eMedicine Specialties > Pediatrics: Surgery > Transplantation
Xenotransplantation
Updated: Jan 14, 2010
Introduction
Organ transplantation has been called a victim of its own success.1 Transplanted human organs can replace failed organs and eliminate the need for insulin administration in patients with insulin-dependent diabetes; however, because of a severe shortage of human donors, less than 1 in 20 individuals who require transplantation are able to undergo the procedure.
Patients who require heart transplantation and are designated on the waiting list as 1A priority (urgent) have a life expectancy of less than a week. If they undergo transplantation, they typically experience more than 10 additional years of active life. Less than 2000 heart transplants are performed annually in the United States.2 The Organ Procurement and Transplantation Network/United Network of Organ Sharing (OPTN/UNOS) waiting list has nearly 3,000 heart transplant candidates. However, this barely explains the true need because these are the candidates of highest priority. The International Heart and Lung Transplant society has estimated that more than 50,000 Americans annually could benefit from heart transplantation if donors were available.3
For primary organs and tissues, 27,958 organ and tissue transplants were performed in the United States in 2008, excluding corneal transplants. More than 101,000 Americans are currently on the OPTN/UNOS waiting list. As with the hearts, the waiting list greatly underestimates the true need. For hearts, kidneys, livers, and pancreatic islets, approximately 500,000 transplants or more could be performed annually in the United States and more than 1.3 million transplants could be performed annually in the developed world if organs were available.
Although stem cell technology and tissue engineering are potential solutions to the organ shortage, xenotransplantation (transplanting organs and tissues from a different species) has generated considerable interest as a potential solution. Pigs are considered the optimal source of xenotransplant organs. Many pig organs are similar to the human counterparts in size, anatomy, and physiology. Large numbers of pigs can be quickly produced under standardized clean conditions. Pigs can be readily modified. Genes can be added or removed. Human cells can be grown in the pig.
Contrary to common belief, pig organs have multiple potential advantages over organs derived from brain-dead human donors. With human organs, little can be done before the donor is declared brain dead. After brain death, organs are procured in an emergency manner and are immediately transported to the medical center performing the transplant. The transplant is also performed with little warning. The transplant organ may come from a suboptimal donor with advanced age and chronic medical conditions or from a carrier with undetected infectious agents or malignant cells. A donor pig is raised under controlled conditions, specifically for use as an organ donor. Potential pathogens can be eliminated from the herd. The donor pig can be extensively analyzed. Organs are procured from young, robust donors. In xenotransplants, the procurement and transplant is performed on a scheduled elective basis.
Xenografts may provide medical advantages as well. These grafts are resistant to many human pathogens specific to human tissues, such as HIV, hepatitis, and human cytomegalovirus. Tumors such as melanoma have also been transferred to the recipient through human allografts. Pigs can be produced that are free of potential pathogens. Xenografts may be resistant to autoimmune reactions, such as the autoimmune destruction of beta cells with type 1 diabetes.
Despite these advantages, relatively few xenotransplants have been successfully performed in experimental models and none have been performed in the clinical arena. This is due to 3 main causes. First, xenotransplantation is subject to severe rejection, involving many different antigen disparities between humans and pigs that elicit multiple mechanisms of immune rejection. Current opinion dictates that severe immune suppression is required to prevent rejection, and this subjects the recipient to a high risk of infection and toxicity. Second, the perceived need for increased immune suppression leads to concern about infectious agents from the pig, including exogenous viruses (eg, circoviruses, hepatitis E) and endogenous viruses (eg, porcine endogenous retrovirus [PERV], which may lead to novel infectious diseases in humans (ie, xenozoonoses). Third, for some tissues such as the liver, the physiological function of the pig organ is insufficiently close to the human to provide long-term support.
Fortunately, significant progress has been made on all fronts. Several xenotransplant technologies are now in clinical trials.
A major goal is to achieve prolonged acceptance of pig xenografts with reduced immune suppression, equivalent to or less than the level of immune suppression used for human allografts. Because human organs must, out of necessity, be reserved for the most ill patients, a defined level of risk and complications is justifiable if life expectancy is threatened. However, if an unlimited supply of donor organs were available, and the morbidity associated with the procedure was minimized, many recipients could be transplanted at a less critical stage in their disease, thereby reducing the burden to the health care system. The acceptable level of risk and complications for this larger group would be reduced.
If a large number of transplants are to be performed annually, attention must also be paid to logistical issues. For example, does the modification of the pig substantially interfere with the litter size or other aspects of breeding? If special clean pigs are required, how long would it take to expand the herd to meet the projected need?
In view of the potential advantages and the progress made to date, a much greater effort is justified for developing xenotransplantation than is currently committed. Compared with other forms of regenerative medicine, xenotransplantation is closer to clinical reality and more cost effective.
Five aspects of xenotransplantation are covered below, including xenograft rejection, xenograft function, xenozoonosis, clinical xenotransplantation, and the future of xenotransplantation. In light of substantial developments, xenotransplantation may address the largely unmet needs in regenerative medicine.
