Pediatric partial heart xenotransplantation as an early use case of xenograft tissue

ABSTRACT

Xenotransplantation is a promising advancement in the field of transplantation that could eliminate deaths on the waitlist and provide an unlimited supply of on-demand organs for those in need of this life-saving therapy. The results of preclinical studies in orthotopic heart xenotransplantation have shown that non-human primate models can consistently survive 9 months post-transplant. However, early clinical results in orthotopic heart xenotransplantation have been subpar compared to traditional orthotopic heart transplantation as the longest surviving patient survived for 60 days with a complicated postoperative course. Partial heart xenotransplantation could serve as an earlier clinical use case of xenotransplantation products due to the many advantages of the neonate and infant population for xenotransplantation as well as the unique immunogenicity of heart valves which is significantly lower than that of whole hearts. The adoption of partial heart xenotransplantation would allow more children to realize the benefits of a valve that tolerates somatic growth without the need for serial reoperation.

1. Introduction

Xenotransplantation involves the use of a non-human donor to provide organs for a human recipient in need of this life-saving therapy. This protocol is a promising answer to the issues of organ supply that currently mean over 100,000 people are on organ transplant lists in the United States, with over 6000 people per year dying while waiting on life-saving organs [1,2]. The first solid organ xenotransplantations primarily utilized non-human primates (NHP) as the donor source and involved the transplantation of kidneys, livers, hearts, and testes into human recipients. Results of these first explorations of xenotransplantation were mixed, and while some success was experienced, they were largely failures [3–5]. These attempts were unsuccessful due to rejection and size differences. An attempt at xenotransplantation in the pediatric population was also performed in the United States, which was unsuccessful [6,7]. An overview of several critical events in the field of xenotransplantation can be seen in Figure 1.

Figure 1.

Timeline of significant achievements and discoveries in xenotransplantation and partial heart transplantation. Events involving humans are written in bold, events related to xenotransplantation are shown in red shades and events related to partial heart transplantation are shown in blue shades.

Abbreviations: XT – Xenotransplantation, WT – Wild type, ISHLT – International Society of Heart and Lung Transplantation, GTKO – Galactosyltransferase Knockout, PHT – Partial Heart Transplantation, NHP – Non-human primate, GE – Gene Edit, PHX – Partial Heart Xenotransplantation.

Recently, porcine donors have become the favored source of both research and clinical use of xenotransplants. This is due to a number of factors, including the significantly increased availability of donors. Swine reach sexual maturity in less than a year compared to NHP, which do not achieve this benchmark until 3–5 years. Swine also have much shorter pregnancies than NHP (114 vs 173 days), which produce more numerous offspring (5–12 vs 1–2). Additionally, swine are significantly cheaper to maintain, and public opinion on the use of these animals is significantly less controversial than the opinion on the use of NHP donors. These animals also carry less risks of zoonotic infections than NHP donors.

Xenotransplantation has several advantageous aspects compared to allotransplantation. The effectively unlimited supply of on-demand organs would mean that wait times would significantly decrease. This not only means that fewer deaths on the transplant list would occur but it is also reasonable that earlier access to organs and the elimination of bridge therapies such as dialysis and ventricular assist devices could lead to improved outcomes [7]. Furthermore, allotransplantation of solid organs is commonly carried out through death by neurological criteria (DNC) donors. The effects of brain death on organs have key disadvantages specifically in heart transplantation, as the depletion of hormones leads to reduced mitochondrial aerobic processes [8]. This means that xeno-sourced organs could theoretically have superior outcomes as negative effects of brain death would not be realized by the donated organs.

While orthotopic heart xenotransplantation (OHX) has made great progress in the last century, results are still significantly subpar when compared to traditional Orthotopic Heart Transplantation (OHT). The primary mechanism of graft failure in both OHX and OHT is rejection of the ventricles [9]. Partial heart transplantation (PHT) is an emerging treatment option that involves the transplant of the valve roots from a donor into a recipient [10]. This procedure has significant advantages, especially in the pediatric population, as it allows for the delivery of a valve that can grow with the patient [11], eliminating the need for incessant reoperation that is currently the standard of care [12,13]. While the transplanted valves require immunosuppressive medication to grow, it is theorized that this treatment could be significantly more conservative than the standard regiment of drugs currently used for OHT. This is due to the immune privilege experienced by heart valves, similar to that experienced by other tissue with limited regenerative capacity such as neural tissue and reproductive organs [14].

