Search

Objective Genetically engineered pigs are thought to be an alternative organ source for patients in end-stage heart failure unable to receive a timely allograft. However, cardiac xenografts exhibit growth and diastolic heart failure within 1 month after transplantation. Grafts function for up to 6 months, but only after administration of temsirolimus and afterload-reducing agents to reduce this growth. In this study we investigated the growth and hemodynamics of growth hormone receptor (GHR) knockout xenografts, without the use of adjuncts to prevent intrinsic graft growth after transplantation. Methods Genetically engineered pig hearts were transplanted orthotopically into weight-matched baboons between 15 and 30 kg, using continuous perfusion preservation before implantation (n = 5). Xenografts included knockout of carbohydrate antigens and knockin of human transgenes for thromboregulation, complement regulation, and inflammation reduction (grafts with intact growth hormone, n = 2). Three grafts contained the additional knockout of GHR (GHR knockout grafts; n = 3). Transthoracic echocardiograms were obtained twice monthly and comprehensively analyzed by a blinded cardiologist. Hemodynamics were measured longitudinally after transplantation. Results All xenografts demonstrated life-supporting function after transplantation. There was no difference in intrinsic growth, measured using septal and posterior wall thickness and left ventricular mass, on transthoracic echocardiogram out to 1 month in either GHR knockout or GHR intact grafts. However, hypertrophy of the septal and posterior wall was markedly elevated by 2 months post transplantation. There was minimal hypertrophy out to 6 months in GHR knockout grafts. Physiologic mismatch was present in all grafts after transplantation, which is largely independent of growth. Conclusions Xenografts with GHR knockout show reduced post-transplantation xenograft growth using echocardiography >6 months after transplantation, without the need for other adjuncts.

Background Mortality for infants on the heart transplant wait list remains unacceptably high, and available mechanical circulatory support is suboptimal. Our goal is to demonstrate the feasibility of utilizing genetically engineered pig (GEP) heart as a bridge to allotransplantation by transplantation of a GEP heart in a baboon. Methods Four baboons underwent orthotopic cardiac transplantation from GEP donors. All donor pigs had galactosyl-1,3-galactose knocked out. Two donor pigs had human complement regulatory CD55 transgene and the other 2 had human complement regulatory CD46 and thrombomodulin. Induction immunosuppression included thymoglobulin, and Anti-CD20. Maintenance immunosuppression was Rapamycin, AntiCD-40 and methylprednisolone. One donor heart was preserved with University of Wisconsin (UW) solution and the other three with del Nido solution. Results All baboons weaned from cardiopulmonary bypass. B217 received a donor heart preserved with UW. Ventricular arrhythmias and depressed cardiac function resulted in early death. All recipients of del Nido preserved hearts easily weaned from cardiopulmonary bypass with minimal inotropic support. B15416 and B1917 survived for 90 days and 241 days respectively. Histopathology in B15416 revealed no significant myocardial rejection but cellular infiltrate around Purkinje fibers. Histopathology in B1917 was consistent with severe rejection. B37367 had uneventful transplant but developed significant respiratory distress with a cardiac arrest. Conclusions Survival of B15416 and B1917 demonstrates the feasibility of pursuing additional research to document the ability to bridge an infant to cardiac allotransplant with a GEP heart.

Galactosyl transferase knock-out pig lungs fail rapidly in baboons. Based on previously identified lung xenograft injury mechanisms, additional expression of human complement and coagulation pathway regulatory proteins, anti-inflammatory enzymes and self-recognition receptors, and knock-down of the β4Gal xenoantigen were tested in various combinations. Transient life-supporting GalTKO.hCD46 lung function was consistently observed in association with either hEPCR (n = 15), hTBM (n = 4), or hEPCR. hTFPI (n = 11), but the loss of vascular barrier function in the xenograft and systemic inflammation in the recipient typically occurred within 24 h. Co-expression of hEPCR and hTBM (n = 11) and additionally blocking multiple pro-inflammatory innate and adaptive immune mechanisms was more consistently associated with survival >1 day, with one recipient surviving for 31 days. Combining targeted genetic modifications to the lung xenograft with selective innate and adaptive immune suppression enables prolonged initial life-supporting lung function and extends lung xenograft recipient survival, and illustrates residual barriers and candidate treatment strategies that may enable the clinical application of other organ xenografts.

