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Clinical Pig : How Close Are We?

David K. C. Cooper,1 Hidetaka Hara ,1 Hayato Iwase,1 Takayuki Yamamoto,1 Abhijit Jagdale,1 Vineeta Kumar,2 Roslyn Bernstein Mannon,2 Michael J. Hanaway,1 Douglas J. Anderson ,1 and Devin E. Eckhoff1

1Division of Transplantation, Department of and 2Division of , Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama

ABSTRACT Patients with ESKD who would benefit from a kidney transplant face a critical and Here we review advances in the ex- continuing shortage of kidneys from deceased human donors. As a result, such perimental laboratory and consider patients wait a median of 3.9 years to receive a donor kidney, by which time approx- which patients might ethically be offered imately 35% of transplant candidates have died while waiting or have been removed the opportunity of receiving a pig kid- from the waiting list. Those of blood group B or O may experience a significantly ney, either to reduce the length of time longer waiting period. This problem could be resolved if kidneys from genetically that they need be supported by chronic engineered pigs offered an alternative with an acceptable clinical outcome. At- before undergoing allotrans- tempts to accomplish this have followed two major paths: deletion of pig xenoanti- plantation or as a means of permanent gens, as well as insertion of “protective” human transgenes to counter the human support. The state of the science is suf- immune response. Pigs with up to nine genetic manipulations are now available. In ficiently advanced to indicate that clini- nonhuman primates, administering novel agents that block the CD40/CD154 cos- cal trials of pig timulation pathway, such as an anti-CD40 mAb, suppresses the adaptive immune are likely to be initiated within the next response, leading to pig kidney survival of many months without features of 2 years. rejection (experiments were terminated for infectious complications). In the ab- sence of innate and adaptive immune responses, the transplanted pig kidneys have generally displayed excellent function. A clinical trial is anticipated within 2 years. HISTORICAL ASPECTS OF KIDNEY We suggest that it would be ethical to offer a pig kidney transplant to selected XENOTRANSPLANTATION patients who have a life expectancy shorter than the time it would take for them to obtain a kidney from a deceased human donor. In the future, the pigs will also be Several attempts were made to trans- genetically engineered to control the adaptive immune response, thus enabling plant NHP kidneys into patients with exogenous immunosuppressive therapy to be significantly reduced or eliminated. ESKD, with little or no success.7–9 When Reemtsma et al. transplanted JASN 31: ccc–ccc, 2020. doi: https://doi.org/10.1681/ASN.2019070651 chimpanzee kidneys into six patients in 1963, although most of them succumbed from either rejection or within 8 weeks, one patient led an active life for There is a continuing shortage of de- The lack of donor kidneys could be 9 months, returning to work as a ceased human donor kidneys for trans- resolved if kidneys from genetically en- plantation in patients with ESKD.1–3 gineered pigs offered an alternative with The overall survival of patients on an acceptable clinical outcome. The po- chronic is 78% at 1 year, tential advantages of xenotransplanta- Published online ahead of print. Publication date 57% at 3 years, and only 42% at 5 years tion are several (Table 1). The results of available at www.jasn.org. (Figure 1).4 Forty percent of waitlisted genetically engineered pig kidney trans- Correspondence: Dr. David K. C. Cooper, Division patients are likely to die within 5 plantation in nonhuman primates of Transplantation, Department of Surgery, ZRB 701, 1720 2nd Avenue South, University of Ala- 4,5 fi years. Importantly, in one analysis, (NHPs) have improved signi cantly in bama at Birmingham, Birmingham, AL 35294. most of those who died on the waitlist recent years (Figure 2), sufficiently to Email: [email protected] had been excellent candidates at the encourage consideration for initial clin- Copyright © 2020 by the American Society of time of listing.6 ical trials. Nephrology