1.1. Outcomes of preclinical orthotopic heart xenotransplantation

In 2000, the International Society of Heart and Lung Transplantation’s Xenotransplantation Committee decided preclinical animal models must achieve a benchmark survival rate of 60% for a period of at least 3 months before clinical trials begin [15]. This benchmark has been achieved by multiple groups in recent years using heterotopic heart transplantation, one cohort of five reaching average survival times of 200 days, with the longest functioning xenograft surpassing 1 year [16]. Another report of porcine to NHP heterotopic transplantation trials have shown short- and long-term success, with the longest surviving recipient lasting for over 900 days [17]. The move from non-life supporting heterotopic heart transplantation into clinically applicable OHX was a significant hurdle to overcome. Two primary drivers of the gradual and iterative improvement in outcomes of porcine to NHP studies have been increased genetic edits to the donor and more sophisticated immunosuppressive regimens in the recipient.

The edits broadly consist of removal of porcine antigens and addition of human antigens that allow for activation of coagulation pathways and reduction of complement or other immune-mediated injury. Three key carbohydrates have been identified as the primary causes of hyperacute rejection of xenotransplanted tissue, these antigens are Galactose-α1,3-galactose (GAL), N-glycolylneuraminic acid, and Sd^a [18]. The removal of these xenoantigens has led to improved outcomes in preclinical trials. Additionally, the addition of several primate antigens has played a key role in the longer-term viability of OHX. Two key additions that reduce complement mediated injury by inactivation of the complement system and accelerated complement decay are hCD46 and hCD55, respectively [19]. The use of triple knockout porcine donors, with the removal of the three major carbohydrate antigens, led to the formation of a neoantigen. The removal of Neu5Gc led to this neoantigen development; however, humans contain a functionally similar mutation of the enzyme that leads to the expression of Neu5Gc, meaning that immune activation by this mechanism is not likely. This highlights a drawback of the porcine to NHP model. The combination of elimination of porcine antigens and addition of human antigens to mediate both coagulation and immune response have allowed for the achievement of long-term survival of animal models of OHX. However, the evolution of the immunosuppression protocol has also had a significant role in the recent advancement of models past the benchmark survival rates.

The iterative improvement of immune suppression has played a key role in the advancement of xenotransplantation toward clinical application. The current state-of-the-art in xenotransplantation immunosuppression, developed by Mohiuddin et al., is an anti-CD40 clone-based protocol that includes MMF and anti-CD20 induction to deplete B-Cells [20]. This combination of continuously improved immunosuppression and genetic editing of donor animals has led to improved survival of life supporting and heterotopic heart xenotransplants that are necessary for the initiation of clinical research of cardiac xenotransplantation.

1.2. Outcomes of clinical orthotopic heart xenotransplantation

Human decedent research has played a vital role in the early development of xenotransplantation. Two OHX were performed at NYU and resulted in no signs of hyperacute rejection or transfer of zoonotic disease in the three-day period of the study [21]. Multi-omics data on these decedent xenotransplantations has provided detailed progression of the immunological response and tissue response to the procedure [22].

The first non-decedent porcine to human cardiac transplantation was performed at the University of Maryland on a 57-year-old male with non-ischemic cardiomyopathy in January 2022. The donor animal had 10 genetic edits that can be broadly classified into knockouts or human transgene expression edits. Among the four knockout edits, three removed enzymes expressing key carbohydrate xenoantigens and one reduced growth factor signaling via the knockout of growth hormone receptors. The remaining six edits were aimed at; regulating the complement system (two edits), reducing coagulation of blood (two edits), and reducing inflammation (two edits).