Background Blood transfusion remains important in the treatment of patients with sickle cell disease (SCD). However, alloimmunization after blood transfusion is associated with patient morbidity and mortality. Triple-knockout (TKO) pigs (i.e., pigs in which the three known xenoantigens to which humans have anti-pig antibodies have been deleted) may be an alternative source of RBCs for these patients because many humans have no preformed antibodies to TKO pig RBCs (pRBCs). Methods and materials In an in vitro study, plasma from alloimmunized (n = 12) or non-alloimmunized (n = 12) SCD patients was used to determine IgM/IgG binding to, and CDC of, TKO pRBCs. In an in vivo study, after an estimated 25% of blood volume was withdrawn from two capuchin monkeys, CFSE-labeled TKO pRBCs were transfused. Loss of TKO pRBCs was monitored by flow cytometry, and 7 weeks later, 25% of blood was withdrawn, and CFSE-labeled monkey RBCs were transfused. Results The in vitro study demonstrated that plasma from neither alloimmunized nor non-alloimmunized SCD patients bound IgM/IgG to, or induced CDC of, TKO pRBCs. In the in vivo study, survival of TKO pRBCs in the two capuchin monkeys was of 5 and 7 days, respectively, whereas after allotransfusion, survival was >28 days. Conclusions In conclusion, (1) in the present limited study, no antibodies were detected that cross-reacted with TKO pRBCs, and (2) TKO pigs may possibly be an alternate source of RBCs in an emergency if no human RBCs are available.

We have seen hydronephrosis (obstructive nephropathy) at necropsy in 3 of 11 (21%) genetically-engineered pig kidneys that functioned in baboons for36 days, even when the clinical and histopathological features of rejection were minimal. We briefly report one such case and illustrate the macroscopic and microscopic appearances of such a kidney and ureter. The causes of the observed changes remain uncertain. In our small experience, there seems to be no correlation between the development of hydronephrosis and (i) the surgical technique, (ii) the genotype of the pig, (iii) the length of the pig ureter, or (iv) the immunosuppressive and anti-inflammatory therapy administered. We suggest that the distal ureteric thickening may be the result of an inflammatory response. In two cases, we resolved the problem by carrying out a secondary side-to-side anastomosis between the proximal pig ureter and the baboon bladder.

We have been testing genetically engineered (GE) pig hearts and optimizing immunosuppression (IS) in non-human primates (NHPs) since 2005. We demonstrate how we translated this preclinical investigation into a US Food and Drug Administration (FDA)-approved clinical cardiac xenotransplantation. First, genetically engineered (GE) pig hearts were transplanted into the abdomen of NHP along with IS, which included anti-CD20 and anti-CD40-based co-stimulation blockade antibodies. We reported 945 days of survival of three gene GE pig hearts in NHPs. Building on this proof-of-concept, we tested 3-10 gene-modified GE pig hearts (in order to improve the immunocompatibility of the xenograft further) in a life-supporting orthotopic model, but had limited success due to perioperative cardiac xenograft dysfunction (PCXD). With novel non-ischemic continuous perfusion preservation (NICP), using the XVIVO Heart solution (XHS), life-supporting survival was extended to 9 months. We approached the FDA under an application for "Expanded Access" (EA), to transplant a GE pig heart in a patient with end-stage non-ischemic cardiomyopathy. He was without other therapeutic options and dependent on VA-ECMO. A team of FDA reviewers reviewed our preclinical research experience and data and allowed us to proceed. This clinical cardiac xenotransplantation was performed, and the patient survived for 60 days, demonstrating the translational preclinical investigation of cardiac xenotransplantation from bench to bedside. The ultimate etiology of graft failure is currently a topic of investigation and lessons learned will progress the field forward.

