Cardiac xenotransplantation: From imagination to achievable practice

Abstract

Allogeneic heart transplantation and durable mechanical circulatory support remain the standard treatments for advanced refractory heart failure, but donor-heart scarcity and device-related complications leave many patients without viable options. Driven by advances in gene editing, immunosuppression, and donor-organ preservation, cardiac xenotransplantation (XTx) has progressed rapidly, culminating in the landmark first-in-human compassionate-use application in 2022. This minireview critically summarizes the development of clinical cardiac XTx. We performed a narrative literature search of PubMed/MEDLINE, Web of Science, and Google Scholar from inception to 2026 using terms including cardiac XTx, gene-edited pig, immunosuppression, and xenograft, prioritizing landmark preclinical studies, human cases, and regulatory or ethical publications, and stratifying the evidence as in vitro or mechanistic studies, non-human primate (NHP) survival studies, living compassionate-use recipients, and deceased-recipient models. The principal findings are: Multigene-edited pig hearts combined with costimulation-blockade immunosuppression have achieved graft survival beyond one year in NHP; two living compassionate-use recipients (2022 and 2023) demonstrated proof-of-concept xenograft function for 40-60 days but died of multifactorial graft failure involving antibody-mediated injury, porcine cytomegalovirus reactivation, and diastolic dysfunction; and deceased-recipient studies confirmed short-term feasibility without hyperacute rejection. Cardiac XTx remains experimental but represents a promising proof of concept whose translation to clinical trials requires resolution of immunologic injury, infectious risk and graft overgrowth.

Key Words: Heart failure; Heart transplantation; Xenotransplantation; Genetic engineering; Pig

Core Tip: Cardiac xenotransplantation has advanced from the failed animal-heart attempts of 1964-1984 to two landmark first-in-human compassionate-use transplants of 10-gene-edited pig hearts in 2022 and 2023. Although both recipients died within 40-60 days, these proof-of-concept cases defined the key remaining barriers-antibody-mediated injury, porcine viral reactivation, and diastolic dysfunction-that must be resolved before gene-edited pig hearts may be explored in the further clinical trials and act as one of reliable and effective means for the treatment of end-stage heart failure.

INTRODUCTION

Heart failure (HF) is a complex clinical syndrome characterized by heterogeneous pathophysiology and multifaceted etiology, affecting more than 64 million people worldwide[1]. It is associated with impaired quality of life, frequent hospitalizations, rising health care costs, and high rates of premature death; although HF-related mortality has declined with advances in medical therapy, the 5-year mortality rate remains as high as 75%[2]. After optimization of conventional treatment, allogeneic heart transplantation (HTx) remains the definitive option for advanced refractory HF[1]. However, the supply of donor organs falls far short of clinical demand. In the United States, 3817 HTx procedures were performed in 2021, yet 3502 patients remained on the waiting list at year’s end; 248 died while waiting, and 946 were removed from the list without receiving a transplant[2]. For patients who cannot receive a human heart in time, left ventricular assist devices (LVADs) or biventricular assist devices are the principal alternatives, and several devices are currently in clinical use. The International Registry for Mechanically Assisted Circulatory Support Third Annual Report reported survival rates of 95%, 87%, 82%, 72%, 62%, and 57% at 1, 6, 12, 24, 36, and 48 months after LVAD implantation, respectively[3]. However, LVAD recipients remain exposed to serious complications, including bleeding (35%), infection (55%), and right ventricular failure (40%)[4-6], which increase short- and long-term mortality and impair the quality of life of surviving patients. These limitations have sustained interest in xenotransplantation (XTx)-the transplantation of organs from genetically engineered non-primate mammals-as a potential source of replacement hearts. Driven by rapid progress in gene-editing technology, cardiac XTx has moved from imagination to the first-in-human compassionate-use applications of 2022 and 2023; nevertheless, it must be emphasized that cardiac XTx remains an experimental, proof-of-concept therapy rather than an established clinical option. The aim of this minireview is to critically synthesize the historical development of clinical cardiac XTx, the genetic engineering (GE) of donor pigs, recipient management strategies, and the early human experience; to distinguish the levels of evidence supporting each advance; and to identify the unresolved scientific, regulatory, and ethical barriers that must be overcome before cardiac XTx can progress from single-patient compassionate-use experiments to regulated clinical trials.

SEARCH STRATEGY AND EVIDENCE SELECTION

This article is a narrative minireview; a systematic-review protocol is beyond its scope, but a structured search and selection strategy was applied to maximize transparency. We searched PubMed/MEDLINE, Web of Science, and Google Scholar from database inception to January 2026 for English-language publications, using the terms “cardiac xenotransplantation”, “heart xenotransplantation”, “gene-edited pig”, “genetically engineered pig”, “immunosuppression”, “xenograft”, “hyperacute rejection”, and “porcine cytomegalovirus”, alone and in combination. The reference lists of retrieved articles and of recent major reviews were screened to identify additional sources. Because the field is rapidly evolving and much of the human experience consists of single-case reports, we prioritized: (1) Landmark preclinical studies that defined the principal immunologic, coagulation, physiologic, and infectious barriers; (2) Peer-reviewed reports of all human cardiac XTx experience; and (3) Regulatory, ethical, and biosecurity literature relevant to clinical translation. Throughout the text, evidence is explicitly stratified into four categories: (1) In vitro and mechanistic studies; (2) Non-human primate (NHP) survival studies; (3) Compassionate-use transplantation in living recipients; and (4) Experiments in recently deceased (brain-dead) human recipients. These categories are not treated as equivalent clinical evidence, and where findings conflict or remain unvalidated, this is stated explicitly.

DEVELOPMENT OF CLINICAL CARDIAC XTX

The pre-genetic era: Clinical attempts from 1964 to 1984

The clinical history of cardiac XTx is instructive precisely because each early failure identified a specific mechanistic barrier. In 1964, Hardy performed the first reported clinical cardiac XTx at the University of Mississippi, transplanting a chimpanzee heart into a moribund patient with terminal HF; the xenograft failed within approximately 2 hours because it was too small to support the recipient’s circulation, with early antibody-mediated injury as a contributing factor[7,8]. This case established two enduring lessons: Donor-recipient size matching is non-negotiable, and the preformed-antibody barrier cannot be ignored. In 1968, Cooley performed an orthotopic transplantation using a sheep heart in Houston, and Ross performed a heterotopic transplantation using a pig heart in London; in both cases, hyperacute rejection (HAR) destroyed the graft within minutes in the operating room[8]. These disasters demonstrated that humans and Old World primates harbor preformed natural antibodies against carbohydrate xenoantigens expressed on discordant vascular endothelium, triggering complement-mediated graft destruction; the resulting skepticism among the public and the medical community effectively halted clinical cardiac XTx for nearly a decade. In 1977, Barnard et al[9] revived interest by using heterotopic xenografts to support two patients with postcardiotomy shock who could not be weaned from cardiopulmonary bypass: A baboon heart failed within hours, whereas a chimpanzee heart supported the patient for 4 days before being rejected before the native heart had recovered. The lesson was that a concordant (NHP) xenograft can provide temporary circulatory support, but without effective immunosuppression it remains acutely vulnerable to rejection. In 1984, Bailey orthotopically transplanted a baboon heart into a neonate (“Baby Faye”) with hypoplastic left heart syndrome at Loma Linda University; under cyclosporine-based immunosuppression the patient survived for 21 days, at that time the longest survival of any cardiac xenograft recipient, before dying of humoral rejection across an ABO-incompatible donor-recipient barrier[10]. The case proved that pharmacologic immunosuppression could delay-but not prevent-antibody-mediated xenograft rejection, and it catalyzed an ethical debate that still shapes the field: Clinical attempts must be grounded in reproducible preclinical evidence rather than surgical enthusiasm alone.