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e.g., human decay-accelerating factor (hCD55) or membrane cofactor protein (CD46), that protected against the recip- ient NHP complement cascade. When the first hCD55 transgenic pigs were in- troduced,17,18 pig kidney graft survival was significantly increased, but graft fail- ure still occurred from delayed anti- body-mediated rejection.19 In 2004, Cozzi and colleagues reported graft survival for a maximum of 90 days (Fig- ure 2), but the mean survival remained at 24 days.20 The identification of the major pig xenoantigen (Gal) against which humans and Old World NHPs (e.g.,ba- boons) have natural (preformed) anti- bodies21 enabled pigs to be bred in which expression of this antigen had been de- leted (GTKO pigs) (Table 2).22–24 The transplantation of kidneys from GTKO pigs into NHPs was associated with pro- longation of graft function, similar to Figure 1. Percentage survival of patients with ESKD by treatment modality in 2010 that achieved by the transplantation of (modified from reference 1, with permission, and on the basis of data from two sources: US Renal Data System [USRDS]4 and Orandi et al.5). CD55- or CD46-transgenic pig or- gans.25,26 The combination of GTKO and a human complement-regulatory schoolteacher, before dying relatively observed in some patients after kidney protein reduced early graft failure fur- suddenly from what was believed to be across the ABO- ther.27,28 an acute electrolyte disturbance.10 The blood group barrier or when sensitized In a GTKO/CD55 pig-to-rhesus transplantation of baboon or monkey to donor HLAs. It was soon clear that the monkey model of kidney transplanta- kidneys was less successful. 80 million years during which humans tion, Higginbotham et al. reported that It was not until the 1980s that it was (and NHPs) and pigs had been evolv- the selection of monkeys with low levels fully appreciated that, despite their phy- ing differently had resulted in a complex of anti-pig antibodies was associated logenetic closeness to humans, NHPs primate immune/inflammatory re- with prolonged graft survival (.4 were for a variety of reasons unlikely to sponse to a pig xenograft.14 However, months),29 as had Yamada et al.,25 provide a solution to the lack of organs importantly, xenotransplantation offers whereas a monkey with a high level of for kidney transplantation, and that pigs us the first real opportunity to modify antibodies rejected the graft within a might make preferable sources of or- the “donor” rather than just treat the week. However, the immunosuppressive gans.11–13 However, the transplantation recipient. regimen had to target the CD40/CD154 of an from a wildtype pig (i.e.,a When the genetic engineering of pigs T cell costimulation pathway, but genetically unmodified pig) was fol- was suggested in the early 1990s,15,16 the not the CD28-B7 (CD80/CD86) path- lowed by almost immediate graft rejec- initial approach was to introduce a hu- way, confirming the observations of tion (hyperacute rejection) (Figure 3), as man complement-regulatory protein, Iwase et al.30

Table 1. Advantages of xenotransplantation over allotransplantation Unlimited supply of “donor” organs. Organs available electively, even when required urgently. Avoids the detrimental effects of brain death on the donor organs. The “donors” will be free of all known infectious microorganisms. “Borderline” transplant candidates, i.e., those with health problems that may be detrimental to prolonged patient survival, e.g., poorly controlled , peripheral or cerebrovascular disease, will be more likely to be acceptable (as they will no longer be competing for scarce organs with other potential transplant candidates). Avoids the “cultural” barriers to deceased human , e.g., in countries such as .

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deletion of pig xenoantigens, and (2) insertion of “protective” human trans- genes.33 The introduction of more sophisticated techniques of genetic engi- neering has increased the speed and fa- cility with which genetic manipulations can be made, and has reduced the costs (Table 3).