BACKGROUND Although improving, survival after pig orthotopic heart transplantation (OHTx) in baboons has been mixed and largely poor. The causes for the high incidence of early failure remain uncertain. MATERIAL AND METHODS We have carried out pig OHTx in 4 baboons. Two died or were euthanized within hours, and 2 survived for 3 and 8 months, respectively. There was evidence of a significant 'cytokine storm' in the immediate post-OHTx period with the elevations in IL-6 correlating closely with the final outcome. RESULTS All 4 baboons demonstrated features suggestive of respiratory dysfunction, including increased airway resistance, hypoxia, and tachypnea. Histopathological observations of pulmonary infiltration by neutrophils and, notably, eosinophils within vessels and in the perivascular and peribronchiolar space, with minimal cardiac pathology, suggested a role for early lung acute inflammation. In one, features suggestive of transfusion-related acute lung injury were present. The 2 longer-term survivors died of (i) a cardiac dysrhythmia with cellular infiltration around the conducting tissue (at 3 months), and (ii) mixed cellular and antibody-mediated rejection (at 8 months). CONCLUSIONS These initial findings indicate a potential role of acute lung injury early after OHTx. If this response can be prevented, increased survival may result, providing an opportunity to evaluate the factors affecting long-term survival.

A 57-year-old man with nonischemic cardiomyopathy who was dependent on venoarterial extracorporeal membrane oxygenation (ECMO) and was not a candidate for standard therapeutics, including a traditional allograft, received a heart from a genetically modified pig source animal that had 10 individual gene edits. Immunosuppression was based on CD40 blockade. The patient was weaned from ECMO, and the xenograft functioned normally without apparent rejection. Sudden diastolic thickening and failure of the xenograft occurred on day 49 after transplantation, and life support was withdrawn on day 60. On autopsy, the xenograft was found to be edematous, having nearly doubled in weight. Histologic examination revealed scattered myocyte necrosis, interstitial edema, and red-cell extravasation, without evidence of microvascular thrombosis - findings that were not consistent with typical rejection. Studies are under way to identify the mechanisms responsible for these changes. (Funded by the University of Maryland Medical Center and School of Medicine.).

BACKGROUND Natural preformed anti-pig IgM/IgG antibodies in primates play an important role in xenograft rejection. As it is not clear how IgE and IgA engage in the immune system in xenotransplantation, we investigated natural preformed and elicited anti-pig IgE/IgA in naive primates and after xenotransplantation in nonhuman primates. METHODS The binding of IgM/IgG/IgE/IgA antibodies to red blood cells (RBCs) from wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and GTKO/cytidine monophospho-N-acetylneuraminic acid hydroxylase gene-knockout/β-1,4 N-acetylgalactosaminyltransferase 2 gene-knockout (ie, triple-knockout pigs) pigs were measured by flow cytometry in naive human (n = 50) and baboon (n = 14) sera. Antibody binding to WT and GTKO pig RBCs (pRBCs) was also measured in the sera of baboons (nonsensitized n = 7, sensitized n = 2) and rhesus monkeys (nonsensitized n = 2, sensitized n = 11) following WT or GTKO pig organ/tissue xenotransplantation. Deposition of IgM/IgG/IgE/IgA in the grafts was detected by immunohistochemistry. RESULTS The majority of humans had natural preformed IgM/IgG/IgE/IgA to WT and GTKO pRBCs. In contrast, IgM/IgG/IgE/IgA to triple-knockout pRBCs were present at lower levels and frequency (P < 0.01). Baboons also had IgM/IgG/IgE/IgA antibodies against WT pRBCs, but fewer to GTKO and triple-knockout (P < 0.01). After xenotransplantation into nonhuman primates, when IgM/IgG increased, IgE/IgA also increased, but to a lesser extent. In addition to IgM/IgG, IgE or IgA deposition was observed in rejected pig xenografts. CONCLUSIONS Primates develop serum anti-pig IgE/IgA antibodies both naturally and during xenograft rejection. The pathophysiological role, if any, of anti-pig IgE/IgA antibodies remains unknown.

Xenografts from genetically modified pigs have become one of the most promising solutions to the dearth of human organs available for transplantation. The challenge in this model has been hyperacute rejection. To avoid this, pigs have been bred with a knockout of the alpha-1,3-galactosyltransferase gene and with subcapsular autologous thymic tissue.We transplanted kidneys from these genetically modified pigs into two brain-dead human recipients whose circulatory and respiratory activity was maintained on ventilators for the duration of the study. We performed serial biopsies and monitored the urine output and kinetic estimated glomerular filtration rate (eGFR) to assess renal function and xenograft rejection.The xenograft in both recipients began to make urine within moments after reperfusion. Over the 54-hour study, the kinetic eGFR increased from 23 ml per minute per 1.73 mGenetically modified kidney xenografts from pigs remained viable and functioning in brain-dead human recipients for 54 hours, without signs of hyperacute rejection. (Funded by Lung Biotechnology.).