The gene-editing era and the first-in-human experience

Two technological advances transformed this discouraging landscape. The first was somatic cell nuclear transfer, demonstrated in 1996 with the cloning of the sheep “Dolly”, which for the first time allowed genetic modifications introduced in cultured cells to be propagated into whole animals[11,12]. The second was the emergence of programmable nucleases-zinc finger nucleases, transcription activator-like effector nucleases, and especially clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-which enabled rapid, multiplexed engineering of the porcine genome[13]. Together, these tools made it possible to delete the genes encoding the three known carbohydrate xenoantigens and to insert human transgenes that regulate complement, coagulation, inflammation, and graft growth, directly targeting rejection, the dominant barrier to cardiac XTx (detailed in subsequent sections).

These advances culminated in the landmark first-in-human compassionate-use case performed on January 7, 2022, at the University of Maryland (Baltimore, United States), in which a heart from a pig with 10 genetic modifications was transplanted orthotopically into a 57-year-old man with end-stage HF who was ineligible for both allotransplantation and LVAD support[14]. Supported by an anti-CD40-based costimulation-blockade immunosuppressive regimen, the xenograft sustained cardiovascular and systemic organ function for 60 days[14]. This case demonstrated proof-of-concept feasibility of gene-edited pig-to-human cardiac XTx; it should not, however, be characterized as a clinical success, because the recipient died on postoperative day (POD) 60, and the mechanisms of graft failure remain only partially understood.

The detailed follow-up analyses of this case constitute the most instructive clinical evidence currently available and deserve explicit emphasis[14,15]. First, regarding porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) reactivation: Although the donor pig had been screened, cell-free DNA analysis and subsequent studies detected rising PCMV/PRV within the xenograft, and autopsy confirmed viral presence in the graft in association with endothelial injury; viral reactivation within the graft is therefore considered a probable contributor to xenograft dysfunction and the recipient’s death, underscoring that conventional screening assays may miss latent infection and that designated pathogen-free (DPF) breeding with more sensitive detection is essential[14,15]. Second, regarding intravenous immunoglobulin (IVIG)-mediated antibody responses: IVIG administered to treat hypogammaglobulinemia was subsequently shown to contain anti-pig antibodies, and an increase in anti-pig xenoantibodies-predominantly IgG-was detected after IVIG administration and during the first plasma exchange, identifying an iatrogenic source of antibody-mediated graft injury that is directly relevant to future protocols[15]. Third, regarding diastolic dysfunction: The graft functioned well echocardiographically until POD 47, when diastolic HF developed; endomyocardial biopsy on POD 50 showed damaged capillaries with interstitial edema, red cell extravasation, rare thrombotic microangiopathy (TM), and complement deposition, and biopsy on POD 56 demonstrated fibrotic changes consistent with progressive myocardial stiffness-an evolution more suggestive of diffuse microvascular endothelial injury than of classic acute cellular rejection[14,15]. The investigative team proposed three potential, non-exclusive etiologies for the endothelial damage: Endogenous xenoantibody-mediated rejection, exogenous IVIG-derived xenoantibodies, and PCMV/PRV reactivation within the xenograft[15]. Critically, these conclusions derive from a single case report and are hypothesis-generating rather than definitive; disentangling their relative contributions will require standardized surveillance protocols and, ideally, controlled clinical-trial data.

A second living compassionate-use recipient was treated at the University of Maryland on September 20, 2023: A 58-year-old man (Lawrence Faucette) with end-stage HF who was likewise ineligible for allotransplantation received an orthotopic heart from a 10-gene-edited pig[16]. Incorporating lessons from the first case, the team selected a clinically less compromised recipient and applied more sensitive donor screening for PCMV/PRV; immunosuppression included costimulation blockade and complement inhibition[16,17]. The xenograft initially demonstrated excellent systolic and diastolic function, but the first biopsy, performed approximately 2 weeks after transplantation, already showed early antibody-mediated rejection, and a subsequent surge in anti-pig antibodies was accompanied by progressive graft injury and renal failure requiring dialysis; the graft ultimately failed, and the patient died on October 30, 2023, approximately 40 days after transplantation[16]. The second case therefore tempers any optimism generated by the first: Even with a healthier recipient, enhanced donor screening, and intensified complement-directed therapy, antibody-mediated rejection still emerged within weeks, indicating that the immunologic barrier to durable cardiac xenograft survival in humans remains unresolved[16-18] (Table 1).

Table 1 Key events in the development of clinical cardiac xenotransplantation.

