Acta vet. scand. 2002, Suppl. 99, 19-23.

Xenotransplantation – Clinical Activities and Regulatory Development

By Annika Tibell and Torbjörn Lundgren

Department of Transplantation Surgery, Huddinge University Hospital, Karolinska Institute, Stockholm, Swe- den.

Clinical xeno-organ transplantation The gap between the number of patients waiting supporting organs such as livers, hearts and for various organ transplantations and the num- lungs die on the waiting list. It has always been ber of available grafts increases steadily. Xeno- especially difficult to find grafts for small chil- transplantation – the use of animal organs and dren in need for size-matched thoracic trans- tissues for transplantation to man – may provide plants. In 1984, an American group therefore a solution to this problem. Small series of xeno- attempted to transplant a baboon heart to a new- transplantations were performed already in the born infant dying from congenital heart malfor- 1960's. In most cases, non-human primates mation (1). The transplantation was technically such as chimpanzees or baboons, were used as successful, and for the first days the graft func- source animals. In one case, a chimpanzee kid- tioned well. However, graft failure occurred af- ney transplant functioned for nine months in a ter 20 days, probably due to rejection. The so- young woman. The result is remarkable, con- called "Baby Fae case" was given great public sidering the primitive immunosuppressive ther- attention, and the ethics behind performing apy available in those days. In most cases, how- such a highly experimental procedure on a dy- ever, organs from non-human primates were ing child was intensively discussed. rejected within days (10). In a few cases, trans- Besides increasing the number of available plantations of organs from pig or sheep were at- grafts, xenotransplants may also provide an ad- tempted, but the function of these organs ceased vantage by being resistant to certain diseases already during the operations. It became obvi- that tend to recur and destroy a human graft. ous that the immunological response was diffi- One such example is, that baboons are resistant cult to control, and that the greater the phyloge- to human hepatitis B, a disease that inevitably netic disparity between source animal and recurs after human liver transplantation. In recipient, the more aggressive was the rejection 1992-1993, two cases of baboon-to-man liver response. When the donation of human organs transplantation were performed in Pittsburgh, from cadaveric donors became established, the USA (8). Both patients suffered from Hepatitis interest for xenotransplantation temporarily B and were in those days not considered eligi- ceased. ble for human liver transplantation. The first pa- The results with human organ transplantation tient had initially a very promising course. He have gradually improved since, and more and was in hepatic coma at the time of transplanta- more patients are opting for a transplant. Wait- tion. After 24 hours he could be taken off the ing times increase and patients waiting for life- ventilator, and after five days he was back at the

Acta vet. scand. Suppl. 99 - 2002 20 A. Tibell & T. Lundgren ward, eating and moving around. As could be transplanted to non-human primates, a model expected, several biochemical parameters that should mimic the immunological barrier changed to a baboon profile, but this did not between pig and man. Transgenic pig hearts seem to cause any problems, at least in the short and kidneys have provided life-supporting run. However, infectious complications devel- function for up to a few months, but the later oped and the patient died at day 70 in a cerebral stages of the rejection process are still difficult bleeding caused by an Aspergillus infection, to control. So far, livers from similar transgenic most probably related to the intense immuno- pigs have in a few cases been used for extracor- suppression. The second patient survived for 26 poreal perfusion in patients with acute liver days, but never regained consciousness or renal failure. The livers were then temporarily con- function. In both cases, there was little nected to the patients circulation and seem to histopathological evidence of rejection in the have provided some detoxifying effects. transplanted livers. Whether they provided any immunological ad- Obviously, non-human primates offer the best vantage compared to wild type pig livers has physiological compatibility, and the disease im- been hard to judge in this model, and so far, munological barrier also seems more manage- transgenic pig organs have not been trans- able. However, due to infectious risk considera- planted as permanent grafts to man. During tions, ethical concerns and the limited supply of 2002 the development of Gal knock-out pigs non-human primates, most groups today con- was reported. The hypothesis is that these or- sider the pig to be the most suitable species for gans will cause a less intense rejection re- clinical xenotransplantation. One major prob- sponse, but so far no data on primate studies are lem is then that vascularized organs trans- available. planted from pig to man undergo hyperacute re- jection, a rapid immunological process that Clinical xeno-cell transplantation destroys the transplanted organ within a few The majority of xenotransplantations per- hours. All humans have preformed antibodies formed during the 1990's have been cellular directed against pig-specific antigens mainly transplantations. In the beginning of 1990's, ten the Gal-epitope. After revascularization of the patients at Huddinge University Hospital, graft, these antibodies bind to the pig endothe- Stockholm, Sweden underwent transplantation lium and complement is activated. The process with porcine foetal pancreatic islets (5). The leads to vascular damage, thrombosis and study was performed in collaboration with the bleeding and more or less immediate organ fail- Biomedical Centre in Uppsala, Sweden, and ure. Prevention of hyperacute rejection re- proceeded with experimental studies showing quires very intense pre-treatment of the recipi- maturation and function of the foetal beta-cells ent with antibody removal or complement in adult recipients. The sows and the foetal tis- inhibition. Such pre-treatment was considered sue also underwent an extensive microbiologi- far too risky for routine clinical use. cal screening. In eight patients, the tissue was In the beginning of the 1990's, a British group injected to the liver via the portal vein. In four produced transgenic pigs specifically designed of these patients, function of the pancreatic tis- as source animals to man (7). These pigs carry sue was observed for a maximum of 450 days as a human complement regulatory factor (e.g. measured by excretion of porcine C-peptide in hDAF) and hyperacute rejection can thus be urine. In the last two patients the porcine grafts avoided. Organs from such pigs have now been were injected under the capsule of a simultane-

