The longest survival of a non-human primate with a life-supporting kidney graft to date has been 90 days, although graft survival > 30 days has been unusual. A baboon received a kidney graft from an α-1,3-galactosyltransferase gene-knockout pig transgenic for two human complement-regulatory proteins and three human coagulation-regulatory proteins (although only one was expressed in the kidney). Immunosuppressive therapy was with ATG+anti-CD20mAb (induction) and anti-CD40mAb+rapamycin+corticosteroids (maintenance). Anti-TNF-α and anti-IL-6R were administered. The baboon survived 136 days with a generally stable serum creatinine (0.6 to 1.6 mg/dl) until termination. No features of a consumptive coagulopathy (e.g., thrombocytopenia, decreased fibrinogen) or of a protein-losing nephropathy were observed. There was no evidence of an elicited anti-pig antibody response. Death was from septic shock (Myroides spp). Histology of a biopsy on day 103 was normal, but by day 136, the kidney showed features of glomerular enlargement, thrombi, and mesangial expansion. The combination of (i) a graft from a specific genetically engineered pig, (ii) an effective immunosuppressive regimen, and (iii) anti-inflammatory agents prevented immune injury and a protein-losing nephropathy, and delayed coagulation dysfunction. This outcome encourages us that clinical renal xenotransplantation may become a reality.

The risk of xenozoonosis mainly by porcine endogenous retrovirus (PERV) has been considered as one of the main hurdles in xenotransplantation and therefore should be elucidated prior to the clinical use of porcine corneal grafts. Accordingly, an investigation was performed to analyze the infectivity of PERVs from porcine keratocytes to human cells, and the long-term risk of transmission of PERVs was determined using pig-to-non-human primate (NHP) corneal transplantation models.

Several immunosuppression (IS) regimens achieve long-term graft survival in non-human primates (NHPs) after porcine islet transplantation (PITx), but their success rates vary. To understand the mechanism of graft loss, we investigated the relationships between graft survival and humoral or inflammatory responses for maintenance IS in NHPs after PITx.

Highly sensitized patients face many barriers to kidney transplantation, including higher rates of antibody-mediated rejection after HLA-incompatible transplant. IdeS, an endopeptidase that cleaves IgG nonspecifically, has been trialed as desensitization prior to kidney transplant, and successfully cleaves donor-specific antibody (DSA), albeit with rebound.

Anti-CD154mAb is a powerful co-stimulation blockade agent that is efficacious in preventing rejection, even in xenogeneic settings. It has been used in the majority of successful long-term pig-to-non-human primate islet transplantation models. However, its clinical use was halted as a result of thromboembolic complications that were also observed in preclinical and clinical organ transplantation models. An anti-CD154mAb was administered to 14 streptozotocin-induced diabetic cynomolgus monkey recipients of porcine islets, some of which received the agent for many months. Monkeys were monitored for complications, and blood monitoring was carried out frequently. After euthanasia, multiple biopsies of all organs were examined for histological features of thromboembolism. Anti-CD154mAb prevented rejection of genetically engineered pig islets in all monkeys. No significant complications were attributable specifically to anti-CD154mAb. There was no evidence of thromboembolism in multiple histological sections from all major organs, including the brain. Our data suggest that in diabetic monkeys with pig islet grafts, anti-CD154mAb would appear to be an effective and safe therapy, and is not associated with thromboembolic complications.

The use of porcine islets to replace insulin-producing islet β-cells, destroyed during the diabetogenic disease process, presents distinct challenges if this option is to become a therapeutic reality for the treatment of type 1 diabetes. These challenges include a thorough evaluation of the microbiological safety of the islets. In this study, we describe a robust porcine islet-screening program that provides a high level of confidence in the microbiological safety of porcine islets suitable for clinical trials.

Islet transplantation is an effective therapy for selected patients with type 1 diabetes with labile glycemic control and hypoglycemic unawareness, but donor organs are limited. Islet xenotransplantation using porcine islets will potentially solve this problem. Although successful proof of concept studies using clinically inapplicable anti-CD154 monoclonal antibody (mAb) in pig-to-non-human primate (NHP) islet xenotransplantation has been demonstrated by several groups worldwide, potentially clinically applicable anti-CD40 (2C10R4) mAb-based studies have not been reported.

Recently, we have shown that an immunosuppression regimen including costimulation blockade via anti-CD154 antibody significantly prolongs the cardiac xenograft survival in a GTKO.hCD46Tg pig-to-baboon heterotopic xenotransplantation model. Unfortunately, many coagulation disorders were observed with the use of anti-CD154 antibody, and recipient survival was markedly reduced by these complications.

The JAK/STAT (Janus Tyrosine Kinase, Signal Transducers and Activators of Transcription) pathway is associated with cytokine or growth factor receptors and it is critical for growth control, developmental regulation and homeostasis. The use of porcine ocular cells as putative xenotransplants appears theoretically possible. The aim of this study was to investigate the response of various porcine ocular cells in vitro to human cytokines in regard to the activation of JAK-STAT signaling pathways.

Pancreatic islet transplantation is currently proven as a promising treatment for type 1 diabetes patients with labile glycemic control and severe hypoglycemia unawareness. Upon islet transplantation, revascularization is essential for proper functioning of the transplanted islets. As IL-6 is important for endothelial cell survival and systemic inflammation related to xenograft, the effect of IL-6 receptor antagonist, tocilizumab, on revascularization of the transplanted islets was examined in pig to non-human primate islet xenotransplantation model. Also, the endothelial cell origin in a new vessel of the transplanted pig islets was determined.

