Xenotransplantation as a model of integrated, multidisciplinary research

Abstract

Xenotransplantation was proposed a long time ago as a possible solution to the world-wide shortage of human organs. For years, researchers in this field have almost exclusively directed their efforts towards combating the immunological barrier that precluded the long-term xenograft survival. Studies have been conducted in both small and large animal models and the most relevant results have been obtained in pre-clincal studies, specifically those utilising the pig-to-nonhuman primate combination. In this context, a better understanding of the immunological mechanisms underlying the rejection of a xenograft have allowed the identification of specific targets of intervention that have resulted in considerable improvements in survival of porcine organs or cells in nonhuman primates. However it has also become apparent that if xenotransplantation has to enter the clinical arena, a multidisciplinary approach will be needed to comprehensively tackle the different issues related to the use of a xenograft to cure human disease.

In this regard, the safety, ethics and regulatory aspects of xenotransplantation are currently being aggressively addressed to enable the initiation of xenotransplantation with a favourable risk/benefit ratio.

Introduction

Xenotransplantation, more commonly defined as the transplantation of organs, tissues or cells from one species to another, has previously been proposed as a possible solution to the world-wide shortage of human organs for patients in terminal organ failure. Whilst considerable progress has been achieved in this field in the last few years, a few issues still need to be satisfactorily addressed to enable xenotransplantation to become a real therapeutic option for our patients.

Considerable advances in this exciting but difficult field took place in the eighties and nineties, both in the European Union and outside Europe. These findings provided important information to enable better understanding of the xenograft rejection process and gain insight into safety aspects relating to xenotransplantation.

During the same period, however, emerging technologies such as regenerative medicine and stem cell research attracted much attention and support from funding agencies, as these approaches were viewed as more promising for patients in organ failure. This resulted in a lack of public and private funding to support xenotransplantation research in many laboratories worldwide, ultimately forcing many research entities to fold their xenotransplant activities and redirect their science towards these approaches with a seemingly higher potential for success.

Whilst it is undeniable that research into stem cells and regenerative medicine has resulted in the generation of some significant results, it has also become apparent that the biological processes involved in such scientific disciplines are much more complex than originally anticipated and will not be immediately offered to those in need. As a consequence of this slower than expected progress, research efforts in xenotransplantation have resumed in many countries world wide, including the United States, the European Union, Australia, Japan and China.

The Advantages of Xenotransplantation and the Use of the Pig as a Potential Source of Organs or Cells

The realisation of xenotransplantation as a clinical reality would result in numerous advantages to the transplant patient (Table 1). Xenotransplantation would provide an unlimited supply of organs of any type or size for human transplantation.1 The availability of such organs would minimise the time spent on the waiting list, avoid progressive clinical deterioration which usually occurs while waiting for an organ and allow elective and programmed surgery in ideal clinical conditions. It would also allow the large number of patients, for whom organs are not currently available, to be transplanted. The reduced time spent on the waiting list would considerably reduce the costs related to the treatment of patients with terminal organ failure, such as those on dialysis. Moreover, xenotransplantation would enable better organisation of the activities of transplant units. Ischaemia time, which is considered an important factor for the long-term survival of an allograft, would be reduced substantially. Finally, the widespread availability of organs would hopefully eliminate the repeatedly denounced illegal trading of human organs.2

Furthermore, xenotransplantation offers an additional, unique advantage over allotransplantation. Indeed, nowadays only minor interventions are possible in deceased donors to improve the health status of a donor organ prior to transplantation and enhance its acceptance by the recipient. Indeed, deceased organ donors are most often in unstable conditions that would preclude manoeuvres to improve the outcome of the transplanted organ. In contrast, xenotransplantation offers the tremendous advantage of donor organ modification to render the graft more “compatible” with human recipients, ahead of time of the actual use of the organ.

The pig is currently viewed as the most appropriate source of organs for the hypothetical clinical application of xenotransplantation. Indeed, several advantages have been associated with the use of this species (Table 2). First, the pig has many anatomical and physiological similarities with man.3 Earlier studies have demonstrated that porcine organs may reach the same size and have the same degree of efficiency as their human counterparts.1 Furthermore, as basic metabolic processes are shared by all mammals, most metabolic activities, such as pH and osmolarity, will not be an obstacle to xenotransplantation in primates using porcine organs.4 Second, the pig has a short gestation period (around 115 days) and produces large litters of 10 or more offspring, that rapidly grow to the size necessary for xenografting into human adults. Third, pigs can easily be bred under qualified pathogen-free condition5 such that the majority of known zoonotic agents can be eliminated. Fourth, this species is widely used for food, and therefore faces fewer ethical objections than the use of nonhuman primates. In this respect, it is worth mentioning that more than 80 million pigs are reared and slaughtered each year in the United States to provide meat and other products.1 For these considerations, a consensus has grown within the scientific community working in this field, that the pig represents the preferred donor species for future clinical xenotransplantation.

