"In a future we would be able to trasplant animal organs"

Joaquin Castilla: You worked for PPL Therapeutics for more than 10 years. What were the long term aims of the team involved in cloning Dolly the Sheep?

Angelika Schnieke: The main goal of the company was to produce therapeutic proteins in the milk of transgenic animals. For this we needed efficient methods of transgenesis. The typical method at the time was DNA microinjection, but this provides only 5% efficiency. That is, to obtain five transgenic animals you must produce one hundred animals, 95 of which are non-transgenic that you don't really want. Our team reasoned that cloning had two things to offer. One advantage of cloning technology is that every animal born is transgenic, making the production of transgenic animals much more efficient. Cloning also allows you to carry out gene targeting, that is knocking out genes or replacing them with their human equivalent. This opened new possibilities such as producing animals for xenotransplantation to fill the shortage of human donated organs.

Why did a company with such a scientific and commercial potential disappear? Is there a clear explanation?

In the end it was a financial problem. The company started small, based on collaboration with scientists at the Roslin Institute. Early promising results allowed us to attract investment and take on new staff. During the 1990s PPL raised significant funding and expanded very rapidly. In addition to our main labs and offices in Roslin we had a farm and a pilot production facility in Scotland, another farm in New Zealand and a subsidiary company in the US. All this increased our running costs. At the time our lead product was alpha 1 antitrypsin produced in sheep. Large amounts of this protein were needed to carry out phase three clinical trials and this required the building of a purification plant at a cost of almost 40 million pounds, perhaps 60 million dollars nowadays. Support for this depended on partnership with the major drug company Bayer. When Bayer decided to pull out, there were simply not enough funds for PPL take the first product through to market.

What are the scientific results in your career for which you feel most proud?

Probably the work that went into my first article in Nature, because at the time I was still working as a technician.

So not too bad for a technician.

No, I had a lot of luck really. We were carrying out experiments to produce transgenic mice using retroviruses; this was before DNA microinjection was developed. In one mouse strain we knew we had introduced an embryonic lethal mutation, but had no idea which gene was affected or what its function might be. My project was to find out where the virus had integrated and identify the gene. This was not an easy task, because at the time there was almost no genomic sequence data available and few genes had been cloned. Almost by chance I hit upon the right gene, type I collagen, and analysed it. Personally I am proud of the way that project turned out, but of course there is also Dolly and the transgenic animals that followed.

You were responsible for cloning the first transgenic animal for pharmaceutical purposes. Now the first product has been commercialised, antithrombin III. How do you think patients will benefit, will the drug be cheaper?

Yes, this first product is probably not something you would call a 'blockbuster drug'. Its launch onto the market is more significant as a proof of principle, demonstrating that a drug produced in large animals can successfully proceed through clinical trials and fulfil all the regulatory requirements of the EMEA and FDA. Production in animals can be more cost efficient than production in tissue culture, and this is important for proteins such as antibodies where large quantities are required. Reduced costs clearly mean more people can benefit.

The first transgenic mammal was a mouse made in 1979. It took about 6 years to extend this result to livestock. In contrast, cloning in livestock occurred before mice. Why was this?

There are two reasons why nuclear transfer in the mouse was difficult. One is that the mouse oocyte is very fragile and doesn't survive the mechanical disruption involved in removal and replacement of the nucleus. The second is that the alternative approach, to use zygotes rather than oocytes, is very inefficient unless the timing is very precisely judged, which was not known at the time.

This problem is now solved?

Yes, the breakthrough came from Wakayama’s group. They used a piezo-electric drill to pierce mouse oocytes. This improved oocyte survival and increased the efficiency of nuclear transfer.

What have you learned from Dolly? For example, how did you finally solve the age problem, did the telomeres finally win?

In the Dolly experiment we used mammary cells from an older sheep. The cells were also grown in culture for quite a while. When we looked at the telomeres in pools of cultured cells they were relatively short, but when we looked at Dolly and other nuclear transfer animals the telomeres seemed normal. We thought that this might have been because each animal comes from a single cell, and the cells that support development are those with longer telomeres. Later experiments showed that telomerase activity in the early embryo restores telomere length. That is why people can use serial nuclear transfer as a method of cell rejuvenation. One can manipulate cells in culture and when they come close to the end of their lifespan carry out nuclear transfer and let them develop to a fetus. Fetal cells are isolated and further manipulations can be carried out. Dolly did not die because she had shorter telomeres. She had a viral infection, and this caused progressive lung disease. To avoid suffering she was euthanised.

