Cardiac Xenografts Research Articles

327.7: Multi-omics profiling in human decedents receiving genetically modified pig hearts reveals hallmarks of perioperative cardiac xenograft dysfunction.

327.7: Multi-omics profiling in human decedents receiving genetically modified pig hearts reveals hallmarks of perioperative cardiac xenograft dysfunction.

Anti‐pig antibodies in swine veterinarian serum: Implications for clinical xenotransplantation

AbstractRecent clinical xenotransplantation and human decedent studies demonstrate that clinical hyperacute rejection of genetically engineered porcine organs can be reliably avoided but that antibody mediated rejection (AMR) continues to limit graft survival. We previously identified porcine glycans and proteins which are immunogenic after cardiac xenotransplantation in non‐human primates, but the clinical immune response to antigens present in glycan depleted triple knockout (TKO) donor pigs is poorly understood. In this study we use fluorescence barcoded human embryonic kidney cells (HEK) and HEK cell lines expressing porcine glycans (Gal and SDa) or proteins (tetraspanin‐29 [CD9], membrane cofactor protein [CD46], protectin, membrane attack complex inhibition factor [CD59], endothelial cell protein C receptor, and Annexin A2) to screen antibody reactivity in human serum from 160 swine veterinarians, a serum source with potential occupational immune challenge from porcine tissues and pathogens. High levels of anti‐Gal IgM were present in all samples and lower levels of anti‐SDa IgM were present in 41% of samples. IgM binding to porcine proteins, primarily CD9 and CD46, previously identified as immunogenic in pig to non‐human primate cardiac xenograft recipients, was detected in 28 of the 160 swine veterinarian samples. These results suggest that barcoded HEK cell lines expressing porcine protein antigens can be useful for screening human patient serum. A comprehensive analysis of sera from clinical xenotransplant recipients to define a panel of commonly immunogenic porcine antigens will likely be necessary to establish an array of porcine non‐Gal antigens for effective monitoring of patient immune responses and allow earlier therapies to reverse AMR.

Integrative multi-omics profiling in human decedents receiving pig heart xenografts.

In a previous study, heart xenografts from 10-gene-edited pigs transplanted into two human decedents did not show evidence of acute-onset cellular- or antibody-mediated rejection. Here, to better understand the detailed molecular landscape following xenotransplantation, we carried out bulk and single-cell transcriptomics, lipidomics, proteomics and metabolomics on blood samples obtained from the transplanted decedents every 6 h, as well as histological and transcriptomic tissue profiling. We observed substantial early immune responses in peripheral blood mononuclear cells and xenograft tissue obtained from decedent 1 (male), associated with downstream T cell and natural killer cell activity. Longitudinal analyses indicated the presence of ischemia reperfusion injury, exacerbated by inadequate immunosuppression of T cells, consistent with previous findings of perioperative cardiac xenograft dysfunction in pig-to-nonhuman primate studies. Moreover, at 42 h after transplantation, substantial alterations in cellular metabolism and liver-damage pathways occurred, correlating with profound organ-wide physiological dysfunction. By contrast, relatively minor changes in RNA, protein, lipid and metabolism profiles were observed in decedent 2 (female) as compared to decedent 1. Overall, these multi-omics analyses delineate distinct responses to cardiac xenotransplantation in the two human decedents and reveal new insights into early molecular and immune responses after xenotransplantation. These findings may aid in the development of targeted therapeutic approaches to limit ischemia reperfusion injury-related phenotypes and improve outcomes.

(87) - Integrative Multi-Omics Profiling in Human Decedents Receiving Genetically Modified Pig Hearts Reveals Early Immune-Cell Responses Indicative of Perioperative Cardiac Xenograft Dysfunction

(87) - Integrative Multi-Omics Profiling in Human Decedents Receiving Genetically Modified Pig Hearts Reveals Early Immune-Cell Responses Indicative of Perioperative Cardiac Xenograft Dysfunction

Hemodynamics in pig-to-baboon heterotopic thoracic cardiac xenotransplantation: Recovery from perioperative cardiac xenograft dysfunction and impairment by cardiac overgrowth.