RECENT PROGRESS IN PIG-TO- NHP KIDNEY TRANSPLANTATION MODELS Figure 2. Reported maximum survivals of nonhuman primates with life-supporting pig kidney grafts, 1989–2017. Details can be found in Lambrigts et al.,19 and Cooper et al.73 In When conventional immunosuppressive some years, no results were reported. therapy was administered, graft survival was short, unless very high blood levels Furthermore, when post-transplant protein was documented (Figure 4)31 of the agents were maintained, which re- dysfunction became obvious, (reviewed in Wang et al.32). sulted in infectious complications. the importance of the additional expres- Subsequently, genetic engineering When novel agents that blocked the sion of a human coagulation-regulatory has followed these two major paths: (1) CD40/CD154 costimulation pathway, e.g., with an anti-CD154 mAb,34 the adaptive immune response was sup- A pressed, leading to prolonged graft sur- vival.25,35 Although anti-CD154 mAbs have been associated with thrombogenic complications, anti-CD40 mAbs, which appear almost equally effective, are not,36 but are not yet approved by the US Food and Drug Administration (FDA) for use in . In contrast, blockade of the CD28-B7 (CD80/CD86) pathway, i.e.,byCTLA4- Ig, is insufficient.30 Asignificant advance was made in 2015, when a baboon with a kidney graft from a GTKO pig that expressed human complement and coagulation-regulatory B proteins functioned for 136 days before the recipient required euthanasia for a systemic infection (Myroides spp.), at which time the graft showed no histo- pathological features of rejection.37 Iwase et al.31 went on to document survival of up to 9 months, with the experiments again being terminated for infectious complications (that would probably have been prevented or treated success- fully in human patients), although good graft function was maintained. Confirmation of the inadequacy of Figure 3. Macroscopic appearances of a wildtype (i.e., genetically unmodified) pig kidney conventional pharmacologic immuno- transplanted into a baboon (A) immediately after reperfusion, and (B) 10 minutes later, suppressive therapy was provided by showing the typical features of hyperacute rejection. Modified from reference 14, with consistent pig kidney graft failure in permission. contrast to consistent prolonged graft

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Table 2. Known carbohydrate xenoantigens expressed on pig cells Carbohydrate (Abbreviation) Responsible Enzyme Gene-Knockout Pig Galactose-a1,3-galactose (Gal) a1,3-galactosyltransferase GTKO N-glycolylneuraminic acid (Neu5Gc) CMAHa CMAHKO Sda b-1,4N-acetylgalactosaminyltransferase b4GalNT2KO aCytidine monophosphate-N-acetylneuraminic acid hydroxylase. survival when the regimen used an anti- NHPs, e.g., baboons, do have antibodies that three grafts underwent antibody- CD40 mAb38 (Figure 5A) with conven- to TKO pig cells.43 This is because Old mediated rejection within a week, and a tional immunosuppressive therapy, and World NHPs, like wildtype pigs, express fourth at 35 days, although two grafts graft failure was associated with histopath- N-glycolylneuraminic acid. When this is functioned for 100 and 435 days, respec- ologic features of a thrombotic glomerul- deleted from the surface of the pig cell, it tively.44 As the pigs did not express any opathy (Figure 5B) despite expression of appears to expose another (a fourth) human protective proteins, these data human coagulation-regulatory proteins, xenoantigen, against which Old World strongly suggest that expression of suggesting that blockade of the CD40/ NHPs, but not humans, have natural an- human complement- and coagulation- CD154 pathway may be critical. tibodies (Figure 6C). In our limited ex- regulatory proteins adds significantly to Two other pig xenoantigens have perience to date, expression of the fourth graft survival. been identified (Neu5Gc39,40 and Sda41) xenoantigen in a pig kidney graft has (Table 3), and have also been deleted from resulted in rejection in four out of five Optimal Genetically Engineered pigs, providing us with triple-knockout cases. Proving the efficacy of both a pig Pigs Today (TKO) pigs42 to which many humans genetic manipulation and/or a novel im- TKO pigs that express no fewer than six have no antibody binding (Figure 6A),43 munosuppressive regimen in the pig-to- human protective proteins (two comple- and no serum cytotoxicity (Figure 6B). Old-World-NHP model is therefore ment-regulatory [CD46, CD55], two We believe that deletion of expression proving more challenging than it is likely coagulation-regulatory [thrombomodu- of the three known pig xenoantigens and to be when pig kidneys are transplanted lin, endothelial protein C receptor], the strong expression of one or more human clinically. anti-inflammatory/antiapoptotic hem- complement- and coagulation-regula- Deletion of both the Gal and Sda an- eoxygenase-1 [HO-1], and CD47 [with tory proteins are essential to the success tigens in the pig (but not of Neu5Gc) immunosuppressive effects]) have re- of clinical organ transplantation. These provides a pig against which some Old cently become available.45,46 Although genetic manipulations do not appear to World NHPs have low antibody binding, most baboons still have antibodies against affect the health of the pigs. thus more closely mimicking the TKO these pig cells, it is hoped that the expres- pig-to-human model. Administering an sion of six protective human transgenes A Remaining Immunologic Hurdle anti-CD154 mAb–based regimen, may prevent rejection from developing. One remaining experimental problem Adams et al. carried out six kidney trans- relates to the fact that most Old World plants in rhesus monkeys, and reported NONIMMUNOLOGIC OBSERVATIONS IN THE PIG-TO-NHP MODEL