And maybe because Dolly was more susceptible to infection?

Yes that's possible, quite a lot nuclear transfer animals seem to have compromised immune systems.

That is a very important point. There has been talk that some test tube babies may have cardiac dysfunction. So what do you expect? When we talk about humans, maybe this has to be much more controlled.

It would not be surprising if some problems were seen in humans, especially in some of the early experiments. In livestock for example, if you simply culture embryos with medium containing serum, the artificial exposure to growth factors results in 'large offspring syndrome' even without cloning. In cloned animals one also has epigenetic changes.

Is there any idea how to solve this problem?

Using medium without serum, improving culture conditions and reducing the time in culture has reduced large offspring syndrome.

But maybe this is not the only syndrome that could be found when they are adults.

In nuclear transfer, one takes an adult nucleus and asks it to revert within a few hours from an adult to an early embryonic pattern of gene expression. If that does not happen quite as quickly or precisely as it should, if for example just one gene undergoes a slight change in its regulation, a whole cascade of consequences can follow.

Yes, it is really amazing that it takes weeks or months of normal development to program cells, then in just a few hours they can be reprogrammed. So it is not surprising that sometimes it is incomplete.

Some cloned animals seem to be entirely healthy but many are slightly compromised. There is a problem with signalling between the embryo and mother and placental abnormalities. Similar problems seem to occur across species due to incomplete programming.

Lets talk about IPS (induced pluripotent stem) cells. What is the most important achievement in relation to IPS cells?

Certainly the IPS cells themselves. Earlier experiments had shown that you can reprogram gene expression by cell fusion. For example, if a somatic cell is fused with an ES cell the somatic cell nucleus starts to express early embryonic genes. Nuclear transfer then showed it was possible to comprehensively and accurately reprogram the whole pattern of gene expression of a cell nucleus. This of course started a search for the factors responsible. What was really surprising with the IPS cell work was the simplicity of the approach; just adding a few transcription factors could achieve comprehensive reprogramming. Brilliant work by Yamanaka.

You mentioned in your talk that it does not matter whether one starts with a fibroblast or another type of cell. The same transcription factors produce basically the same IPS cell. How is this possible? How can just a few transcription factors control any kind of cell?

Oct is one of the master genes controlling pluripotency and is expressed in every pluripotent cell type, for example germ cells or the inner cell mass cells of the early embryo. Oct is part of a complex circuit of regulation involving other key genes, such as Sox2 and nanog. They regulate each other as well as other transcription factors. These ensure expression of pluripotency genes and inhibit expression of genes that initiate differentiation. Addition of exogenous Oct and Sox2 activates the endogenous Oct / Sox2 / nanog circuit and with it the pluripotent state.

So now we can go from any kind of cell to an IPS cell and then to any kind of tissue or cell, just by controlling transcription factors. Some day we will maybe see personalised tissues or complete organs?

Whole complex organs are probably still quite a way off, but smaller structures such as pancreatic islets to treat diabetes are likely to be produced in the near future. There have also been some very nice experiments regenerating relatively simple larger structures. An organ is of course a three dimensional structure. So an important part of this work is developing the matrices to build the scaffolding to which cells attach. At the moment there are basically two different approaches to this. One way is to use synthetic matrices and this has been used to produce a new bladder. The second way is to take an existing organ and decellularise it, leaving only the extra cellular matrix. There has been some very nice work where this has been done with rat heart and most recently with a human trachea here in Spain. The matrix was seeded with cells from a patient who had suffered damage to her airway because of tuberculosis; the reconstructed airway was successfully transplanted with no rejection problems and functioned well I understand.

Do you think IPS cells will solve the controversy of using embryonic stem cells?

Not in the short term, because there is as yet no absolute proof that IPS cells are identical to embryonic stem cells. It will first be necessary to show that ES cells and IPS cells behave the same way under a wide variety of conditions and that IPS cells do not bring any special risks or adverse effects. That said, all indications at the moment are that IPS cells can replace ES cells and they certainly make regenerative procedures a lot easier.

You mentioned that definitive IPS cells have not been produced for large animals such as the pig, even though people are working on it. Do you have an idea why it is so complicated? What kind of change or experiment do you carry out to modify something that has been done before and not worked?

