Research opportunities and ethical considerations for heart and lung xenotransplantation research: A report from the National Heart, Lung, and Blood Institute workshop.

Xenotransplantation offers the potential to meet the critical need for heart and lung transplantation presently constrained by the current human donor organ supply. Much was learned over the past decades regarding gene editing to prevent the immune activation and inflammation that cause early organ injury, and strategies for maintenance of immunosuppression to promote longer-term xenograft survival. However, many scientific questions remain regarding further requirements for genetic modification of donor organs, appropriate contexts for xenotransplantation research (including nonhuman primates, recently deceased humans, and living human recipients), and risk of xenozoonotic disease transmission. Related ethical questions include the appropriate selection of clinical trial participants, challenges with obtaining informed consent, animal rights and welfare considerations, and cost. Research involving recently deceased humans has also emerged as a potentially novel way to understand how xeno-organs will impact the human body. Clinical xenotransplantation and research involving decedents also raise ethical questions and will require consensus regarding regulatory oversight and protocol review. These considerations and the related opportunities for xenotransplantation research were discussed in a workshop sponsored by the National Heart, Lung, and Blood Institute, and are summarized in this meeting report.

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.

Partial heart xenotransplantation: A research protocol in non-human primates.

Partial heart transplantation is a new type of transplant that delivers growing heart valve replacements for babies. Partial heart transplantation differs from orthotopic heart transplantation because only the part of the heart containing the heart valve is transplanted. It also differs from homograft valve replacement because viability of the graft is preserved by tissue matching, minimizing donor ischemia times, and recipient immunosuppression. This preserves partial heart transplant viability and allows the grafts to fulfill biological functions such as growth and self-repair. These advantages over conventional heart valve prostheses are balanced by similar disadvantages as other organ transplants, most importantly limitations in donor graft availability. Prodigious progress in xenotransplantation promises to solve this problem by providing an unlimited source of donor grafts. In order to study partial heart xenotransplantation, a suitable large animal model is important. Here we describe our research protocol for partial heart xenotransplantation in nonhuman primates.

Intraoperative Management of an Orthotopic Porcine-to-Human Cardiac Xenotransplant.

We report the intraoperative management of an orthotopic cardiac xenotransplant in a 57-year-old man with nonischemic cardiomyopathy requiring venoarterial extracorporeal membrane oxygenation. Transesophageal echocardiography was used for preharvest assessment. Continuous ex vivo perfusion of the heart was performed. Steps were taken to avoid potential xenozoonosis transmission to other patients and staff. Preclinical experience guided our intraoperative management in controlling hemodynamics and using prophylactic antiarrhythmic medications. Echocardiography aided in the diagnosis of aortic dissection in the patient after transplant. Intraoperative cardiac function was excellent. The patient was weaned from all mechanical support 4 days after transplant.

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.

Anatomical Differences Between Human and Pig Hearts and Their Relevance for Cardiac Xenotransplantation Surgical Technique.

Cardiac xenotransplantation has been proposed to bridge the gap between supply and demand for patients with end-stage heart failure requiring transplantation. However, differences in pig anatomy compared with human anatomy require modification of the surgical approach. In addition, careful consideration should be given to size matching before transplantation. (Level of Difficulty: Advanced.).

Recent advances in porcine cardiac xenotransplantation: from aortic valve replacement to heart transplantation.

INTRODUCTION: Cardiac xenotransplantation presents significant potential to the field of heart failure by addressing the high demand for donor organs. The availability of xenograft hearts would substantially augment the number of life-saving organs available to patients and may ultimately liberalize eligibility criteria for transplantation. AREAS COVERED: In this review, we will discuss the need for cardiac xenotransplantation and the history of research and clinical practice in this field. Specifically, we address immunologic concepts and clinical lessons learned from heart valve replacement using xenogeneic tissues, the advancement of xenotransplantation using organs from genetically modified animals, and the progression of this research to the first-in-man pig-to-human heart transplantation. EXPERT OPINION: Cardiac xenotransplantation holds tremendous promise, but the indications for this new treatment in adults will need to be clearly defined because mechanical support with ventricular assist devices and total artificial hearts are increasingly successful alternatives in heart failure. Cardiac xenotransplantation will also serve as temporary bridge to allotransplantation in babies with complex congenital heart disease who are too small for the currently available mechanical assist devices and total artificial hearts. Moreover, xenotransplantation of the part of the heart containing a heart valve could deliver growing heart valve implants for babies with severe heart valve dysfunction.

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.