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.

Transthoracic echocardiography is a simple tool for size matching in cardiac xenotransplantation.

BACKGROUND: Preoperative size matching is essential for both allogeneic and xenogeneic heart transplantation. In preclinical pig-to-baboon xenotransplantation experiments, porcine donor organs are usually matched to recipients by using indirect parameters, such as age and total body weight. For clinical use of xenotransplantation, a more precise method of size measurement would be desirable to guarantee a "perfect match." Here, we investigated the use of transthoracic echocardiography (TTE) and described a new method to estimate organ size prior to xenotransplantation. METHODS: Hearts from n = 17 genetically modified piglets were analyzed by TTE and total heart weight (THW) was measured prior to xenotransplantation into baboons between March 2018 and April 2022. Left ventricular (LV) mass was calculated according to the previously published method by Devereux et al. and a newly adapted formula. Hearts from n = 5 sibling piglets served as controls for the determination of relative LV and right ventricular (RV) mass. After explantation, THW and LV and RV mass were measured. RESULTS: THW correlated significantly with donor age and total body weight. The strongest correlation was found between THW and LV mass calculated by TTE. Compared to necropsy data of the control piglets, the Devereux formula underestimated both absolute and relative LV mass, whereas the adapted formula yielded better results. Combining the adapted formula and the relative LV mass data, THW can be predicted with TTE. CONCLUSIONS: We demonstrate reliable LV mass estimation by TTE for size matching prior to xenotransplantation. An adapted formula provides more accurate results of LV mass estimation than the generally used Devereux formula in the xenotransplantation setting. TTE measurement of LV mass is superior for the prediction of porcine heart sizes compared to conventional parameters such as age and total body weight.

Genetic diversity, growth and heart function of Auckland Island pigs, a potential source for organ xenotransplantation.

One of the prerequisites for successful organ xenotransplantation is a reasonable size match between the porcine organ and the recipient's organ to be replaced. Therefore, the selection of a suitable genetic background of source pigs is important. In this study, we investigated body and organ growth, cardiac function, and genetic diversity of a colony of Auckland Island pigs established at the Center for Innovative Medical Models (CiMM), LMU Munich. Male and female Auckland Island pig kidney cells (selected to be free of porcine endogenous retrovirus C) were imported from New Zealand, and founder animals were established by somatic cell nuclear transfer (SCNT). Morphologically, Auckland Island pigs have smaller body stature compared to many domestic pig breeds, rendering their organ dimensions well-suited for human transplantation. Furthermore, echocardiography assessments of Auckland Island pig hearts indicated normal structure and functioning across various age groups throughout the study. Single nucleotide polymorphism (SNP) analysis revealed higher runs of homozygosity (ROH) in Auckland Island pigs compared to other domestic pig breeds and demonstrated that the entire locus coding the swine leukocyte antigens (SLAs) was homozygous. Based on these findings, Auckland Island pigs represent a promising genetic background for organ xenotransplantation.

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

INTRODUCTION: 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. METHODS: 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. RESULTS: 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 48 h under support of the recipient's heart and there was no difference in survival compared to the other 14 ones. After 48 h, graft pressure increased up to 200 mmHg 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. CONCLUSION: 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.

An Approach to Controlling Inflammation and Coagulation in Pig-to-Baboon Cardiac Xenotransplantation.