Xenotransplantation: The Need for an Integrated, Multidisciplinary Approach to Tackle Currently Outstanding Issues

Xenotransplantation research undertaken in the nineties has clearly taught us that success in this field is dependent on a vast array of knowledge that goes far beyond understanding and addressing the rejection process. Certainly, the immunological barrier has historically constituted a major obstacle to the long-term survival of pig xenografts transplanted into primates and still needs to be appropriately overcome in order to allow clinical studies. However, other issues, previously unknown or possibly underestimated, have now arisen. These include the need to broaden knowledge regarding biosafety aspects of this practice, to deepen understanding of the physiological compatibility between pigs and primates and clearly define the Ethical and Regulatory requirements that will need to be adequately addressed and satisfied prior to allowing the clinical application of xenotransplantation.

In this context, it is beyond any doubt that, at this stage, advancements in xenotransplantation that will allow the clinical application and success of this field will be entirely dependent on a multidisciplinary approach that will integrate very diverse skills. In this light, the European Union has recently promoted a xenotransplantation research programme across Europe entitled “Xenome”. Xenome is one of the EU funded “Integrated Projects” spread over up to 5 years. The project, launched at the end of 2006, is a research effort that includes 22 academic/private institutions in 11 countries with a fairly balanced representation and distribution of funding between academic and private entities within EU member states. The key areas of research pursued in Xenome include immunology, safety, genetic engineering and physiology, as well as ethics, and social and regulatory aspects of xenotransplantation. In addition, in accordance with the mission of any Integrated Project, effort has been directed towards the dissemination of knowledge and the training of new generations of investigators in Europe. For financial and ethical reasons, all the in vivo studies in Xenome are confined to a limited number of carefully planned preclinical xenotransplantation studies in the primate.

The Immunological Barrier

The immunological response to xenografts remains a central issue that will need to be dealt with satisfactorily if clinical xenotransplantation trials are to be initiated. In particular, a complete understanding of the immunological responses leading to xenograft rejection has yet to be fully acquired. Nonetheless, significant advances have been achieved in the understanding of such processes and at least five approaches are being pursued to overcome the immune obstacles to the long-term survival of a porcine xenograft in a primate (Table 3). The first approach entails the development of user-friendly pharmacological immunosuppression, finely adapted to specifically counteract the immunological events underlying the xenograft rejection process.6,7 Secondly, much research has been directed towards the development of tolerance inducing regimens, i.e., the development of strategies that would allow long-term survival of porcine xenografts in the absence of immunosuppression.8–10 The establishment of accommodation, defined as long-term graft survival, notwithstanding the continuing presence of xenoreactive antibodies and complement,11 is regarded as a third strategy which may enable long-term graft survival. Furthermore, excellent results have recently been achieved by utilising encapsulation as a strategy to protect cellular xenografts from immune damage, whilst allowing optimal metabolic function of the transplanted cell mass.12 Finally, the last approach designed to tackle the rejection process is that obtained via genetic engineering of the donor source, to render the xenograft more immunologically compatible with man.13

In any case, as for the development of any novel strategy to cure human diseases, the success of xenotransplantation is intimately linked to the fine comprehension of the mechanisms underlying the pathological insult, the rejection process in our case, allowing the identification of novel and specific targets for interventions. In the case of xenotransplantation, this may ultimately translate into the specific engineering of the pig genome to introduce (or subtract) traits that will enhance graft survival (Fig. 1).

As the rejection process differs considerably between solid organ and cellular xenotransplantation, these two types of xenografts will be reviewed separately, focussing specifically on the context of pig-to-nonhuman primate xenografts.

Solid organ xenotransplantation.

Our understanding of the immunological processes which occur following pig-to-nonhuman primate xenotransplantation has grown considerably in the last few years. With specific regard to solid organ xenotransplantation, the mechanisms of hyperacute rejection (HAR) and acute humoral xenograft rejection (AHXR) [also called acute vascular rejection (AVR)14 or delayed xenograft rejection (DXR)15], the two key immunological hurdles affecting the long-term survival of solid pig organs in the primate, have now been fairly well elucidated.