Xenograft Rejection and Prevention of Rejection
The primary obstacle to successful xenotransplantation is xenograft rejection, which is more vigorous and complex than rejection of human allografts and is therefore more difficult to prevent and reverse.
Allotransplants have few antigen disparities, principally the human leukocyte antigen (HLA) or histocompatibility antigens. These disparities can be minimized through tissue typing and matching. The major immune response is cellular rejection. In contrast, pig tissues express multiple antigens that cannot be matched to human recipients. These antigens elicit multiple reactions, not only through the adaptive immune system but also through the innate immune system.
Strategies that are effective with allografts are not effective with xenografts. Moreover, the rejection barrier of vascular xenografts (eg, hearts, kidneys, livers, lungs) is greater than for cellular grafts (eg, islets, hepatocytes, neural tissue). With vascular grafts, injury to the endothelial cells lining the vessels leads to thrombosis, hemorrhage, and prompt loss of the graft. This is due to the vulnerability of the endothelial cells that line the blood vessels, which become activated. Xenografts are lost to an immediate hyperacute and acute vascular rejection days after transplant.
The major antigen or epitope on the vascular endothelium attacked by the human immune system is galactose a-1,3-galactose (alphaGal). AlphaGal is expressed on many tissues, but especially endothelial cells. Humans and old world primates lack this particular oligosaccharide and therefore have preformed natural antibodies to the alphGal epitope. These antibodies bind to the vascular endothelium and fix complement, leading to lysis and apoptosis of the vascular endothelial cells. Hyperacute rejection may occur within minutes or hours.
Transgenic knockout pigs have been generated with deleted galactosyl transferase, which is responsible for the synthesis of the alphaGal within the pig cells. Pigs homozygous for this deletion do not express alphaGal antigens (double knockout pigs). Hyperacute rejection is greatly reduced or eliminated.
Besides alphaGal, additional antigenic differences are noted between primate recipients and donor pigs. Kidney xenotransplants from double knockout pigs into nonhuman primates eliminated hyperacute rejection but not acute xenograft rejection. Cytotoxic antibodies to non-Gal antigens are observed pretransplant.4,5 With lung xenografts, hyperacute rejection was delayed but still observed with alphaGal knockout pigs.6 The contribution of antibodies against non-Gal antigens towards hyperacute rejection has not been excluded. Hyperacute rejection was also eliminated with pig heart xenotransplants using double knockout pigs.7
Hyperacute rejection can be prevented, either through removal of the preformed anti-pig antibodies, removal of the antigens, complement inhibition, or tissue accommodation. However, the xenograft is not yet out of danger. Indeed, acute vascular rejection presents an even greater challenge than hyperacute rejection. Endothelial cells are injured by multiple mechanisms, including cytotoxic T cells, innate immunity, and complement-dependent cytotoxicity from induced antibodies. The immune suppression required to block all of these reactions substantially exceeds that required for prevention of allograft rejection.
Acute xenograft rejection represents a reaction against both alphaGal and non-Gal antigens. Acute rejection with GalT-knockout pigs is associated with a disordered coagulopathy.8 The pathology shows thrombotic microangiopathy consisting of platelet rich thrombi filling the microvasculature.9,10 However, the primary event still appears to be endothelial cell injury. Although the GalT-knockout transgenic pigs have nearly eliminated hyperacute rejection, the xenografts typically undergo acute rejection.4,11 The multiple antigens other than alphaGal trigger a robust acute rejection.
In addition to natural antibodies to alphaGal, vascular endothelial cells are injured by multiple mechanisms, including cytotoxic T cells, natural killer (NK) cells, and antibodies produced de novo after transplantation. The immune suppression required to block all of these reactions substantially exceeds that required for prevention of allograft rejection.
Cellular xenografts, such as porcine hepatocytes, neural tissue, islets and islet cell clusters, differ from vascular grafts. Cells of the endocrine pancreas express little or no alphaGal12,13 and are less susceptible to hyperacute rejection. Nonetheless, instant blood-mediated inflammatory reactions (IBMIR), an innate immune response that depends on release of tissue factor and binding of complement, can destroy much of the islet cell mass through thrombosis. The immediate loss of islet tissue can prevent the achievement of euglycemia in diabetic recipients, even if rejection is avoided. IBMIR results from factors in the host as well as in the islet xenograft.14 The host can be treated. Alternatively, the graft can be modified.
The process of procuring the islets from the pancreas induces proinflammatory factors in the islets, such as monocyte chemotactic protein (MCP)-1, interleukin (IL)-1 β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-6, and IL-8. The pig or the islets can be also modified to prevent IBMIR. Acute rejection is principally cellular rejection15 and induced antibody mediated rejection.16 Prolonged engraftment of porcine islets in cynomolgus monkeys was achieved using wild-type pigs rather than alphaGal knockout pigs in combination with T-cell suppression.17
Prolonged insulin-free survival was achieved in diabetic cynomolgus macaques with transplanted pig islets. The donor pigs were not genetically modified. Rejection was prevented with antibodies to CD25 and CD154, FTY720 (or tacrolimus), everolimus, and leflunomide. In this study, eliminating antibodies to alphaGal or using pigs modified to eliminate the alphaGal antigen was not necessary.
