Slide 1

Thank you very much Mr Chairman. I would like to thank the organisers of this meeting that have allowed me to be here to bring you up to speed with whatever has been going on recently in the field of pig to primate, in the field of xenotransplantation and I will immediately get everybody on the same page reminding that those working in this field at this very moment have identified the pig as the most likely source that could at some stage get into the clinical arena.

Slide 2

Why the pig? The pig has undoubtedly several advantages in terms of anatomical and physiological similarities, short gestation periods, large litters, rapid growth rates that would make a large number of organs potentially suitable for recipients of any size very, very quickly. The pig breeding is obviously not as costly as breeding other species, there is the possibility of breeding in an autobiotic or specific pathogen free conditions the pig and there are fewer ethical concerns compared to using organs from other species.

Slide 3

Now, another very important concept which is strictly linked to what my colleagues were just saying a few minutes ago in terms of advancement in the new technologies, which is also the topic of this session, the pig puts us in a very privileged situation in the sense that when we do allotransplantation these days, we can hardly manipulate in any way the source of the organ, we can hardly manipulate a human kidney we plan to use for clinical purposes. Xenotransplantation using pigs put us in a completely different scenario. Indeed we do have certainly the possibility to genetically engineer the pigs and make it more compatible, more closer to what we would expect in order to be functional and useful for pig to primate xenotransplantation. This genetic engineering exercise can be done quite ahead, quite before eventually the use of this organ for clinical purposes and as you know, it is not an option for our patients.

Slide 4

Now, the genetic engineering that is ongoing currently in the labs working in xenotransplantation regards either the insertion of one or new genes into the pig genome, as we said the intention is to get a pig organ more compatible with a human recipient, so we can either add new genes in the genome and at this stage people are working very hard in trying to address the new genes that will give the organ a better profile as far as resistance to complement cascade activation or a better profile as far as the modulation of the clotting cascade but there are other genes that are being produced that are perhaps better as far as the capacity of immunomodulating the immune response as well.
As opposed to inserting new genes to have an organ which is closer to human organs we can delete one or more genomic sequences in the pig genome as we have, for instance, achieved in the production of Gal-KO pigs that do not express anymore the galactosyltransferase and therefore, the α1, 3 Gal-KO sugar residue is no longer present on pig endothelial cells.

Slide 5

Xenotransplantation today I will go over the key main points for those who are working in the field of xenotransplantation that need to be adequately addressed, if we want to propose a clinical xenotransplantation program. We will deal briefly with the immunological issues, physiology issues, biosafety and ethical issues of xenotransplantation.

Slide 6

The interesting point is that 3 of these 4 points can be managed, can be handled, can be improved by the genetic engineering of the pigs that today are available. So that is a very encouraging starting point obviously.

Slide 7

As far as immunology is concerned

Slide 8

a cartoon that very briefly reminds you that we are talking specifically about xenotransplantation using pig organs, that’s an important point. If we look at the immune response I’ve drawn here what we have today to face for the long-term survival of a human allograft into a human recipient and today we deal primarily with cellular rejection which we have learnt how to handle and it’s something that we’re quite good at these days. Certainly the major problem today is chronic rejection and I don’t want to go into the details can chronic rejection or whatever is responsible for preventing the long-term survival of our organs beyond the survival that Professor Remuzzi has just mentioned to you a few minutes ago.

Slide 9

Now as far as xenotransplantation in addition to these two barriers we do have two additional an obstacles which are hyperacute rejection and acute vascular rejection. So we have to be able from an immunological standpoint to get rid, get over these two immunological barriers, if we want to fall into a form of rejection we’re more familiar with or to fall into a situation where people doing allotransplantation, as some of you are facing everyday in your clinics.
Now just very, very quick slides to remind you that this is the way an organ appears prior to being transplanted and this is the fate of a porcine organ transplanted into a non-human primate and this is the picture of hyperacute rejection, the first barrier we have to handle. So it’s a very violent, a very severe immune response that is very, very early after transplantation, a matter of a few minutes or a few hours and this is an irreversible graft damage.