Until recently, HAR was the primary immunological barrier to the xenotransplantation of pig organs in the primate. HAR is characterised by diffuse interstitial haemorrhage, oedema and thrombosis of small vessels and capillaries.16 These changes are secondary to endothelial cell activation and damage caused by pre-existing anti-pig antibodies. Clarification of the mechanisms responsible for the onset of HAR has demonstrated the central role of complement and antibodies in its pathogenesis,17 allowing the development of approaches which interfere with either the interaction of xenoreactive natural antibodies with their primary target, Galα1-3Galβ1-4GlcNAc-R structures (known as αGal epitopes)18,19 or, alternatively, with the complement cascade.

In this context, the emphasis of research has been on the production of genetically engineered pigs expressing inhibitors of the human complement cascade such as human decay accelerating factor (hDAF; CD55),13,20 membrane cofactor protein (MCP; CD46,21) or CD59.22 The validity of this approach has now been convincingly demonstrated by several groups20,23,24 who have shown that similarly engineered pig organs usually do not undergo HAR once transplanted into primates.

However, xenografts which survive HAR almost inevitably fail eventually as a consequence of AHXR. Several elements have been implicated in the pathogenesis of AHXR. Its pathology is primarily characterised by vascular thrombosis, blood extravasation and oedema.16 Deposits of fibrin, immunoglobulins and complement in the graft do not differ substantially from those observed in HAR. Cellular infiltrates include neutrophils, macrophages, CD8⁺ T cells and few NK cells.16 Recently, however, with the use of xenografts deriving from αGalT^−/− pigs and the introduction of novel immunosuppressive strategies, the pattern of AHXR is changing towards a picture compatible with a prominent thrombotic microangiopathy.25 As for HAR, the identification of specific targets of intervention for AHXR should allow scientists to overcome this immune barrier, ultimately further extending xenograft survival. To this end, at least three different approaches are currently being explored. First, ongoing research regarding the role of complement in AHXR has demonstrated that complement deposition is consistently observed in the majority of xenografts rejected due to AHXR and is considered a hallmark of this process.23 In this light, interventions have been designed to completely prevent complement activation that is only partially abrogated when using organs from the existing pig lines transgenic for human complement regulators.26 Indeed, whilst it is clear that such lines can dramatically reduce complement-mediated damage and prevent HAR, it is also certain that such complement control is incomplete, possibly as a consequence of the overwhelming complement activation that at some stage supersedes the capacity of transgenically expressed proteins to control complement activation. Secondly, efforts are underway to reduce the immunogenicity of transplanted organs. To this end, several laboratories have recently obtained pig lines lacking the galactosyltransferase gene (αGalT^−/−). These animals do not express αGal epitopes in the majority of cases, although the reported presence of low residual levels of αGal epitopes in αGalT^−/− pigs, possibly added to lipids by iGb3 synthase, have been described by Sharma and colleagues.27 However, despite the absence of αGal residues, AHXR is still observed following the transplantation of αGalT^−/− organs and in many cases, it has been demonstrated that AHXR of αGalT^−/− organs is associated with the presence of circulating elicited anti-non-αGal antibodies.28 Nonetheless, a direct causative role of such non-αGal antibodies has not yet been proven and additional studies to further investigate these observations are eagerly awaited. Engineering of pig lines that are deficient in one or more of the target molecules recognised by anti-non-αGal antibodies may indeed be necessary to improve current survival rates. Finally, molecular incompatibilities between the coagulation systems of the pig and primate have previously been reported.29 These are currently being studied as possible additional targets of intervention to overcome frequently observed coagulopathy and fibrin deposition in xenografts explanted in the presence of AHXR.

In addition to HAR and AHXR, a third category of acute rejection, termed acute cellular xenograft rejection (ACXR) has also been described. Notwithstanding in vitro demonstrations of strong cellular responses,30 and in contrast to in vivo results in the hamster-to-rat model,31 it is important to underline that ACXR per se, does not lead to solid organ failure following pig-to-primate organ xenotransplantation. Therefore, at this stage, it would seem that graft damage directly mediated by the cell-mediated immune response can be largely prevented by the immunosuppressive regimens currently available.30 On the other hand, a contribution of T cells to the development of the elicited anti-xenograft humoral immune response cannot be ruled out.

With regards to the phenomenon of chronic rejection, very little information is available in the pig-to-primate context. This is most likely due to the fact that long-term survival of xenografts is not routinely obtained due to the problems posed by AHXR. However, recently, a phenomenon described as chronic xenograft vasculopathy was reported in a pig-to-baboon heterotopic cardiac transplantation model, despite chronic immunosuppression, in primate recipients of αGalT^−/− hearts.32 In these animals, both humoral and cellular features of rejection were associated with the development of chronic xenograft vasculopathy, characterised by intimal thickening, fibrin exudation, complement and immunoglobulin deposition and cellular infiltration. This preliminary data warrants further investigation to determine if chronic rejection will impact significantly on long-term xenograft rejection as observed in allotransplantation.

