BACKGROUND:Severe thrombocytopenia and coagulation dysregulation remain major barriers to survival in pig-to-primate liver xenotransplantation models. METHODS:Porcine livers with five genetic modifications (GTKO, CMAHKO, β4GalNT2KO, hCD55, and hTBM) were orthotopically transplanted into four Tibetan macaques under two conventional immunosuppressive protocols. Longitudinal monitoring included graft and multi-organ function, hematology, coagulation, and immune responses. Histopathological and immunohistochemical analyses were performed to characterize graft and recipient pathology. RESULTS:High expression of human thrombomodulin (hTBM) in donor livers effectively prevented early rapid and severe thrombocytopenia, with three of four recipients maintaining platelets >100 × 109/L. Thrombotic microangiopathy (TMA) was absent in grafts and recipient organs despite transient hypercoagulability. No spontaneous bleeding occurred in three of four cases. However, porcine livers failed to synthesize functional coagulation factor II, protein C, and protein S, contributing to progressive coagulopathy. While early graft function and coagulation remained stable for four posttransplant days, late dysfunction ensued, characterized by impaired coagulation factor synthesis and progressive graft failure. Antibody-mediated rejection, accompanied by IgM/IgG/C4b deposition, anti-TKO antibody increase, and pvWF upregulation, triggered injury that was amplified by innate and T cell-mediated responses. CONCLUSIONS:GTKO/CMAHKO/β4GalNT2KO/hCD55/hTBM donor liver transplantation was associated with an absence of early severe thrombocytopenia, transfusion requirement, and TMA, but long-term survival was limited by immune-mediated rejection and cross-species incompatibilities in coagulation factor synthesis. Optimizing (i) donor genetic modifications and (ii) immunosuppressive therapy, with (iii) replacement of targeted coagulation factors will be crucial for achieving durable survival in pig liver xenotransplantation.

Persistent proteinuria remains a major barrier to successful pig-to-nonhuman primate (NHP) kidney xenotransplantation and may become an important issue in clinical renal xenotransplantation. However, published human renal xenotransplant reports to date have not established severe proteinuria as a consistent clinical finding, and its relevance in living human recipients should therefore be regarded as an important unresolved question.In pig-to-NHP models, severe proteinuria may also be accompanied by urinary loss of therapeutic monoclonal antibodies, including anti-CD154, potentially compromising rejection prophylaxis. Proteinuria in this setting may occur in association with heterogenous and overlapping patterns of immune-mediated and non-immune mediated injury, including rejection-associated endothelial, microvascular, and humoral lesions such as thrombotic microangiopathy, capillaritis, C4d deposition, and xenograft glomerulopathy. Hemodynamic mismatch should currently be regarded as a possible but unproven contributor rather than a dominant initiating trigger.We therefore propose a unifying hypothesis based on an evidence-informed, testable framework rather than a definitive causal cascade, which we hope will stimulate discussion. Clarifying the dominant mechanisms in individual cases, correlating proteinuria with biopsy findings, and prospectively monitoring urinary drug loss will be essential for developing rational preventive and therapeutic strategies.

Background Systemic inflammation in xenograft recipients (SIXR) has been suggested to contribute to gene-edited pig xenograft failure after transplantation into nonhuman primates. Inflammation is associated with high levels of proinflammatory cytokines. We investigated selected proinflammatory cytokines (IL-6, IL-8, IL-1b, IL-12, and tumor necrosis factor [TNF]) after pig-to-baboon kidney xenotransplantation. Methods Cytokines were measured during the first 60 days after the transplantation of kidneys from either (i) α1,3-galactosyltransferase gene-knockout (GTKO) pigs (n = 3) or (ii) triple-knockout pigs with additional knockout of growth hormone receptors and transgenic expression of 6 human ‘protective’ proteins (10GE pigs) (n = 4): All recipients received an immunosuppressive regimen based on anti-CD154mAb, IL-6R blockade (with tocilizumab), rapamycin, and methylprednisolone. Results Baboons with GTKO pig kidneys required euthanasia on days 47, 61, and 69 (mean 59 days) for a variety of unexplained complications (loss of mobility, loss of weight, recurrent severe ascites), but all with functioning grafts and no anti-donor pig antibodies. All recipients of 10GE pig kidneys survived for >3 months. Serum creatinine in both groups remained within the near-normal range for >3 months. IL-6 and IL-8 levels increased significantly in the GTKO group but not in the 10GE group. There was no increase in any other cytokine in either group. Conclusions There was a greater response of IL-6 and IL-8 to a GTKO kidney than to a 10GE kidney. Although our data are limited, it appears that multiple genetic modifications (e.g., triple knockout and/or human protective transgenes) might be necessary to overcome high levels of IL-6 and IL-8 in the early phase after xenotransplantation.