Orthotopic cardiac xenotransplantation has seen notable improvement, leading to the first compassionate use in 2022. However, it remains challenging to define the clinical application of cardiac xenotransplantation, including the back-up strategy in case of xenograft failure. In this regard, the heterotopic thoracic technique could be an alternative to the orthotopic procedure. We present hemodynamic data of heterotopic thoracic pig-to-baboon transplantation experiments, focusing on perioperative xenograft dysfunction and xenograft overgrowth. We used 17 genetically modified piglets as donors for heterotopic thoracic xenogeneic cardiac transplantation into captive-bred baboons. In all animals, pressure probes were implanted in the graft's left ventricle and the recipient's ascending aorta and hemodynamic data (graft pressure, aortic pressure and recipient's heart rate) were recorded continuously. Aortic pressures and heart rates of the recipients' hearts were postoperatively stable in all experiments. After reperfusion, three grafts presented with low left ventricular pressure indicating perioperative cardiac dysfunction (PCXD). These animals recovered from PCXD within 48h under support of the recipient's heart and there was no difference in survival compared to the other 14 ones. After 48h, graft pressure increased up to 200mmHg in all 17 animals with two different time-patterns. This led to a progressive gradient between graft and aortic pressure. With increasing gradient, the grafts stopped contributing to cardiac output. Grafts showed a marked weight increase from implantation to explantation. The heterotopic thoracic cardiac xenotransplantation technique is a possible method to overcome PCXD in early clinical trials and an experimental tool to get a better understanding of PCXD. The peculiar hemodynamic situation of increasing graft pressure but missing graft's output indicates outflow tract obstruction due to cardiac overgrowth. The heterotopic thoracic technique should be successful when using current strategies of immunosuppression, organ preservation and donor pigs with smaller body and organ size.

Applying integrative multiomic profiling in two human decedents receiving pig heart xenografts reveals early immune cell responses indicative of perioperative cardiac xenograft dysfunction

Applying integrative multiomic profiling in two human decedents receiving pig heart xenografts reveals early immune cell responses indicative of perioperative cardiac xenograft dysfunction

107.3: Recovery from perioperative cardiac xenograft dysfunction and consequences of cardiac overgrowth in pig-to-baboon heterotopic thoracic cardiac xenotransplantation

107.3: Recovery from perioperative cardiac xenograft dysfunction and consequences of cardiac overgrowth in pig-to-baboon heterotopic thoracic cardiac xenotransplantation

311.8: Preservation of cardiac xenografts in a model of infant human cardiac xenotransplantation

311.8: Preservation of cardiac xenografts in a model of infant human cardiac xenotransplantation

A Standardized Approach to Orthotopic (Life-supporting) Porcine Cardiac Xenotransplantation in a Nonhuman Primate Model.

Cardiac xenotransplantation from swine has been proposed to "bridge the gap" in supply for heart failure patients requiring transplantation. Recent preclinical success using genetically modified pig donors in baboon recipients has demonstrated survival greater than 6 mo, with a modern understanding of xenotransplantation immunobiology and continued experience with large animal models of cardiac xenotransplantation. As a direct result of this expertise, the Food and Drug Administration approved the first in-human transplantation of a genetically engineered cardiac xenograft through an expanded access application for a single patient. This clinical case demonstrated the feasibility of xenotransplantation. Although this human study demonstrated proof-of-principle application of cardiac xenotransplantation, further regulatory oversight by the Food and Drug Administration may be required with preclinical trials in large animal models of xenotransplantation with long-term survival before approval of a more formalized clinical trial. Here we detail our surgical approach to pig-to-primate large animal models of orthotopic cardiac xenotransplantation, and the postoperative care of the primate recipient, both in the immediate postoperative period and in the months thereafter. We also detail xenograft surveillance methods and common issues that arise in the postoperative period specific to this model and ways to overcome them. These studies require multidisciplinary teams and expertise in orthotopic transplantation (cardiac surgery, anesthesia, and cardiopulmonary bypass), immunology, genetic engineering, and experience in handling large animal donors and recipients, which are described here. This article serves to reduce the barriers to entry into a field with ever-growing enthusiasm, but demands expertise knowledge and experience to be successful.

Accommodation in allogeneic and xenogeneic organ transplantation: Prevalence, impact, and implications for monitoring and for therapeutics

Accommodation refers to acquired resistance of organs or tissues to immune or inflammatory reactions that might otherwise cause severe injury or rejection. As first observed in ABO-incompatible kidney transplants and heterotopic cardiac xenografts, accommodation was identified when organ transplants continued to function despite the presence of anti-graft antibodies and/or other reactants in the blood of recipients. Recent evidence suggests many and perhaps most organ transplants have accommodation, as most recipients mount B cell responses specific for the graft. Wide interest in the impact of graft-specific antibodies on the outcomes of transplants prompts questions about which mechanisms confer protection against such antibodies, how accommodation might be detected and whether and how rejection could be superimposed on accommodation. Xenotransplantation offers a unique opportunity to address these questions because immune responses to xenografts are easily detected and the pathogenic impact of immune responses is so severe. Xenotransplantation also provides a compelling need to apply these and other insights to decrease the intensity and toxicity of immunosuppression that otherwise could limit clinical application.

