still work when we do this nuclear transfer.
This is Polly. This is the first animal we have produced by nuclear transfer, who was transgenetic. She carries the gene for human factor 9, the clotting factor, and produces that in her milk. She was born in 1997. As I said, you can make many flocks...you can just make a few of these animals, and they are all still kicking around and quite healthy.
You just need to look at this bottom line here. This is this pro-nuclear injection method. We needed 50 animals to make 1 transgenetic, and this is the nuclear transfer--we only used 20 animals. So [the research process] is also a boon to animal welfare.
Just to show you a few others--these produce an enzyme called extracellular superoxidedismutase. This animal here produces a neuroactive peptide called calcitonin, which may be useful in preventing the onset of osteoporosis. And we have a number of others. But that is just adding a gene. What about gene targeting?.
Gene Targeting:
Normally, gene targeting is done in the mass. And you use these things called embryonic stem cells. You can take these embryonic stem cells, and you can do this gene targeting or precise modification, inject them into a blastocyst, and you end up with a chimera which is basically a mass which has four parents.
You can then breed, and you end up at the end of this time with a mass which is just derived from these cells. Now that is all very well and good in the mass, but you try doing that in a calf, it is 9 months' gestation. It takes over a year to get to sexual maturity. It is a very good experiment if you want to sit down on your backside for a long time but the mass, remember, breeds very quickly.
We tried to do the same thing in sheep using our fetal cytoblast and we did two things. First of all, we tried to knock out a gene and we chose a collagen locus, which is a highly expressed gene. Secondly, we tried to add a gene to a precise location in the genome, and we went back to our old friend alpha 1 antitrypsin because we knew quite a lot about it. These are the first gene-targeted sheep, Cupid and Diana. Cupid is actually a boy's name but they are both females. It has to do with bows and arrows and hitting a target..
And what sort of genes do we want to knock out in animals? These are just a few examples: The mad cow disease gene--we could get rid of mad cow disease, but mainly to do a lot of research on it so we could understand it. To make less allergenic milk. Take out alpha 1 three (inaudible) transferase which is an enzyme which puts a carbohydrate (inaudible) onto pig cells involved in graft rejection. To replace the bovine serum albumin gene with a human serum albumin gene so we can produce lots of human serum albumin for treating burn victims. Also to make human antibodies in calves, again for treating human diseases.
Thoughts on Human Cloning:
Those are some of the things I have been doing with animals. [Now] I want to talk about the thing that we all don't like talking about, which is cloning in humans. This is a great magazine. I don't know if anybody reads it, Time for Kids. It is a very good magazine. So, now that scientists have cloned a sheep, a human is next..
Well, can we clone humans? The answer is, probably. Nobody has tried yet...not that I know of...but it is probably true. It may be slightly different than doing it in sheep because the biology is slightly different, but there is no reason we can't clone humans.
But there aren't any medical reasons for cloning humans that I can think of, although I will give you one a little bit later. Everybody thinks when you talk about cloning that this is what we are after. The Boys from Brazil scenario where we are cloning lots of identical people to carry out nasty and nefarious deeds.
But in fact, if we did clone humans, would we produce the same person? Well, it depends how much you think that genes are responsible for personality and behavior. And I don't want to get into this, but there is a big nature/nuture debate. It appears that we do have some genetic component to personality, but I don't think it is the be all and end all, so that it depends on your interaction with environmental, familial, socioeconomic, or culture and other factors. If we produce a bunch of Adolph Hitlers would they all behave like Adolph Hitler? They might all be quite nice people. We don't know.
But there are lots of hazards. As I have said, [cloning] is not very efficient. There are lots of abnormalities. We don't know what is going to happen in the long term with these clones. There will be a lot of pressure on the child to behave like the person they were supposedly cloned from, and I don't know what it would be like. I can't imagine bringing myself up. It is bad enough living with myself.
Anyway, are there any uses of cloning in humans? Could we use it to remove genetic defects from embryos? Possibly. At the moment we have pre-implantation embryo screening. You take out a cell from the embryo, you check if it has got the disease. If it has, you just squish the embryo and it doesn't get transplanted.
People have suggested that we should use it to revive dead people. But would they be the same? And don't forget they come out as babies, not as full-grown adults. To treat infertility and for non-heterosexual relationships. I think this all needs debate. I have my personal views and I am sure everybody else does..
I am opposed to reproductive cloning and can think of no medical reasons for it. Apart from this one--but then you could say this isn't reproductive cloning because we are not cloning or copying someone. If we were to take a zygote, which is the product of a sperm and an oocyte, so it would be from sexual reproduction, it would be a unique individual. But it has got a genetic defect. We grow this embryo in a dish for a while, take some cells out, fix the genetic defect, clone it, make the fetus, transplant it back into it's mother and then throw all the cells away. We have ended up with one baby, which is the product of sexual reproduction, so it is a unique individual, but we have corrected a germ line defect or a genetic defect along the way..