Using islet cell clusters from neonatal pigs (aged 1-2 d) and immune suppression, prolonged insulin-free survival was achieved in diabetic rhesus macaques.18 Preformed antibodies to alphaGal also did not prevent engraftment and function. The induction immune suppression included antibodies to CD25 and CD154. Maintenance immune suppression included belatocept and sirolimus or mycophenolate.
Encapsulation of porcine islets has long been pursued as a method to prevent acute rejection by protecting the graft from invading lymphocytes and antibodies. The initial formulations of polyalginate encapsulation led to fibrosis surrounding the capsules and breakdown of the capsules. Efforts are now focused on eliminating those problems.
Using encapsulation, prolonged survival of porcine islet xenografts has been realized in diabetic monkeys without the need for antirejection drugs. Using a macroencapsulation device and implanting it within the abdominal fat, islet xenograft function has been achieved as long as 26 weeks.19
Prolonged function of vascularized pig xenografts in nonhuman primates is possible if rejection is prevented. Heterotopic pig heart xenografts have survived for up to 6 months in baboons.20 The donor pigs were transgenic, expressing the CD46 human complement inhibitor. The baboons received anti-CD20 (Rituximab), thymoglobulin for induction, splenectomy, removal of anti-Gal antibodies, and tacrolimus, sirolimus, and steroids.
Unfortunately, these experiences illustrate how the strategy used with allografts (ie, tissue matching and selective immune suppression) only succeeds with xenografts if the immune system is compromised to an unacceptable level.
Thus, the challenge of xenotransplantation is to prevent xenograft rejection without severely suppressing the recipient’s immune competence. Two processes promote engraftment without the need for immune suppression: specific immune tolerance and tissue accommodation.
A comprehensive approach that resolves multiple antigen disparities and immune responses without severely compromising the recipient’s ability to fight infection is needed. Immune tolerance to self is centrally maintained through clonal deletion of self-reactive lymphocytes in the thymus and peripherally in suppressor or regulatory cells that block self-reactive lymphocytes that escape thymic censure.21 Specific immune tolerance is very difficult to achieve in adult recipients. One approach is to induce mixed hemopoietic chimerism (eg, with pig bone marrow transplanted into primates).22 This can only succeed if the recipient is subjected to a period of severe immune deficiency. Furthermore, tolerance induced in this way is limited to antigens expressed on hematopoietic cells.
T-regulatory cells play a major role in preventing autoimmune diseases, including autoimmune diabetes. Autoimmune diabetes develops in animal models with thymic abnormalities and deficiencies of T-regulatory cells. Autoimmunity to β cells can be prevented and reversed with induction of T-regulatory cells.
Natural T-regulatory cells are circulating CD4+/CD25+ T cells that facilitate the maintenance of tolerance to self.23 These cells express the transcription factor foxP3, which is integral to the function of inhibiting self-reactive T cells. The cells are maintained by IL-2. They mature within the thymus and are positively selected for cells with a T-cell receptor reactive to class II antigen expressed on the thymic dendritic cells. The presence of such cells within the chimeric pigs is justified. Human cells grow within the thymus of the fetal chimeric pig. Transplantation of pig thymic tissue into nonhuman primates leads to engraftment of T-cell precursors within the pig thymus.24
Relevant to xenotransplantation, human CD4+/CD25+ T-regulatory cells suppress the reaction of human responder cells to pig stimulator cells.25 Initial studies showed increased circulating human CD4+ T cells in chimeric pigs.26 In the preliminary data below, human CD4+/CD25+/foxP3+ cells are present in the spleen of a chimeric pig.
Preliminary data identify increased numbers of human CD4+/CD25+ lymphocytes from the spleens of chimeric pigs. A large subpopulation of these cells coexpresses foxP3.
CD4+/CD25+ regulatory T cells develop early in human fetal development.27 Regulatory T cells prevent graft versus host disease after allogeneic in utero allogeneic transplants of hematopoietic stem cells.28
Large numbers of CD4+/CD25+ regulatory T cells have been cultured from baboon peripheral blood that specifically block the reaction of baboon responder lymphocytes to pig stimulator cells.26
Ideally, regulatory cells would lead to specific inhibition of both adaptive and innate immunity,29,30 protecting the xenograft but leaving the recipient sufficiently immune competent to defend against pathogens. Previous work with pigs that were chimeric with human lymphocytes demonstrated that the chimeric lymphocytes specifically inhibited the human versus pig mixed lymphocyte reaction.31
Generating T-regulatory cells from chimeric pigs can provide specific immune tolerance to the pig xenograft. The tolerance is induced prior to organ transplantation and outside of the recipient, effectively avoiding the risk and complications often associated with the induction of tolerance. Using this approach, in 2 preclinical trials of diabetic monkeys, 5 of 6 transplants demonstrated prolonged survival and function of the islet xenografts without any posttransplant immune suppression. One monkey was insulin free 31 weeks posttransplant when he died of unrelated causes.32
Accommodation is the term applied to the phenomenon whereby cells, principally vascular endothelial cells, adapt to resist injury by complement. It is typically seen in allograft recipients with antigraft antibodies, such as with an AB mismatch.33 If rejection can be prevented for 2 or more weeks, such as with plasmapheresis, the graft then becomes resistant to injury, even in the presence of antigraft antibodies. Multiple protective proteins, such as heme oxygenase-1, protein inhibitor of apoptosis, and A20, are induced which protect the cells from apoptosis.34 The protection is antigen nonspecific, providing protection to multiple antibodies.