Cellular xenotransplantation.

Pig islets represent the most studied model of cell xenotransplantation in the primate and will be here briefly discussed. In comparison to solid organs, islets have a particular advantage when it comes to rejection. Indeed, islets are not immediately vascularised and express low to negligible levels of the αGal epitope, thereby making them intrinsically resistant to the majority of the natural antibody repertoire.33 Moreover, the isolation and pre-transplant culture procedures result in the loss of most of the islet vasculature, essentially eliminating those few porcine islet vascular endothelial cells which do express αGal epitopes and could otherwise present a target for the xenogeneic humoral response. Therefore, islets are largely resistant to the process of HAR. Nonetheless, islets are not resistant to rejection. Essentially, two immunological barriers have been demonstrated to be the most important in islet xenograft rejection. The first of these has been termed the Instant Blood-mediated Inflammatory Reaction (IBMIR), and occurs immediately following the contact of xenogeneic islets with human blood. Indeed, the exposure of porcine islets to whole blood results in an inflammatory reaction, characterised by macroscopic coagulation, the activation of complement, consumption of platelets and infiltration of leukocytes.34 Although the reaction occurs with a kinetics similar to HAR, failure to observe antibody deposition on the graft is a distinguishing feature.

The onset of the IBMIR may account for the premature loss of grafted islets following intraportal islet infusion and the consequent large tissue volume required to achieve a functional islet mass following transplantation via this route. Whilst heparin treatment is standard procedure for intraportal islet transplantation, the use of novel approaches such as dextran sulfate,35 or surface-coating islets with heparin36 may prove to be beneficial in further abrogating this response.

In the absence of immunosuppression, islets which are not destroyed by the IBMIR, primarily undergo cellular rejection mediated in large part by T cells and macrophages.37,38 Following the initial recruitment of CD4⁺ T cells and macrophages to the graft, the secretion of a cascade of chemokines leads to a massive influx of macrophages and helper T cells, followed by the infiltration of eosinophils, CD8⁺ T cells and neutrophils, ultimately resulting in graft destruction.39

Islet Xenotransplantation as the Most Advanced Model of Pig-to-Primate Xenotransplantation

As islet rejection mechanisms involve a primarily cellular immune response, the principal immunosuppressive strategies applied have preferentially targeted this rejection process. Recently, three independent research groups have obtained porcine islet xenograft survival for 6 months in nonhuman primate transplantation models via the development of novel immunosuppression or encapsulation strategies.40–42 These studies will be briefly discussed below. The first strategy entailed the use of multi-agent immunosuppression regimens, principally focused on the prevention of T-cell-dependent rejection by the impairment of costimulatory pathways. Interestingly, using such an approach, sustained normoglycemia in diabetic nonhuman primates was achieved for more than 6-months by two different groups with immunosuppressive therapy which included anti-T cell agents interfering with the CD154/CD40 pathway. In particular, Hering and colleagues transplanted adult porcine islets into streptozotocin-diabetic cynomolgus macaques applying an immunosuppression regimen consisting of anti-IL2R and anti-CD154 monoclonal antibodies, FTY720, everolimus and leflunomide.40 Porcine islets functioned for over 180 days and a 100% xenograft survival rate was observed at day 100 after transplant.

Using a similar multi-agent immunosuppression approach, Cardona and colleagues transplanted neonatal porcine islets to pancreatectomized rhesus macaques with an immunosuppression regimen consisting of anti-IL2R and anti-CD154 monoclonal antibodies, sirolimus and belatacept (a high-affinity derivative of CTLA4-Ig).41 In this study, sustained insulin independence was achieved with a median survival time greater than 170 days, with the longest surviving graft having a duration of 345 days.43

Despite these pre-clinical successes, the complex immunosuppression protocols and the reported association of anti-CD154 antibodies with thromboembolic events,44 make these immunosuppression regimens clinically inapplicable. For this reason, the latter group is currently studying the efficacy of an alternative multi-drug immunosuppressive regimen, with a chimeric anti-CD40 antibody replacing the prothrombotic anti-CD154 antibody for the engraftment of neonatal porcine islets in nonhuman primates.43 Encouraging ongoing experiments indicate that this CD28/CD40 costimulatory blockade provides adequate initial xenograft function with improved glucose control, suggesting that with further refinements, this immunosuppressive therapy might become a valid alternative to anti-CD154 based immunosuppressive regimens.