INTRODUCTION: Inflammatory responses and coagulation disorders are a relevant challenge for successful cardiac xenotransplantation on its way to the clinic. To cope with this, an effective and clinically practicable anti-inflammatory and anti-coagulatory regimen is needed. The inflammatory and coagulatory response can be reduced by genetic engineering of the organ-source pigs. Furthermore, there are several therapeutic strategies to prevent or reduce inflammatory responses and coagulation disorders following xenotransplantation. However, it is still unclear, which combination of drugs should be used in the clinical setting. To elucidate this, we present data from pig-to-baboon orthotopic cardiac xenotransplantation experiments using a combination of several anti-inflammatory drugs. METHODS: Genetically modified piglets (GGTA1-KO, hCD46/hTBM transgenic) were used for orthotopic cardiac xenotransplantation into captive-bred baboons (n = 14). All animals received an anti-inflammatory drug therapy including a C1 esterase inhibitor, an IL-6 receptor antagonist, a TNF-α inhibitor, and an IL-1 receptor antagonist. As an additive medication, acetylsalicylic acid and unfractionated heparin were administered. The immunosuppressive regimen was based on CD40/CD40L co-stimulation blockade. During the experiments, leukocyte counts, levels of C-reactive protein (CRP) as well as systemic cytokine and chemokine levels and coagulation parameters were assessed at multiple timepoints. Four animals were excluded from further data analyses due to porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) infections (n = 2) or technical failures (n = 2). RESULTS: Leukocyte counts showed a relevant perioperative decrease, CRP levels an increase. In the postoperative period, leukocyte counts remained consistently within normal ranges, CRP levels showed three further peaks after about 35, 50, and 80 postoperative days. Analyses of cytokines and chemokines revealed different patterns. Some cytokines, like IL-8, increased about 2-fold in the perioperative period, but then decreased to levels comparable to the preoperative values or even lower. Other cytokines, such as IL-12/IL-23, decreased in the perioperative period and stayed at these levels. Besides perioperative decreases, there were no relevant alterations observed in coagulation parameters. In summary, all parameters showed an unremarkable course with regard to inflammatory responses and coagulation disorders following cardiac xenotransplantation and thus showed the effectiveness of our approach. CONCLUSION: Our preclinical experience with the anti-inflammatory drug therapy proved that controlling of inflammation and coagulation disorders in xenotransplantation is possible and well-practicable under the condition that transmission of pathogens, especially of PCMV/PRV to the recipient is prevented because PCMV/PRV also induces inflammation and coagulation disorders. Our anti-inflammatory regimen should also be applicable and effective in the clinical setting of cardiac xenotransplantation.

Current Status of Cardiac Xenotransplantation: Report of a Workshop of the German Heart Transplant Centers, Martinsried, March 3, 2023.

This report comprises the contents of the presentations and following discussions of a workshop of the German Heart Transplant Centers in Martinsried, Germany on cardiac xenotransplantation. The production and current availability of genetically modified donor pigs, preservation techniques during organ harvesting, and immunosuppressive regimens in the recipient are described. Selection criteria for suitable patients and possible solutions to the problem of overgrowth of the xenotransplant are discussed. Obviously microbiological safety for the recipient and close contacts is essential, and ethical considerations to gain public acceptance for clinical applications are addressed. The first clinical trial will be regulated and supervised by the Paul-Ehrlich-Institute as the National Competent Authority for Germany, and the German Heart Transplant Centers agreed to cooperatively select the first patients for cardiac xenotransplantation.

[Xenotransplantation of solid organs]. / Xenotransplantation von Organen.

Transplantation of genetically modified porcine hearts and kidneys could become a solution to the persistent shortage of human organ donors. Progress has been made in genetic engineering of donor pigs, preservation techniques after organ harvesting and immunosuppression using co-stimulation blockade with anti-CD40/CD40L monoclonal antibodies. Progress has also been made in in the development of methods that detect pathogenic porcine viruses and prevent their transmission to the recipient. As normal land breed pig organs continue to grow in the recipient to their original size, different pig breeds (such as Auckland Island pigs) are now used which reach a final size suitable for humans. Alternatively, a knock-out of the growth hormone receptor gene has been established, e.g., in the 10GM genetically modified pigs from Revivicor/United Therapeutics, USA. The first clinical pilot studies including patients suffering from terminal heart failure are expected to start in Germany in about 2 years.

The Endothelial Glycocalyx in Pig-to-Baboon Cardiac Xenotransplantation-First Insights.

Cardiac xenotransplantation has seen remarkable success in recent years and is emerging as the most promising alternative to human cardiac allotransplantation. Despite these achievements, acute vascular rejection still presents a challenge for long-term xenograft acceptance and new insights into innate and adaptive immune responses as well as detailed characterizations of signaling pathways are necessary. In allotransplantation, endothelial cells and their sugar-rich surface-the endothelial glycocalyx-are known to influence organ rejection. In xenotransplantation, however, only in vitro data exist on the role of the endothelial glycocalyx so far. Thus, in the current study, we analyzed the changes of the endothelial glycocalyx components hyaluronan, heparan sulfate and syndecan-1 after pig-to-baboon cardiac xenotransplantations in the perioperative (n = 4) and postoperative (n = 5) periods. These analyses provide first insights into changes of the endothelial glycocalyx after pig-to-baboon cardiac xenotransplantation and show that damage to the endothelial glycocalyx seems to be comparable or even less pronounced than in similar human settings when current strategies of cardiac xenotransplantation are applied. At the same time, data from the experiments where current strategies, like non-ischemic preservation, growth inhibition or porcine cytomegalovirus (a porcine roseolovirus (PCMV/PRV)) elimination could not be applied indicate that damage of the endothelial glycocalyx also plays an important role in cardiac xenotransplantation.