We report orthotopic (life-supporting) survival of genetically engineered porcine cardiac xenografts (with six gene modifications) for almost 9 months in baboon recipients. This work builds on our previously reported heterotopic cardiac xenograft (three gene modifications) survival up to 945 days with an anti-CD40 monoclonal antibody-based immunosuppression. In this current study, life-supporting xenografts containing multiple human complement regulatory, thromboregulatory, and anti-inflammatory proteins, in addition to growth hormone receptor knockout (KO) and carbohydrate antigen KOs, were transplanted in the baboons. Selective "multi-gene" xenografts demonstrate survival greater than 8 months without the requirement of adjunctive medications and without evidence of abnormal xenograft thickness or rejection. These data demonstrate that selective "multi-gene" modifications improve cardiac xenograft survival significantly and may be foundational for paving the way to bridge transplantation in humans.

INTRODUCTION: Platelet sequestration, inflammation, and inappropriate coagulation cascade activation are prominent in liver xenotransplant models and are associated with poor outcomes. Here, we evaluate a cassette of six additional genetic modifications to reduce anti-pig antibody binding (α-1,3-galactosyl transferase knockout [GalTKO]) and target coagulation dysregulation (human endothelial protein C receptor [hEPRC] and thrombomodulin [hTBM]), complement pathway regulation (human membrane cofactor protein, hCD46), inflammation heme oxygenase 1 [hHO-1]), and a self-recognition receptor (integrin-associated protein [hCD47]), as well as donor pharmacologic treatments designed to blunt these phenomena. METHODS: Livers from GaltKO.hCD46 pigs (“2-gene,” n = 3) and GalTKO.hCD46 pigs also transgenic for hEPRC, hTBM, hCD47, and hHO-1 (“6-gene,” n = 4) were perfused ex vivo with whole human blood. Six-gene pigs were additionally pretreated with desmopressin (DDAVP) and clodronate liposomes to deplete vWF and kupffer cells, respectively. RESULTS: The average perfusion times increased from 304 (±148) min in the 2-gene group to 856 (±61) min in the 6-gene group (p= .010). The average heparin administration was decreased from 8837 U/h in the 2-gene to 1354 U/h in the 6-gene group (p= .047). Platelet sequestration tended to be delayed in the 6-gene group (p= .070), while thromboxane B2 (TXB2, a platelet activation marker) levels were lower over the first hour (p= .044) (401 ± 124 vs. 2048 ± 712 at 60 min). Thrombin production as measured by F1+2 levels tended to be lower in the 6-gene group (p= .058). CONCLUSIONS: The combination of the hEPCR.hTBM.hCD47.hHO-1 cassette along with donor pig DDAVP and clodronate liposome pretreatment was associated with prolonged function of xenoperfused livers, reduced coagulation pathway perturbations, and decreased TXB2 elaboration, and reflects significant progress to modulate liver xenograft injury in a pig to human model.

Post-transplantation cardiac xenograft growth in an orthotopic pig to baboon model is a life-limiting phenomenon that is poorly understood. Possible causes of growth include both intrinsic and extrinsic etiologies. Extrinsic causes are thought to be attributed to maladaptive hypertrophy as a result of increased mean arterial pressure experienced by the cardiac xenograft after transplantation. Intrinsic causes are thought to be a result of discordant growth between pig xenografts and recipients. This results in intrinsic xenograft growth that parallels the donor and continues in a recipient in which growth is relatively minimal, controlled in part by the growth hormone receptor, IGF-1 axis. Recently, Zaman, et al. published a study titled, "Selective loss of resident macrophage-derived insulin-like growth factor-1 abolishes adaptive cardiac growth to stress," in Immunity, Volume 54; Issue 9, pp. 2057-2071. They demonstrated that insulin growth factor-secreting resident macrophages that sense hypertensive stress are a mechanistic link to hypertension and maladaptive hypertrophy in the setting of hypertension. While notable in its own right, we comment on how this work may shed light on a new underlying mechanism for the use of growth hormone receptor knockout (GHRKO) pig donors and its role in addressing post-transplantation xenograft growth. We hypothesize that GHRKO pig donors contain syngeneic resident cardiac macrophages that abrogate IGF-1 mediated maladaptive hypertrophy from hypertension. Futures studies in post-transplantation cardiac xenotransplantation growth should examine this mechanism as a potential contributor.