Slide 10

Now, what are the strategies that we can think about, if we want to have a long-term survival of a porcine xenograft and if we want to potentially propose this to the clinician? So, basically the 4 strategies and approaches that can be used to tackle the immunological obstacles are first of all we just spoke about it the genetic engineering of the pig, a pig that is more compatible, that will not hurt and that is where a lot of emphasis is being put in. Clearly we can extend pig survival by using an adequate pharmacological immunosuppression either with conventional or more likely with novel immunosuppressants. Another approach is to induce tolerance, to devise regimens that would lead to tolerance and as you have heard, this is quite a challenge already for allotransplantations, so that’s going to be possibly even harder for xenotransplantation. Then another approach that is being pursued from an immunological standpoint to extend the survival of a xenograft is encapsulation and this is put into brackets because this only applies to islet xenotransplantation where these islets would be coated by a capsule.

Slide 11

Now, we have learnt the immunological processes underlying hyperacute rejection which as we said, is the first barrier. We have learnt it, immunologists have understood which are the 3 players that are responsible, the key players responsible for this violent immune rejection process and the players are the naturally present antibodies that each of us have in this room and these are antibodies directed against some sugar residues, so we can either tackle this by removing the antibodies, by removing the sugar residues as we say by genetic engineering of the pigs or we can work on what follows to the binding of antibodies to pig endothelial cells which is the activation of the complement cascade.

Slide 12

This is what has been tackled and done by most laboratories working in this field, so we have today a transgenic pig whose endothelial cells express hDAF which is a human complement regulator so we did not work on antibodies, we did not work on the sugars but we did work on the generation of pigs that express an inhibitor of the human complement activation.

Slide 13

We did that by producing a minigene for this human protein that was this minigene microinjected into a pig embryo. We had eventually, we, I mean the people working in Cambridge where I was at the time but the other people working in this field we had the --- genetically engineered animals that look absolutely normal and they grow naturally and normally and they reach sexual maturity and just this to point out that the first line of such pigs was generated 15 years ago and is still available. So they matured, they reproduced and they are quite good.

Slide 14

These were studies that were undertaken in transgenic pigs.

Slide 15

I just wanted to show you on the y axis you see the amount of this transgenic protein on the heart of endothelial pigs and these are human controls and as you can see, we have been able to reach, to have genetically engineered pigs that express an amount of this protein hDAF, which is at least as good as the amount of hDAF expressed by 2 cadaveric human donors.

Slide 16

These slides want to show us that the genetic engineering of the pigs has led to hDAF transgenic pig that indeed expresses the transgene and they express it exactly where the immune system attacks the organs I mean the immune reaction that happens at the surface of the porcine endothelial cells. Does it work? Just a couple of slides.

Slide 17

This one is a paper published in 2002 with the group in Cambridge that indicates that out of 326 transplants either heart or kidney transplanted in 2 species either the Cynomologous monkeys or the baboon as you can see, hyperacute rejection occurred in only 7% of the xenografted organs so obviously this technology only with the addition of a single gene in the pig genome is there to bring us a substantial step ahead and we have today pigs that have been generated, that have 2 or 3 human complement regulators that have a much further reduced incidence of hyperacute rejection or even prevention.

Slide 18

Another way to tackle the issue, as I said initially we all work on complement regulators but in fact, where we can work is in trying to delete on the porcine endothelial cells in the porcine genome the gene that leads to these sugar residues, as you would expect for instance, the incompatibility of sugar residues as we have when we do a transfusion between patients.

So we were able to generate pigs that are knockout for this galactosyltransferase.

Slide 19

Also in this case hyperacute rejection was not seen. So the bottom line is that today we all agree that hyperacute rejection does not exist anymore.

Slide 20

And we are working on acute vascular rejection.

Slide 21

What do we know? This is another very important immunological obstacle we have to overcome, if we want to have a long-term survival of pig organs into a primate. This is a very severe rejection episode also here it is primarily the humoral immune response that is responsible for these immunological problems.

Slide 22

So again, as we did 15 yeas ago actually even earlier than that, people have been working very hard in trying to understand the immunological principals underlying the rejection of the xenograft and acute vascular rejection. Again, for the identification of specific targets to tackle to obtain long-term survival of pig organs transplanted into primates.

Slide 23

In this context I think I should certainly remind all of you an important paper that was published 9 years ago by Professor Platt who was at the time very active in pig to primate xenotransplantation and he gives us this very important piece of information. If we immunologists are good enough to prevent the formation of antibodies, you will not see rejection, you will not see the activation of the clotting cascade that is considered today an obstacle associated with the acute vascular rejection process. So from this paper it would seem that if we are able to adequately handle the humoral immune response, we should be able to overcome most of our problems.