415.4: Ischemia Minimization Improves Cardiac Function in an Ex Vivo Xeno Working Heart Model

Introduction: Previous studies showed that ischemia minimization (IM), accomplished by perfusing the heart xenograft during storage, prevents ‘initial cardiac xenograft dysfunction’ (ICXD) and enables prolonged survival following orthotopic cardiac xenotransplantation. Here we report initial observations in a model designed to evaluate effects on heart performance associated with IM and genetic modifications targeted to address xeno-injury mechanisms in a working ex vivo model of ICXD. Method: Hearts from genetically modified and wildtype (WT) pigs were procured after flushing with cold preservation solution (UW, 4oC) and stored for 3 hours either in cold saline (0.9%, 4oC: cold storage (CS)) or were perfused with oxygenated STEEN Solution with RBCs (IM). IM perfusion at 40 mmHg was initiated at room temperature for 20 minutes to facilitate homogeneous graft perfusion before cooling to 4oC for the remainder of the storage period. The genetically modified hearts either had combined knockouts of three specific xenogeneic carbohydrate genes (GTKO, CMAHKO, b4GALNT2KO: TKO) with variable expression of human transgenes (hTG) including complement and thrombo-regulatory proteins (n=29); or GTKO with additional expression of hCD55 (GTKO.hCD55; n=3). Heart function and laboratory parameters were assessed at specific timepoints on a working heart rig while perfused with freshly collected heparinized whole human blood. Troponin I was used as a marker for myocardial injury. Results: In total, 41 hearts were perfused ex vivo, 23 after CS (TKO + hTG n=14, GTKO.hCD55 n=2, WT n=7), and 18 after IM (TKO + hTG n=15, GTKO.hCD55 n=1, WT n=2). Cardiac output (mL/min) was significantly improved for the IM group at all loading conditions relative to CS, (see figure 1). There was a strong trend toward reduced troponin I release, particularly within the first 60 minutes of perfusion (see figure 2). Conclusion: IM is protective with respect to cardiac function following pig heart ischemia and subsequent reperfusion with human blood in an ex vivo model of heart xenotransplantation. The hypothesis that variation in physiologic and biochemical outcomes within groups is influenced by variable expression of complement and coagulation pathway regulatory genes is being investigated.

The road to the first FDA-approved genetically engineered pig heart transplantation into human.

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.

Preclinical rationale and current pathways to support the first human clinical trials in cardiac xenotransplantation

Recent initiation of the first FDA-approved cardiac xenotransplantation suggests xenotransplantation could soon become a therapeutic option for patients unable to undergo allotransplantation. Until xenotransplantation is widely applied in clinical practice, consideration of benefit versus risk and approaches to management of clinical xenografts will based at least in part on observations made in experimental xenotransplantation in non-human primates. Indeed, the decision to proceed with clinical trials reflects significant progress in last few years in experimental solid organ and cellular xenotransplantation. Our laboratory at the NIH and now at University of Maryland contributed to this progress, with heterotopic cardiac xenografts surviving more than two years and life-supporting cardiac xenografts survival up to 9 months. Here we describe our contributions to the understanding of the mechanism of cardiac xenograft rejection and development of methods to overcome past hurdles, and finally we share our opinion on the remaining barriers to clinical translation. We also discuss how the first in human xenotransplants might be performed, recipients managed, and graft function monitored.

Significant Reduction of Myocardial Injury Markers After Ischemia Minimization in the First Hour of an Ex Vivo Xeno Working Heart Model

<h3>Purpose</h3> Previous studies showed ischemia minimization (IM), accomplished by perfusing the heart xenograft during storage, prevented ‘initial cardiac xenograft dysfunction' (ICXD) and prolonged survival following <i>in vivo</i> orthotopic cardiac xenotransplantation. We report initial observations in a model designed to evaluate effects associated with IM and genetic modifications on heart performance in a working <i>ex vivo</i> model. <h3>Methods</h3> Hearts from genetically modified (gm) and wildtype (WT) pigs were procured and stored for 3 hours either in cold saline (cold storage (CS)) or were perfused with oxygenated Steen's solution with RBCs (IM). The gm hearts either had combined knockouts of 3 xenogenic carbohydrate genes (GTKO, CMAHKO, β4GALNT2KO: TKO) with variable expression of human transgenes (n=13); or GTKO with expression of hCD55 (GTKO.hCD55; n=2). Heart function and troponin I as a marker for myocardial injury were assessed on a working heart rig while perfused with whole human blood. <h3>Results</h3> 19 hearts were perfused <i>ex vivo</i>, ten with CS (TKO n=6, GTKO.hCD55 n=1, WT n=3), 9 with IM (TKO n=7, GTKO.hCD55 n=1, WT n=1). Mean troponin I levels were significantly reduced after 1 hour of <i>ex vivo</i> perfusion in the IM group (70.2 ng/mL vs. 279.5 ng/mL; p = .038), but not at final time points (see fig. 1a-c). Cardiac function (cardiac output in response to increased filling pressures) decreased over time after CS, whereas the IM-stored heart function tended to improve over time (Fig. 2). <h3>Conclusion</h3> IM significantly decreased myocardial injury during the first hour of <i>ex vivo</i> working heart perfusion relative to CS hearts: heart injury (troponin release) was attenuated, and graft failure was delayed in some TKO hearts treated with IM. Evaluation of whether variation in expression of human transgenes may have influenced TKO graft protection from ICXD is in progress. We provisionally conclude that IM attenuates ICXD of genetically modified pig hearts during initial exposure to human blood.