Acta vet. scand. Suppl. 99 - 2002 NKVet Proceedings 2002 21 ously transplanted human kidney. Surviving pig Xenotransplantation is usually defined as any cells were then found in a kidney graft biopsy contact between a human patient's body fluids taken three weeks after transplantation. The and animal organs or tissue. Using this defini- finding was remarkable as this was the first time tion several hundreds of patients in both Europe pig tissue was shown to survive in a human be- and the United States have been "xenotrans- ing. In no case, however, could the patients stop planted" when treated with a bioartificial liver. their insulin injections. The postoperative Most patients suffered from acute liver failure, courses were uneventful, and there were no and the treatment was an attempt to keep the pa- signs of infectious complications. tients alive until a human graft for transplanta- In the United States, a biotech company has tion became available. Their plasma was passed performed transplantations of foetal pig neu- through a "liver dialysis machine" containing rons to treat patients with Parkinson's disease, encapsulated pig cells (6). Improvements in Huntington's disease, and in a few cases, hepatic encephalopathy and certain biochemi- epilepsy or sequel after stroke (4). In the pilot cal parameters have been observed. study on Parkinson's disease, several patients reported remarkable improvements. The treat- Regulatory development ment has since then been analysed in a ran- The scientific development of xenotransplanta- domised placebo-controlled trial. Survival of tion during the 1990's led to several national the cellular graft occurred in several patients. and international initiatives to regulate the clin- However, no clear therapeutic effect was noted ical use of the procedure. The main concern in the controlled trial. was the risk for infectious complications, af- Bovine adrenal cells have also been evaluated fecting not only the patient, but also potentially as treatment for patients with morphine-resis- introducing new pathogens into the general tant cancer pain (3). The cells were placed in a population. Two land-mark reports were pub- semi-permeable membrane, and put into the lished in the United Kingdom. The first was spinal canal. No immunosuppressives were produced by the Nuffield Council on Bioethics given in this study. The cells were protected by (13) and was within a year followed by a gov- the membrane, and by being placed behind the ernmental report "Animal tissue into humans – blood-brain barrier where the immunological a report from the advisory group on the ethics response is weaker than in the rest of the body. on xenotransplantation" (14). The ethical basis Secretion of therapeutic substances, and sur- for these reports is the traditional weighing of vival of cells for more than six months, were benefits against risks. They conclude that the observed, but the therapeutic effect was ques- scientific basis at that time was insufficient for tionable. In Switzerland, xenotransplantation entering clinical trials. The term source animal has been attempted as a treatment for amy- is recommended instead of donor as the ani- otrophic lateral sclerosis (ALS) (11). In this mals obviously do not donate their organs. Pig study, baby hamster cells were genetically mod- is considered acceptable as source animals ified to secrete a human neurotrophic factor. while non-human primates are not considered Encapsulated cells were placed in the spinal acceptable for the reasons given earlier in this canal, and again secretion of the therapeutic article. The production of transgenic pigs is protein was observed, and cells survived for also considered acceptable as long as the pigs several months even without immunosuppres- "remain pigs", and are not caused to suffer from sion. the genetic modification. The potential infec-