Year	Surgeon/team	Institution, country	Donor	Transplant type	Recipient profile	Survival/observation	Key outcome/Lesson	Ref.
1964	Hardy	University of Mississippi Medical Center, Jackson, United States	Chimpanzee	OHT	Moribund man with terminal heart failure	About 2 hours	Graft failure from donor-recipient size mismatch with early antibody-mediated injury; established the need for size matching and recognition of the preformed-antibody barrier	[7,8]
1968	Cooley	Texas Heart Institute, Houston, United States	Sheep	OHT	48-year-old man with terminal ischemic cardiomyopathy	Minutes	Hyperacute rejection in the operating room; demonstrated the preformed natural antibody/complement barrier against discordant species	[8]
1968	Ross	National Heart Hospital, London, United Kingdom	Pig	HHT	48-year-old man with terminal heart disease	Minutes (about 4 minutes)	Immediate hyperacute rejection; confirmed the hyperacute barrier for pig-to-human grafts and redirected the field toward concordant donors and, later, genetic engineering	[8]
1977	Barnard	Groote Schuur Hospital, University of Cape Town, South Africa	Baboon; chimpanzee	HHT (circulatory support)	Two patients with postcardiotomy shock unable to be weaned from cardiopulmonary bypass	< 1 day (baboon); 4 days (chimpanzee)	Heterotopic xenografts can provide temporary mechanical support, but rejection occurred before native-heart recovery, highlighting the need for effective immunosuppression	[9]
1984	Bailey	Loma Linda University Medical Center, Loma Linda, United States	Baboon	OHT	Neonate (“Baby Faye”) with hypoplastic left heart syndrome	21 days	Longest survival of the pre-genetic era under cyclosporine-based immunosuppression; death from humoral rejection across an ABO-incompatible barrier; catalyzed scientific and ethical debate	[10]
2022	Griffith/Mohiuddin	University of Maryland Medical Center, Baltimore, United States	10-gene-edited pig1	OHT	57-year-old man with end-stage heart failure, ineligible for allotransplantation or LVAD; FDA compassionate use (living recipient)2	60 days	Proof-of-concept xenograft function; late graft failure associated with PCMV/PRV reactivation, IVIG-associated anti-pig IgG, and progressive diastolic dysfunction; recipient died on POD 60	[14,15]
2022	Moazami	NYU Langone Health, New York, United States	10-gene-edited pig1	OHT	Two deceased (brain-dead) recipients on ventilatory support3	Observation period (66 hours)3	No hyperacute, cellular, or antibody-mediated rejection and no zoonotic transmission during observation; dysfunction in one graft attributed to donor-recipient size mismatch	[18]
2023	Griffith/Mohiuddin	University of Maryland Medical Center, Baltimore, United States	10-gene-edited pig1	OHT	58-year-old man with end-stage heart failure, ineligible for allotransplantation; FDA compassionate use (living recipient)2	About 40 days (died October 30, 2023)	Early biopsy evidence of antibody-mediated rejection by about 2 weeks despite a less compromised recipient, enhanced donor screening, and complement inhibition; graft failure led to withdrawal of support	[16,17]

¹The 10-gene-edited donor pigs carried knockouts of three carbohydrate xenoantigen genes (GGTA1, CMAH, B4GALNT2) and of the growth hormone receptor gene, together with human transgenes encoding complement-regulatory (CD46, CD55), coagulation-regulatory (thrombomodulin, endothelial protein C receptor), and immunomodulatory/anti-inflammatory proteins (CD47, heme oxygenase-1).

²Living compassionate-use recipients: Single-patient, United States Food and Drug Administration-authorized experimental procedures; outcomes represent patient survival.

³Deceased (brain-dead) recipient research model: The 66-hours figure denotes the protocol-defined observation period, not patient survival; this model provides short-term mechanistic evidence only.

OHT: Orthotopic heart transplantation; HHT: Heterotopic (auxiliary) heart transplantation; LVAD: Left ventricular assist device; FDA: United States Food and Drug Administration; POD: Postoperative day; PCMV/PRV: Porcine cytomegalovirus/porcine roseolovirus; IVIG: Intravenous immunoglobulin.

In parallel, investigators at the New York University Langone Transplant Institute used a fundamentally different human model: In 2022, 10-gene-edited pig hearts were transplanted into two deceased (brain-dead) recipients maintained on ventilatory support, and xenograft function, hemodynamics, and systemic responses were monitored over a 66-hour observation period[18]. Both xenografts functioned immediately, and there was no evidence of HAR, cellular or antibody-mediated rejection, or zoonotic transmission during the observation period, although cardiac function declined in one case owing to a donor-recipient size mismatch[18]. Importantly, these experiments constitute short-term mechanistic observations in deceased-recipient models rather than survival evidence in living patients, and they should be interpreted as complementary to-not equivalent with-the living compassionate-use experience.

Taken together, the human evidence base for cardiac XTx currently comprises two living compassionate-use recipients and a small number of deceased-recipient experiments (Table 2); no controlled clinical trial has yet been performed. The proof-of-concept cases establish short-term technical feasibility but simultaneously define the dominant unresolved problems-antibody-mediated endothelial injury despite triple-xenoantigen-knockout donors, latent viral reactivation, iatrogenic antibody exposure, progressive diastolic dysfunction, and the absence of validated long-term immunosuppressive strategies. Whether the encouraging NHP survival data can be reproduced in humans with clinically tolerable regimens remains an open and contested question.

Table 2 Comparative summary of the human clinical evidence in cardiac xenotransplantation.

Case	Evidence model	Donor genotype	Preservation strategy	Immunosuppression	Follow-up duration	Dominant complications	Key limitations	Ref.
University of Maryland, January 2022	Living compassionate-use recipient	10-gene-edited pig (GGTA1/CMAH/B4GALNT2 knockout, GHR knockout; hCD46, hCD55, hTBM, hEPCR, hCD47, hHMOX1 transgenes)	Not fully reported in the primary publication	Investigational anti-CD40 costimulation blockade combined with mycophenolate mofetil and corticosteroids	60 days (recipient died on POD 60)	Progressive diastolic dysfunction from POD 47; capillary injury with complement deposition; PCMV/PRV reactivation within the graft; IVIG-associated rise in anti-pig IgG	Single compassionate-use case; non-approved investigational agents; latent donor viral infection missed by conventional screening; mechanism of graft failure multifactorial and incompletely resolved	[14,15]
University of Maryland, September 2023	Living compassionate-use recipient	10-gene-edited pig (same editing platform)	Not fully reported	Costimulation blockade with intensified complement-directed therapy	Approximately 40 days (died October 30, 2023)	Early antibody-mediated rejection on biopsy at 2 weeks; subsequent anti-pig antibody surge with progressive graft injury and renal failure	Single compassionate-use case; antibody-mediated rejection emerged despite a healthier recipient, enhanced donor screening, and complement inhibition	[16,17]
NYU Langone, 2022 (two cases)	Deceased (brain-dead) recipient research model	10-gene-edited pig	Standard clinical procurement and preservation	Conventional allotransplant-style immunosuppression without investigational agents	Protocol-defined observation period of 66 hours (not patient survival)	Functional decline in one graft attributed to donor-recipient size mismatch; no rejection and no zoonotic transmission observed	Very short observation window; brain-death physiology does not model living recipients; cannot inform on long-term survival, chronic rejection, or infection risk	[18]

POD: Postoperative day; PCMV/PRV: Porcine cytomegalovirus/porcine roseolovirus; IVIG: Intravenous immunoglobulin; GHR: Growth hormone receptor; hTBM: Human thrombomodulin; hEPCR: Human endothelial protein C receptor; hHMOX1: Human heme oxygenase-1; NYU: New York University.

SELECTION OF DONOR SOURCES FOR CARDIAC XTX

Several animal species have been used or considered as potential organ sources for XTx. Old World NHP were once regarded as advantageous donors because humans carry few natural antibodies against their organs[7]; however, the only NHPs large enough to supply a human-sized heart-chimpanzees and great apes-are endangered species, and NHP viruses may be transmissible to humans with unpredictable pathogenicity[14]. NHPs were therefore abandoned as source animals for solid-organ XTx.