Slide 24

This shows basically data that was reported a couple of years ago by the group of Fitzback where he shows that using protocols that also include a novel immunosuppressant which is the anti CD154MAb you can considerably extend survival of pig xenograft. However, you seem to shift the emphasis of the rejection process where you see a thrombotic microangiopathy taking place.

Slide 25

I’m showing you other very important data that shows you where we are in terms of survival and this is important data published a year ago in Nature Medicine and it shows you that if you render your primates diabetics and you transplant them with porcine islets with a series of different immunosuppressive combinations, 2 different groups were able to keep alive diabetic primates with porcine islets xenografted. So, that’s a very important piece of information that we have here so basically certainly a pig organ can sustain the life of a primate for several weeks, several months but here we have evidence that we can keep 100% of the primates alive using porcine islets.

Slide 26

In this study from Bernard Hering you can see that we have excellent insulin producing cells in the graft at autopsy and there is no infiltration of CD3 cells.

Slide 27

These animals as we said are normoglycemic. The porcine c-peptide is measurable. They survive for longer than 180 days and there is no rejection. I would like you just to remember this data because that is very important when we will talk about ethics of xenotransplantation. This is an important paper we will come back to.

Slide 28

Now, certainly people are able to sustain the life of primates for several months primarily in islet xenotransplantation using a pig organ. As far we know, besides the humoral immune response that needs to be better handled, we certainly know that there is the potential for a cell-mediated anti-pig immune response but this is fairly well-controlled. Indeed, we feel that at this stage there is no evidence that a cell-mediated injury, so T cell, NK cell mediated injury is not sufficient per se to lead to organ failure unless we do have also humoral damage. So that is an important point. We feel that as far as T cell immunosuppression is concerned, these days that’s not where our problem is, we have to find a better way to handle the antibody mediated, the B cell mediated immune response.

Slide 29

So basically it is what I said, the humoral rejection then remains the principal obstacle to the long-term survival of pig organs transplanted into primates. As you see here, these are the longer survival of primates reported to date. 90 days for a primate. A bilaterally nephrectomised primate that was kept alive on a pig organ. 3 months is the survival. A heterotopic cardiac xenograft was kept alive for 6 months. 8 weeks for an autotopic heart xenograft and as we said, at least 2 groups are reporting long-term survival of diabetic primates that are xenografted with porcine islets and these primates are normoglycemic.

Slide 30

So we have seen the problems from an immunological standpoint where it is unquestionable that we have greatly improved survival in our primates. We have only been able to achieve a 3 month survival for a renal xenograft in a primate but we are going ahead and progress is there and we have new lines of pigs that are being generated.

Slide 31

In terms of physiology here we see for 40 days survival in a group of 8 primates, these primates are maintained alive with a normal sodiemia, with a normal creatinine.

Slide 32

And this is in the paper of David Sachs the survival where you can see that we have a normal creatinine until we do have rejection episodes. So there is no rejection and there is good physiological compatibility, a pig kidney does work in a non-human primate milieu.

Slide 33

Certainly from a physiological standpoint there are incompatibilities that have been shown, this regards the incompatibilities as far as the coagulation is concerned. There are also incompatibilities possibly as far as the stimulation of the primate bone marrow to produce red blood cells and possibly the porcine erythropoietin may not be as efficient as we would like to stimulate a pig bone marrow.

Slide 34

But here what you can see is that with recombinant erythropoietin we can reverse the trend and we can keep the primates long-term, xenografted primates long-term alive with a normal haemoglobin using human recombinant erythropoietin as we do for our patients.

Slide 35

So from a physiological standpoint what can we say? We can certainly say that there are some incompatibilities between humans and primates that is no question but the important thing is that none of the physiological incompatibilities demonstrated have prevented the long-term survival at least medium-term survival of non-human primates and this is reported both for life supporting renal cardiac and islet xenotransplantation model.
So we now know today that basically we can sustain the life of a primate with a cardiac, with a renal or islet xenograft without any major physiological incompatibilities.

An important point to remember is that certainly the paper as we said from the group of Geoffrey Platt that is saying that if you have good immunologists, also the coagulation should be fairly under control.