It is worth taking a step back and looking at the early days of mouse ES cells. For a long time the only ES cells available were from the 129 strain. It was some time before ES derivation was successful from a broader range of strains

Just changing the genetic background made it difficult ?

The genetic background clearly has a strong influence on how easy it is to isolate ES cells. Even now it is more difficult from some mouse strains than others. Also consider that mouse ES cells were isolated almost 30 years ago, but in rats, which are very close to mice, the first ES cells were only published last year.

So basically do you think is a matter of time. So what kind of experiment do you do differently?

First ES cells from mice and humans are not identical. After the protocol for mouse ES isolation and culture was developed, it was thought that it could be applied to other species. Now it is clear that human ES cells have a different phenotype, they do not need LIF, they need different growth factors, and expression some characteristic genes differently. It seems probable that large animal ES cells again have different requirements. For example, in pig we can culture inner cell masses and obtain ES-like cells that passage once or twice, but then they differentiate. So whatever it takes to keep them undifferentiated is still missing.

The generation of IPS cells requires genetic manipulation of these cells by transferring new transcription factors. Will it be possible in the future to do it chemically to avoid genetic manipulation?

People have already done that, with recombinant proteins.

There is no way to use something more chemical?

Scientists have used mRNA, proteins and tried to substitute transgenes with small molecules such as MEK inhibitors to block differentiation pathways. There is a huge ongoing effort to minimise or eliminate the need for transgenes

In addition to the interest in generating animals to produce pharmaceutical drugs, I see at least another two areas of interest: xenotransplantation and the generation of disease resistant animals. How do you see the future of these two areas?

I think xenotransplantation may be a realistic option in the near future. I must admit that I was very sceptical when we first got into this area. If a normal pig organ is transplanted into a primate host, it is destroyed within three minutes. The extent of the genetic changes needed to obtain long-term survival of a pig graft in a human seemed incredibly daunting. But results have shown that even inactivation of one gene, alpha 1,3 GT, causes a clear reduction of the hyperacute rejection response. The next step is to tackle acute vascular rejection. Work is going on using transgenes that reduce activation of the endothelium. It has also been discovered that the pig and human blood coagulation system are incompatible in some ways. So protective genes such as thrombomodulin offer further improvement in graft survival. There have been reports of pig organs surviving for four or even six months transplanted into baboons.

That is amazing, six months.

It is amazing. That could help someone on a waiting list survive until a suitable human heart became available.

However one of the major problems of xenotransplantation is the possible transmission of zoonotic diseases, particularly retroviruses. Could this slow down its development?

It is a real possibility, but when pig and human cells were co-cultured there was no evidence that virus was transferred. However the patients likely to receive a xenotransplant will of course be very sick and possibly immune compromised. So the risk is perhaps greater that virus transmission could occur. Several researchers are looking into strategies to inactivate porcine viruses and it may be possible to identify pigs with no active retrovirus in their genome

Do you think that governments should intervene in the use of germ cells or embryos for cloning or similar purposes? Do you think governments should control this kind of study or that the scientific community should control them?

There has to be a compromise that balances the interests and needs of all concerned. Reproductive cloning can probably be carried out in almost every mammalian species, so theoretically one could clone a human. A few years ago some people even claimed to be doing so, but no serious scientist would support it. Government regulation is necessary, but there has to be a distinction between human reproductive cloning and the production of embryonic stem cells to help people.

What is your answer to the growing rejection of the use of transgenic plants and animals in Europe?

I have worked with transgenic animals for so long that they are in no way unusual. I do understand concerns about transgenic products where the main advantage seems to be to make more money for a company. On the other hand the world population is increasing at an enormous rate, and there is real uncertainty how food demand will be met without destroying the environment with ever more use of agricultural chemicals such as pesticides. This is made more acute with increasing affluence and changing eating habits in big countries such as China and India. I also feel that people in extremely wealthy countries in Europe really have no right to dictate food policy to those less privileged in the world. Genetically modified plants are here to stay. Over thousands of years food plants have been modified by selective breeding and many have little resemblance to their wild ancestors. GM is basically a more precise and controlled continuation of the same process.

It is true that society is not always prepared for new scientific discoveries. However, why do you think people are scared by the words 'transgenic' and 'clone' when we are talking about humans? Do you think that perhaps scientists do not know how to explain their discoveries to the public?