In pig-to-baboon heart/artery patch transplantation models, adequate costimulation blockade prevents a T-cell response. After heart transplantation, coagulation dysfunction (thrombocytopenia, reduced fibrinogen, increased D-dimer) and inflammation (increased C-reactive protein [CRP]) develop. We evaluated whether coagulation dysfunction and/or inflammation can be detected following pig artery patch transplantation.

Xenotransplantation has the potential to alleviate the organ shortage that prevents many patients with end-stage renal disease from enjoying the benefits of kidney transplantation. Despite significant advances in other models, pig-to-primate kidney xenotransplantation has met limited success. Preformed anti-pig antibodies are an important component of the xenogeneic immune response. To address this, we screened a cohort of 34 rhesus macaques for anti-pig antibody levels. We then selected animals with both low and high titers of anti-pig antibodies to proceed with kidney transplant from galactose-α1,3-galactose knockout/CD55 transgenic pig donors. All animals received T-cell depletion followed by maintenance therapy with costimulation blockade (either anti-CD154 mAb or belatacept), mycophenolate mofetil, and steroid. The animal with the high titer of anti-pig antibody rejected the kidney xenograft within the first week. Low-titer animals treated with anti-CD154 antibody, but not belatacept exhibited prolonged kidney xenograft survival (>133 and >126 vs. 14 and 21 days, respectively). Long-term surviving animals treated with the anti-CD154-based regimen continue to have normal kidney function and preserved renal architecture without evidence of rejection on biopsies sampled at day 100. This description of the longest reported survival of pig-to-non-human primate kidney xenotransplantation, now >125 days, provides promise for further study and potential clinical translation.

Recent advancements in gene editing techniques have increased in number and utility. These techniques are an attractive alternative to conventional gene targeting methods via homologous recombination due to the ease of use and the high efficiency of gene editing. We have previously produced cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) knockout (KO) pigs in a Minnesota miniature pig genetic background. These pigs were generated using zinc-finger nucleases (ZFNs) in combination with donor DNA containing a total homology length of 1600 bp (800-bp homology on each arm). Our next aim was to introduce the targeted disruption of alpha-1,3-galactosyltransferase (GGTA1) in the CMAH KO genetic background and evaluate the effect of donor DNA homology length on meganuclease-mediated gene targeting.

Islet allotransplantation is a promising way to treat some type 1 diabetic (T1D) patients with frequent hypoglycemic unawareness, and islet xenotransplantation is emerging to overcome the problem of donor organ shortage. Our recent study showing reproducible long-term survival of porcine islets in non-human primates (NHPs) allows us to examine whether autologous regulatory T-cell (Treg) infusion at peri-transplantation period would induce transplantation tolerance in xenotransplantation setting.

Patients in fulminant hepatic failure currently do not have a temporary means of support while awaiting liver transplantation. A potential therapeutic approach for such patients is the use of extracorporeal perfusion with porcine livers as a form of "liver dialysis". During a 72-h extracorporeal perfusion of porcine livers with human blood, porcine Kupffer cells bind to and phagocytose human red blood cells (hRBC) causing the hematocrit to decrease to 2.5% of the original value. Our laboratory has identified porcine sialoadhesin expressed on Kupffer cells as the lectin responsible for binding N-acetylneuraminic acid on the surface of the hRBC. We evaluated whether blocking porcine sialoadhesin prevents the recognition and subsequent destruction of hRBCs seen during extracorporeal porcine liver xenoperfusion.

Some patients with acute or acute-on-chronic hepatic failure die before a suitable human liver allograft becomes available. Encouraging results have been achieved in such patients by the transplantation of human hepatocyte progenitor cells from fetal liver tissue. The aim of the study was to explore survival of hepatocytes from genetically engineered pigs after direct injection into the spleen and other selected sites in immunosuppressed baboons to monitor the immune response and the metabolic function and survival of the transplanted hepatocytes.

Parkinson's disease (PD) features the motor control deficits resulting from irreversible, progressive degeneration of dopaminergic (DA) neurons of the nigrostriatal pathway. Although intracerebral transplantation of human fetal ventral mesencephalon (hfVM) has been proven effective at reviving DA function in the PD patients, this treatment is clinically limited by availability of hfVM and the related ethical issues. Homologous tissues to hfVM, such as porcine fetal ventral mesencephalon (pfVM) thus present a strong clinical potential if immune response following xenotransplantation could be tamed. Olfactory ensheathing cells (OECs) are glial cells showing immunomodulatory properties. It is unclear but intriuging whether these properties can be applied to reducing immune response following neural xenotransplantation of PD.

Pigs deficient in three glycosyltransferase enzymes (triple-knockout [TKO] pigs, that is, not expressing the three known carbohydrate xenoantigens) and expressing 'protective' human transgenes are considered a likely source of organs for transplantation into human recipients. Some human sera have no or minimal natural antibody binding to red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) from TKO pigs. However, all Old World monkeys exhibit natural antibody binding to TKO pig cells. The xenoantigen targets of Old World monkey natural antibodies are postulated to be carbohydrate moieties exposed when the expression of the carbohydrate N-glycolylneuraminic acid (Neu5Gc) is deleted. The aim of this study was to compare the survival in baboons and histopathology of renal grafts from pigs that either (a) expressed Neu5Gc (GTKO pigs; Group 1) or (b) did not express Neu5Gc (GTKO/CMAHKO [DKO] or TKO pigs; Group 2).