Other new technologies are being actively explored to reduce the immune suppression required for prolonged xenograft function. The need for immune suppression could be reduced by reducing the overall antigenicity of the xenograft. Fetal pig tissues have a reduced density of antigens. These could be transplanted into the recipient, where it later matures.35 Another is to induce specific immune tolerance to the pig tissue by reconstituting the immune system through a pig thymus within the recipient.36,37
Another approach is to block co-stimulation of the mature T-effector cells by inhibiting the second signal with monoclonal antibody, such as anti-CD154. Co-stimulator blockade causes the T-effector cells to undergo apoptosis. When combined with donor specific transfusions, prolonged acceptance of allografts has been achieved; unfortunately, anti-CD154 antibodies appear to be associated with thromboembolic complications. Alternatives to anti-CD154 are being developed to disrupt costimulation, such as antibodies to CD40.38
Another strategy for using pigs as organ donors is to genetically modify the donor pigs. Innovative genetically modified pigs and tissues have been designed and produced to prevent rejection. The transgene would modify the target pig tissue, interfering with the pathophysiology of xenograft rejection. In designing such pigs, care must be taken to not interfere with the pigs' immune response. That could produce immune deficient pigs that are difficult to raise and breed. It could also produce organ grafts that are more susceptible to infection.
As acute vascular rejection ultimately leads to organ destruction through thrombotic microangiopathy, donor pigs have are being designed and produced that produce inhibitors of human coagulation factors.39
Accommodation is associated with an induced expression of heme oxygenase I (HO-1). Transgenic pigs have been produced on a background of CD55 that overexpress human HO-1. Compared with wild-type pigs, the kidneys resist hyperacute rejection when perfused with human blood ex vivo.40
Fas ligand inhibits CD8+ cytotoxic T cells. A promising new technology transfects pig islets with FasL and related molecules. Initial studies demonstrate prolonged survival in rats and inhibition of human CD8+ T cells.41,42
All efforts to induce immune tolerance within the xenograft recipient are limited by the need to induce severe immune deficiency, at least during the time of induction, and this renders this unattractive as a viable clinical strategy. A more original approach to avoid xenograft rejection is to generate regulatory cells by engrafting bone marrow from the recipient into the fetal pig destined to be the organ donor. These cells can then be harvested and used to condition the recipient prior to the transplant. Preliminary evidence suggests, not only that regulatory cells are produced in such models, but that the organs retrieved from the fetally injected piglets exhibit accommodation.43 Such an approach, if successfully transferred to the clinic, could spare the patient the risks associated with therapeutic immunosuppression.
Preclinical studies showed that prolonged engraftment could be achieved with pig hearts transplanted into sheep using modest immune suppression44 and with pig hearts transplanted into sensitized sheep that have preformed antipig antibodies.45 Using a similar approach, pig islet cell clusters were successfully transplanted into diabetic macaques, providing prolonged function without the need for immune suppression.32
Xenograft Function
The ability of a pig xenograft to replace the function of a failed human organ depends on multiple physiological factors. In general, organs that are simple in function are the best candidates for xenotransplantation. Hearts pump blood through the lungs and system. Lungs provide gas exchange of oxygen and carbon dioxide. Pancreatic islets provide insulin, glucagon, and somatostatin. If rejection can be prevented, these organ xenografts are likely to provide long-term support for the human recipients.
Pig and human kidneys are similar with respect to renal blood flow, glomerular filtration rate, and creatinine clearance.46 Porcine erythropoietin is about 80% homologous with human erythropoietin.47
Pig hearts orthotopically transplanted into nonhuman primates provide adequate circulation until the graft is rejected.48
Porcine insulin differs from human insulin by just a single amino acid, and provides excellent control of glucose metabolism. However, the connecting C-peptide of humans and pigs differs at 11 of 31 amino acids, which may affect microvascular blood flow49 and the development of chronic vascular pathology.50
On the other hand, complex organs such as the pig liver might be inadequate for long-term support of humans.51 Among other functions, the hepatocytes produce many proteins. Porcine albumin has less than 65% homology with human albumin.52 Several complement and coagulation factors, such as coagulation factor 6, are species-specific. The species-specific proteins produced by the pig liver do not provide long-term function in the human recipient. One innovation under development could produce hybrid pig livers in which native pig hepatocytes have been replaced with human hepatocytes. Lines of transgenic pigs have been produced with suicide genes under the control of hepatocyte specific promoters. These could allow for the selective and conditional removal of the native pig hepatocytes.
If the challenge of immune rejection and function can be overcome, the antigen disparity of xenografts may provide some advantages for the recipient by avoiding the primary pathology that injured the native organ.