Baboons with well functioning pig renal grafts remain alert and active, and are indistinguishable from healthy baboons without transplants. In the absence of innate and adaptive immune responses (which have only recently been over- come, and have affected renal graft func- tion), function of the transplanted pig kidneys in NHPs (on the basis of mea- surements of serum , albumin, fi Figure 4. Rapid development of thrombocytopenia (consumptive coagulopathy, a reli- electrolytes, and speci c gravity) has gen- able indicator of graft rejection/failure) in two baboons with life-supporting GTKO/hCD46 erally been excellent, although low phos- pig kidney grafts (indicated in red), and maintenance of normal platelet counts in two phate levels have been observed.31,47 baboons (treated identically) with life-supporting GKTO/hCD46/hTBM pig kidney grafts In particular, there is no or minimal pro- (indicated in black). Modified from reference 31, with permission. teinuria, and polyuria has not been

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Table 3. Timeline for application of evolving techniques for genetic engineering of The first is rapid growth of the kidney pigs employed in xenotransplantation for the first 2–3 months after trans- Year Technique plantation, during which period it can 31,37,47 1992 Microinjection of randomly integrating transgenes double in length and/or mass 2000 Somatic cell nuclear transfer (SCNT) (Figure 7). This can also occur after al- 2002 Homologous recombination lotransplantation when a kidney from a 2011 Zinc finger nucleases (ZFNs) rapidly growing pig is transplanted into a 2013 Transcription activator-like effector nucleases (TALENs) miniature swine that grows much more 2014 CRISPR/Cas9a slowly.48 There appears to be an intrinsic aCRISPR/Cas9, clustered randomly interspaced short palindromic repeats and the associated protein 9. factor that drives growth for the first few weeks. As there is space within the abdo- observed. Because blood is drawn fre- adequate hematocrit. It is therefore un- men for the expanding kidney, we have quently from the (immunosuppressed) certain whether pig erythropoietin func- not found this to be problematic (al- baboons, recombinant human erythro- tions in primates. However, two potential though it could be for a rapidly growing poietin is administered to maintain an problems have been identified. heart within the restricted confines of the chest). If it proves problematic, there are ways to genetically engineer the pigs to A reduce the rate of growth. A second phenomenon that requires confirmation and investigation is the in- termittent development of hypovolemia that we have observed in baboons with well functioning pig kidney grafts.47,49 Preliminary evidence suggests that this may be related to the inability of pig re- nin to cleave angiotensinogen in pri- mates. This results in a reduced ability for vasoconstriction and retention of fluid. In the pig-to-primate experi- ments, we excise both baboon native kidneys to ensure that life is being sup- ported only by the pig graft. In contrast, B in humans, both native kidneys generally remain in situ, and so the problem should not arise. (If necessary, the organ-source pig could be genetically engineered to ex- press human as well as pig renin.) Progress in the experimental labora- tory has been sufficiently encouraging to consider a clinical trial.

