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Xenotransplantation Scientific American July 97
Robert Lanza, David Cooper and William Chick

Early morning, sometime in the near future. A team of surgeons removes the heart, lungs, liver, kidneys and pancreas from a donor, whereupon a medical technician packs these organs in ice and rushes them to a nearby airport. A few hours later the heart and liver land in one city, the two kidneys in another, and the lungs and pancreas arrive in a third. Speedily conveyed to hospitals in each city, these organs are transplanted into patients who are desperately ill. The replacements function well, and six people receive a new lease on life. Back at the donor center, surgeons repeat the procedure several times, and additional transplants take place at a score of facilities distributed around the country. In all, surgical teams scattered throughout the U.S. con6uct more than 100 transplant operations on this day alone. How could so many organ donors have possibly been found. Easily - by obtaining organs not from human cadavers but from pigs. Although such a medical miracle is not vet possible, we and orher researchers are taking definite steps toward it. Our efforts are driven bv the knowledge that the supply will always be insufficient to satisfy demand. Within just the U.S., thousands of patients await transplants of the heart, liver, kidney, lung and pancreas, and millions struggle wlith diseases that may one day be curable with other kinds of donations. Notably, hemophilia, diabetes and even Alzheimer's and Parkinson's diseases might well be treated using transplanted cells. So the pressure to devise ways to transplant animal cells and organs into patients-'xenotransplantation"-steadily mounts.