Progressive genetic modifications of porcine cardiac xenografts extend survival to 9 months.

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.

Cardiac Xenotransplantation: Progress in Preclinical Models and Prospects for Clinical Translation.

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.

An intrinsic link to an extrinsic cause of cardiac xenograft growth after xenotransplantation

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, etal. 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.

The growth of xenotransplanted hearts can be reduced with growth hormone receptor knockout pig donors

ObjectiveGenetically 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. MethodsGenetically 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. ResultsAll 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. ConclusionsXenografts with GHR knockout show reduced post-transplantation xenograft growth using echocardiography >6 months after transplantation, without the need for other adjuncts.

Blood Cardioplegia Induction, Perfusion Storage and Graft Dysfunction in Cardiac Xenotransplantation.

BackgroundPerioperative cardiac xenograft dysfunction (PCXD) describes a rapidly developing loss of cardiac function after xenotransplantation. PCXD occurs despite genetic modifications to increase compatibility of the heart. We report on the incidence of PCXD using static preservation in ice slush following crystalloid or blood-based cardioplegia versus continuous cold perfusion with XVIVO© heart solution (XHS) based cardioplegia.MethodsBaboons were weight matched to genetically engineered swine heart donors. Cardioplegia volume was 30 cc/kg by donor weight, with del Nido cardioplegia and the addition of 25% by volume of donor whole blood. Continuous perfusion was performed using an XVIVO © Perfusion system with XHS to which baboon RBCs were added.ResultsPCXD was observed in 5/8 that were preserved with crystalloid cardioplegia followed by traditional cold, static storage on ice. By comparison, when blood cardioplegia was used followed by cold, static storage, PCXD occurred in 1/3 hearts and only in 1/5 hearts that were induced with XHS blood cardioplegia followed by continuous perfusion. Survival averaged 17 hours in those with traditional preservation and storage, followed by 11.47 days and 15.03 days using blood cardioplegia and XHS+continuous preservation, respectively. Traditional preservation resulted in more inotropic support and higher average peak serum lactate 14.3±1.7 mmol/L compared to blood cardioplegia 3.6±3.0 mmol/L and continuous perfusion 3.5±1.5 mmol/L.ConclusionBlood cardioplegia induction, alone or followed by XHS perfusion storage, reduced the incidence of PCXD and improved graft function and survival, relative to traditional crystalloid cardioplegia-slush storage alone.

Leflunomide Inhibits rat-to-Mouse Cardiac Xenograft Rejection by Suppressing Adaptive Immune Cell Response and NF-κB Signaling Activation

Xenotransplantation is a potential solution for the severe shortage of human donor organs and tissues. The generation of humanized animal models attenuates strong innate immune responses, such as complement-mediated hyperacute rejection. However, acute vascular rejection and cell mediated rejection remain primary barriers to xenotransplantation, which limits its clinical application. In this study, we systematically investigated the immunosuppressive effect of LEF using a rat-to-mouse heart xenotransplantation model. SD rat xenogeneic hearts were transplanted into C57BL/6 mice, and survived 34.5 days after LEF treatment. In contrast, BALB/c allogeneic hearts were transplanted into C57BL/6 mice, and survived 31 days after LEF treatment. Compared to normal saline treatment, LEF treatment decreased xenoreactive T cells and CD19+ B cells in recipient splenocytes. Most importantly, LEF treatment protected myocardial cells by decreasing xenoreactive T and B cell infiltration, inflammatory gene expression, and IgM deposition in grafts. In vivo assays revealed that LEF treatment eliminated xenoreactive and alloreactive T and B lymphocytes by suppressing the activation of the NF-κB signaling pathway. Taken together, these observations complement the evidence supporting the potential use of LEF in xenotransplantation.