SELECTION OF PATIENTS FOR THE FIRST CLINICAL TRIAL

Patients with various conditions have been discussed (Table 4).1,50 An impor- tant point is that, because xenotrans- Figure 5. (A) Pig kidney graft survival in baboons receiving either conventional (tacroli- plantation will be offered to patients mus-based; group A) or anti-CD40 mAb–based (group B) immunosuppressive therapy. Median pig kidney graft survival in group B (186 days) was significantly longer than in group who are unable to receive a timely allo- A(13days)(P,0.01). Reproduced from reference 38, with permission). (B) Histopathology graft, the results of the initial pig kidney of thrombotic glomerulopathy in a pig kidney of group A. Glomerular histopathology in- xenotransplants should be compared cluded thrombotic microangiopathic glomerulopathy, glomerular thrombi, mesangial with those for comparable patients thickening, and glomerular edema (expansion of Bowman’s space). Hematoxylin and eosin maintained on chronic dialysis, but not stain, original magnification 3400. Reproduced from reference 38, with permission. with those for patients receiving kidney

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A allografts. A second major point is that the patient’s general physical state should make him or her an acceptable candidate for allotransplantation. To se- lect patients who are unlikely to survive after receiving an allograft (e.g.,from general frailty, chronic infection, or pre- vious or current neoplasia) would not prove to be an adequate trial of xeno- transplantation, as the patient would be equally unlikely to survive. We suggest it would be ethical to offer a pig kidney transplant to selected B patients whose life expectancy is less than the time it will take for them to obtain a deceased human donor organ. The median waiting period for a patient with ESKD to obtain a human donor kidney is 3.9 years,4 by which time ap- proximately 35% of transplant candi- dates may have died or removed from the waitlist (Figure 1). Those of blood group B or O may experience a wait of $7years,4 even when the patient has no antibodies directed to HLA. Many patients might choose to receive a life- supporting pig kidney as long as a rea- sonable period of graft function could be C anticipated without the need for exces- sive immunosuppressive therapy (com- pared with that required to maintain an allograft). Other parameters that may be important are listed in Table 5. At present, the FDA recommends that a patient should only be considered for a pig organ transplant if his or her life expectancy is anticipated to be ,2 years. It may be difficult to predict the exact survival of a patient, and this guideline, if followed, might rule out many of the Figure 6. (A) Human IgM (left) and IgG (right) antibody binding to wildtype (WT), GTKO, double patients we have suggested above. Fur- knockout (DKO; i.e., knockout of Gal and Sda), and triple knockout (TKO) pig red blood cells thermore, some patients who are un- 5 fl (RBCs). Human serum antibody binding to pig RBCs (n 14) was measured by ow cytometry likely to survive for 2 years have already using the relative geometric mean (GM), which was calculated by dividing the GM value for each been supported by dialysis for several sample by the negative control (see Gao et al.,74 or Li et al.43). Negative controls were obtained by incubating the cells with secondary anti-human antibodies only (with no serum). Binding to years and have developed comorbidi- TKO pig RBCs was not significantly different from human IgM and IgG binding to human RBCs of ties and are no longer ideal transplant O. **P,0.01. Modified and reproduced from reference 45, with permission. (B) candidates. In this case, patients in Pooled human serum complement-dependent cytotoxicity (hemolysis) to WT, GTKO, and TKO (GTKO/b4GalKO/CMAHKO) pig RBCs was performed (Cooper et al.46). Briefly, RBCs were in- human O blood type cells. Reproduced from cubated with diluted serum for 30 minutes at 37°C. After washing, RBCs were incubated with reference 46, with permission. (C) Human (left) rabbit complement (Sigma, St. Louis, MO) (final concentration 20%) for 150 minutes at 37°C. and baboon (right) IgM and IgG binding to After centrifugation, supernatant was collected, and hemolysis was evaluated using a Multi-Label TKO pig red blood cells. Baboon sera contain Microplate Reader (Victor3; Perkin Elmer, Waltham, MA). The absorbance of each sample at 541 significantly more IgM directed to TKO pig nm was measured. Cytotoxicity of the same serum to autologous human blood type O RBCs cells than human sera. Modified from ref- was tested as a control. Serum cytotoxicity to TKO pig cells was not significantly different to erence 43, with permission.