Are there any other uses? There are some cloning-related techniques. There are some maternally inherited barriers to development, mitochondrial disease and cytoplasmic dysfunction. It is not really nuclear transfer, but if we start off with a defective oocyte...we could take some cytoplasm from another oocyte and put it in to help rescue it. Or we could take the nucleinase and put them into another oocyte. It is cloning-related but it isn't cloning. It is simply rescuing a defective zygote, but the product in terms of genetics, is still a unique individual..
Stem Cells:
Now, I am not going to talk a lot about stem cells, but what is a stem cell? It is an undifferentiated cell that has the ability to become a specific differentiated cell type. I have mentioned embryonic stem cells, and these have the ability to contribute to all of the tissues of the conceptors.
So, embryonic stem cells come from an early embryo, from the blastocyst stage, and we can keep them indefinitely in culture. In a dish, in vitro, they can differentiate into all different cell types, but we know they can contribute to all of the tissues of the conceptors, because if we take some of these cells and we inject them into another blastocyst stage embryo, we end up with a mass with 4 parents, again, which we can then breed on. We end up with a chimera, and we can find contributions of this ES cell in every tissue in that mass.
Now there are lots of different types of stem cells. The embryonic stem cell comes from the embryo. There are some that are derived from the fetus, derived from the early germ cells and they have been termed EG cells. There are stem cells that are found in the cord blood, and there are organ and tissue-specific stem cells. More recently we have also been finding a lot of adult stem cells.
But what role would nuclear transfer have in stem cells? What if we could take a cell from someone [with] a particular disease (I will come back to a few examples in a minute). We take some differentiated cells, go through a nuclear transfer, and make embryonic stem cells. That would be quite interesting. But why would we want to do that? Well, we could then differentiate them into specific cell types, and transplant them back into the person they came from. So hopefully we wouldn't have any problems of immune rejection. Before we differentiated them we could genetically correct whatever the defect was, or could use these cells as a vehicle for genetic therapy..
So what applications are there for production of personalized stem cells? Immunological disorders, viral diseases. It may be a way of treating HIV and hepatitis. Fixing various genetic disorders. Fixing some autoimmune diseases, for instance, diabetes.
People have been very successful in taking pancreatic islet cells, injecting them into [the] liver and curing diabetes. We now can take certain cells and turn them into cells similar to these pancreatic islet cells. If we inject those, will they also cure people of diabetes? Then there are the age-related disorders. The potential for actually causing some growth or fixing nerve damage in spinal cords...vectors for gene therapy. Until we do these things, we don't know..
We have done some of that. Through nuclear transfer, we can take cells from an adult--in farm animals anyway...and grow them for a while in a dish and produce chimeras, like in the mass. But the real thing that was done in the masses...they did some somatic cell nuclear transfer, made a blastocyst, made some nuclear transfer-derived ES cells. This blastocyst, this cell, was taken from an adult, made a blast on embryo, made some embryonic stem cells, injected them into a blastocyst and actually got a chimera and some of the embryos they actually transferred, and actually got mice. So it was working both ways..
Adult Stem Cells:
So this proves the principle [that] it can be done in the mass. But there are some problems with cell therapies. And particularly if we are thinking of stem cells from adult tissues, or for mother. We don't know how many.
The numbers of adult stem cells in the body are very, very low and I don't know, I tell awful jokes, I suppose. I was saying to someone the other day, "Well it is all very well and good you know the percentage of adult stem cells in a tissue is very low. It is all very well and good saying, 'I had the treatment for my Parkinson's disease but they had to chop my arm of to get the stem cell.'"
Now, I don't know how this is all going to go. There is the possibility we may just be able to have banks of stem cells, like we have blood banks, but we don't know what is going to happen with the rejection problem, unlike blood. If you have a blood transfusion it is only in your body for a few weeks, so you don't have any of these problems of long term rejection. We don't know, with stem cells, what the problems will be. So it may be that using these autologous stem cells through nuclear transfer may overcome that, and of course, we have to worry about longevity and the control of function. Obviously, we may get over some of that with our autologous stem cells via nuclear transfer, particularly the rejection. But we can also do genetic manipulations and selections, as we have said.
And finally the thing that really excites me nowadays, is the fact that as we understand more, we may not have to look for stem cells anymore. It may be we can take differentiated cells and de-differentiate them. Rather than actually going out and finding a cell that has this capacity, we may be able to intervene and not have to go through this nuclear transfer route, but actually get cells grown in vivo--or to cause redifferentiation or dedifferentiation, trans differentiation, whatever you want to call it--in cells from an adult.
I have given you a quick run around some of the issues of nuclear transfer, and what it is. I just mentioned that all of this work was done at the Roslin Institute, and then at PPL Therapeutics in Scotland, and in Virginia, and just acknowledge a lot of the people that have been involved in this work, first at the Roslin Institute and then at PPL Therapeutics. Some of them you may see wandering around here, teaching in the course as well. I thank you all for listening.
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