Pig xenografts may resist infection with native viruses from the patient. Human cytomegalovirus infections often infect human allografts and cause disease, including enhancement of rejection and accelerate chronic rejection, a major problem with allografts.53 Cytomegalovirus is a species specific virus. In a study of pig-to-baboon heart transplantation, baboon and porcine cytomegalovirus were evaluated in the transplant and native tissues. Although low levels of infection were detected in the xenogeneic tissues (ie, baboon cytomegalovirus in the pig heart), cytomegalovirus infection was observed only in the corresponding tissues.
Xenografts may also provide resistance to autoimmune disease. For example, porcine islets are partially resistant to the autoimmune reaction to islets in nonobese diabetic mice.54 Antibodies to human glutamic acid decarboxylase (GAD) in patients at risk for type 1 diabetes are more predictive of clinical progression to insulin dependence than antibodies to porcine GAD.55
Xenozoonoses
One of the most discussed risks of porcine xenotransplantation has been the potential to pass infectious agents from the donor pig to the patient. At one time, the discussion led to a moratorium against xenotransplantation in Europe and to US Food and Drug Administration (FDA) guidelines outlining extensive measures for monitoring xenograft recipients. Although some caution is appropriate with any new technology, the extensive media exposure has given pig xenotransplantation an undeserved reputation as a high-risk procedure. In fact, pig herds can be produced that are free of pathogens and that carry much less risk of infection than a human allograft.
Most of the concern of xenozoonosis comes from the perceived need for high levels of immune suppression to prevent xenograft rejection. With a severe immune deficiency, the recipient would be more susceptible to acquiring an agent from the donor pig. The problem is compounded if xenotransplantation is done on a widespread basis. As technology reduces the need for severe immune suppression, and as experience accumulates demonstrating the relative safety of porcine xenotransplantation, the concern about xenozoonosis is likely to fade.
The viral zoonotic agents can be divided into endogenous and exogenous viruses. The endogenous viruses are encoded within the genome and, therefore, cannot be eliminated from the herd using conventional technology. In 1997, coculture of human and porcine cells led to porcine endogenogenous retroviruses (PERV) appearing within the human cells. Speculation about PERV progressed to a concern that it could potentially become a public health hazard. Despite considerable research, no pathology has ever been observed related to PERV. Indeed, although a major portion of the world’s population either consumes or prepares pork, no known PERV-related disease has ever been described.
In a retrospective study of patients transplanted or transfused with viable pig tissue, no evidence of infection was observed. A few subjects had detectable PERV RNA, but it was consistent with RNA from circulating pig cells. In humanized mouse models infused with porcine cells, a few mice were described in which the human cells were initially thought to contain PERV. However, subsequent studies attributed this apparent infection to murine leukemia virus.
The risk of PERV becoming a public health hazard is infinitesimal. PERV would need to undergo a series of improbable transformations to make it both a pathogen and contagious. Many herds of pigs have been described in which PERV is not passed to human cells in coculture. Some strains of pigs have very limited copies of PERV in their genome. The risk is further reduced by the extensive monitoring of patients and cohorts required and by the sensitivity of PERV to antiviral agents. The minimal potential risk of PERV is far outweighed by the potential medical value of xenotransplants and should not be a barrier to xenotransplantation.
Recognizing the potential benefit of xenotransplantation outweighs the risks, the FDA (Center for Biologics Evaluation and Research) has chosen a pathway of proceeding with caution. Guidelines have been published that recommend source herds that do not transmit PERV to human cells, prolonged monitoring of recipients, and archiving of fluids and tissues from recipients. PERV is sensitive to antiviral agents. The FDA has allowed clinical xenotransplantation trials to programs following the guidelines. The number of investigational new drug applications for clinical trials has recently increased.Whether PERV is a true public health risk or not, the discussion has had a major negative impact on the development of xenotransplantation. Corporate support of xenotransplantation has nearly disappeared, in large part because of a fear of liability of the PERV risk and the requirement for prolonged monitoring of patients. Large pharmaceutical corporations that have abandoned xenotransplantation include Novartis, Roche, and Genzyme. Smaller corporations dedicated to xenotransplantation have been unable to obtain the investments needed to proceed and have folded. These include Immerge Biotherapeutics, Imutran, Diacrin, and Circe.
The one observation of concern remains the transmission of PERV to human cells in tissue coculture. However, not all pigs transmit PERV to human cells. The herd of VitalPure Designated Pathogen Free (VP-DPF) pigs is one of the herds that fail to transmit PERV in coculture. PERV is defined at 3 loci in the porcine genome, termed PERV-A, PERV-B, and PERV-C. The transmission of PERV is thought to require a homologous recombination between PERV-A and PERV-C.56,57 The recombined viruses are able to adhere to human cells. PERV-A and PERV-B are nearly ubiquitous. PERV-C is present in 97% of the pigs surveyed. Cells from pigs that are PERV-C–negative fail to transmit to human cells.58 Using only PERV-C–negative pigs for clinical xenotransplantation has been recommended.
Lines of transgenic pigs have been produced that synthesize silencing interference RNA for PERV.59,60 These pigs have substantially reduced PERV expression (>20%). These pigs are termed PERV knock-down pigs rather than PERV knockout pigs. As many as 30 copies of each PERV type per cell may be present; thus, eliminating PERV by genetic modification may be difficult.