Pigs are now widely accepted as the most suitable donor species, for six principal reasons: (1) Physiological and immunological similarities to humans; (2) The availability of efficient and precise GE techniques to overcome rejection and physiological incompatibilities; (3) High fecundity and a short developmental period, permitting rapid production of optimized donor lines; (4) A natural life expectancy of 15-20 years, supporting potential xenograft longevity; (5) A low risk of zoonotic transmission when animals are maintained under DPF conditions; and (6) Broad ethical acceptance, given the long-standing safe clinical use of porcine-derived materials (e.g., bioprosthetic valves).

These advantages should not obscure important residual caveats. The porcine heart is anatomically and physiologically similar to, but not identical with, the human heart. Synthesizing the available comparative data, Flowers et al[19] documented differences in coronary artery distribution, valve geometry, cardiac orientation within the thorax, hemodynamic behavior, and myocardial growth trajectory, all of which may influence graft function after orthotopic XTx. Size matching remains a further unresolved problem: To approximate the dimensions of a primate or human heart, donors are usually young, rapidly growing animals, which complicates both perioperative fitting and post-transplant growth control (as discussed in the section on control of growth of porcine xenohearts). In the deceased human recipient model, size mismatch was directly implicated in early functional decline, underscoring that donor-recipient size matching is a genuine clinical decision parameter rather than a theoretical concern (as discussed in the clinical practice section). Whether hearts from specific breeds (e.g., miniature pigs) or from growth hormone receptor-deficient lines can resolve this constraint without introducing new liabilities remains an open question, and the preclinical evidence summarized in the following sections should be read against these anatomical limitations.

DEVELOPMENT OF GENETICALLY MODIFIED PORCINE HEARTS

The immune response to a xenograft is driven largely by the genetic dissimilarity between donor and recipient. Two complementary genetic strategies are currently used to attenuate rejection: (1) Deletion of porcine genes encoding xenoantigens against which humans have preformed (natural) antibodies; and (2) Transgenic expression of human complement-regulatory, coagulation-regulatory, or anti-inflammatory proteins. For clarity, we distinguish below between donor modifications that are supported by reproducible NHP survival data or have been used in human recipients (“established” edits), and modifications that remain at the in-vitro or early preclinical stage (“exploratory” edits).

Deletion of carbohydrate xenoantigens (established)

The principal xenoantigen is the carbohydrate epitope galactose-α-1,3-galactose (αGal), a terminal sugar residue present on porcine cell-surface glycoproteins and glycolipids and synthesized by the enzyme α1,3-galactosyltransferase, encoded by the GGTA1 gene. Humans and Old World primates lack a functional GGTA1 gene and therefore produce natural anti-αGal antibodies that trigger hyperacute, antibody-mediated rejection. Knockout of GGTA1 (GTKO), first achieved in 2003, was a milestone that largely abolished HAR in pig-to-primate models[20]. Two additional carbohydrate xenoantigens were subsequently identified: N-glycolylneuraminic acid (Neu5Gc), a sialic acid synthesized by the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase (encoded by CMAH, which is non-functional in humans), and the Sda blood group antigen, synthesized by the enzyme β1,4-N-acetylgalactosaminyltransferase 2 (encoded by B4GALNT2). A triple-knockout (TKO; GGTA1/CMAH/B4GALNT2-deficient) pig was established in 2015[21] and now constitutes the accepted antigen-reduction platform; residual human antibody binding to TKO pig cells is low, although non-αGal/Neu5Gc/Sda antibody targets have not been fully characterized.

Human complement-regulatory proteins (established)

Antigen deletion alone is insufficient, because complement is activated not only by antibody binding to residual xenoantigens but also during ischemia-reperfusion injury (IRI), and because complement intersects with coagulation, inflammation, and adaptive immunity[22]. Transgenic expression of human complement-regulatory proteins-membrane cofactor protein (hCD46), decay-accelerating factor (hCD55), or protection (hCD59)-on the porcine endothelium inhibits complement-mediated injury at defined steps of the cascade, including membrane attack complex formation[4]. Expression of at least one human complement-regulatory protein (most commonly hCD46 and/or hCD55) is now a standard component of clinically oriented donor genotypes.

Human coagulation-regulatory proteins (established)

Dysregulation of coagulation is a further barrier: Pig hearts transplanted into baboons develop TM driven by endothelial activation and by molecular incompatibilities between porcine and primate coagulation regulators[23]. Because systemic anticoagulation has proven of limited efficacy (as discussed in the anticoagulation management subsection), transgenic correction at the graft level has been pursued. Donor-pig expression of human thrombomodulin (hTBM) provides an independent protective effect on cardiac xenograft survival within a costimulation blockade-based regimen[24], and human endothelial protein C receptor (hEPCR) is incorporated to reinforce activation of the protein C pathway (as discussed in the anticoagulation management subsection). The GTKO.hCD46.hTBM genotype is the best-validated example of this strategy in pig-to-baboon cardiac XTx; the survival outcomes achieved with this genotype under specific immunosuppressive regimens are detailed in the recipient management section[25].

Growth hormone receptor knockout (established)

Because intrinsic xenograft growth can contribute to post-transplant dysfunction, knockout of the porcine growth hormone receptor gene (GHR-KO) has been introduced to blunt post-transplant xenograft growth, as confirmed by echocardiographic follow-up beyond six months without growth-restricting adjuncts[26]. The rationale, alternatives, and limitations of this approach are analyzed in the section on control of growth of porcine xenohearts.

Exploratory modifications (not yet clinically validated)

Several additional modifications target innate cellular immunity and inflammation, but the supporting evidence remains confined to in-vitro assays, ex-vivo perfusion, or short-term preclinical experiments, and none has been validated in NHP long-term survival studies or human recipients; they should therefore be regarded as investigational rather than established components of clinical donor pigs. Transgenic human CD47 (hCD47) engages signal regulatory protein-α on recipient macrophages to deliver a “do not eat me” signal and attenuate phagocytosis[27], and hematopoietic-cell chimerism was enhanced in baboons receiving cells from hCD47-transgenic pigs[28,29]; however, hCD47 expression alone does not fully abrogate macrophage-mediated injury[27]. Pigs transgenic for HLA-E with human β2-microglobulin were protected against human natural killer (NK) cell-mediated cytotoxicity in vitro[30]. Transgenic expression of the anti-inflammatory and cytoprotective proteins human TNF-α-induced protein 3 (also known as A20)[31] and human heme oxygenase-1 (HMOX1)[32] conferred resistance against apoptotic and inflammatory stimuli in cellular and ex-vivo models. Other proposed strategies-including endothelial expression of T-cell modulatory molecules, ectonucleotidases (CD39/CD73), or tissue factor pathway inhibitor—remain at similarly early stages of validation[4].