The third group applied an immunosuppression-free approach to protect transplanted islets from the cell-mediated immune response. Whilst successful transplantation of encapsulated porcine islets was described by Sun and colleagues in 1996,45 these results have proven difficult to reproduce. Using a novel approach to encapsulation, outstanding results were recently obtained by Gianello and colleagues with alginate encapsulated adult pig islets. As a first step, a proof of concept study demonstrated the stability and biocompatibility of alginate-encapsulated pig islets and indicated that xenografts can survive for up to 6 months in non-diabetic nonhuman primates, maintaining the ability to respond to an in vitro glucose challenge.42 More recently, this group has been able to achieve sustained normoglycemia following the subcutaneous transplantation of a monolayer cellular device composed of alginate in four diabetic nonhuman primates. In the complete absence of immunosuppression, normoglycemia was maintained in these recipients for up to 6 months. These impressive preliminary results which await confirmation, indicate that the device developed can control the diabetic status for up to 6 months without any immunosuppression, an important feature which will be of critical significance in a possible future application of such technology in diabetic patients.12

It is of interest that these excellent results obtained in the islet transplantation field have utilised unmodified pigs (both neonatal and adult). In this light, it is not unreasonable to expect that islet xenotransplantation will further benefit from genetic modification of the donor pig. As a consequence of the inherently low to negligible expression of αGal on pig islets, the αGalT^−/− pigs may not have a great impact on islet xenotransplantation. In contrast, the use of transgenic pigs possessing high-level expression of antithrombotic molecules could lead to considerable protective effects, mitigating the IBMIR and allowing further islet resistance following exposure to the bloodstream. In this regard, encouraging results have been obtained with CD39 transgenic mice showing a delay in clotting time when CD39 transgenic islets were exposed to human blood.46 Similarly, the use of transgenic pigs expressing the NK and T-cell apoptotic inducer TRAIL47 as islet sources, may also prove beneficial to islet xenotransplantation.

Xenotransplantation and Tolerance

A number of approaches have been applied in efforts to induce tolerance in xenotransplant models. These include mixed hematopoietic chimerism, costimulatory blockade, donor-specific transfusion and thymic transplantation.48,49 Whilst tolerance has been achieved in xenotransplant models utilising rodent recipients, achieving complete tolerance in large animal models is still elusive. Furthermore, achieving xenogenic tolerance appears to be a much more difficult process than that required for allografts, most likely due to the significant role of the humoral response in all phases of xenograft rejection. In this light, the effective establishment of xenograft tolerance in large animals will require approaches to tackle both B and T cell-mediated xenoimmune responses.50

B-cell tolerance to xenografts has been achieved in rodents following bone-marrow transplantation to induce B-cell anergy and deletion, preventing the generation of newly-formed and pre-existing antibody responses towards xenoantigens.51 Additional strategies to induce B-cell xenograft tolerance have included gene-therapy and treatment with leflunomide (reviewed in ref. 52). However, the effects of such procedures in nonhuman primate responses have yet to be evaluated.

T-cell tolerance inducing strategies have been extensively assessed in rodent xenograft models, however, few have shown efficacy in large animal models. These strategies include co-stimulatory blockade, thymic transplantation and hematopoietic chimerism. Co-stimulation blockade therapy aims to render T-cells unresponsive to antigenic stimulation by inhibiting necessary co-stimulator molecule engagement during binding of the T-cell receptor with antigen. Whilst both the CD80/86-CD28 and CD40-CD40L pathways have been successfully targeted in rodent xenotransplant models, providing markedly prolonged xenograft survival, tolerance using these agents has not been achieved in pig-to-primate models. Nonetheless, the inclusion of these agents in immunosuppressive protocols has resulted in significant enhancements in graft survival in xenograft recipients.32,40,41,53

Prolonged survival of xenografts has been reported following co-transplantation of porcine kidneys with donor thymic tissue. Studies in rodent models have demonstrated the ability of the porcine thymus to generate donor-specific tolerance of both rodent and human T-cells towards the pig.54–56 Using this approach, Yamada et al. were able to extend the survival of pig kidney xenografts in the nonhuman primate, demonstrating sustained normal graft function in the absence of rejection for up to 83 days.53 In this study, the authors were able to demonstrate pig-specific unresponsiveness via mixed lymphocyte reaction in two long-term surviving recipients, and the absence of a pig-specific cytotoxic T lymphocyte response in one case. However, the role of an immune-mediated mechanism behind the mild and focal thrombotic microangiopathy observed in these grafts has yet to be ruled out. Furthermore, the requirement for continuous administration of a human anti-CD154 monoclonal antibody, occasionally associated with thrombotic complications, suggests that further refinements will be necessary before complete tolerance can be achieved.