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have been better utilized. For the first clinical trial, however, we suggest that these patients, particularly those in whom the disease might recur rapidly, e.g., FSGS, may not be ideal candidates. At present, we would exclude patients with any sensitization to HLA. Although our own studies have repeatedly indi- cated that sensitization to HLA would not be detrimental to survival of a pig kidney graft,52,53 several other studies have indicated that there can be cross- Figure 7. Increases in the lengths of the kidneys in four baboons with genetically reactivity between anti-HLA antibodies engineered pig kidney grafts that functioned for 90, .136, .237, and .260 days, re- and some swine leukocyte antigens spectively. There were similar increases in the width and depth of the kidneys. A 2-month- (SLA).53,54 Methods are being devel- old pig weighing 20 kg will grow rapidly to approximately 75–90 kg by 6 months of age, oped to delete or replace specificSLA whereas a recipient 8 kg baboon may grow by only 1 kg within the following 6 months. against which there might be crossreac- tivity.55,56 Although HLA-sensitized patients may be those who ultimately whom vascular access for dialysis is be- it with the predicted survival after kid- benefitmostfrompigkidneyxeno- coming difficult could be considered, but ney allotransplantation, A similar ap- transplantation, we submit that no risks again many of these will have been un- proach is worth exploration in relation in this respect should be taken in the dergoing dialysis for a prolonged period to xenotransplantation. first clinical trial. of time and, for this and other reasons, A second group of patients who could In addition, the kidney allocation sys- may not be suitable candidates for inclu- be considered as possible candidates for tem introduced in the in sion in a clinical trial. Selection of the xenotransplantation is those with an un- December 2014 prioritizes allocation of initial patients, therefore, will require derlying disease that has recurred in a donor kidneys to HLA highly sensitized re- very careful consideration. second or even third allograft (Table cipients if there are no preformed anti- A method of predicting the potential 4).50 However, some of these patients bodies against that specific donor. This benefit of a pig kidney transplant would will likely have developed antibodies to has resulted in increased rates of transplan- be valuable. This could possibly be indi- HLA, and therefore, if we follow our cur- tation for highly sensitized patients, some cated by a similar method to that sugges- rent criteria (see below), would be ex- of whom may now have a shorter waitlist ted by Bae et al.51 On the basis of the cluded. It is unknown whether any of time than less-sensitized patients.50,57 Kidney Donor Profile Index and the Es- these diseases will recur in a pig kidney Although a pre-emptive transplant timated Post-Transplant Survival, this but, even if they do, the xenograft will might be most beneficial, for the first group calculated the predicted survival have allowed for the allocation of an al- clinical trial we suggest that recipient of a patient on the waitlist and compared lograft to another recipient where it may selection should be limited to those

Table 4. Potential conditions for which initial clinical trials of pig kidney xenotransplantation may be justified Elderly patients without significant concomitant disease Patients of blood group B or O often wait for .5 yr for a suitable donor. The mortality of waitlist patients is 40% at 5 yr, and so many of these patients, particularly in the age range 55–65 yr, will not survive until a deceased human kidney becomes available. Recurrent Recurrent FSGS Recurrence can be very rapid in some patients. If recurrence occurs rapidly in the pig kidney, this may not be a valid test of xenotransplantation. Other potentially recurrent diseases Recurrence is slower in several other disease states, e.g., Ig A nephropathy, membranoproliferative GN type 2, and so these patients might possibly be candidates for a trial of pig kidney transplantation. High sensitization to HLA There is evidence for some cross-reactivity between anti-HLA antibodies and swine leukocyte antigens, suggesting that patients sensitized to HLA should be excluded from the first clinical trials. Loss of vascular access for dialysis These patients have often been on dialysis for some time (years rather than months) and may have diseased blood vessels making kidney transplantation technically difficult. They are frequently less than ideal candidates even for allotransplantation. Modified from reference 50, with permission.

