Xenotransplantation

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XX International Congress of The Transplantation Society (ICTS)Forging Ahead With New Technologies: Progress, Obstacles, Issues, and Controversies

Release Date: October 15, 2004; Xenotransplantation

Sachs, MD

The field of xenotransplantation provides verification of the old adage "Necessity is the mother of invention." Indeed, the need for xenotransplantation has become more and more acute as the success of allogeneic transplantation has increased. Most of the advances in transplantation medicine presented at this Congress have been directed at alleviating 2 of the major limitations to the field of transplantation today: treatment-related complications and chronic rejection. However, none of these advances can address the third major limitation of our field, the limited number of human tissues and organs available for patients in need of transplantation.

Thus, paradoxically, as the treatment successes continue to build in the field of transplantation, the number of patients on waiting lists for transplantation is growing and the number of available organs has plateaued. An example of this limitation is illustrated in the Figure, which shows the number of heart transplants performed worldwide over the past 3 decades. As illustrated in this figure, the number of procedures increased exponentially in the early 1980s, coinciding with the introduction of cyclosporine as an immunosuppressive drug, but quickly reached a plateau and has not increased (in fact has even shown a small decrease) over the past 15 years. The same limitation is true for other organs as well as for islet cells, thus making this limitation the most severe obstacle to progress in the field of transplantation today.

Figure 1. Number of heart transplants performed worldwide over 3 decades.

At this Congress, 1 symposium, 3 oral presentation sessions, 2 poster sessions, a luncheon debate, and a plenary talk were devoted to subjects in the field of xenotransplantation. In a brief review such as this, it will not be possible to cover all of the work presented, and I apologize in advance for numerous valuable contributions that I have not been able to summarize. I have tried, however, to provide a flavor of the various areas of research in xenotransplantation that were covered at the meeting, with examples of some of the recent research efforts in these areas.

Advances in Survival of Pig-to-Primate Organs and Islets

Over the past 2 years, 2 groups have reported the successful production of pigs that do not express the Gal epitope.[1-3]These pigs have been produced by disrupting the gene for alpha-1-3 galactosyltransferase (Gal-T) through homologous combination, followed by nuclear transfer to produce knockout (KO) animals (GalT-KO animals). One GalT-KO line was produced by PPL Therapeutics, using outbred swine as the source of the modified DNA for nuclear transfer,[2] while the other was produced by Immerge BioTherapeutics, using our inbred miniature swine as the DNA source.[1-3]

Several papers were presented at the meeting from the research center of the author, at the Massachusetts General Hospital, utilizing the Immerge GalT-KO animals as donors for heart and kidney transplantation into primates. Surprisingly, no papers were presented utilizing the other line of GalT-KO animals produced by PPL, which is now being studied at the University of Pittsburgh.

Utilizing GalT-KO miniature swine, and colleagues[4] reported a marked increase in survival of heterotopic heart transplants into baboons, utilizing a relatively standard immunosuppressive regimen. A survival of 6 months in 1 animal reported by this group is the longest survival of a pig heart into a primate thus far reported. In all cases, the hearts were eventually lost through the occurrence of a thrombotic microangiopathy observed as a marked feature of the rejection process. The relative role of antibody in the eventual rejection of these organs is under study.

The second series of organ transplants from GalT-KO animals to baboons was reported by Yamada and colleagues,[5,6] and involved the utilization of a preparative regimen designed toward the induction of transplantation tolerance. Utilizing combined vascularized thymus plus kidney transplantation, these investigators showed a marked increase in organ graft survival over previous studies utilizing the same regimen, but without GalT-KO donors. Unlike the heart transplants, which were heterotopic, these transplants involved life-supporting organs. One kidney continued to function with a normal creatinine for over 80 days, and the kidney still looked normal grossly and histologically when the recipient baboon died of a myocardial infarction.

Studies of orthotopic heart transplants were presented at this meeting by Brenner and colleagues.[7] Survival was measured in weeks, but the fact that these grafts were orthotopic and, therefore, life-supporting is very encouraging from the point of view of physiologic compatibility. Clearly, organ survival using the new GalT-KO animals and new methods of immunosuppression and/or tolerance induction remains a fertile area for advances in the coming years.

Another major advance was reported by Jie and colleagues.[8] These investigators have been studying porcine islet xenotransplantation in cynomolgus monkeys made diabetic by treatment with streptozotocin. Islets were prepared from either inbred miniature swine or outbred swine and were cultured in vitro and then injected intraportally into the diabetic monkeys. Using several different immunosuppressive regimens, the investigators were able to obtain graft function of these islets for more than 187 days. Insulin was administered early in the posttransplant course, but it was later discontinued and the animals showed normal islet function as well as numerous islets by liver histology, without any sign of rejection. Because Gal is not expressed on adult islets, it is apparently unnecessary to use GalT-KO donor for these studies, although testing of the possible advantage of these donors is something that will undoubtedly be attempted in the future. At this point, Hering and colleagues conclude that their immunosuppressive regimen is still too toxic to be routinely used for clinical islet transplants, but their outstanding results go a long way toward suggesting that such xenografts will eventually provide a solution to the shortage of islets for clinical use.