Blending Species

The thought of combining parts from different species is not at all new. Greek lore of more than 3,000 years ago featured centaurs creatures that were half man, half horse - and the Chimera, a combination of lion, goat and serpent. As early as 1682 a Russian physician reportedly repaired the skull of a wounded nobleman using bone from a dog. But it was not until after the tum of the 20th century that doctors attempted with some regularity to graft tissues from animals into humans. For instance, in 1905 a French surgeon inserted slices of rabbit kidney into a child suffering from kidney failure. 'The immediate results,' he wrote, 'were excellent." Nevertheless, the child died about two weeks later. During the next two decades, several other doctors tried to transplant organs from pigs, goats, lambs and monkeys into various patients. These grafts all soon failed, for reasons that seemed puzzling at the time. Before the pioneering investigations of Nobel laureate Sir Peter Medawar at the University of London during the 1940s, physicians had little inkling of the imunologic basis of rejection. So, with only failures to show, most doctors lost interest in transplantation. But some medical researchers persevered, and in 1954 Joseph E. Murray and his colleagues at Peter Rent Brigham Hospital in Boston performed the first truly successful kidney transplant. They avoided immunologic rejection by transplanting a kidney between identical twin brothers (whose organs were indistinguishable to their immune systems). Subsequently, Murray and others were able to transplant kidneys from more distantly related siblings and, finally, from unrelated donors, by administering drugs to suppress the recipient's innate immune response. Medical practice has since grown to include transplantation of the heart, lung, liver and pancreas. But these accomplishments have brought tragedy with them: because of the shortage of donated organs, most people in need cannot be offered treatment. Of the tens of thousands of patients in the U.S. every year deemed good candidates for a transplant, less than half receive a donated organ. The shortfall will become even more dire once doctors perfect methods to treat diabetes by transplanting pancreatic islet cells, which produce insulin. Islet replacement is simpler than transplanting the whole pancreas, but it may require harvesting cells from several donors to treat each patient. Fortunately, scientists did not entirely abandon the possibility of using animal tissues in patients after human organ transplants came into vogue. During the 1960s, medical researchers continued to investigate exactly why organs transplanted between widely different species fail so rapidly. A major cause, they learned, is that the recipient's blood harbors antibody molecules that bind to the donated tissues. (These antibodies are normally directed against infectious microbes but can also respond to components of transplanted organs.) The attachment of these antibodies then activates special - complement proteins in the blood, which in turn trigger destruction of the graft. Such hyperacute rejection of foreign tissue-which begins within minutes o; at most, hours after the surgery - destroys the capillaries in the transplanted organ, causing it to hemorrhage massively. Although this reaction presents an imposing barrier to xenotransplantation, recent experiments suggest that scientists may yet overcome it. For example, in 1992 Dallid J. G. White and his colleagues at the University of Cambridge managed to create "transgenic' pigs, bearing on the inner walls of their blood vessels prc!teins that can prevent human complement proteins from doing damage. They did this by introducing into pig embryos a human gene that directs the production of a human complement-inhibiting protein [see 'Transgenic livestock as Drug Factories,' by W H. Nielander, Henryk Lubon and William N. Drohan; Sci Am Jan. White and his co-workers have not yet tested how tissues from these pigs fare in a human host, but organs from such genetically engineered pigs havc functioned for as long as two months in monkeys, because the pig cells that are in direct contact with the host's immune system are able to quash the first wave of attack. Other methods may also serve to thwart hyperacute rejection. In 1991 one of us (Cooper), along with several other investigators, identified the specific pi molecular fragments, or antigens, on pig tissues that human antibodies target. The cells lining pig vasculature have on their surfaces antigens made up of a particular sugar group. So it may tv pos'ble to breed (or 'ndeed to clone) a line of genetically engineered pigs that lack this troublesome sugar group. One plan would eliminate the enzyme that adds the sugar in the first place. Altematively, scientists could provide pigs with a gene specifying an enzyme that would replace the problematic sugar wiEh some other carbohydrate structure. For example, they could give pigs the gene for an enzyme that replaced existing antigens with the human type 0 blood group antigen, which does not elicit an immune response. Or, in principle, the offensive groups could be removed by introducing into a pig the gene for an enzyme that degrades the undesirable sugar. Yet another strategy to prevent hyperacute rejection would be to alter the recipient's immune s,.,stem so that it cannot destroy the transplanted tissue. For example, using standard apparatus, doctors can remove from the patient's blood all the antibodies to pig tissue, It is also possible to deplete the complement proteins temporarily or otherwise interfere with their activation. Remarkably, animal studies suggest that if surgeons transplant a pig organ while the patient's immune system is so suppressed, the organ may-for reasons that remain largely mysterious-achieve accommodation, a state that enables it to survive even after the host's antibodies and complement return to non-nal levels. T'he transplanted organ then continues to work despite a distinct lack of tolerance from the host's immune system. Unfortunately, researchers have not yet managed to induce accommodation reliably in animals undergoing xenotransplantation. But Guy Alexandra and his colleagues at the University of Louvain Nledical @chool in Belgium have achieved it in certain patients who received human organs from donors with incompatible blood rypes-a situation that, like xenotransplantation, normally sparks hyperacute rejection.