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Table 5. Factors considered in selection of patients for first clinical trials of pig kidney xenotransplantation Age 55–65 yr. As the anticipated period of pig graft survival remains uncertain, younger patients, who are more likely to survive until a suitable allograft becomes available, should perhaps be excluded from the initial trials. No significant health problems except ESKD. A patient with a pig xenograft may possibly (1) require more immunosuppressive therapy than one with an allograft, and (2) may need to return to chronic dialysis, which may be associated with a higher morbidity than initially. It is therefore important that the patient should have no other health problems except ESKD. A patient with isolated polycystic kidney disease might be a preferred candidate. Blood type B or O, as these patients spend longer on the waiting list for a deceased human donor kidney. No anti-HLA antibodies (to avoid any risk of cross-reactivity of anti-HLA antibodies with swine leukocyte antigens). Supported by dialysis, but for ,12 mo. Initiation of dialysis will confirm to the patient and his/her family that ESKD has advanced sufficiently to warrant kidney transplantation, but the period of dialysis has not been so long to be associated with complications or lead to general debility. Fulfill all other criteria for allotransplantation, e.g., absence of potentially life-threatening or malignant disease. Vascular access may be problematic, or is likely to become limited. already on dialysis. This removes the ad- REGULATORY, PSYCHOSOCIAL, potential. The genetic manipulations ditional variable of native renal function AND ETHICAL ASPECTS OF that have been introduced in the pigs from the interpretation of the study re- CLINICAL to date have largely been directed to sults. Furthermore, if the patient is al- XENOTRANSPLANTATION overcome the innate immune response, ready requiring dialysis, there will be for which effective drug therapy is lim- no doubt that ESKD had advanced Clinical trials will clearly be overseen by ited. In future, however, the pigs will also enough to warrant a kidney transplant. national regulatory authorities. Archiv- be manipulated to control the adaptive Importantly, the current (limited) ev- ing of specimens (blood, tissues) from immune response, thus enabling exoge- idence is that a failed pig xenograft, even the organ-source pig will be essential so nous immunosuppressive therapy to be if anti-pig antibodies are developed, that, in the event that an unusual com- significantly reduced or, indeed, ulti- would not be detrimental to a subse- plication develops in the human mately unnecessary. For example, pigs quent allograft.54 recipient (e.g.,infection,neoplasia), have already been produced in which ex- suitable testing can be carried out to de- pression of SLA class 1 has been deleted65 termine whether this can be attributed to or SLA class 2 has been downregulated,66 THE POTENTIAL RISK OF the presence of the pig xenograft. or in which PD-L1,67–69 the immuno- XENOZOONOSIS Several studies of the attitudes to clin- suppressive agent CTLA4-Ig,70,71 or ical xenotransplantation of patients HLA-E or G (providing protection from Breeding and housing of the organ- awaiting organ transplantation, their NK cell activity)72 have been expressed. source pigs in a biosecure, designated family members, medical and nursing The ultimate goal of both allotrans- pathogen-free environment should rid staff, or of other groups within the com- plantation and xenotransplantation is them of all potentially pathogenic mi- munity (e.g.,religiousgroups62)have the induction of immunologic toler- croorganisms. Nevertheless, expert been undertaken, with those personally ance, in which the recipient no longer opinion is that the immunosuppressed affected by terminal disease (e.g.,ESKD) attempts to reject the graft. Although recipients will be susceptible to the being more positive in accepting this po- efforts in this respect in xenotrans- same infectious complications as recipi- tential new form of therapy than those plantation have to date been unsuc- ents of allografts, but that these will be with no immediate threat to life (W.D. cessful, in view of the potential offered manageable.58 Paris, unpublished data). by genetic engineering of the donor, it There has, however, been special con- Ethical aspects of xenotransplantation would seem it is more likely to be cern about the transfer of porcine en- have been discussed for many years,63 achieved in xenotransplantation than dogenous retroviruses (PERVs, that are with particular consideration on whether in allotransplantation. present in every pig cell). Although not a patient with a xenograft (who, in the pathogenic in pigs, whether these will be event of developing a porcine infection, pathogenic in humans is unknown, but might present a risk to society) can with- ACKNOWLEDGMENTS considered unlikely. Furthermore, there draw from a clinical trial.64 are drugs that can be administered Prof. Hara, Prof. Iwase, Dr. Yamamoto, Dr. to prevent or treat PERV activation. Jagdale, Prof. Anderson, Prof. Eckhoff, and Among the numerous genetic manipu- THE FUTURE Prof. Cooper carried out the preclinical lations in pigs that have been explored is studies outlined in the manuscript. Prof. inactivation or deletion of PERVs,59–61 The transplantation of organs, tissues, Kumar, Prof. Mannon, Prof. Hanaway, Prof. although the present opinion is that and cells from genetically engineered Eckhoff, and Prof. Cooper contributed to the this will not be necessary. pigs has immense clinical therapeutic section on selection of potential patients.