Finally, the ability of mixed hematopoietic chimerism to generate xenograft tolerance has been successfully demonstrated in pig-to-mouse models.9,57,58 However, a recipient conditioning regimen able to induce successful stable engraftment of porcine bone marrow in the primate has yet to be identified. This is largely due to the inability to achieve complete T-cell depletion in primates and the need to overcome hematopoietic cell rejection by natural antibodies, NK cells and macrophages.52 However, as transient chimerism has proven sufficient for the establishment of T-cell tolerance to allografts in large animals,49,59 it has yet to be established whether tolerance may indeed be induced in the pig-to-primate combination following the application of such a procedure.

Physiology

Future clinical applications of xenotransplantation can only be envisaged if adequate compatibility between pigs and primates can be demonstrated. Whilst physiological incompatibilities between these two species have been reported,29,60,61 these do not appear to represent an insurmountable challenge to the long-term survival of porcine renal, cardiac xenografts or islet xenografts. Reported incompatibilities include molecular differences between the coagulation system of the pig and primate.29 Indeed, porcine von Willebrand Factor (vWF) has been shown to interact with human platelet receptors with high affinity. Porcine vWF is able to activate quiescent platelets, resulting in platelet aggregation in the absence of shear stress, possibly resulting in elevated pro-coagulant activity.62 Porcine tissue factor pathway inhibitor (TFPI) is not able to neutralize human factor Xa, and is therefore unable to inhibit the direct activation of human prothrombin to thrombin.63,64 In addition, although porcine thrombomodulin has been shown to bind human thrombin and Protein C, the human thrombin-porcine thrombomodulin complex is a poor activator of Protein C. As a consequence, the insufficient production of activated Protein C contributes to enhanced levels of thrombin, favouring the initiation of clotting.64,65

In order to combat such incompatibilities, transgenic modulation of the clotting cascade by de novo expression or induction of anticoagulants, or the elimination of pro-coagulant molecules on the xenogenic vascular endothelium, may represent a potential therapeutic strategy. In this context, several target gene candidates for transgenic expression (e.g., CD39, TFPI, thrombomodulin, hirudin, CD73), or knock-out (e.g., Tissue Factor, PAR3, PAR4, Fgl-2) in pig tissues have been identified. Encouraging results, although only obtained in vitro66 and in small animal models,67–71 have provided a basis for the future genetic manipulation of porcine organs possibly able to overcome thrombotic events that compromise xenograft survival.

Notwithstanding the physiological differences identified, in vivo studies in nonhuman primates suggest that porcine hearts, kidneys and islets are able to function in primates and sustain their life for up to several months.6,32,72 During this time, the organs support normal levels of activity, with the recipients exhibiting normal social behaviour. Together, these in vivo observations suggest that, to date, no insurmountable physiological incompatibility has been identified between the pig and primate.

Safety Aspects

Transplantation per se carries the intrinsic risk of transmitting donor-derived infectious agents to the recipients. This is irrespective of whether donor and recipient are from the same species or not. Indeed, even in the case of clinical allotransplantation, we are still unable to guarantee protection of human recipients from existing donor-derived pathogens. This was clearly demonstrated earlier this year when a newly identified Arena virus was found to be responsible for a cluster of transplant-associated deaths.73

As far as the safety aspects related to xenotransplantation are concerned, considerable progress has been achieved.74 It is beyond any doubt that the exclusion of known exogenous pathogens represents a necessary step toward clinical xenotransplantation. This can be achieved by direct caesarian derivation of founder animals, followed by barrier rearing and regular screening of sentinel animals for the presence of specific pathogens. Whilst such an approach can, in theory, eliminate all known exogenous pathogens from the breeding colony, such an approach cannot eliminate agents in a latent or intracellular state such as porcine endogenous retrovirus (PERV), circoviruses and other agents, or as yet unidentified pathogens.

It is nonetheless encouraging that PERV have not been associated with any current, past or latent infection in xenografted patients75 and that PERV are susceptible to some of the currently available antiviral agents.76 Furthermore, pig lines have recently been identified which are incapable of transmitting PERV to human cells in vitro.77,78 In addition, genetic manipulation of the porcine genome may provide an additional strategy to remove the viral risk. In this regard both specific knock outs of endogenous retroviruses79 or the use of short interfering RNAs specific for PERV sequences80 have been proposed and preliminary data in engineered pigs are very encouraging.81

From a diagnostic standpoint, the development of microarray-based technology capable of rapidly identifying known and as yet unidentified potential infectious agents82 will allow their timely identification and control in the xenotransplantation setting. In this regard, it is expected that advances in this field will significantly contribute to enhance the safety profile of xenotransplantation.