Another approach to creating PERV-free pigs is selectively breeding pigs based on PERV load.61 A defined pathogen herd from New Zealand has been assessed for PERV. Although the boars had an average of about 15 copies of PERV per cell, and the sows averaged about 10 copies of PERV, one pig in the herd had only 4 copies of PERV per cell. Two piglets of the herd were PERV-C negative. Unfortunately, even if both the sow and boar had only 4 copies of PERV per cell, producing PERV-free pigs could take many breedings.
The authors have screened the pathogen-free pigs in Nebraska for PERV-A, PERV-B, and PERV-C.62 One sow was found to be totally free of all 3 types of PERV. This has been confirmed with different sets of primers. Most of the herd was free of PERV-C, and half of the herd was free of PERV-A as well. In quantifying the PERV types, 5 pigs (1 boar, 4 sows) were identified as PERV-A-negative and PERV-C negative, with 1 copy of PERV-B per cell. PERV-free breeders could potentially be realized within one generation.
On the other hand, some ubiquitous exogenous viruses pose a real danger to xenograft recipients but have received relatively less attention.63,64 Examples of viruses that potentially could be passed from swine to humans include the H1N1 virus, Nipah virus, Menangle virus, hepatitis E virus, encephalomyocarditis virus, and Japanese encephalitis virus.
Some viruses may have a low potential to infect the recipient tissues but could adversely affect the graft. Porcine lymphotropic herpes viruses may lead to posttransplant lymphoproliferative syndrome in recipients receiving porcine lymphocytes.65,66 Porcine cytomegalovirus and porcine encephalomyocarditis virus may adversely affect vascular xenografts such as hearts.67
Some porcine exogenous viruses adversely affect the health of the donor herd, leading to other adverse effects. Porcine circoviruses cause wasting in postweaned piglets. The piglets are immune deficient, making them susceptible to additional infections. Porcine parvovirus is associated with late-term abortions. Different strains of porcine coronaviruses lead to chronic diarrhea, respiratory disease, and encephalitis.
The exogenous viruses can be eliminated from the pig donor herd, through intense careful husbandry and persistent monitoring of the herd. In producing a clean herd, some have proposed starting with a hysterotomy delivered, colostrum deprived piglets. This eliminates the bacterial and fungal pathogens but does not eliminate pathogens that pass the placental barrier such as circovirus, Arterivirus, and parvoviruses.61,68 Porcine lymphotropic herpesviruses and encephalomyocarditis virus may also escape this procedure.69,70
The production of a herd of donor pigs that is virus pathogen–free requires considerable time and effort. The pigs must be housed in a filtered environment and protected from pathogens from human caretakers, food, insects, and rodents. The pigs need to be screened for the agents and for serological evidence of agents. All infected and exposed animals need to be removed.
Although the effort to produce a biomedical-grade herd of pathogen-free donor pigs is a responsibility for xenotransplantation, it reflects a major opportunity that is not available for human allografts. The elimination of exogenous pathogens from the donor pig herd combined with a reduced need for immune suppression could make xenotransplantation much safer than allotransplantation regarding infectious diseases.
Clinical Xenotransplantation
The use of animals as organ and tissue donors is not a new idea. At a time when medical technology and understanding of immunology and physiology were primitive, animals were the preferred source.
Jean Babtiste Deny performed the first blood transfusion into a patient in 1667 using blood taken from a sheep.71 In 1906, Jaboulay performed the first vascular xenotransplants, transplanting kidneys from a pig and a dog into patients with renal insufficiency.72 In 1963, Hitchcock transplanted a kidney from a baboon into 65-year-old woman; it functioned for 4 days.73 Reemtsma and Starzl achieved a measure of clinical success transplanting kidneys from nonhuman primates into human recipients,.74,75
However, over the next 25 years, focus turned to transplanting organs and tissues from human donors. In the early 1990s, porcine islets prepared from fetal pigs were transplanted into diabetic patients with modest immune suppression. Porcine C-peptide was monitored in the urine until the grafts eventually rejected.76 In 1992 and 1993, 2 orthotopic xenotransplants were performed placing baboon livers into patients with liver failure related to hepatitis B virus infection.77 Multidrug therapy was administered to prevent cellular and antibody-mediated rejection. The patients survived 70 and 26 days, respectively. The grafts provided at least partial function. Although the grafts did not undergo rejection, one of the patients developed a terminal aspergillosis related to the immune suppression.
