The Young Investigator Committee of the International Xenotransplantation Association-Perspective of advancements in the field in 2023.

The 2023 IXA conference, hosted in San Diego, CA, brimmed with excitement against the backdrop of recent innovations in both the pre-clinical and clinical realms with several first-in-human applications of xenotransplantation. The theme, "Pigs are flying," alluded to the adage that xenotransplantation would only become a clinical reality "when pigs fly," suggesting a day that might never come. The event witnessed significant attendance, with 600 participants-the highest in the history of an IXA-IPITA joint congress. Among the attendees were members of the Food and Drug Administration (FDA), the National Institutes of Health (NIH), and corporate sponsors deeply engaged in the field. We summarize the latest topics from the congress, ranging from the pros/cons of decedent models of xenotransplantation and genetic engineering of porcine heart valves, solid organs, and cells for clinical translation and their regulatory and ethical landscape.

Genetic Engineering of Donor Pig for the First Human Cardiac Xenotransplantation: Combatting Rejection, Coagulopathy, Inflammation, and Excessive Growth.

PURPOSE OF REVIEW: The first successful pig to human cardiac xenotransplantation in January 2022 represented a major step forward in the fields of heart failure, immunology, and applied genetic engineering, using a 10-gene edited (GE) pig. This review summarizes the evolution of preclinical modelling data which informed the use of each of the 10 genes modified in the 10-GE pig: GGTA1, Β4GalNT2, CMAH, CD46, CD55, TBM, EPCR, CD47, HO-1, and growth hormone receptor. RECENT FINDINGS: The translation of the 10-GE pig from preclinical modelling to clinical compassionate xenotransplant use was the culmination of decades of research combating rejection, coagulopathy, inflammation, and excessive xenograft growth. Understanding these 10 genes with a view to their combinatorial effects will be useful in anticipated xenotransplant clinical trials.

Graft dysfunction in compassionate use of genetically engineered pig-to-human cardiac xenotransplantation: a case report.

BACKGROUND: A genetically engineered pig cardiac xenotransplantation was done on Jan 7, 2022, in a non-ambulatory male patient, aged 57 years, with end-stage heart failure, and on veno-arterial extracorporeal membrane oxygenation support, who was ineligible for an allograft. This report details our current understanding of factors important to the xenotransplantation outcome. METHODS: Physiological and biochemical parameters critical for the care of all heart transplant recipients were collected in extensive clinical monitoring in an intensive care unit. To ascertain the cause of xenograft dysfunction, we did extensive immunological and histopathological studies, including electron microscopy and quantification of porcine cytomegalovirus or porcine roseolovirus (PCMV/PRV) in the xenograft, recipient cells, and tissue by DNA PCR and RNA transcription. We performed intravenous immunoglobulin (IVIG) binding to donor cells and single-cell RNA sequencing of peripheral blood mononuclear cells. FINDINGS: After successful xenotransplantation, the graft functioned well on echocardiography and sustained cardiovascular and other organ systems functions until postoperative day 47 when diastolic heart failure occurred. At postoperative day 50, the endomyocardial biopsy revealed damaged capillaries with interstitial oedema, red cell extravasation, rare thrombotic microangiopathy, and complement deposition. Increased anti-pig xenoantibodies, mainly IgG, were detected after IVIG administration for hypogammaglobulinaemia and during the first plasma exchange. Endomyocardial biopsy on postoperative day 56 showed fibrotic changes consistent with progressive myocardial stiffness. Microbial cell-free DNA testing indicated increasing titres of PCMV/PRV cell-free DNA. Post-mortem single-cell RNA sequencing showed overlapping causes. INTERPRETATION: Hyperacute rejection was avoided. We identified potential mediators of the observed endothelial injury. First, widespread endothelial injury indicates antibody-mediated rejection. Second, IVIG bound strongly to donor endothelium, possibly causing immune activation. Finally, reactivation and replication of latent PCMV/PRV in the xenograft possibly initiated a damaging inflammatory response. The findings point to specific measures to improve xenotransplant outcomes in the future. FUNDING: The University of Maryland School of Medicine, and the University of Maryland Medical Center.

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.

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

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.

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.

CD40-CD40L Blockade: Update on Novel Investigational Therapeutics for Transplantation.

Effective immune responses require antigen presentation by major histocompatibility complexes with cognate T-cell receptor and antigen-independent costimulatory signaling for T-cell activation, proliferation, and differentiation. Among several costimulatory signals, CD40-CD40L is of special interest to the transplantation community because it plays a vital role in controlling or regulating humoral and cellular immunity. Blockade of this pathway has demonstrated inhibition of donor-reactive T-cell responses and prolonged the survival of transplanted organs. Several anti-CD154 and anti-CD40 antibodies have been used in the transplantation model and demonstrated the potential of extending allograft and xenograft rejection-free survival. The wide use of anti-CD154 antibodies was hampered because of thromboembolic complications in transplant recipients. These antibodies have been modified to overcome the thromboembolic complications by altering the antibody binding fragment (Fab) and Fc (fragment, crystallizable) receptor region for therapeutic purposes. Here, we review recent preclinical advances to target the CD40-CD40L pair in transplantation.

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.

Genetically Modified Porcine-to-Human Cardiac Xenotransplantation.

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.).

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.

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.

The immunobiology and clinical use of genetically engineered porcine hearts for cardiac xenotransplantation.

A summary of the scientific rationale of the advancements that led to the first genetically modified pig-to-human cardiac xenotransplantation is lacking in a complex and rapidly evolving field. Here, we aim to aid the general readership in the understanding of the gradual progression of cardiac (xeno)transplantation research, the immunobiology of cardiac xenotransplantation (including the latest immunosuppression, cardiac preservation and genetic engineering required for successful transplantation) and the regulatory landscape related to the clinical application of cardiac xenotransplantation for people with end-stage heart failure. Finally, we provide an overview of the outcomes and lessons learned from the first genetically modified pig-to-human cardiac heart xenotransplantation.