Congestion, granular platelet debris both within macrophage and extracellularly, and neutrophil infiltration in the spleen of a baboon that was euthanized with profound thrombocytopenia.

A radical solution is needed for the organ supply crisis, and the domestic pig is a promising organ source. In preparation for a clinical trial of xenotransplantation, we developed an in vivo pre-clinical human model to test safety and feasibility tenets established in animal models. After performance of a novel, prospective compatible crossmatch, we performed bilateral native nephrectomies in a human brain-dead decedent and subsequently transplanted two kidneys from a pig genetically engineered for human xenotransplantation. The decedent was hemodynamically stable through reperfusion, and vascular integrity was maintained despite the exposure of the xenografts to human blood pressure. No hyperacute rejection was observed, and the kidneys remained viable until termination 74 h later. No chimerism or transmission of porcine retroviruses was detected. Longitudinal biopsies revealed thrombotic microangiopathy that did not progress in severity, without evidence of cellular rejection or deposition of antibody or complement proteins. Although the xenografts produced variable amounts of urine, creatinine clearance did not recover. Whether renal recovery was impacted by the milieu of brain death and/or microvascular injury remains unknown. In summary, our study suggests that major barriers to human xenotransplantation have been surmounted and identifies where new knowledge is needed to optimize xenotransplantation outcomes in humans.

There are 5.7 million people in the United States with heart failure, which is life-limiting in 20% of patients.1 While data is most robust in the United States for this cohort, it is known to be a global problem with over 23 million people carrying the diagnosis.1 For end-stage heart failure, many require a heart transplantation, however, there is a shortage in the supply of organ donors. Cardiac xenotransplantation has been proposed to “bridge the gap” in supply for these patients requiring transplantation. Recent pre-clinical success using genetically modified pig donors in baboon recipients has demonstrated survival greater than 6 months.2–5 First-in-human transplantation of a genetically modified pig kidney demonstrated 54 hour rejection-free function when perfused by a deceased human recipient, demonstrating the feasibility of cross-species transplantation and invigorating enthusiasm further to utilize this new organ source for a population that would otherwise die waiting for a human organ.6 While this human study demonstrated proof-of-principle of overcoming hyperacute rejection, further regulatory oversight by Food and Drug Administration (FDA) may be required with pre-clinical trials in large animal models of xenotransplantation with long-term survival. These studies not only require a multi-disciplinary team and expertise in orthotopic transplantation (cardiac surgery, anesthesia and cardiopulmonary bypass), immunology and genetic engineering; but also, specifically handling large animal recipients that cannot communicate their symptoms. Here we detail our approach to pig-to-primate large animal model of orthotopic cardiac xenotransplantation perioperatively and in the months thereafter in long-term surviving animals. We also detail xenograft surveillance methods and common issues that arise in the postoperative period specific to this model and ways to overcome them.

Survival of pig cardiac xenografts in a non-human primate (NHP) model has improved significantly over the last 4 years with the introduction of costimulation blockade based immunosuppression (IS) and genetically engineered (GE) pig donors. The longest survival of a cardiac xenograft in the heterotopic (HHTx) position was almost 3 years and only rejected when IS was stopped. Recent reports of cardiac xenograft survival in a life-sustaining orthotopic (OHTx) position for 6 months is a significant step forward. Despite these achievements, there are still several barriers to the clinical success of xenotransplantation (XTx). This includes the possible transmission of porcine pathogens with pig donors and continued xenograft growth after XTx. Both these concerns, and issues with additional incompatibilities, have been addressed recently with the genetic modification of pigs. This review discusses the spectrum of issues related to cardiac xenotransplantation, recent progress in preclinical models, and its feasibility for clinical translation.