Xenotransplantation offers a promising solution to the shortage of human organs for transplantation but requires overcoming numerous immune responses-particularly those mediated by the innate immune system through natural killer (NK) cells and macrophages. This review examines advances demonstrating that the expression of human leukocyte antigen E (HLA-E) on porcine cells contributes to reduce cellular xenograft rejection. HLA-E expression partially inhibits both direct NK cell cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), while also attenuating macrophage-mediated lysis. Furthermore, perfusion of transgenic porcine organs expressing HLA-E with human blood resulted in significantly less tissue damage compared to wild-type counterparts, thereby confirming the protective effect of HLA-E against innate immunity. Inhibition of NK cell activation can be further enhanced by co-expression of HLA-G and HLA-E. These findings confirm the potential of HLA-E and HLA-G expression as a complementary strategy in the design of immune-compatible porcine organs for clinical xenotransplantation.

Recent progress has been made toward introducing gene-edited (GE) pig heart and kidney xenotransplantation into clinical practice. In particular, the outcomes of GE pig kidney transplantation have been promising, with three recipients currently surviving between 3 and 9 months after transplantation. These encouraging results highlight the growing feasibility of xenotransplantation as a solution to the shortage of human donor organs. In the United States and many other countries, nearly half of all patients on kidney transplant waiting lists are diabetic. Individuals with diabetic nephropathy often achieve better outcomes when they receive both kidney and islet allotransplants, as this combination restores renal function and improves glucose control. Therefore, such patients would be ideal candidates for combined GE pig kidney and islet xenotransplantation. Because immunosuppressive therapy is essential to maintain a kidney graft, performing a simultaneous islet transplant is clinically justifiable. Studies in nonhuman primates have demonstrated that porcine islet transplantation can achieve long-term glucose regulation, supporting its translational potential. For the foreseeable future, until complications such as post-transplant proteinuria are fully resolved, we propose that patients with diabetic nephropathy receive a human kidney allotransplant in combination with a GE pig islet xenotransplant to optimize outcomes and improve long-term metabolic stability.

BACKGROUND:Complement activation is critical in xenograft rejection, but the relative efficacy of pharmacological complement inhibitors remains unclear. METHODS:Peripheral blood mononuclear cells (PBMCs) from wild-type (WT) pigs were exposed to human serum in the presence or absence of complement inhibitors. Complement-dependent cytotoxicity (CDC) and complement deposition were measured by flow cytometry. A C1s inhibitor (sutimlimab, 12.5-800 µg/mL), a C1-esterase inhibitor (berinert, 1.25-10U/mL), a C3/C3b inhibitor (pegcetacoplan, 0.25-4.0 mg/mL), and a C5 inhibitor (tesidolumab, 500-800 µg/mL) were applied to dose-response assays. RESULTS:Only pegcetacoplan (C3/C3b inhibitor) achieved complete inhibition of CDC with minimal C3b/iC3b and C5b-9 deposition. Sutimlimab (C1s inhibitor) produced partial inhibition of CDC (hitting a plateau) and complement deposition, while C1-esterase inhibitor had limited CDC effect and did not reduce C3b/iC3b. Tesidolumab (C5 inhibitor) suppressed C5b-9 but failed to prevent CDC or upstream opsonization. CONCLUSIONS:Although these results might reflect experiments conducted at concentrations lower than clinical doses, our data suggest that, of the limited number of agents tested, pegcetacoplan may be the most effective in inhibiting xenoreactive human complement activation in this experimental setting (i.e., CDC of human complement in the pooled-serum against WT pig PBMCs). However, the results we obtained are puzzling because the C5 inhibitor, eculizumab, has been proven to be highly effective in inhibiting human complement activation in the in vitro CDC model. Furthermore, tesidolumab was reported to be effective in an in vivo NHP model. To obtain the additional information required for clinical application, further investigation would be needed. We would need to (i) ensure that the tesidolumab we were using had not deteriorated in any way and lost its efficacy, and (ii) compared pegcetacoplan with other C5 inhibitors (e.g., ravulizumab, eculizumab).