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

Background: Perioperative 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. Methods: Baboons 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. Results: PCXD 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. Conclusion: Blood 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.

Heterotopic Porcine Cardiac Xenotransplantation in the Intra-Abdominal Position in a Non-Human Primate Model.

Heterotopic cardiac transplantation in the intra-abdominal position in a large animal model has been essential in the progression of the field of cardiac transplantation. Our group has over 10 years of experience in cardiac xenotransplantation with pig to baboon models, the longest xenograft of which survived over 900 days, with rejection only after reducing immunosuppression. This article aims to clarify our approach to this model in order to allow others to share success in long-term survival. Here, we demonstrate the approach to implantation of a cardiac graft into the intra-abdominal position in a baboon recipient for the study of transplantation and briefly highlight our model's ability to provide insight into not only xenotransplantation but across disciplines. We include details that have provided us with consistent success in this model; performance of the anastomoses, de-airing of the graft, implantation of a long-term telemetry device for invasive graft monitoring, and ideal geometric positioning of the heart and telemetry device in the limited space of the recipient abdomen. We additionally detail surveillance techniques to assess long-term graft function.

Early Experience With Preclinical Perioperative Cardiac Xenograft Dysfunction in a Single Program.

BACKGROUND: Perioperative cardiac xenograft dysfunction (PCXD) was described by McGregor and colleagues as a major barrier to the translation of heterotopic cardiac xenotransplantation into the orthotopic position. It is characterized by graft dysfunction in the absence of rejection within 24 to 48 hours of transplantation. We describe our experience with PCXD at a single program. METHODS: Orthotopic transplantation of genetically engineered pig hearts was performed in 6 healthy baboons. The immunosuppression regimen included induction by anti-CD20 monoclonal antibodies (mAb), thymoglobulin, cobra venom factor, and anti-CD40 mAb, and maintenance with anti-CD40 mAb, mycophenolate mofetil, and tapering doses of steroids. Telemetry was used to assess graft function. Extracorporeal membrane oxygenation was used to support 1 recipient. A full human clinical transplantation team was involved in these experiments and the procedure was performed by skilled transplantation surgeons. RESULTS: A maximal survival of 40 hours was achieved in these experiments. The surgical procedures were uneventful, and all hearts were weaned from cardiopulmonary bypass without issue. Support with inotropes and vasopressors was generally required after separation from cardiopulmonary bypass. The cardiac xenografts performed well immediately, but within the first several hours they required increasing support and ultimately resulted in arrest despite maximal interventions. All hearts were explanted immediately; histology showed no signs of rejection. CONCLUSIONS: Despite excellent surgical technique, uneventful weaning from cardiopulmonary bypass, and adequate initial function, orthotopic cardiac xenografts slowly fail within 24 to 48 hours without evidence of rejection. Modification of preservation techniques and minimizing donor organ ischemic time may be able to ameliorate PCXD.

Intra-Abdominal Heterotopic Cardiac Xenotransplantation: Pearls and Pitfalls.

Heterotopic cardiac xenotransplantation in the intra-abdominal position has been studied extensively in a pig-to-baboon model to define the optimal donor genetics and immunosuppressive regimen to prevent xenograft rejection. Extensive investigation using this model is a necessary stepping stone toward the development of a life-supporting animal model, with the ultimate goal of demonstrating suitability for clinical cardiac xenotransplantation trials. Aspects of surgical technique, pre- and post-operative care, graft monitoring, and minimization of infectious risk have all required refinement and optimization of heterotopic cardiac xenotransplantation over time. This review details non-immunologic obstacles relevant to this model described by our group and in the literature, as well as strategies that have been developed to address these specific challenges.

Cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs.

A combination of genetic manipulations of donor organs and target-specific immunosuppression is instrumental in achieving long-term cardiac xenograft survival. Recently, results from our preclinical pig-to-baboon heterotopic cardiac xenotransplantation model suggest that a three-pronged approach is successful in extending xenograft survival: (a) α-1,3-galactosyl transferase (Gal) gene knockout in donor pigs (GTKO) to prevent Gal-specific antibody-mediated rejection; (b) transgenic expression of human complement regulatory proteins (hCRP; hCD46) and human thromboregulatory protein thrombomodulin (hTBM) to avoid complement activation and coagulation dysregulation; and (c) effective induction and maintenance of immunomodulation, particularly through co-stimulation blockade of CD40-CD40L pathways with anti-CD40 (2C10R4) monoclonal antibody (mAb). Using this combination of manipulations, we reported significant improvement in cardiac xenograft survival. In this study, we are reporting the survival of cardiac xenotransplantation recipients (n = 3) receiving xenografts from pigs without the expression of hTBM (GTKO.CD46). We observed that all grafts underwent rejection at an early time point (median 70 days) despite utilization of our previously reported successful immunosuppression regimen and effective control of non-Gal antibody response. These results support our hypothesis that transgenic expression of human thrombomodulin in donor pigs confers an independent protective effect for xenograft survival in the setting of a co-stimulation blockade-based immunomodulatory regimen.

Consideration of appropriate clinical applications for cardiac xenotransplantation.

The field of cardiac xenotransplantation has entered an exciting era due to recent advances in the field. Although several hurdles remain, the use of rapidly evolving transgenic technology has the potential to address current allogeneic donor pool constraints and mechanical circulatory system device limitations. The success of xenotransplantation will undoubtedly be dependent on specific patient selection criteria. Defining these particular indications for xenotransplantation is important as we approach the possibility of clinical applications.