The transplantation of organs across species offers the potential to solve the shortage of human organs. While activation of human platelets by human von Willebrand factor (vWF) requires vWF activation by shear stress, contact between human platelets and porcine vWF (pvWF) leads to spontaneous platelet adhesion and activation. This non-physiologic interaction may contribute to the thrombocytopenia and coagulation pathway dysregulation often associated with xenotransplantation of pig organs in nonhuman primates. Pigs genetically modified to decrease antibody and complement-dependent rejection (GTKO.hCD46) were engineered to express humanized pvWF (h*pvWF) by replacing a pvWF gene region that encodes the glycoprotein Ib-binding site with human cDNA orthologs. This modification corrected for non-physiologic human platelet aggregation on exposure to pig plasma, while preserving in vitro platelet activation by collagen. Organs from pigs with h*pvWF demonstrated reduced platelet sequestration during lung (p ≤ .01) and liver (p ≤ .038 within 4 h) perfusion ex vivo with human blood and after pig-to-baboon lung transplantation (p ≤ .007). Residual platelet sequestration and activation were not prevented by the blockade of canonical platelet adhesion pathways. The h*pvWF modification prevents physiologically inappropriate activation of human or baboon platelets by porcine vWF, addressing one cause of the thrombocytopenia and platelet activation observed with xenotransplantation.

Existing strategies for repair of major peripheral nerve injury (PNI) are inefficient at promoting axon regeneration and functional recovery and are generally ineffective for nerve lesions >5 cm. To address this need, we have previously developed tissue engineered nerve grafts (TENGs) through the process of axon stretch growth. TENGs consist of living, centimeter-scale, aligned axon tracts that accelerate axon regeneration at rates equivalent to the gold standard autograft in small and large animal models of PNI, by providing a newfound mechanism-of-action referred to as axon-facilitated axon regeneration (AFAR). To enable clinical-grade biomanufacturing of TENGs, a suitable cell source that is hypoimmunogenic, exhibits low batch-to-batch variability, and able to tolerate axon stretch growth must be utilized. To fulfill these requirements, a genetically engineered, FDA-approved, xenogeneic cell source, GalSafe(®) neurons, produced by Revivicor, Inc., have been selected to advance TENG biofabrication for eventual clinical use. To this end, sensory and motor neurons were harvested from genetically engineered GalSafe day 40 swine embryos, cultured in custom mechanobioreactors, and axon tracts were successfully stretch-grown to 5 cm within 25 days. Importantly, both sensory and motor GalSafe neurons were observed to tolerate established axon stretch growth regimes of ≥1 mm/day to produce continuous, healthy axon tracts spanning 1, 3, or 5 cm. Once stretch-grown, 1 cm GalSafe TENGs were transplanted into a 1 cm lesion in the sciatic nerve of athymic rats. Regeneration was assessed through histological measures at the terminal time point of 2 and 8 weeks. Neurons from GalSafe TENGs survived and elicited AFAR as observed when using wild-type TENGs. At 8 weeks postrepair, myelinated regenerated axons were observed in the nerve section distal to the injury site, confirming axon regeneration across the lesion. These experiments are the first to demonstrate successful harvest and axon stretch growth of GalSafe neurons for use as starting biomass for bioengineered nerve grafts as well as initial safety and efficacy in an established preclinical model—important steps for the advancement of clinical-grade TENGs for future regulatory testing and eventual clinical trials. IMPACT STATEMENT: Biofabrication of tissue engineered medical products requires several steps, one of which is choosing a suitable starting biomass. To this end, we have shown that the clinical-grade, genetically engineered biomass—GalSafe(®) neurons—is a viable option for biomanufacturing of our tissue engineered nerve grafts (TENGs) to promote regeneration following major peripheral nerve injury. Importantly, this is a first step in clinical-grade TENG biofabrication, proving that GalSafe TENGs recapitulate the mechanism of axon-facilitated axon regeneration seen previously with research-grade TENGs.