The ten-gene-edited pig used in the first clinical case

The donor pig used in the landmark first-in-human cardiac XTx at the University of Maryland in January 2022 carried ten genetic modifications[14]: Knockouts of GGTA1, CMAH, and B4GALNT2 (eliminating the three carbohydrate xenoantigens described above), knockout of GHR (to limit xenograft growth), and transgenic expression of six human proteins-the complement-regulatory proteins hCD46 and hCD55, the coagulation-regulatory proteins hTBM and hEPCR, the anti-phagocytic protein hCD47, and the anti-inflammatory protein HMOX1. This genotype thus combined established edits with modifications whose individual contributions had not been isolated in large-animal cardiac studies.

Selection logic for gene-editing combinations: How many modifications are enough?

The optimal number and identity of edits remains an unresolved and actively debated question. On one side, the 10-gene Maryland donor demonstrated short-term clinical feasibility[14]; on the other, Reichart and colleagues have argued that donors with a minimal number of essential modifications should be preferred[33]. The arguments for parsimony are practical and regulatory: (1) Each modification requires its own evidence of safety and efficacy to satisfy regulatory review, and the evidentiary burden grows with every added locus; (2) Breeding strategies become progressively more complex and time-consuming as the number of segregating modified loci increases; (3) Interactions among multiple modifications-e.g., between transgenes affecting coagulation, inflammation, and innate immunity-are largely unpredictable and may be deleterious; and (4) Cost and development time constrain iterative optimization. Conversely, advocates of expanded editing note that residual barriers (innate cellular immunity, inflammation, coagulation) are mechanistically distinct and may not be addressable by a minimal genotype, and that incremental genetic modifications have shown additive survival benefit in NHP models[34]. Applicability and decision parameters for selecting an editing combination therefore include: The target organ and its dominant failure mode (for the heart, TM and humoral rejection); the intended immunosuppressive regimen; the recipient’s risk profile (e.g., sensitization status); the stage of the program (first-in-human proof of concept vs a standardized clinical-trial product); and the regulatory pathway. A comprehensive catalog of candidate modifications has been compiled by Reichart et al[4], but the field currently lacks head-to-head comparisons of donor genotypes under standardized conditions; defining a minimal sufficient genotype-and validating the contribution of each additional edit in NHP studies before clinical translation-should be considered a priority for future investigation.

DEVELOPMENT OF MANAGEMENT OF CARDIAC XTX RECIPIENTS

Genetic modification of the donor organ addresses only part of the barrier. Recipient management-immunosuppression, control of inflammation, and regulation of coagulation-remains decisive for xenograft survival, and most current protocols have been developed empirically in NHP models rather than in controlled studies. The three pillars are considered below, with attention to what is mechanistically plausible, what has been demonstrated in NHP survival studies, and what has actually been attempted in human recipients.

Immunosuppression

Protocols tested in pig-to-NHP cardiac XTx generally combine induction therapy-an anti-CD20 monoclonal antibody (B-cell depletion), anti-thymocyte globulin (T-cell depletion), and a costimulation-blocking antibody-with maintenance therapy comprising mycophenolate mofetil, corticosteroids, and, variably, calcineurin or mTOR inhibitors[26,33,35]. Regimens built on blockade of the CD40-CD154 costimulation pathway have proved the most effective to date: Using induction with anti-thymocyte globulin and anti-CD20 antibody followed by mycophenolate mofetil and an intensively dosed anti-CD40 antibody (2C10R4), Mohiuddin et al[25] achieved reproducible survival of GTKO.hCD46.hTBM cardiac xenografts in baboons beyond 900 days, without consumptive coagulopathy.

The theoretical importance of this pathway deserves emphasis. CD154 (CD40 ligand), expressed on activated CD4+ T cells, engages CD40 on B cells, dendritic cells, and macrophages, and this interaction is required for T cell-dependent B-cell activation, germinal-center formation, immunoglobulin class switching, and the generation of memory B cells and plasma cells. Blocking CD40-CD154 signaling therefore suppresses the elicited anti-pig antibody response at its origin rather than downstream-a property of particular value in XTx, where antibody-mediated rejection and its attendant coagulation activation are the dominant causes of graft loss. First-generation anti-CD154 antibodies were associated with thromboembolic complications in clinical trials, plausibly because CD154 is also expressed on activated platelets, where it contributes to thrombus stabilization; targeting the ligand thus carried an intrinsic prothrombotic liability. This experience motivated the switch to antibodies against CD40 itself (e.g., 2C10R4 and related agents), which interrupt the same signaling axis without directly engaging the platelet-expressed ligand. An additional practical advantage is that costimulation blockade is non-nephrotoxic: Unlike calcineurin inhibitors, which cause vasoconstrictive and cumulative renal injury, biologic costimulation blockers have no direct hemodynamic or tubular toxicity, an important consideration for recipients who often have marginal renal function. These potential advantages must be weighed against a decisive limitation: No anti-CD40 or anti-CD154 agent is currently approved by regulatory authorities for use in humans, and the agents used in NHP studies and in the 2022 clinical case remain investigational. This unresolved regulatory gap, and the infection-risk implications of intensive immunosuppression, are discussed further in the section on the dilemma in the field of cardiac XTx. Whether long-term xenograft survival will require indefinite costimulation blockade, and at what infectious and oncologic cost in humans, is unknown; the evidence base remains, at best, robust NHP data plus a single short-term human experience.

Anti-inflammatory management

Even under effective T cell-directed immunosuppression, recipients of GE pig hearts exhibit elevated pro-inflammatory cytokines and chemokines. Iwase et al[36] showed that systemic administration of combined anti-inflammatory agents and complement regulation can mitigate this systemic inflammatory response in xenograft recipients. Two caveats warrant emphasis. First, the inflammatory cascade after XTx is not a single pathway but an overlap of IRI at implantation, innate immune activation, and antibody-mediated endothelial injury; interventions directed at one component may leave others untouched, and dissecting their relative contributions experimentally is difficult. Second, most evidence derives from NHP studies with heterogeneous regimens, so the incremental value of any single agent is uncertain. Corticosteroids remain the pragmatic mainstay, but their long-term use conflicts with strategies to limit xenograft growth (as discussed in the growth control section). Targeted anti-cytokine therapy-for example, interleukin-6 blockade with agents such as tocilizumab, which is being explored in allo- and XTx settings-is a rational future direction but cannot yet be recommended on the basis of XTx-specific evidence. A priority for future work is the systematic, time-resolved characterization of the inflammatory response after cardiac XTx in both NHP and human recipients, so that anti-inflammatory therapy can be matched to defined molecular targets rather than applied empirically.