Coagulation Disturbances

Several papers presented at the meeting demonstrated coagulation disturbances as a prominent feature of discordant xenotransplantation in the pig-to-primate combination. Since, in many cases, these disturbances did not appear immediately, but rather toward the end of the organ xenograft survival, the question was raised as to how much of the problem is related to incompatibilities of coagulation factors vs induction of the disturbance by the appearance of anti-pig antibodies.

D'Apice[9] concluded that one approach to overcoming these coagulation problems may involve further genetic engineering of the donor pigs. His data in mice indicate that transgenic animals bearing xenogeneic analogs of some coagulation-related proteins show marked correction of the defects.

Schroeder and colleagues[10] presented preliminary data in an ex-vivo lung perfusion system study. GalT-KO lungs showed marked protection from many of the vascular and coagulation problems seen in the same system using lungs from pigs expressing Gal. Nevertheless, delayed platelet activation and intravascular thrombosis did occur, suggesting that even in this system, coagulation problems will need to be addressed. Again, the relative contribution of antibody to the problem needs to be resolved.

Infectious Disease Risks

There was a marked decrease in the number of papers investigating porcine endogenous retrovirus (PERV) as a potential pathogen in pig-to-primate transplantation compared with the number of papers devoted to this topic at the last Congress 2 years ago. This decrease is undoubtedly due, at least in part, to the fact that most investigators in the field have now concluded that PERV will not pose a major health risk, and that the potential risk can be adequately addressed by heeding the precautions recommended by the United States Food and Drug Administration.[11] Most of the studies presented at this meeting supported this viewpoint.

For example, Zhang and colleagues[12] demonstrated that even a human cell line that could be infected by PERV in vitro could not be infected in vivo in a nude mouse after coadministration of a pig cell line producing PERV. They concluded that the possibility of PERV transmission from pig tissues to humans following xenotransplantation would be "remote."

A study from Ziolkowski and colleagues[13] indicated that PERV could serve as an antigen for accelerated cellular xenograft rejection in immunized mice, suggesting that elimination of PERV may have additional benefits with respect to xenotransplantation. It remains clear, however, that infectious disease risks are still an important topic in the field of xenotransplantation and will continue to be studied as the field progresses toward potential clinical applications.

New Approaches Relevant to Xenotransplantation

Several papers on the potential effects of NK cells in xenograft rejection and ways of avoiding these effects were presented at the meeting. Forte and colleagues[14] showed that the aggregation of interactions between NK activation receptors and their respective ligands on pig cells might potentially overcome NK xenoreactivity. These findings have implications for the potential genetic engineering of donor pigs by transgenic and KO technologies to avoid NK reactivity.

Sharland and colleagues[15] provided evidence that certain pig cells are capable of expressing ligands for human NK cells only after the onset of rejection, suggesting that these ligands might contribute to the intensity of the xenoresponse and may therefore be important targets for elimination by genetic engineering.

In a symposium devoted to xenotransplantation, Bach[16] reviewed the potential effects of protective genes in xenotransplantation. Most of the data involved the role of such genes in protection from allogeneic rejection, but a convincing argument was made for the potential usefulness of this approach for xenotransplantation.

Milland and colleagues[17] demonstrated that another gene producing alpha-1,3-Gal is present in both mice and pigs and, therefore, the knockout of alpha-1,3-galactosyltransferase may not be sufficient to fully eliminate Gal from donor animals. However, the nature of the determinants produced and the fact that they are not readily detectable on the cell surface of normal cells suggest that these determinants may not be of great relevance to xenotransplantation.

Finally, McKenzie and colleagues[18] presented evidence that sperm-mediated gene transfer may be a powerful new method for rapidly introducing multiple genes into transgenic pigs. In the studies presented, the genes utilized were only markers easily detectable by florescence of different colors. However, it was suggested that this proof of principle may pave the way for producing transgenic pigs with multiple human genes for future xenotransplantation studies.

How Close Are We to Clinical Xenotransplantation?

This question was the subject of a debate between Sykes and Wallwork[19] at a luncheon workshop, which was followed by a lively discussion among attendees. In essence, both debaters were in favor of the eventual clinical application of xenotransplantation to the treatment of human disease. Wallwork expressed the sentiment that the time for such applications is now. He believes that the kinds of results that have been obtained in preclinical pig-to-primate experiments justify treatment of critically ill patients even before long-term survival has been achieved in preclinical studies. In his opinion, this would be the only way to really determine which problems need to be further addressed without delay. Sykes, on the other hand, expressed that it is extremely important for workers in xenotransplantation to be cautious and not to attempt clinical trials until there is a reasonable expectation of success. Her main concern is to prevent workers in this field from being seen by the public as taking unnecessary risks, especially in view of the cautions that have been raised about potential PERV transmission and infection. She believes that only groups with extensive experience in preclinical models and with approval from a competent regulatory body should be advised to go forward with clinical trials.