Regulatory and Ethical Frameworks

To date, a single legal document regulating clinical xenotransplantation procedures world-wide has yet to be developed. Still, several well-written documents have been released in the last few years that should constitute the basis for an international regulatory framework within which clinical xenotransplantation should take place. These documents include the U.S. Public Health Service (PHS) Guideline on Infectious Disease Issues in Xenotransplantation (January 19, 2001),83 the Guidance for Industry—Source Animal, Product, Preclinical and Clinical Issues Concerning the Use of Xenotransplantation Products in Humans—FDA April 2003,84 the Recommendation Rec (2003)10 of the Committee of Ministers to EU member states on xenotransplantation85 and the document prepared following the Xenotransplantation Advisory Consultation held in Geneva in April 2005.86 The aspects of xenotransplantation addressed by these documents are diverse and include efficacy and safety aspects, ethical principles and regulatory frameworks for xenotransplantation. In particular, it comes across very clearly that the initiation of clinical trials must not be exclusively based on the generation of efficacy data in preclinical models. Indeed, beside the demonstration that porcine xenografts are not rejected for a significant period of time using a clinically applicable immunosuppression strategy and meet the physiological requirements of the nonhuman primate recipient, no infectious agents should be identified in porcine xenograft recipients.

Several ethical requirements common to all clinical trials and some specific to xenotransplantation should clearly be satisfied in order to initiate and conduct clinical xenotransplantation studies. The ethical principles which need to be met have recently been outlined by the Ethics Committee of the IXA87,88 and encompass ethical considerations for both the human recipient and the donor source animal. As far as the human recipient is concerned, it should be underlined that minors should be denied participation in such trials, and an appropriate informed consent should be prepared that clearly outlines all the caveats and restrictions that could be applied to xenograft recipients involved in initial clinical studies. Furthermore, with regard to source animals, these should be bred in barriered facilities in accordance with the highest standards of animal husbandry, respecting the requirements of the species.

With respect to the preparation of an internationally accepted and applied regulatory framework, a report which formed the basis of the World Health Assembly Resolution WHA57.18 adopted in May 2004 represents a first important step towards the harmonization of xenotransplantation procedures and policies.89 This resolution urged Member States to “allow xenotransplantation only when effective national regulatory control and surveillance mechanisms overseen by National Health Authorities are in place.”

More recently, the Xenotransplantation Advisory Consultation held in Geneva in April 2005, released a statement where the role of each Member State was more comprehensively defined. In particular, the role of Member States in the implementation of such a Resolution could be summarized as follows:

	Undertake an inventory of xenotransplantation practices in their country.
	Allow xenotransplantation only if there is an effective regulatory system in place.
	Ensure that the regulatory authorities properly weigh the risks and potential benefits of any clinical trials or procedures before giving authorization.
	Ensure that first class regulatory standards are in place with regards to source animals; authorization of procedures, ethical approval and consent procedures; education of patients, intimate contacts and health-care workers, including those in public health; quality management and auditing of outcomes.
	Ensure that there are effective surveillance systems in place that would identify and manage events that pose a potential danger to public health.
	Ensure transparency about xenotransplantation activities.
	Promote public awareness.

Taken together, these considerations indicate that all the principles are being put in place to resume clinical xenotransplantation efforts, when appropriate, under the best auspices for its success and minimizing any risks of failure.

Conclusion

The data discussed above clearly indicate that considerable progress has been achieved in this field in the last few years. This primarily includes advancements from immunological, safety and physiological points of view. However, it is the authors opinion and that of many, that it is only when a favourable risk/benefit ratio can be clearly demonstrated in convincing and relevant pre-clinical studies that well designed clinical xenotransplantation trials should be initiated in countries where an appropriate ethical and regulatory framework is in place.

SanjayJain, M.D Ph.D. Assistant Professor of Medicine, Washington University School of Medicine:

In your genetic engineering approaches, it would appear that the most efficient way to produce a pig that would be a good organ donor for humans would be to make a multi-transgenic animal that has genes targeting different beneficial biological aspects for graft survival. What is the experience or technical status of that in pigs?