Fostering Tolerance

Investigators studying xenotransplantation are optimistic that, with some combination of these methods, immediately harmful immune reactions can be overcome. Yet grafts of animal tissues in patients would still fall prey to more delayed forms of immune rejection, which can take days or weeks to develop. In particular, the so-called cellular inunune response to grafts from animals is likely to be at least as strong as the robust attacks that ,N,hite blood cells of the immune system often mount against organs transplanted from one person to another. Avoiding such dela),ed reactions might require massive doses of immunosuppressive drugs, such as cvclosporine, to be given indefinitely, and the risks of toxicity, infections and other complications would be excessive. Newly devised immunosuppressive agents should help, but it would clearly be more desirable if the human body could be induced to accept animal tissues without requiring ongoing drug therapy. That happy condition might seem impossibly difficult to arrange. But hope springs from the observation that long-term organ acceptance, or inununologic tolerance, has occurred spontaneously in a few people who have received human organs. Doctors of these patients were able to reduce, and ultimatel), eliminate, the normal regimen of immunosuppressive drugs. Though still an elusive goal, the induction of immunologic tolerance is an area of vigorous research, and advances are sure to come. Curiously, it may ultimately prove easier to achieve tolerance with xenotransplantation than with traditional organ transplants. Donated human organs need to be procured urgently under emergency conditions, but animal organs would be available on demand. Ilat flexibillty might give phvsicians adequate time to reprogram the immune system of the recipient. One way to create tolerance involves modifN'ing the immune system of the patient with bone marrow cells from the donor animal. (Bone marrow is the source of all components of the blood, including the white blood cells of the immune system.) Once introduced, the donated cells spread and mature, creating a "chimeric" immune system that is part donor, part recipient. The aim is to alter the patient's immune system so that it does not recognize as foreign either the donated cells or subsequently transplanted tissues from the same animal. Following this strategy, David H. Sachs and his colleagues at Massachusetts General Hospital injected bone marrow cells from-donor pigs (along with substances to stimulate proliferation of the cells) into baboons. These animals had undergone a course of radiation to deplete their immune systems temporarily and prevent rejection of the pig bone marrow cells. The researchers also filtered from the blood of the baboons those antibodies directed against pig tissues and administered a brief course of inununosuppressive drugs. Although the baboons' immune systems evenrualiv killed most of the transplanted cells, some pig DNA survived in one of the baboons for almost a year. What is more, an important component of this chimeric baboon's immime system-the aggressive killer T cells - no longer reacted to the pig cells as foreign. Such research may yield ways to prevent inunune rel'ection of organs transplanted from animals, but truly effective measures are probably still some years away. Another scheme for evading rejection is, however, already undergoing clinical trials: immunoisolation. Following this approach, physicians physically sequester transplanted tissue within a membrane that allows small molecules (such as nutrients, oxygen and certain therapeutic agents) to cross it while blocking large molecules (such as antibodies) and white blood cells from reaching the graft. This tactic is feasible only for protecting isolated cells or small packages of tissues, not for whole organs. So it does not address the needs of someone who requires, for example, a new heart or kidney. It should nonetheless be valuable for treating many disorders. And it offers some practical advantages: physicians can manipulate cells or small masses of tissue comparatively easily and can maintain them outside the body for longer periods than are possible when working with intact organs. Recent attempts at using encapsulated cells from animals to treat liver failLire, chronic pain and amyotrophic lateral sclerosis (Lou Gehrig's disease) have all shown promise in cliniccal trials. Medical researchers may soon try to implant immunoisolated cells from animals to prol.11 'de the blood-clotting factors hemoph-diacs need or to produce nerve growth factors that might help reverse certain neurodegenerative disorders. Some investigators are especially eager to treat diabetes with isolated pig islet cells. Although one of us (Chick) pioneered the use of "perfused' devices (large sheathed implants connected to a supply of blood) for this purpose, it is easy to see some disadvantages to that particular technique. Most important, the patient requires major surgery, and the device is apt to become cloned. Engineering hollow plastic fibers or chambers unconnected to the bloodstream to isolate cells from the recipient's immune system also has drawbacks: although the surgery needed would be less traumatic than for a perfused device, it is unclear how well a patient could tolerate the plastic materials or having the implant replaced many times-a likely requirement of long-term therapy. In an effort to overcome these difficulties, two of us (Lanza and Chick), along with colleagues at BioHybrid Technologies, have developed ways to encase cells in small, biodegradable capsules that can be inj6cted under the skin or placed in the abdominal cavity with a syringe. Less than a gram of encapsulated islets from pigs should supply a diabetic patient with normal amounts of insulin. Although a vast number of cells are involved, the total volume required for these implants would be oray a few dozen cubic centimeters. In recent tests, encapsulated islet cells from cows remained alive in dogs for six weeks (the point at which the experiment ended). These results, and others from studies of mice, rats and rabbits, indicate that encapsulated pig islets would most likely survive in panents for anywhere from several months to more than a yeal Eventually the tiny packages would degrade, so no surgery would be needed to remove the old capsules when the supply of islet cells needed to be replenished. [email protected] trials of this technique should begin within a year.