Acta vet. scand. Suppl. 99 - 2002 22 A. Tibell & T. Lundgren tious risk for the public is emphasised and con- and a number of reports and recommendations sidered as a major area for further investiga- on clinical xenotransplantation trials can be tions. The importance of education of and ex- found on the FDA web site. As in the UK, pri- tensive information to future xenograft mates are not considered acceptable as source recipients and their families to make it possible animals. to obtain a truly informed consent is under- The infectious risks connected with xenotrans- lined. The reports led to the foundation of the plantation were highlighted a few years ago, but United Kingdom Xenotransplantation Interim more data are now available, and most experts Regulatory Authority (UKXIRA). The in the field today agree that the risk to the pub- UKXIRA has produced a number of reports lic is minimal provided that strict safety pre- within the field, which can be found on their cautions are followed. Obviously, microbes do web site. The UKXIRA has set high standards not respect geographical borders, and countries for approving a clinical xenotransplantation having a moratorium on clinical xenotransplan- trial, and so far, they have not received any ap- tation, may still have to face the effects of trials plications. performed elsewhere. With this background, The regulatory development in the rest of Eu- several international organisations, including rope varies (9). Some countries have decided the WHO (12) and the OECD have organised on, or are considering a moratorium for clinical workshops and published reports in the field. xenotransplantation e.g. Switzerland and The The European Council has also appointed a Netherlands. Others, such as the UK and Spain, working group producing a state of the art re- have issued reports suggesting a regulatory port and draft guidelines for clinical xenotrans- framework within which applications for xeno- plantation trials. The international perspective transplantation trials could be considered. is also important to prevent so-called xeno- Among the Nordic countries, Norway and Den- tourism. This term refers both to patients trav- mark at present have moratoriums for clinical elling to countries were the treatment is offered, xenotransplantation trials, while Finland and but lacking proper follow-up at home, and to Iceland have no official stand on the subject. In companies or researchers having applications Sweden, a governmental report has suggested a turned down in their own country and will then legal framework making it possible to perform perform xenotransplantations in countries lack- limited trials, but the report has not yet been ing regulatory framework and the resources to presented to the Parliament. control the trials. The international regulatory The United States authorities have taken a more initiatives will hopefully provide all countries liberal standpoint to clinical xenotransplanta- with baseline recommendations for clinical tion trials. Several studies on cellular xeno- xenografting and promote rapid exchange of transplantation have been approved. They are experiences across borders. performed within a strict regulatory framework and extensive microbiological testing and fol- References low-up are required (2). Samples from source 1. Bailey LL, Nehlson-Cannarella SL, Concepcion animals and recipients are to be stored for 50 W, Jolley WB: Baboon-to-human cardiac xeno- transplantation in a neonate. JAMA 1985, 254, years. A central registry of xenotransplant re- 3321-3329. cipients and a central archive for the safety 2. Bloom ET: Regulating clinical xenotransplanta- samples is being developed. Applications are tion in the United States. Graft 2001, 4, 160-162. handled by the Food and Drug Administration, 3. Buchser E, Goddard M, Heyd B, Joseph JM,

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Favre J, de Tribolet N, Lysaght M, Aebischer P: 8. Starzl TE, Fung J, Tzakis A, Todo S, Demetris Immunoisolated xenogenic chromaffin cell ther- AJ, Marino IR, Doyle H, Zeevi A. Warty V, apy for chronic pain. Initial clinical experience. Michaels M, et al.: Baboon-to-human liver Anesthesiology 1996, 85, 1005-1012; discus- transplantation. Lancet 1993, 341, 65-71. sion 29A-30A. 9. Tibell A: Regulating clinical xenotransplantation 4. Fink JS, Schumacher JM, Ellias SL, Palmer EP, in Europe. Graft 2001, 4, 157-159. Saint-Hilaire M, Shannon K, Penn R, Starr P, 10. Traeger J: Transplantations of kidneys from VanHorne C, Kott HS, Dempsey PK, Fischman chimpanzees to man. Primates Med. 1969, 3, 52- AJ, Raineri R, Manhart C, Dinsmore J, Isacson 54. O: Porcine xenografts in Parkinson's disease and 11. Zurn AD, Henry H, Schluep M, Aubert V, Winkel Huntington's disease patients: preliminary re- L, Eilers B, Bachmann C, Aebischer P: Evalua- sults. Cell Transplant. 2000, 9, 273-278. tion of an intrathecal immune response in amy- 5. Groth CG, Korsgren O, Tibell A, Tollemar J, otrophic lateral sclerosis patients implanted with Moller E, Bolinder J, Ostman J, Reinholt FP, encapsulated genetically engineered xenogeneic Hellerstrom C, Andersson A: Transplantation of cells. Cell Transplant. 2000, 9, 471-484. porcine fetal pancreas to diabetic patients. 12. WHO Guidance on Xenogenic Infection/Disease Lancet 1994, 344, 1402-1404. Surveillance and Response: A Strategy for In- 6. Pascher A, Sauer IM, Hammer C, Gerlach JC, ternational Cooperation and Coordination. Neuhaus P: Extracorporeal liver perfusion as Geneva: WHO, 2001. hepatic assist in acute liver failure: a review of 13. Animal-to-human transplants: The ethics of world experience. Xenotransplantation 2002, 9, xenotransplantation. Nuffield Council of Bio- 309-324. ethichs, UK, 1996. 7. Rosengard AM, Cary NR, Langford GA, Tucker 14. Animal tissue into humans: The Advisory Group AW, Wallwork J, White DJ: Tissue expression of on the Ethics on Xenotransplantation. Depart- human complement inhibitor, decay-accelerat- ment of Health, London, UK, 1999. ing factor, in transgenic pigs. A potential ap- proach for preventing xenograft rejection. Transplantation 1995, 59, 1325-1333.

Acta vet. scand. Suppl. 99 - 2002