Slide 36

Biosafety. Certainly there has been a lot of concern related to the presence in the pig genome of porcine retroviral particles and at the time it was of major concern really, so there were a lot of scientists that embarked in trying to study patients that were exposed to living porcine tissues, organs or cells.

Slide 37

The important thing is that in these studies published in Science a few years back is that in these patients there were no signs of PERV-related infection neither current, past or latent or disease in these patients that had been exposed to porcine organs or porcine tissues, for instance, the severely burnt patients that were exposed to pig skin. No signs of PERV infection. Other advancements regard, for instance, the isolation in Boston Massachussetts of a line of mini pigs whose PERV do not seem to have the possibility in vitro to infect human cells. So that would be a line, a pig line that would have PERV somehow defective that would not be able to infect humans. We have been able now to generate data that shows that PERV replication can be inhibited by drugs and we can always remember that the generation of PERV specific vaccine could be also something that we could think about in case this was necessary.

Slide 38

But the important point is once again here we can work on the genoma of our pigs and we could, for instance, produce a siRNA specific technology for pigs or, for instance, pigs that are knockout for PERV. Nonetheless, there maybe some pathogens that are yet unknown who could be there and we should be able to handle this in case they had to appear. So, clearly the search for potential unknown pathogens has to go ahead and people working in the field of xenotransplantation, they’re not just working on immunology, they’re not just working on physiology but certainly on safety aspects of xenotransplantation. We have been fortunate enough in the last few years that there have been serious advancements in the biomolecular technology front and new techniques could be applied for an early identification of potentially unknown pathogens arising as a consequence of xenotransplantation.

Slide 39

Finally, last point but not the least important is the ethical issue of xenotransplantation.

Slide 40

When we talk about xenotransplantation, we know that there are several ethical concerns, some are obvious some are not as obvious. Certainly we have always to remember that when we talk about transplantation, we have always to remember that ethics require also, do not forget, the prevention that can be done in a large number of the patients waiting for an organ.
The ethical issues are related of course to the use of animals, to the use of genetic manipulation, the resource allocation, the commercial pressure that is behind any kind of funding. Those working in the field of xenotransplantation should not create expectations, it’s another concern. There are other issues related to the informed consent, the risk of zoonoses. Clearly the public engagement is unknown, the risk of failure and the risk of the unknown.

Slide 41

Basically xenotransplantation should progress into the clinical contest only in the presence of a favourable risk/benefit ratio. However, and I’ve almost finished, we do have several concerns. Those working in the field of xenotransplantation are very concerned that some studies are today ongoing in several places in the world. We’re not talking about renal xenotransplantation but we’re talking about islet xenotransplantation and the Ethics Committee of the Xenotransplantation Association has been acting very aggressively to try to stop this study at the Hospital Infantile de Mexico where an Australian company was providing Australian pig islets to transplant children, minors affected by Type 1 diabetes. They were transplanting these children in the complete absence of efficacy data generated in another species. So, basically an Australian company was running a study that was not allowed in New Zealand, that was not allowed in Australia and these islets were sent to this hospital for doing these studies whose data quite frankly are very disappointing.

Slide 42

Strictly linked to these slides and I’ve finished, are several other worrying rumours and I would like to remind you these two rumours, they are very recent and they can be verified by any of you in this room is that again the same company, the Australian company LCT and you can visit the company’s website is claiming that the company is initiating a clinical trial of islet xenotransplantation in Russia this year and the rumour around is that this study is ongoing. There is another company which is MicroIslet that is claiming the same on their website at the moment. The concern that we have is that these are 2 companies that are either based in Australia or based in the United States and they’re using countries that are neither Australia, neither United States, neither Europe to run these trials and they’re going to countries like Russia and China where possibly the regulatory framework is not as satisfactory as we would like, as I said possibly.

Slide 43

To conclude I’ve shown you that there has been quite extensive work ongoing in this field in the last few years that has allowed a prolonged survival of pig organs in primates.
New immunosuppressive regimens are being developed. We have said that it does not appear that there are insurmountable physiological incompatibilities between pigs and primates.
Biosafety is a key point. There is a reborn interest for xenotransplantation research that should take place only in the presence of a favourable risk/benefit ratio and for those working in this field as myself for the time being we believe that the human organ remains the only real clinical option. I would like to thank you for your attention.