Baboon marrow was transplanted into a patient with AIDS with the knowledge that the baboon CD4+ lymphocytes were resistant to HIV. Although the patient rejected the baboon cells, his clinical condition improved, and he continued to do well at the time of publication.78
Dopaminergic neurons from a fetal pig were transplanted into the brain of a patient with Parkinson’s disease.79 The transplant significantly improved the clinical course of the patient. Seven months later, the fetal pig neurons were identified. The implantation of pig neural tissue into an immune privileged environment of the brain reduced the risk of rejection. Unfortunately, a subsequent controlled trial failed to demonstrate a statistically significant difference with the control group,.80,81
Patients in acute liver failure have been supported for a few hours to days with extracorporeal liver perfusion (ECLP) while a human liver donor is sought.82,83 Blood from the patient is perfused through the pig liver and returned. These procedures indicate that the pig liver is functional on a short-term basis. Patients typically show clinical improvement with reduction of blood ammonia and lactic acid levels, conjugation and excretion of bilirubin, and stabilization of prothrombin time.84
Devices that incorporate cells or tissue from animals or incorporate human cells or tissues that have been cocultured with animal cells are considered xenografts. One promising device provides short-term support for patients with acute liver failure.85 Initial clinical trials were promising, providing time to bridge to a human liver transplant. Others showed spontaneous recovery during the support period.
Following a series of public hearings by the National Institutes of Health (NIH), the Centers for Disease Control and Prevention (CDC), and the FDA, the FDA published guidelines for xenotransplantation to address concerns raised about infectious diseases from donor animals. The latest guidelines were published in 2003.86 For a clinical trial to be allowed, the investigator must demonstrate evidence of efficacy of the xenotransplants in nonhuman primates. The investigator must also demonstrate compliance with the safety guidelines, including a certified source herd, prolonged archiving of tissues and records, and current good manufacturing practice (cGMP) facilities and procedures.
Most likely, the first successful clinical trials will be with cellular transplants, such as pancreatic islets, neural cells, or hepatocytes.87 Because vascular xenografts are sensitive to rejection of the endothelial cells, the threshold is set higher. Heart xenografts and kidney xenografts will likely be the first tested. Lung xenografts are presently the most challenging xenografts because of the extensive capillary network and sensitivity of endothelial cells to hyperacute rejection.88
The Future of Xenotransplantation
Regenerative medicine urgently needs an alternative technology to supplement the transplantation of human allografts for the cure of organ and tissue failure. The leading technologies to provide tissues and organs include xenotransplantation, stem cell technology, and tissue engineering. Xenotransplantation is the first such technology to be pursued and the most advanced.
Compared with the current status of stem cell technology and tissue engineering, xenografts provide multiple advantages. First, xenograft function of fully formed porcine tissues and organs provide function that most closely resembles the human counterpart. In particular, xenotransplantation is the closest of the 3 regenerative medicine technologies to providing fully functional replacement organs for heart failure, kidney failure, liver failure, respiratory failure, and fully functional pancreatic islets for the cure of type 1 diabetes.
A second advantage of xenotransplantation is that porcine cells and tissues resist the pathological processes that adversely affect human cells, such as viral infections, metabolic processes, and autoimmunity. For many transplant candidates, these processes are the very reason that they need a transplant. Replacement organs made of allogeneic human cells or of cells derived from the patient would be vulnerable to the same injury process.
A third major advantage of porcine xenografts is that organs and tissues could be provided on a cost effective basis for all who need the transplant.
Until recently, the major drawback to xenotransplantation has been vigorous rejection. That challenge may explain the commonly held view of pigs as a second-rate alternative source of transplant organs and tissues. Until rejection can be prevented with minimal immune suppression, the transplantation of viable pig tissues will not be done on a large scale. As the need for immune suppression drops, so will the concern about zoonotic infections such as porcine endogenous retroviruses (PERV). In recent years, the challenge of rejection is quietly being resolved through innovative methods for inducing tolerance and accommodation to the pig tissues, as well as improvements in antirejection agents. Prolonged survival of porcine xenografts in nonhuman primates is becoming commonplace. The potential for long-term functional xenografts with little or even no immune suppression is now a realistic possibility.
The large-scale clinical application of xenotransplantation is threatened by 3 fiscal and logistical barriers: unrealistic federal regulation, inadequate funding by industry and government, and inadequate qualified source herds of clean swine.
The federal guidelines that regulate xenotransplantation were formed following workshops that were concerned about the potential public health hazards of zoonotic infections, particularly concern about the potential threat of PERV. At the time of those discussions, prolonged acceptance of pig xenografts in preclinical studies could be achieved only with high doses of antirejection drugs. Speculation held that if xenotransplantation was performed on a large scale, recombination of the PERV subtypes could lead to a new virus that was contagious, pathological, and a threat to the public health. The resulting guidelines called for patient followup for as long as 50 years and severe restrictions on xenograft recipients, such as travel. Although the guidelines were appropriate for the information at that time, they unfortunately discouraged large pharmaceutical and medical device companies from investing in xenotransplantation.
In addition to the stringent requirements for monitoring recipients, companies were concerned about the liability of pursuing a technology perceived to be a potential hazard to the public health. However, since PERV was initially described, numerous studies have shown no evidence of PERV becoming contagious or being pathological. Many strains fail to pass PERV to human cells in coculture. The molecular virology of PERV passage is now understood. Swine strains have been produced that are free of the PERV-C that is needed for passage. Indeed, in the near future, swine strains will likely be produced with no genomic PERV.