Anticoagulation management

Coagulation dysregulation after cardiac XTx manifests histologically as TM within the graft and systemically as consumptive coagulopathy, and it arises from two converging sources: Immune-mediated activation and injury of the porcine endothelium, and molecular incompatibilities across species[23,37]. The best-characterized incompatibilities include: (1) Porcine von Willebrand factor, which binds human platelet glycoprotein Ib spontaneously, even in the absence of shear stress, thereby promoting platelet adhesion and aggregation; (2) Porcine thrombomodulin, which binds human (and primate) thrombin but is an inefficient cofactor for activation of human protein C, crippling a central endogenous anticoagulant pathway; and (3) Porcine tissue factor pathway inhibitor, which poorly neutralizes human factor Xa[37]. This dual etiology explains why purely pharmacologic anticoagulation has been disappointing: In pig-to-baboon heterotopic cardiac XTx, neither warfarin nor low-molecular-weight heparin prolonged xenograft function[38], and intensified immunosuppression proved more effective than intensified anticoagulation in delaying TM, indicating that endothelial immune injury-not a primary hemostatic defect-is usually the initiating event.

Against this background, several more targeted strategies have been explored, and the evidence for each should be graded carefully. (1) Antithrombin III. Recombinant human antithrombin was administered (in combination with heparin) to baboon recipients of GTKO porcine hearts, yet TM still developed in association with humoral rejection[39]; in a pig-to-primate renal xenograft model, even high-dose recombinant human antithrombin failed to improve survival or prevent intra graft fibrin deposition and was associated with an increased bleeding tendency[40]. The antithrombin data therefore do not support routine systemic supplementation, although combination regimens have not been formally tested in cardiac models; (2) Activated protein C and the EPCR pathway. Because the thrombomodulin-protein C axis is the weakest cross-species link, transgenic compensation is mechanistically attractive: Donor-pig expression of hTBM restores protein C activation and independently prolongs cardiac xenograft survival[24], and hEPCR-which augments thrombomodulin-dependent protein C activation-is incorporated in current multi-transgenic donors. Notably, in-vitro data indicate that endogenous porcine EPCR is itself largely compatible with the human protein C pathway, suggesting that the principal benefit of hEPCR expression is amplification of an already functional axis rather than correction of an absolute incompatibility[41]; and (3) Target-specific (direct) oral anticoagulants. Direct thrombin and factor Xa inhibitors are theoretically appealing in this setting-oral administration, predictable pharmacokinetics, and no requirement for routine monitoring-but, to our knowledge, no published study has evaluated any direct oral anticoagulant in organ XTx, and their efficacy against an endothelial surface whose key regulators (thrombomodulin, tissue factor pathway inhibitor, von Willebrand factor) are species-mismatched cannot be extrapolated from human experience; this represents a concrete and testable research priority rather than an evidence-based option.

Taken together, the current consensus favors transgenic coagulation regulation in the donor organ as the primary strategy, with systemic anticoagulation relegated to an adjunctive role. The benefit-risk balance of sustained systemic anticoagulation-particularly the bleeding hazard in immunosuppressed, often thrombocytopenic recipients-has not been established in any model, and the occasional de-novo emergence of TM triggered by immunosuppressive drugs or acute antibody-mediated rejection[42] argues that anticoagulation cannot substitute for adequate control of the immune response. Defining which recipients, if any, derive net benefit from pharmacologic anticoagulation should be addressed in future NHP studies before clinical protocols are fixed.

TREATMENT OF PERIOPERATIVE CARDIAC XENOGRAFT DYSFUNCTION

Perioperative cardiac xenograft dysfunction (PCXD) denotes early, typically transient impairment of systolic function occurring in up to 40%-60% of experimental cardiac XTx, and it phenotypically mimics the myocardial stunning observed after clinical allotransplantation[43]. The porcine heart appears intrinsically more vulnerable than the primate heart to cardiopulmonary bypass and ischemia-reperfusion (I/R) injury[44], although the mechanistic basis-species-specific metabolic responses, denervation, the young age of donors, or a combination-is incompletely defined. Because PCXD can be lethal when the xenograft is life-supporting, preservation strategy is a genuine clinical decision point rather than a logistical detail.

To minimize I/R injury between explantation and implantation, Steen et al[45] developed a non-ischemic preservation system in which the explanted heart is continuously perfused at 8 °C with an oxygenated, hyperoncotic, albumin-containing cardioplegic solution supplemented with washed red blood cells (target hematocrit approximately 15%), metabolic substrates, hormones, and antibiotics. Under these conditions the cardioplegic-perfused porcine heart maintains a measurable but markedly reduced oxygen consumption (approximately 1.1 mL/minute/100 g at normothermia, falling by approximately 85% at 8 °C)[46], i.e., the heart is metabolically supported rather than arrested in ischemia. This system was an integral component of the Munich group’s consistent long-term success in life-supporting orthotopic pig-to-baboon XTx: Xenohearts were continuously perfused and oxygenated from explantation to transplantation, with intermittent perfusion during implantation until cross-clamp release, and non-ischemic preservation was judged beneficial for subsequent xenograft survival[47].

How does this approach compare with the systems available for clinical heart procurement? The SherpaPak Cardiac Transport System (Paragonix Technologies, MA, United States) provides standardized, monitored static hypothermic storage: Simple, portable, and cleared for clinical use, but ischemic by design and therefore unable to protect an I/R-sensitive xenoheart beyond limited cold ischemic times. The Organ Care System (TransMedics, Andover, MA, United States) maintains the heart in a warm, beating, blood-perfused state: Physiologically attractive and clinically validated in allotransplantation, but normothermic perfusion is technically demanding, resource-intensive, and itself associated with metabolic and hemolysis-related challenges. The Steen-type system occupies an intermediate position-cold enough to suppress metabolism by about 85%, yet continuously oxygenated and substrate-supplied to avoid ischemia-at the price of considerable circuit complexity. Decision parameters in practice therefore include: (1) The anticipated interval between explantation and implantation (short intervals with immediate implantation may tolerate simpler hypothermic storage; prolonged or logistically complex procurements favor continuous perfusion); (2) The vulnerability of the graft-for porcine xenohearts, the accumulated preclinical evidence currently favors continuous oxygenated perfusion whenever feasible[45,47]; (3) Institutional resources and expertise, since perfusion systems require dedicated personnel and fail safe only with experience; and (4) Regulatory availability, as none of these platforms is specifically approved for xenograft preservation. Important evidence gaps remain: PCXD has no standardized definition, its incidence in human recipients is unknown, no head-to-head comparison of preservation strategies exists for cardiac XTx, and reliable intraoperative predictors of PCXD (e.g., biomarkers or perfusion parameters) have not been validated. Standardizing definitions and comparing preservation modalities under controlled conditions should be a near-term research priority.