Dr.Cozzi:

Indeed, successful xenotransplantation will require an engineered source animal with multiple genetic modifications. Specific modifications will ultimately result in a better control of the immune response and of the coagulation cascade, and result in a better safety profile of the potential donor pig. At this stage, we certainly have multi-transgenic pigs that co-express, for instance, inhibitors of the coagulation cascade and inhibitors of complement.90 Furthermore, complement regulators have been added to the αGalT^−/− background that renders the donor more immunologically “compatible” with man. Pigs with knocked-down PERV expression by PERV-specific shRNA have also been reported.81

Basically, we are independently tackling all the different aspects that need to be addressed to allow long-term and safe survival of xenotransplanted organs. What we need to do now is bring the different traits together in a single donor animal. This will be achieved using cloning technologies and conventional breeding strategies to ensure fertility of the newly generated line.91 Alternatively, sperm-mediated gene transfer could represent another tool.92

Dr.Jain:

I would like to hear more about your experience with cell-based therapies for neurodegenerative diseases such as Parkinson's. The relevance of this technology is often questioned, because in order for it to work, neuronal axons must find their targets, and re-establish functional synapses that will remain stable throughout life. What do you see in your models?

Dr.Cozzi:

These long-lasting experiments are underway. These experiments can be grossly divided into 2 groups. In the first set of experiments, we will only verify whether genetically engineered neuroblasts obtained from CTLA4-Ig transgenic pigs93 can survive in the brain of immunosuppressed nonhuman primates. These studies will also have to verify whether surviving xenografts are able to establish appropriate synaptic connections with the relevant recipient parenchyma. In the second set of experiments, we will verify whether graft survival can be associated with graft function, resulting in some sort of functional benefit. These studies will include neuroradiological investigations (PET-scan imaging with F-Dopa) and motor tests.

Dr.Jain:

Can you develop a transgenic pig with organs that are less susceptible to ischemia- reperfusion injury?

Dr.Cozzi:

We do not plan to undertake this in the context of XENOME, but it is obviously a very important issue and we will perform assessments of ischemia/reperfusion injury in the context of our xenotransplantation experiments.

DanBrennan, M.D, Professor of Medicine, Washington University School of Medicine:

Does XENOME have a data safety monitoring mechanism to verify pre-clinical results prior to proceeding into human trials? Also does XENOME require that results be verified by another laboratory prior to proceeding with clinical trials? The reason I ask is that you have described successful xenotransplantation of islets in non-immunosuppressed hosts using alginate encapsulation. However, the history of this approach is that it is not uniformly successful.

Dr.Cozzi:

XENOME has no plan to proceed with clinical trials but only to get xenotransplantation closer to its clinical application. To this end, a wide range of experiments in the most diverse fields have been planned, which involve a multidisciplinary team of scientists, experts in ethics and experts in law. Furthermore, there will be extensive interaction with the public to make sure that all potential stakeholders are involved, bringing them up to speed with the advancements in this field. In all cases, as already stated, the initiation of clinical trials will only be possible when a favourable risk/benefit ratio exists, and in the context of an appropriate ethical and regulatory framework. With regard to the results generated by Gianello and colleagues,12 we are all intrigued by the very promising results of their latest alginate experiments. We still do not know why they are substantially different from previous studies using alginate technology. However, it is my understanding that with this new approach, oxygen diffusion is improved, and pore size or molecular weight cut-off is more efficient which somehow leads to such significant results.

Dr.Brennan:

It seems to me that XENOME should require that results have to be concurrent in another laboratory before proceeding into a clinical trial.

Dr.Cozzi:

We have not formally specified independent replication. However, in principle we could accommodate such a requirement as the program utilises two nonhuman primate species. If a strategy is successful in one species, for instance in cynomolgus monkeys, the concept could be demonstrated in the other species available to the network (baboon). We already have this internal arrangement but the important thing to understand is that XENOME does not have the intention to go into man.

Figures and Tables

Figure 1 Definition of novel targets for engineering more compatible porcine xenografts.

Table 1 The advantages of xenotransplantation

Unlimited supply of organs of choice

Organs of any size

Reduction of the time spent on the waiting list

Avoidance of clinical deterioration while waiting for the organ

Reduction of the costs related to terminal organ failure

Improved organisation of the activities in the transplant units

Substantial reduction of ischaemia time

Disappearance of illegal trading of human organs

Table 2 The advantages of the pig as a source of organs for clinical xenotransplantation

Physiological and anatomical similarities with man

Short gestation period

Production of large litters

Offspring rapidly grow

Organs reach the size necessary for xenografting into human adults

Can easily be bred under qualified pathogen-free conditions

Ethical considerations

Table 3 Strategies to extend xenograft survival

Pharmacological immunosuppression

Tolerance inducing regimen

Accommodation

Genetic engineering of the source animal

Encapsulation

Acknowledgements

This work was supported by CORIT (Consorzio per la Ricerca sul Trapianto d'Organi, Padua, Italy), the Italian Ministry of Health, the Veneto Region and the EU FP6 Integrated Project “Xenome”, contract # LSHB-CT-2006-037377.