The aims of this study were to evaluate the efficacy of US Food and Drug Administration-approved drugs in genetically engineered pig-to-baboon kidney xenotransplantation and compare the results with those using an anti-CD40 monoclonal antibody (mAb)-based regimen.Ten life-supporting kidney transplants were carried out in baboons using α1,3-galactosyltransferase gene-knockout/CD46 pigs with various other genetic manipulations aimed at controlling coagulation dysregulation. Eight transplants resulted in informative data. Immunosuppressive therapy consisted of induction with antithymocyte globulin and anti-CD20mAb, and maintenance based on either (1) CTLA4-Ig and/or tacrolimus (+rapamycin or mycophenolate mofetil) (GroupA [US Food and Drug Administration-approved regimens], n = 4) or (2) anti-CD40mAb + rapamycin (GroupB, n = 4). All baboons received corticosteroids, interleukin-6R blockade, and tumor necrosis factor-α blockade. Baboons were followed by clinical and laboratory monitoring of kidney function, coagulation, and immune parameters. At euthanasia, morphological and immunohistochemical studies were performed on the kidney grafts.The median survival in GroupB was 186 days (range 90-260), which was significantly longer than in GroupA; median 14 days (range 12-32) (P0.01). Only GroupA baboons developed consumptive coagulopathy and the histopathological features of thrombotic microangiopathic glomerulopathy and interstitial arterial vasculitis.Recognizing that the pig donors in each group differed in some genetic modifications, these data indicate that maintenance immunosuppression including anti-CD40mAb may be important to prevent pig kidney graft failure.

PURPOSE OF REVIEW: To review the impact of a new technology, 3D-bioprinting, in xenotransplantation research. RECENT FINDINGS: Genetically-engineered pigs, beginning with human (h) CD55-transgenic and Gal-knockout pigs, have improved the outcomes of xenotransplantation research. Today, there are >30 different genetically-engineered pigs either expressing human gene(s) or lacking pig gene(s). CRIPSR/cas9 technology has facilitated the production of multigene pigs (up to 9-genes in a single pig) which lack multiple pig xenoantigens, and express human transgenes, such as hCD46, hCD55, hThrombomodulin, hCD39, etc. Although recent studies in nonhuman primates (NHPs) have demonstrated prolonged survival after life-supporting pig kidney, heart, and islet xenotransplantation, researchers have difficulty determining the best genetic combination to test in NHPs due to a potential >100,000 genetic combinations. 3D-bioprinting of genetically-engineered pig cells (i) is superior to 2D in vitro testing, (ii) enables organ-specific testing, (iii) helps to understand differences in immunogenicity between organs, and (iv) is faster and cheaper than testing in NHPs. Moreover, 3D-bioprinted cells can be continuously perfused in a bioreactor, controlling for all variables, except the studied variable. SUMMARY: 3D-bioprinting can help in the study of the impact of specific genes (human or pig) in xenotransplantation in a rapid, inexpensive, and reliable way.

INTRODUCTION: Pig heart xenotransplantation might act as a bridge in infants with complex congenital heart disease (CHD) until a deceased human donor heart becomes available. Infants develop antibodies to wild-type (WT, i.e., genetically-unmodified) pig cells, but rarely to cells in which expression of the 3 known carbohydrate xenoantigens has been deleted by genetic engineering (triple-knockout [TKO] pigs). Our objective was to test sera from children who had undergone palliative surgery for complex CHD (and who potentially might need a pig heart transplant) to determine whether they had serum cytotoxic antibodies against TKO pig cells. METHODS: Sera were obtained from children with CHD undergoing Glenn or Fontan operation (n = 14) and healthy adults (n = 8, as controls). All of the children had complex CHD and had undergone some form of cardiac surgery. Seven had received human blood transfusions and 3 bovine pericardial patch grafts. IgM and IgG binding to WT and TKO pig red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) were measured by flow cytometry, and killing of PBMCs by a complement-dependent cytotoxicity assay. RESULTS: Almost all children and adults demonstrated relatively high IgM/IgG binding to WT RBCs, but minimal binding to TKO RBCs (p < 0.0001 vs WT), although IgG binding was greater in children than adults (p < 0.01). All sera showed IgM/IgG binding to WT PBMCs, but this was much lower to TKO PBMCs (p < 0.0001 vs WT) and was greater in children than in adults (p < 0.05). Binding to both WT and TKO PBMCs was greater than to RBCs. Mean serum cytotoxicity to WT PBMCs was 90% in both children and adults, whereas to TKO PBMCs it was only 20% and < 5%, respectively. The sera from 6/14 (43%) children were cytotoxic to TKO PBMCs, but no adult sera were cytotoxic. CONCLUSIONS: Although no children had high levels of antibodies to TKO RBCs, 13/14 demonstrated antibodies to TKO PBMCs, in 6 of these showed mild cytotoxicity. As no adults had cytotoxic antibodies to TKO PBMCs, the higher incidence in children may possibly be associated with their exposure to previous cardiac surgery and biological products. However, the numbers were too small to determine the influence of such past exposures. Before considering pig heart xenotransplantation for children with CHD, testing for antibody binding may be warranted