CONTROL OF GROWTH OF PORCINE XENOHEARTS FOLLOWING CARDIAC XTX

Intrinsic growth of the xenograft is a distinct, non-immune cause of post-transplant dysfunction. Because donor pigs are typically three to four weeks old at procurement-and thus in a rapid growth phase-to achieve approximate size matching with NHP recipients, the transplanted heart retains substantial growth potential driven by the intact somatotropic (growth hormone) axis of the young donor[48]. Excessive growth is not merely hypothetical: A doubling of cardiac mass with the evolution of a restrictive cardiomyopathy has been documented after experimental XTx[26]. Recipient hemodynamics may add a further stimulus, because systemic blood pressure in primate (and human) recipients substantially exceeds porcine physiological levels, imposing a chronic afterload mismatch on the graft.

Three countermeasures have been tested, each with a different applicability profile. (1) Blood-pressure management. Lowering recipient blood pressure toward porcine physiological levels attenuates the afterload-driven component of hypertrophy; it is immediately available and inexpensive, but it treats only one growth stimulus, may conflict with recipient perfusion requirements, and has no effect on intrinsic, growth-hormone-driven growth; (2) Pharmacologic growth restriction. In the Munich orthotopic model, early tapering of corticosteroids combined with the mTOR inhibitor temsirolimus (a sirolimus prodrug) successfully counteracted cardiac overgrowth[47]. This approach is clinically translatable-both drug classes are familiar to transplant physicians-but it carries the metabolic, wound-healing, and infectious liabilities of mTOR inhibition, its efficacy beyond the first post-transplant months is unproven, and mTOR signaling interfaces with immune regulation in ways that are incompletely understood in XTx; and (3) Genetic growth restriction. Donor pigs with GHR knockout exhibit intrinsically restrained organ growth; echocardiographic follow-up beyond six months confirmed reduced xenograft growth without growth-restricting adjuncts[26], and GHR-deficient pigs show no apparent deleterious effects on health or fertility, offering a single-step means of generating smaller, growth-limited donor lines[49]. The trade-offs are those of any added genetic modification-extended breeding, regulatory evidentiary burden per edit (as discussed in the genetic modification section)-and the fact that GHR-KO donors are smaller at procurement, which may shift rather than solve the size-matching problem for adult human recipients.

From a decision-making standpoint, pharmacologic and hemodynamic measures are the only options once a given donor heart has been transplanted, and are therefore the pragmatic default for current NHP experiments and any near-term clinical use of conventionally bred donors; genetic growth restriction is an upstream, potentially definitive solution but presupposes validated GHR-KO donor lines and is most attractive when donor size reduction is independently desirable. Critically, almost all evidence derives from NHP models with juvenile donors; whether clinically significant xenograft growth occurs in adult human recipients of hearts from older or genetically growth-restricted donors is unknown, and systematic echocardiographic and morphometric surveillance should be embedded in any future clinical protocol so that the choice among these strategies can eventually be evidence-based rather than empirical.

PREVENTION OF TRANSMISSION OF XENOZOONOSES

The possibility that porcine microorganisms could cause disease in xenograft recipients-collectively referred to as xenozoonoses-remains a central safety concern in XTx. PCMV a PRV, is of particular importance. Although PCMV does not productively infect human cells, its reactivation within the graft has been associated with rejection, consumptive coagulopathy, and early graft loss in pig-to-NHP kidney and orthotopic HTx[48]. The detection of PCMV/PRV in the first living human cardiac xenograft recipient, and its probable contribution to graft dysfunction, constituted one of the most consequential lessons of the University of Maryland case (analyzed in detail in the section on the clinical development of cardiac XTx) and has directly prompted an upgrading of microbiological surveillance standards for donor herds[14,15].

Current practice: Donor screening and biosecurity

The established-and currently the only clinically validated-approach to infectious safety relies on stringent husbandry and screening rather than on genetic or pharmacological intervention. Donor pigs are derived from closed herds maintained in DPF barrier facilities[13]. Most exogenous porcine microorganisms, including PCMV, can be eliminated from such herds through cesarean derivation of piglets, early weaning, colostrum diversion, vaccination, antiviral treatment where indicated, and strict isolation[48]. Complementing these measures, Denner[50] proposed comprehensive detection systems that standardize sample collection, preparation, origin, timing, and the inclusion of appropriate negative and positive controls, together with validated detection methods for each agent. Otabi et al[51] subsequently developed a panel of 76 highly sensitive PCR assays covering 41 viruses, one protozoan, and a broad range of bacteria, providing a practical template for release testing of donor animals. It should be emphasized that the effectiveness of this biosecurity strategy depends entirely on the completeness of the screening panel and the rigor of herd management; the Maryland experience illustrates that a single undetected agent can substantially alter clinical outcome[14,15].

Investigational strategies: Porcine endogenous retroviruses

Porcine endogenous retroviruses (PERVs) pose a distinct problem because, as germline-integrated elements, they cannot be removed by husbandry measures. PERV-A and PERV-B are polytropic and can infect human cells in vitro, whereas PERV-C is ecotropic and infects only pig cells[52]. Several strategies have been proposed to reduce the theoretical risk of PERV transmission: (1) Selecting donor pigs with low PERV expression or a PERV-C-negative genotype; (2) Vaccinating recipients before XTx; (3) Administering antiretroviral drugs guided by screening results; and (4) Inhibiting PERV expression by RNA interference[52]. More recently, pigs with genome-wide inactivation of PERVs have been generated using CRISPR/Cas9 technology[53]. All of these approaches remain investigational and must not be represented as established clinical practice: Multiplex genome editing is associated with off-target effects[54] and with reduced viability of the edited animals[46], and none of these interventions has been required to date, because no PERV transmission to recipients of porcine cells, tissues, or organs has been documented in preclinical or clinical studies[52]. A pragmatic reading of the evidence is that rigorous donor screening and DPF biosecurity-continuously refined in light of the PCMV/PRV lesson-constitute the present standard of care, whereas PERV-directed interventions remain a contingency should transmission ever be demonstrated.

CLINICAL PRACTICE IN CARDIAC XTX

Clinical evidence in cardiac XTx currently derives from two fundamentally different settings that should not be treated as equivalent: (1) Compassionate-use transplantation in living patients, which tests life-sustaining graft function and clinical management over weeks; and (2) Experiments in brain-dead (recently deceased) human recipients, which permit controlled, invasive physiological and immunological monitoring but are limited to short observation periods and cannot inform on long-term outcomes. Findings from the two models are complementary rather than interchangeable.

After receiving expanded-access (“compassionate use”) authorization from the United States Food and Drug Administration (FDA), the University of Maryland team performed the first genetically modified pig-to-human HTx in a 57-year-old patient with end-stage HF on January 7, 2022. The recipient had been declined for allotransplantation and durable mechanical circulatory support (MCS), and XTx represented his last viable option. A heart from a pig carrying ten genetic modifications, combined with an anti-CD40-based immunosuppressive regimen, sustained the patient’s life for 60 days[14]. The detailed clinical course-including PCMV/PRV detection in the graft, the temporal association between IVIG administration and rising anti-pig antibodies, and the development of diastolic dysfunction-is analyzed in the section on the clinical development of cardiac XTx and is not repeated here[14,15].