I think that was probably true in the past, but now there seem to be plenty of good informative articles on television and in the press. Nevertheless there is still a high level of scepticism and misunderstanding among the public. Unfortunately this is not helped by powerful lobby groups who play on people's fears. I also know that in Germany, some politicians make public statements against genetic modification, but express different opinions in private.

I would ask you an effort of imagination. So we are in 29 January 2060, 50 years from now. You will be probably retired. So where are your PhD students working? Can you imagine the kind of study they are doing?

I doubt I will be around then and hesitate to speculate so far ahead, but I think well before fifty years we will have a DNA sequence revolution. There have been predictions that soon it may cost just €1000 to sequence a whole human genome, and there is even talk of a €100 genome. This would allow real individualised medicine. Within a decade or so IPS cell technology will also become reality, and I think we would be able to transplant animal organs. Farther ahead I think we will see a significant extension of healthy lifespan through an increasing understanding of the aging process and sophisticated regenerative medicine and tissue engineering techniques. Totally new areas will also emerge, for example from the integration of biology with microengineering and nanotechnology.

You mentioned in your talk that a part of the problem in humans is the difficulty of obtaining enough oocytes. Leaving aside the ethical reasons, what is the problem? Are there not enough donors?

Oocyte collection is an invasive procedure.

Yes but can you recover oocytes that have been already taken and nobody has used?

Fertility clinics store many early embryos, but very few oocytes. Their usual practice is to fertilise all the oocytes required for a woman receiving IVF treatment, identify the best looking embryos for transfer and store the rest. That is why people have tried using oocytes from cattle or rabbits for human therapeutic cloning. There was a publication from a Chinese group who claimed they could do human nuclear transfer using rabbit oocytes and then derive ES cells but this could not be reproduced.

So you do not have any limitations, you can choose any kind of species?

Well, other people have tried it and it doesn't work.

So this is going to open the Jurassic Park possibility?

No, at the moment it seems to be very difficult, especially for full development, you cannot cross most species barriers.

But now, if you are in the same species you can use different families and…

That is working.

So it is a question of time that you can move from one species to another?

The problem is incompatibility between the nuclear and mitochondrial genomes of distantly related species. There is a lot of interaction between the mitochondria and the nucleus and this is very sensitive to evolutionary distance. For example human and chimpanzee are compatible but human and orang-utan are not. Mouse and rat are compatible but if you go further apart say to hamster it does not work. The general understanding is that cross-specific nuclear transfer will support some early development, but nothing further.

Theoretically would it be possible to create a new individual using just two ova from the same species? Can you use two pronuclei from females without using a male?

These experiments were carried out some time ago and it does not work. Embryos containing either two male, or two female genomes do not develop normally. With male only embryos, the extra-embryonic structures such as the placenta develop but there is almost no embryo proper. This is because of imprinted genes, which are specifically expressed from either the male or female parental genome. There is only one publication in mouse, where they first inactivated an imprinted gene, and then obtained live mice using just female genomes.

What would you change about European scientific funding policy?

A large proportion of EU funding is for very specific predetermined areas of research, a specific 'call', and is designed for large numbers of participants that necessarily involve considerable administration and coordination. I would like to see more open calls and encourage projects from individual groups or smaller collaborations. I believe some of this is being implemented.

What do you consider the major scientific challenge that our society faces in this current major economic crisis? In this environment what is the most important challenge that we should worry more about? Because we have to choose, this economic crisis will force us to choose.

Speaking of course only about biomedical research, I worry that in recent years there has been such a strong emphasis on IPS cells, and there is a risk of 'over hyping'. This area is certainly exciting and opens a lot of possibilities but it would be a real pity if other fields of research suffered because of economic constraints. There is a considerable need for basic knowledge.

In conclusion, do you think in general the proper use of the stem cells, therapeutic cloning or related techniques, will make a better world for living?

I think we should always try to reduce human suffering wherever we can. Populations around the world are aging and the typical diseases of age are becoming more important. Of course it would help if people took more exercise and improved their diet to reduce cardiovascular diseases and diabetes and so on, but science and medicine must do whatever is possible to ensure people continue living a healthy and active life into old age.

The last question is, what do you think about this 5 year old CIC bioGUNE?

I was certainly impressed I must admit. It is quite an achievement to bring together all these various groups, who seem to interact and collaborate well. To do this in five years and produce top quality science is certainly something everyone here can be proud of.