Troublemaldng Htchhikers

The growing sense that xenotransplantation may be near at hand raises some critical concerns. In particular, many experts worry that animal donors might harbor diseases that, like the Ebola virus or 'mad cow' disease, can harm people. After infecting a transplant patient, such pathogens might spread into the general population and spark 'dem'c. Indeed, scientists now bean epi I lieve HW (the human immunodefficiency virus, which causes AIDS) originated in monkeys and somehow jumped the species barrier to infect humans. Thus, widespread transplantation of tissues from monkeys or baboons could conceivably put the general health at risk. Fortunately, the threat of such a catastrophe is markedly less with pigs as donors. People have lived in close association with pigs for thousands of years, and yet, except for the possibility of some flu strains, few serious diseases of swine origin appear ever to have arisen in humans. Pigs could be especially good donors for other reasons as well. Thev are relatively easy to raise and have organs that are comparable in size and ph-, s'ology to human organs. Breeds of pigs already exist that are free from cerram known pathogens. And, unlike the case with primates, few voice ethical concerns about killing an animal that people routinely slaughter for food. , Still, many questions need to be;olved before the transplantation of pig tissues into ailing patients becomes a realirv. In addition to the challenge of inunune re'action, scientists must also make sure that transplanted pig organs perform properly in their new hosts. Pig hearts and kidneys have ftmctioned adequately ab in some primates for several weeks, and it seems likely that such organs would tie work in humans as weu. But a pig liver po would probably not be able to carry out the myriad functions of a human liver, although the pig organ may be so able to sustain life for a short period of t time, allowing, for example, the patient's own liver to recover from a temporary shutdown. It may take many years before physiclans can routinely outwit evolution - as some have labeled the goal of xenotransplantation - and replace any faging organ with an animal substitute. But the transplantation of isolated cens and tissues appears poised on the threshold of modern medical practice. And we are optimistic that soon there wifl be some true successes to report.

Dealing with Viral Stowaways

The observation that pigs have passed only a few pathogens to humans despite centuries of close contact assures many people that transplanting organs from these animals would not give rise to any new and dangerous diseases. Still, there are reasons to be cautious. Certain retroviruses-viruses that incorporate their genetic blueprint directly into the host's DNA-pose a possible threat. Pigs, as well as all other mammals, contain within their genetic stores so-called proviruses-sequences of genetic code that can potentially direct the production of infectious viral particles. (As much as 1 percent of the DNA people carry is made of such viral genes.) These sequences owe their presence in modern animals to past episodes Of retroviral infection in their ancestors, when the viruses inserted their genetic cdde into sperm or egg cells. The offspring ofthe infected animals retained these viral genes, which were then passed from generation to generation. Over time, most of these vestigial viruses have evolved into forms harmless to their hosts. Yet some remain capable of activity that can cause disease in other species. So scientists naturally worry about what the proviruses of pigs would do when conveyed into a patient along with tissues transplanted from these animals. Not only does this transfer offer the resulting virus direct access to human cells, it may also present it with a uniquely susceptible victim-one having, for the sake of preserving the transplanted organ, a compromised immune system. Under these circumstances, pig proviruses might be able to give rise to active retroviruses, perhaps causing illness. It is also conceivable that these pig retroviruses could mutate in human hosts or combine with human retroviruses to produce a completely new pathogen. The result might be quite dangerous: unlike the viruses that bring on a short-lived swine flu, certain retroviruses are potentially cancer-causing and produce lifelong infection. Molecular biologists are working hard to identify troublesome proviruses lurking within pig DNA and eliminate them from breeding herds. The genetic manipulations involved may yet require significant effort, but the results of this work should help quell any remaining fears that xenotransplantation could cause new human diseases. -R.RL, D.KCC and W.LC