Following the transplant up to 49 days postoperatively, the xenograft functioned fairly normally, with an endomyocardial biopsy on post-op day 34 showing no signs of rejection. Rejection first detected on day 49 eventually led to a withdrawal of life support on day 60 due to atypical antibody mediated rejection [23,24]. A second clinical cardiac xenotransplantation was performed with the same donor model, with similar atypical antibody mediated endothelial damage that led to a transition to comfort care on post-op day 40 [25]. While these procedures have provided a great deal of insight into cardiac xenotransplantation, the limited time of xenograft survival before failure means that further research and modifications to either the gene edits of the donor animal or changes in the pre, peri, and postoperative care are needed to improve results of clinical OHX in the future. However, partial heart xenotransplantation may serve as an early use case of xenotransplantation.

1.3. Outcomes of preclinical partial heart transplantation

The primary animal model for preclinical investigation of partial heart transplantation have been swine models. These animals are well suited for the study of PHT as they share many immunologic, anatomic, and physiological similarities [26]. The results of these studies have been positive and showed good outcomes with the growth of the valves and doubling in weight of the animal model after a period of approximately 2 months [27]. Preclinical models of PHT have also utilized green-fluorescent protein positive swine that allows additional data to be gathered on the viability and proliferation of donor cells throughout the recipient lifecycle [28]. A variety of rodent models have also been used to gain insight into the immune response to PHT through heterotopic transplantation of valve leaflets, which have shown reduced immune response in comparison to full heart transplantation, supporting the idea that heart valves are subject to immune privilege [29].

1.4. Outcomes of clinical partial heart transplantation

The first clinical PHT was performed in 2022 on a neonate with irreparable truncal valve dysfunction [30]. Early results show that the valve is growing, and no signs of rejection have been reported. Since this first procedure, PHTs have been performed at many centers across the country, including the first domino PHT which was done in 2023 [31–33]. At this time approximately 25 PHTs have been performed, with the majority of these cases so far unreported in the literature. The limited reports to this date show that the valves grow and maintain excellent function through the short-term [34]. These valves could solve the problem of valve reoperation in children with irreparable heart disease as the valves have shown in both preclinical models and early clinical outcomes [35].

1.5. Partial heart xenotransplantation

In light of the current state of clinical OHX, Partial Heart Xenotransplantation (PHX) could serve as an earlier routine clinical use case of xeno-sourced cardiac tissue due to the reduced immunogenicity of heart valves as well as the maintenance of function during rejection seen in large cohorts of patients receiving homovital valves as well as animal models of PHTs. Furthermore, the population of patients currently receiving PHT are ideal for xenotransplantation. The inducibility of children to different antigens is significantly higher than adults. It has been shown that early antigen exposure can modify immune response in children [36]. This has led to the transplant of ABO incompatible hearts in children with outcomes comparable to ABO compatible, and importantly children receiving ABO incompatible hearts do not develop antibodies to the foreign antigen [37,38].

Children undergoing cardiac surgery, including PHT, routinely undergo complete or partial thymectomy during surgery. While this is done for technical considerations as the position of the thymus significantly impedes access to the heart, it also significantly reduces T-cell counts with no increase in infection risks observed through decades of this practice [39–41]. This thymectomy, along with the greatly increased inducibility, are theorized to be the cause of significantly favorable outcomes of OHT in the infant population compared to the adult population, with incidence of retransplantation under 15% at 20 years post transplant [42]. These outcomes in the pediatric population are far superior to the outcomes of adult heart transplants, which have a half-life of just 13 years [43]. The case for children as the best population for the early clinical use of xenotransplantation is further supported by in-vitro work on the native response to porcine antigens, which showed that young children do not have a native response to antigens, while older children and adults show an immune response [44].

Another strength of PHX compared to OHX is the outcome in the event of rejection. While OHX failure inevitably leads to death, as mechanical support and allotransplantation have necessarily been deemed non-options, PHX failure due to rejection does not have nearly as serious consequences. Evidence of this can be found in the large cohort report of homovital valve outcomes by Yacoub et al. Homovital valves are fresh tissue valves; however, patients do not receive immunosuppressive medications like patients undergoing PHT do. In a cohort of 275 patients receiving homovital valves, there were 0 reports of accelerated degeneration caused by rejection [45]. Furthermore, many currently used bioprosthetic valves contain xenoantigen carbohydrates such as alpha-Gal and Neu5Gc; these antigens have been linked to the deterioration of valves overtime [46]. Despite not receiving immunosuppressive medications, these valves last over a decade on average. This lack of rapid degenerative failure, even without immunosuppression, supports PHX as an early clinical use of xenografts. The first use of xenografts being in tissue with redundant options in the event of failure is an idea previously supported by Cooper et al. who recommended that kidneys be the current goal for clinical xenotransplantation as the kidney xenograft can be removed and dialysis resumed in the event of graft failure [47]. Rejection mediated failure of PHX would likely result in outcomes similar to those seen in Yacoub’s large cohort of homovital valves, in preclinical animal models of PHT, and in the use of bioprosthetic valves containing the major carbohydrate antigens [48].