The current evidence is that sensitization to a pig xenograft does not result in the development of antibodies that cross-react with alloantigens, and therefore, sensitization to a pig xenograft would not be detrimental to the outcome of a subsequent allograft. This evidence relates almost entirely to the transplantation of cells or organs from wild-type or α1,3-galactosyltransferase gene-knockout (GTKO) pigs. However, it is not known whether recipients of triple-knockout (TKO) pig grafts who become sensitized to TKO pig antigens develop antibodies that cross-react with alloantigens and thus be detrimental to a subsequent organ allotransplant. We identified a single baboon (B1317) in which no (or minimal) serum anti-TKO pig antibodies could be measured-in our experience unique among baboons. We sensitized it by repeated subcutaneous injections of TKO pig peripheral blood mononuclear cells (PBMCs) in the absence of any immunosuppressive therapy. After TKO pig PBMC injection, there was a transient increase in anti-TKO pig IgM, followed by a sustained increase in IgG binding to TKO cells. In contrast, there was no serum IgM or IgG binding to PBMCs from any of a panel of baboon PBMCs (n = 8). We conclude that sensitization to TKO pig PBMCs in the baboon did not result in the development of antibodies that also bound to baboon cells, suggesting that there would be no detrimental effect of sensitization on a subsequent organ allotransplant.

Pigs deficient in three glycosyltransferase enzymes (triple-knockout [TKO] pigs) and expressing "protective" human transgenes are likely sources of organs for transplantation into human recipients. Testing of human sera against red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) from TKO pigs has revealed minimal evidence of natural antibody binding. However, unlike humans, baboons exhibit natural antibody binding to TKO pig cells. The xenoantigen specificities of these natural antibodies are postulated to be one or more carbohydrate moieties exposed when N-glycolylneuraminic acid (Neu5Gc) is deleted. The aim of this study was to compare the survival of renal grafts in baboons from pigs that either expressed Neu5Gc (GTKO pigs; Group1, n = 5) or did not express Neu5Gc (GTKO/CMAHKO [DKO] or TKO pigs; Group2, n = 5). An anti-CD40mAb-based immunosuppressive regimen was administered in both groups. Group1 kidneys functioned for 90-260 days (median 237, mean 196 days), with histopathological features of antibody-mediated rejection in two kidneys. Group2 kidneys functioned for 0-183 days (median 35, mean 57), with all of the grafts exhibiting histologic features of antibody-mediated rejection. These findings suggest that the absence of expression of Neu5Gc on pig kidneys impacts graft survival in baboon recipients.

We report orthotopic (life-supporting) survival of genetically engineered porcine cardiac xenografts (with 3-9 progressive gene modifications) for almost 9 months in baboon recipients. This work builds on our previously reported heterotopic cardiac xenograft (3 gene modifications) survival up to 945 days with an anti-CD40 monoclonal antibody-based immunosuppression. In this current study, life-supporting xenografts containing multiple human complement regulatory, thromboregulatory, and anti-inflammatory proteins, in addition to growth hormone receptor knockout (KO) and carbohydrate antigen KOs, were transplanted. Selective "multi-gene" xenografts demonstrate survival greater than 8 months without the use of adjunctive medications and without evidence of abnormal xenograft thickness or rejection. These data demonstrate that selective “multi-gene" modifications improve cardiac xenograft survival significantly and may be foundational for paving the way to bridge transplantation in humans.