Sentiment among the other attendees was mixed on this subject, but no one was against the idea that xenotransplantation should be pursued for its potential clinical benefit. In Wallwork's view, trials should start immediately, while Sykes envisions starting such trials only after another 1 or 2 years of preclinical studies.

One concern raised, to which no immediate solution was obvious, was the need for additional funding if preclinical trials are to be continued and extended. The recent withdrawal of financial support by Novartis for xenotransplantation research has left an enormous void, with investigators scrambling to find other sources of support. While public funding continues -- and many of the attendees felt that this is a reasonable source for preclinical funding -- the establishment of clinical trials will be expensive and will probably require commitment by industry. No immediate solution was apparent, but general optimism prevailed that as positive results continue to accumulate, additional industrial sponsors will become involved.

In summary, the enormous increase in xenotransplantation research activity that occurred during the past decade has now leveled off, and perhaps even diminished over the past 2 years. Nevertheless, judging from the exciting studies presented and the enthusiasm among investigators still in this field, as well as the continuing clinical need, it is clear that as a field, xenotransplantation remains alive and well.

References

Lai L, Kolber-Simonds D, Park KW, et al. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science. 2002;295:1089-1092. Phelps CJ, Koike C, Vaught TD, et al. Production of alpha 1,3-galactosyltransferase-deficient pigs. Science. 2003;299:411-414. Kolber-Simonds D, Lai L, Watt SR, et al. Production of alpha-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations. Proc Natl Acad Sci U S A. 2004;101:7335-7340. Knosalla C, DJ, Moran K, et al. Initial experience with the human anti-human CD154 monoclonal antibody, ABI793, in pig-to-baboon xenotransplantation. Xenotransplantation. 2004;11:353-360. Barth RN, Yamamoto S, LaMattina JC, et al. Xenogeneic thymokidney and thymic tissue transplantation in a pig-to-baboon model: I. Evidence for pig-specific T-cell unresponsiveness. Transplantation. 2003;75:1615-1624. LaMattina JC, Kumagai N, Barth RN, et al. Vascularized thymic lobe transplantation in miniature swine: I. Vascularized thymic lobe allografts support thymopoiesis. Transplantation. 2002;73:826-831. Brenner P, Schmoeckel M, Reichenspurner H, et al. Mean xenograft survival of 16,6 days in a small group of HDAF-transgenic pig hearts transplanted orthotopically into baboons. Transplantation. 2004;78 (Suppl 2):21. Jie T, Graham M, Wijkstrom M, et al. Pancreatic islet xenotransplants restore normoglycemia and insulin independence in immunosuppressed non-human primates. Transplantation. 2004;78 (Suppl 2):185. D'Apice A. Regulation of coagulation. Xenotransplantation. Program and abstracts from the XX International Congress of The Transplantation Society; September 5-10, 2004; Vienna, Austria. Schroeder C, Allan J, Nguyen B-N, et al. Xenogenic ex vivo perfusion of lungs from GALT K/O pigs: Initial results. Transplantation. 2004;78 (Suppl 2):20. Bloom ET. Xenotransplantation--federal regulatory considerations. Curr Top Microbiol Immunol. 2003;278:239-251. Zhang L, Yu P, Li SF, et al. Phylogenetic relationship of porcine endogenous retrovirus (PERV) in Chinese pigs with other pathogenic type C retroviruses. Transplantation. 2004;78 (Suppl 2):185. Ziolkowski A, Milburn P, Gibbs A, Simeonovic C. Porcine endogenous retrovirus (PERV) is a major xenoantigen for porcine cell xenografts in mice. Transplantation. 2004;78 (Suppl 2):584. Forte P, Baumann BC, Lilienfeld B, Schneider MKJ, Seebach JD. Prevention of NK cell-mediated cytotoxicity in pig-to-human xenotransplantation. Transplantation. 2004;78 (Suppl 2):21. Sharland AF, Christiansen D, Tran P, Winterhalter A, A, Sandrin MS. Porcine cells express ligands for the human NK cell activating receptor NKG2D. Transplantation. 2004;78 (Suppl 2):159. Bach F. Protective Genes in Transplantation. Xenotransplantation. Program and abstracts from the XX International Congress of The Transplantation Society; September 5-10, 2004; Vienna, Austria. Milland J, Christiansen D, Li YQ, Sandrin MS. The molecular basis for Galα(1,3)GAL expression in alpha,1,3galactosyltransferase knockout animals. Transplantation. 2004;78 (Suppl 2):186. Mckenzie IFC, Webster N, Forni M, et al. SMGT: Production of multigene transgenic pigs with high efficiency. Transplantation. 2004;78 (Suppl 2):187. 19. Wallwork J, Sykes M. Xenotransplantation: is it around the corner? Program and abstracts from the XX International Congress of The Transplantation Society; September 5-10, 2004; Vienna, Austria.