The risk of a public health hazard from PERV needs to be re-examined in light of current information and developments. The risk of a public health hazard from PERV in pigs is not measurably greater than a public health hazard from technologies based on human cells and tissues. Indeed, the risk for transmitting exogenous pathogens such as hepatitis, HIV, and malignant cells to patients is much less for porcine xenografts than with human based transplants. One could speculate that some unknown virus or mutational event could still produce a public health hazard. Totally ruling out the unknown is impossible. Fear of the unknown, however, is irrational and effectively blocks all technological development. Should blood transfusions and human tissue transplants also be severely restricted because of this unknown factor? The infinitesimal risk of the unknown must be balanced against the tremendous potential for regenerative medicine.
The second barrier is lack of funding. Although xenotransplantation is the closest to clinical reality of the 3 major regenerative technologies, it is also the least-supported technology. Large corporations have ceased their support of xenotransplantation. The NIH supports xenotransplantation at a level far below that for stem cell and tissue engineering. For example, the recent economic stimulation package (American Recovery and Reinvestment Act of 2009) provided $200 million for 200 challenge grants. Although multiple challenge topics address issues with stem cell and tissue-engineering technology, no challenges are posted for improving xenotransplantation.89 As stem cell and tissue engineering are at a speculative stage of development with major technical hurdles to overcome, abandoning xenotransplantation, which is at an advanced stage of development, is absurd.
The third major barrier to xenotransplantation is lack of sufficient qualified source herds. At this time, only a handful of swine herds are qualified to be used for clinical trials. All of these are small herds with less than 100 pigs. Several small clinical trials are currently being pursued. The likelihood that at least one of these trials will be successful and lead to a new device approval is great. However, when such approval is achieved, not nearly enough qualified pigs will be available to satisfy the unmet need. Maintaining and developing qualified herds with the appropriate barrier facilities and husbandry is very expensive. The current herds need to be greatly expanded for widespread clinical application. This will take several years and much more support.
Regrettably, the 3 regenerative medicine technologies are usually considered in competition with each other. The underlying assumption is that one will "win" and become the standard technology for alternate tissues whereas the others will "lose" and be abandoned. Considering the enormity of the unmet need in regenerative medicine, this assumption is unfortunate. Most likely, each technology will prove to be optimal for different select diseases.
A more promising approach would be to combine these technologies. Xenotransplantation could provide a cost-effective and sterile bioreactor for maturing stem cells into tissues that can be transplanted. Xenotransplantation could also provide the scaffolding for tissue engineering.
Several developments in recent years support such hybrid technologies.
Our program has been growing human hematopoietic lymphocytes and stem cells from the transplant recipient, with the goal of producing human antigen-specific T-regulatory cells that prevent rejection of pig xenografts after transfer from the chimeric pig back into the recipient. Prolonged survival and function of pig islet cell clusters has been realized in nonhuman primates without posttransplant immune suppression.29
As an example, the pig liver differs from human livers in several critical aspects. Although pig livers have provided short-term life support, they are unlikely to be effective for long-term support. Human hepatocytes grow within the fetal pig liver and demonstrate normal sinusoidal architecture. Expansion is limited though by competition by the native pig hepatocytes. Transgenic pigs have been produced that express suicide genes (thymidine kinase or cytosine deaminase) in the pig hepatocytes. By providing the chimeric pig with a prodrug such as ganciclovir for thymidine kinase, selectively and conditionally destroying the pig hepatocytes is possible, giving the human hepatocytes an edge to expand.90
Human hepatocytes may not be essential to develop hybrid livers. For example, when fetal lambs were injected with human CD34-positive bone marrow cells, the livers of the newborn lambs were shown to contain human hepatocytes.91
With stem cell technology, the pluripotent stem cells would not be directly transplanted into the recipient because of the risk of developing teratomas. The challenge has been to differentiate the stem cells outside of the patient. Although the production of human islets or insulin producing glucose sensitive beta cells remains a challenge, the transplantation of pancreatic primordia can provide good glucose control to diabetic recipients. Fetal pigs could be used as a cost effective bioreactor to expand human stem cells or primordia and differentiate them into mature beta cells or islets.
Tissue engineering involves repopulating a collagenous scaffolding with new cells, preferably from the organ recipient. This has been applied to heart tissue. Rat hearts were decellurized with detergents. Although perfused under pressure on a Langendorff apparatus, the collagenous scaffolding was repopulated with neonatal rat cardiocytes.92 The repopulated exograft was partially functional for as long as 28 days. The clinical application could use hearts from human cadavers to prepare the scaffolding. Alternately, it could use pig hearts from young, healthy, and fresh donor pigs. The decellurized pig heart scaffold would be similar to the collagenous pig heart valves that are already commonly implanted.
With sufficient support, xenotransplantation will address the large unmet need for many of the diseases requiring replacement of failed tissues and organs. In some devices, xenotransplantation may be a stand-alone technology. In other technologies, it may be combined with stem cell or tissue engineering technology. The future of xenotransplantation and regenerative medicine could potentially be very exciting.
Keywords
xenotransplantation, diabetes, kidney transplant, heart transplant, lung transplant, liver transplant, regenerative medicine, organ donors, organ transplant, pig organs
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References
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Keywords
xenotransplantation, diabetes, kidney transplant, heart transplant, lung transplant, liver transplant, regenerative medicine, organ donors, organ transplant, pig organs