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Prof. Cooper wrote the original draft of the 9. Cooper DKC: A brief history of cross-species 23. Phelps CJ, Koike C, Vaught TD, Boone J, manuscript. All authors reviewed the man- organ transplantation. Proc Bayl Univ Med Wells KD, Chen SH, et al.: Production of al- Cent 25: 49–57, 2012 pha 1,3-galactosyltransferase-deficient pigs. uscript, after which revisions were made and 10. Reemtsma K, McCracken BH, Schlegel JU, Science 299: 411–414, 2003 approved by all authors. Pearl MA, Pearce CW, Dewitt CW, et al.: 24. Kolber-Simonds D, Lai L, Watt SR, Denaro M, Renal heterotransplantation in man. Ann Surg Arn S, Augenstein ML, et al.: Production of 160: 384–410, 1964 alpha-1,3-galactosyltransferase null pigs by DISCLOSURES 11. Lexer G, Cooper DKC, Rose AG, Wicomb means of nuclear transfer with fibroblasts WN,ReesJ,KeraanM,etal.:Hyperacute bearing loss of heterozygosity mutations. Proc rejection in a discordant (pig to baboon) Natl Acad Sci U S A 101: 7335–7340, 2004 Prof. Cooper reports grants from National Insti- cardiac xenograft model. J Heart Transplant 25. Yamada K, Yazawa K, Shimizu A, Iwanaga T, tutes of Health National Institute of Allergy and 5: 411–418, 1986 Hisashi Y, Nuhn M, et al.: Marked prolonga- Infectious Diseases during the conduct of the 12. Cooper DKC, Human PA, Lexer G, Rose AG, tion of porcine renal xenograft survival in study. Prof. Eckhoff reports grants from United Rees J, Keraan M, et al.: Effects of cyclo- baboons through the use of a1,3-gal- Therapeutics, during the conduct of the study. sporine and antibody adsorption on pig actosyltransferase gene-knockout donors Prof. Kumar reports receiving honoraria from Mal- cardiac xenograft survival in the baboon. J and the cotransplantation of vascularized linckrodt, outside the submitted work. All remain- Heart Transplant 7: 238–246, 1988 thymic tissue. Nat Med 11: 32–34, 2005 ing authors have nothing to disclose. 13. Cooper DKC, Gollackner B, Sachs DH: Will 26. Shimizu A, Yamada K, Robson SC, Sachs DH, the pig solve the transplantation backlog? Colvin RB: Pathologic characteristics of Annu Rev Med 53: 133–147, 2002 transplanted kidney xenografts. JAmSoc FUNDING 14. Cooper DKC, Ezzelarab MB, Hara H, Iwase H, Nephrol 23: 225–235, 2012 Lee W, Wijkstrom M, et al.: The pathobiology 27. Azimzadeh AM, Kelishadi SS, Ezzelarab MB, Work on xenotransplantation at the University of of pig-to-primate xenotransplantation: A Singh AK, Stoddard T, Iwase H, et al.: Early Alabama at Birmingham is supported in part by Na- historical review. Xenotransplantation 23: graft failure of GalTKO pig organs in ba- tional Institutes of Health National Institute of Allergy 83–105, 2016 boons is reduced by expression of a human and Infectious Diseases U19 grant AI090959, and in 15. 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