This result is comparable to the current use of biological valves which do not have regenerative capacity nor the capacity for growth. Furthermore, valve replacement is a routinely performed procedure that can be done in the event of xenograft failure without the need for waitlist time, which is a requirement in the event that retransplantation is needed with OHX. This treatment plan in the event of failure is significantly preferable to the failure of OHX xenograft tissue which would necessitate retransplantation, and supports the use of PHX as an early clinical application of xenotransplantation to deliver growing heart valves to children. An overview of benefits and challenges facing PHX can be seen in Table 1.

Table 1.

Overview of benefits and challenges facing partial heart xenotransplantation.

Benefits	Challenges
– Lowered Immunogenicity of heart valves – Replacement valves are available off-the-shelf in case of xenograft failure – Unlimited donor supply could increase adoption of partial heart procedures	– Ethical issues associated with xenotransplantation in pediatric population – Potential zoonotic risks – Limited funding for research from both industry and public funders

2. Conclusion

The key advantages of PHX over OHX in both the immunogenicity of the transplanted tissue and the unique suitability of the patient population to xenotransplantation mean that PHX could serve as the first routine clinical use of xenotransplantation. PHX shows much promise to deliver on-demand growing heart valves to infants, and is under active investigation. A protocol has been established for porcine to NHP animal model studies [49]. These studies are of vital importance to establish whether the theorized benefits of PHX over OHX can be realized and translated into long-term survival of animal models. Following the achievement of the 60% benchmark set in 2000, the clinical trials of PHX could pull from the already published work on trials for both OHX in the pediatric population and established clinical trial protocols for PHT [50,51].

2.1. Future perspective

The widespread adoption of partial heart transplantation to treat neonates and infants with irreparable heart disease is limited by existing donor supply constraints. Xenotransplantation could serve to alleviate these concerns and increase the number of children that can benefit from not only the partial heart transplants but also novel applications of partial heart transplantation such as pulsatile conduits derived from donor atrial tissue to improve the long-term outcomes of Fontan palliated patients [52]. These innovations in the use of donor tissue from both xeno and allograft sources are imperative in finding creative solutions to the current shortcomings in the treatment of congenital heart disease and ensure that outcomes in the most vulnerable populations continue to improve. Furthermore, novel research into the storage of PHT grafts could allow for significantly favorable distribution and logistical management of PHT or PHX in the future, which could serve to improve access to this emerging technology [53,54].

Funding Statement

This research is supported by the National Institutes of Health/National Heart, Lung, and Blood Institute [grant R41 hL169059], the American Association for Thoracic Surgery, the Brett Boyer Foundation, the Saving tiny Heart Society, the Emerson Rose Heart Foundation, philanthropy from senator Campbell and the Arkansas Children’s Research Institute.

Article highlights

• Partial Heart Transplants have proven the ability to allow for somatic growth of neonatal recipients without the need for reoperation.

• Partial heart transplant operative volume is limited by the very small donor pool.

• Xenotransplantation promises to provide an unlimited source of donor organs.

• Clinical xenotransplantation is limited by the immune rejection that has been seen in early use cases.

• Valvular grafts have lower immunogenicity then full solid organ transplants.

• Partial Heart Xenotransplantation could be used to provide on-demand growing valve replacements to children.

• PHX would alleviate the donor supply concerns of traditional partial heart transplantation.

Author contributions

E.J.C. – Original Draft, Revisions, Figures. H.J. – Original Draft, Figures, Revisions. B.R.- Supervision, Revisions, Review. T.K.R. – Figures, Revisions, Supervision.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.