Diabetes mellitus and end-stage renal disease (ESRD) are major causes of morbidity and mortality worldwide.Pancreatic islet transplantation offers potential insulin independence. Different sites and their suitability for islet transplantation have been investigated, including renal subcapsular and intraportal sites, among others. Various limitations and complications have been reported, such as the number of donor islet species, islet isolation, and site of transplantation. Combining islets and kidney transplantation in diabetic patients suffering from ESRD has several advantages.This review compares the experimental experience of the renal subcapsular and intraportal sites for islet allo- and xeno-transplantation. The subcapsular site appears preferable in rodent models. The intraportal site has been more successful in large animals. Combined islet-kidneys have been suggested as a treatment targeting both ESRD and type 1 diabetes. With our increasing ability to genetically-engineer pigs, and the availability of novel immunosuppressive agents, xenotransplantation is an emerging therapeutic option.

One-third of patients with diabetes will develop end-stage kidney disease, and approximately one-half of the patients requiring kidney transplantation in the U.S. suffer from diabetes. In 2002, Yamada, Sachs, and their colleagues explored the renal subcapsular site for islet implantation in a partially inbred miniature swine model. All pancreatectomized pigs that received autologous islets under the renal capsule maintained normoglycemia. The transplantation of a composite islets-kidney (i.e., the kidney with the revascularized autologous islets) into another pancreatectomized, bilaterally nephrectomized pig provided successful renal and islet function. In 2011, this model was extended successfully to nonhuman primates (NHPs). There are several hurdles to be overcome if this model is to be successfully translated into clinical practice, and xenotransplantation might resolve this problem. Pancreases harvested from gene-edited pigs could be sources of islets that could be isografted beneath the renal capsule of an identical cloned littermate. After allowing the islets to become vascularized over a period of 6-12 weeks, during which no immunosuppressive therapy would be required, the composite islets-kidney would then be transplanted into an immunosuppressed NHP or human recipient. On reperfusion of the graft, both renal and islet function should rapidly return. If sufficient islets have been transplanted with the kidney and the immunosuppressive regimen is successful, normoglycemia and normal renal function should be quickly achieved. However, a large number of genetically engineered neonatal donor piglets will be required to manufacture one therapeutic patient dose of islets, which may prove to be a logistical problem. Alternative approaches are discussed. ARTICLE HIGHLIGHTS:Autologous islet implantation in the kidney subcapsular space of pigs and baboons and subsequent allotransplantation of the composite islet-kidney graft have been demonstrated to be successful, but the limited availability of human islets limits this approach in clinical practice. The implantation of neonatal pig islets under the kidney capsule of an identical cloned piglet and subsequent transplantation into a human patient with diabetic nephropathy could potentially correct both diabetes and renal failure. Neonatal pig islets can become revascularized in cloned littermates in the absence of immunosuppressive therapy, but a very large number of donors may be required.

BACKGROUND:Pig-to-human kidney xenotransplantation offers a potential solution to the organ shortage. However, the ability of the transplanted pig kidney to regulate blood pressure and fluid balance remains uncertain. The renin-angiotensin-aldosterone system (RAAS) plays a crucial role in these functions, but species differences may impair its effectiveness in xenotransplantation. METHODS:Gene-edited pig kidneys were transplanted into six immunosuppressed baboons. Group A (n = 2) underwent bilateral native nephrectomy, while Group B (n = 4) had unilateral nephrectomy with one native kidney remaining in situ (with its ureter ligated) to avoid it contributing to salt and volume regulation. Plasma creatinine, potassium, renin, angiotensinogen, angiotensin I, and aldosterone levels were measured. RESULTS:Group A, but not Group B, exhibited increases in plasma creatinine and potassium levels, indicating hypovolemia that could be corrected by frequent fluid administration. Pig-specific renin was undetectable at all post-transplant time points in both groups. Baboon renin concentration and activity were measurable only in Group B, indicating that the native kidney contributed to renin production. Aldosterone levels remained unchanged in both groups. CONCLUSIONS:The absence of detectable pig renin highlights a potential physiological challenge in xenotransplantation. However, the retention of a native kidney may help maintain RAAS function and mitigate fluid and electrolyte imbalances. In clinical pig-to-human kidney transplantation, both native kidneys are usually retained, thus minimizing the development of hypovolemia.

Since January 2022, eleven clinical xenotransplants into living patients involving gene-edited pig hearts (n = 2) or kidneys (n = 9) have provided initial data on patient selection, organ sourcing, and immunosuppression. Lessons from cardiac cases indicate that (i) patient selection must prioritize candidates with a realistic recovery potential from preexisting debility, (ii) immunosuppressive regimens should be initiated 5-7 days pretransplant to ensure therapeutic blood levels, (iii) the administration of products that potentially contain anti-pig antibodies should be avoided, and (iv) highly sensitive assays are essential to ensure the graft is free of pathogenic microorganisms. In renal cases, evidence suggests that (i) pig organs with multiple gene edits may provide superior protection against the human immune response, and (ii) while immunosuppression targeting the CD40/CD154 co-stimulation pathway effectively prevents the adaptive immune response, it currently fails to eliminate the development of thrombotic microangiopathy or proteinuria. It remains to be determined whether the current gene editing and immunosuppressive protocols are sufficient to enable truly long-term survival of patients and grafts.

Porcine kidney xenotransplantation for end-stage renal disease (ESRD) has reached the stage of clinical testing following major advances in donor pig genetic modifications and effective immunosuppressive strategies through decades of rigorous translational research. Reports of pig kidney xenograft survival beyond 1 year post-transplant in nonhuman primate (NHP) models justify optimism for its potential as an alternative to allotransplantation. In the United States, experimental transplantations of genetically engineered (GE) porcine kidneys into brain-dead subjects and a small number of ESRD patients have shown no evidence of hyperacute rejection and adequate pig kidney function for up to several months. Here we discuss pre-clinical/clinical results, infectious disease, ethical, and regulatory considerations, and propose evidence-based recommendations. For initial clinical trials in kidney xenotransplantation, we make the following recommendations: (i) transplantation with organs from a triple knockout (TKO) donor pig, preferably with added human transgenes, (ii) an immunosuppressive regimen with induction therapy to deplete T (and possibly B) cells, and maintenance therapy based on a cluster of differentiation (CD)40/CD154 co-stimulation pathway blockade, (iii) the patient should be fully acceptable as a candidate for allotransplantation but should be unlikely ever to receive an allograft. Patients aged 60–69 years (extendable to 40-75 years, if one of the criteria mentioned below is present), of blood group B or O, and with diabetes are most at risk in this regard. Other patients who could be considered are (i) those who have lost two or more previous kidney allografts from recurrent disease in the graft, (ii) those with broad human leukocyte antigen (HLA)-reactivity but no evidence of anti-pig antibodies, including swine leukocyte antigen (SLA), and (iii) those with failing vascular access. Clinical pilot studies in carefully and highly selected patients with no alternative therapy will provide the foundation upon which to base subsequent formal expanded clinical trials.

The nature and severity of the inflammatory response influences the outcome of organ allotransplantation and xenotransplantation. In allotransplantation, the source of the allograft, for example, from a living, brain-dead, or circulatory death donor, influences the inflammatory response, as do such factors as the preexisting comorbidities and the length of the period of chronic kidney disease in the recipient and the management he/she has received. There is also inflammation associated with the transplant surgery, for example, as a result of ischemia-reperfusion injury. In xenotransplantation, inflammation associated with donor factors will be reduced and, as the patients will receive a pig graft at a much earlier stage of their chronic organ failure, the contribution of recipient factors should also be reduced. However, there is a well-documented systemic inflammatory response to the presence of a pig xenograft (probably associated with species molecular differences) that plays a role in activating the innate immune response. Indeed, there is a complex interaction between inflammation, coagulation dysfunction, and the innate and adaptive immune responses. Suppression of the inflammatory response, for example, by interleukin-6 receptor blockade, would appear to be beneficial after xenotransplantation. Several biomarkers of inflammation have been identified that may be valuable in assessing the response to therapy.

BACKGROUND:In recent years, gene-edited pigs have become sources of organs for clinical xenotransplantation. They have the potential to be sustainable sources of red blood cells (pRBCs). We investigated in vitro the effect of human complement inhibition by using (i) human CD55-expressing pRBCs from pigs with 10 gene-edits (10GE) and (ii) a C1-esterase inhibitor (C1-INH). METHODS:RBCs were collected from pigs (triple-knockout [TKO] with or without expression of "protective" human transgenes [10GE] on peripheral blood mononuclear cells [PBMCs], including two complement-regulatory proteins, hCD46 and hCD55). hCD46 and hCD55 expression, anti-pRBC antibody binding, and C3b/iC3b deposition were measured by flow cytometry. Hemolysis by complement-dependent cytotoxicity (CDC) was measured by a plate reader. A C1-INH was added to the hemolysis assay. RESULTS:HCD46 was not expressed on either TKO or 10GE pRBCs. hCD55 was expressed at low levels on 10GE pRBCs. Hemolysis induced by human complement and anti-pRBC antibodies was less when pRBCs were from 10GE pigs than from TKO pigs (57.3% ± 2.2% vs. 26.2% ± 3.8%, p < 0.01). C3b/iC3b deposition of 10GE pRBCs under nonhemolytic conditions was also lower. C1-INH decreased hemolysis (No C1-INH = 18.6% ± 2.3% vs. 2.5U/mL C1-INH = 7.0% ± 1.1%, p < 0.05). C3b/iC3b deposition on pRBCs was also decreased (gMFI: No C1-INH = 2680 ± 82 vs. 2.5 U/mL C1-INH = 719 ± 57, p < 0.01). CONCLUSIONS:Even low expression of hCD55 contributes to the protection of pRBCs from hemolysis by CDC, but the possibility of phagocytosis still remains. However, C1-INH partially protects pRBCs from hemolysis and C3b/iC3b deposition. Therefore, higher hCD55 expression and the administration of a complement inhibitor are likely to prolong pRBC survival after clinical xenotransfusion.

In vitro studies indicate that kidney transplantation from gene-edited pigs in which expression of all 3 of the known glycan xenoantigens has been deleted may be more challenging in nonhuman primates (NHPs) than it will be in human recipients. Furthermore, pig-to-human xenotransplantation offers several other advantages-(1) the patient can communicate with the surgical team; (2) recipient microbiological monitoring and environment will be clinical-grade; and (3) sophisticated graft monitoring and imaging techniques, (4) therapeutic interventions, eg, dialysis, plasmapheresis, and (5) intensive care can be deployed that are not easily available in NHP laboratory models. We suggest, therefore, that progress to develop safe, informative human clinical trials will be accelerated if pilot clinical cases are initiated. The selection of patients for kidney xenotransplantation can include those who are at high risk of dying imminently, for example, those experiencing increasing vascular access challenges with no realistic alternative therapy available, and those who have been accepted onto the waitlist for an allograft, but who are unlikely ever to receive one. Patients with an increased risk of dying include those with (1) age above 60 years, (2) blood groups O or B, and (3) diabetic nephropathy. UNOS data indicate that an average of 25 patients on the kidney waitlist in the United States die or are removed from the list every day (ie, >9000 each year). Given the improved xenograft survival observed in preclinical studies, we suggest that it is time to plan a small pilot clinical trial for healthy dialysis patients who understand the risks and potential benefits of kidney xenotransplantation.

Recent advances in gene-edited pig organ xenotransplantation offer a promising solution to the critical shortage of human donor organs. Differences in physiology, drug metabolism, excretion, and responses between species require careful consideration for posttransplant medication management in human recipients. This brief review compares the anatomical, physiological, and biochemical features of pig and human organs, particularly in relation to the liver, kidney, heart, and lungs, and their potential implications on drug exposure, response, and efficacy after xenotransplantation. The transplantation of gene-edited organs from pigs to humans will result in a complex metabolic interplay, depending on the organ being transplanted. For example, if a pig liver is transplanted into a human, the drugs metabolized by the pig liver may be excreted in the bile or by the human kidney. There may be breed-specific metabolism of drugs, and genetic polymorphism in pigs may contribute to variability in drug exposure similar to what is observed among humans. Furthermore, there may be significant size differences between pigs and human organs, and their functional capabilities may change over time as the pig organs age within the recipient's body. In this article, we review how pig organ xenotransplantation may impact physiology, drug exposure, and response to immunosuppressive agents, anti-infective drugs, and other medications commonly used for posttransplant medical conditions. There is limited data available on the specific breeds of pigs used in xenotransplantation, and further research is required to ensure appropriate drug dosing, minimize toxicity, and optimize long-term graft function in recipients of pig organ xenografts.