Subsequently, investigators at the New York University Langone Transplant Institute transplanted hearts from 10-gene-edited pigs into two brain-dead human recipients and monitored xenograft function, hemodynamics, and systemic responses over 66 hours. Although both xenografts demonstrated excellent cardiac function immediately after transplantation, cardiac function declined postoperatively in one case, an event attributed to size mismatch between the donor pig and the recipient. For both hearts, transgene expression was confirmed, and there was no evidence of cellular or antibody-mediated rejection on histology, flow cytometry, or cytotoxic crossmatch assays; nor was there any evidence of zoonotic transmission from the donor pigs to the human recipients during the observation period[18]. These findings demonstrate the short-term immunological feasibility of pig-to-human HTx in a controlled decedent model, but they cannot be extrapolated to predict outcomes in living recipients.

Beyond the individual case reports, the most instructive common denominator of current clinical practice is the near-universal reliance on blockade of the CD40-CD154 co-stimulation pathway. Mechanistically, this pathway is arguably the central node of the anti-xenograft immune response: CD154 expressed on activated CD4 T cells engages CD40 on B cells and antigen-presenting cells, thereby licensing T-cell-dependent antibody production, germinal-center reactions, and memory B-cell generation, while indirectly amplifying innate effectors such as macrophages and NK cells[25,35]. Consistent with this central role, NHP studies have shown that maintenance regimens anchored on anti-CD40 blockade achieve xenograft survival far exceeding that obtained with conventional immunosuppression alone[25]. The preference for anti-CD40 over anti-CD154 antibodies is historically contingent: Early clinical experience with anti-CD154 antibodies was halted by thromboembolic complications, prompting the development of antibodies directed against CD40, which-unlike CD154-is not expressed on activated platelets[35]. Nevertheless, near single-agent dependence on this pathway carries unresolved risks. Sustained co-stimulation blockade may predispose recipients to infection; the anti-CD40 antibodies administered to human recipients to date remain investigational agents that have not been approved by regulatory authorities; and their long-term availability, optimal dosing, and affordability outside research settings are uncertain. Diversifying maintenance immunosuppression beyond a single-pathway strategy should therefore be regarded as a priority for the field[14,25,35].

SELECTION OF CANDIDATES OF CARDIAC XTX

Given the large number of waitlisted patients at high risk of death and the continuing advances in gene-editing technology, Reichart proposed the compassionate implantation of genetically modified pig hearts in carefully selected candidates, arguing that consistent survival of up to six months in preclinical models, without irreversible rejection or infection, would justify progression to clinical trials. Under this framework, XTx could initially be offered as a bridge therapy-for example, for several months-followed by cardiac allotransplantation if clinically indicated. Early candidates might include patients who are poor candidates for MCS, such as those with hypertrophic cardiomyopathy, prior mechanical valve replacements, deteriorated aortic bioprostheses, or post-infarction ventricular septal defects, particularly when they face a high risk of death before an allograft becomes available because of unstable arrhythmias or escalating inotrope requirements, and especially in the presence of a high panel-reactive antibody (PRA) level[4]. Brenner similarly identified cardiac XTx as an option for patients who are highly sensitized after prior allotransplantation, those requiring biventricular mechanical support, patients with relative or absolute contraindications to allotransplantation, and those unsuitable for ventricular assist device therapy because of infection-related concerns or inability to undergo anticoagulation[48]. For some elderly patients with high PRA and risk factors for poor outcomes after allotransplantation-such as reoperative status, potentially reversible renal or hepatic dysfunction, or progressive debility primarily attributable to HF-XTx has also been discussed as a potential destination therapy[4].

This framework, although reasonable, requires critical qualification. The bridge-to-allotransplantation strategy rests on the assumption that xenograft exposure will not compromise subsequent allotransplantation. In vitro and baboon data suggest that sensitization to triple-knockout pig cells are unlikely to increase reactivity against human alloantigens, and thus may not be detrimental to subsequent allotransplantation[55]. However, this reassuring conclusion derives from mechanistic and preclinical evidence rather than from clinical experience: A failed xenograft bridge could still leave a critically ill patient sensitized to non-Gal porcine antigens, clinically deteriorated, and effectively excluded from both therapeutic arms. Candidate selection should therefore weigh the plausibility of rescue allotransplantation against the irreversibility of a failed bridge, and destination-therapy candidates should be clearly distinguished from bridge candidates in future protocols.

XTx may also be particularly relevant for pediatric patients. Children with complex congenital heart disease, especially single-ventricle physiology, face a high risk of death while awaiting a suitably sized human heart, and MCS options for small children remain limited. Cardiac XTx as a bridge therapy in pediatric patients appears ethically more defensible than its use as destination therapy, because the expected lifespan of the child greatly exceeds any plausible xenograft durability, making bridging the only proportionate objective; preclinical data further suggest a low risk of additional sensitization to alloantigens[55]. The favorable long-term outcomes of neonatal heart allotransplantation-nearly 60% of recipients alive with a functioning graft 25 years after surgery-nonetheless set a high benchmark that any pediatric xenograft bridge must be designed to preserve rather than jeopardize[56].

ETHICAL, REGULATORY, AND PUBLIC-HEALTH CONSIDERATIONS

Cardiac XTx raises ethical and public-health questions that extend beyond infectious safety, because its risks-however small-are borne not only by the recipient but also by close contacts and, potentially, the wider community[57,58]. These considerations should be integrated into candidate selection and trial design rather than treated as an afterthought.

First, the quality of informed consent obtained from patients with end-stage disease warrants particular scrutiny. Candidates for XTx are, by definition, patients who have exhausted conventional options and face imminent death; such circumstances may compromise the voluntariness of consent and foster a therapeutic misconception, in which an experimental intervention is perceived as established treatment. Consent processes must therefore convey explicitly the experimental nature of the procedure, the uncertainty of benefit, and the long-term obligations that follow[57,59]. Second, the eligibility criteria themselves must be transparent and equitable, so that access to this scarce and high-risk intervention is not determined by non-medical considerations[57].

Third, recipients assume an obligation to prolonged-potentially lifelong-surveillance, including serial clinical and microbiological sampling, archiving of specimens for retrospective analysis, and adherence to precautionary measures such as deferral of blood and tissue donation[58,60]. Ensuring long-term adherence to such surveillance, particularly after graft failure or explantation, is both an ethical and a logistical challenge that protocols must anticipate. Fourth, the potential public-health impact extends to household members, caregivers, and other close contacts, whose exposure risk, although low, exceeds that of the general public; their education, voluntary engagement in screening, and protection of their own autonomy require dedicated procedures[58,61]. Centralized or international data registries have been repeatedly recommended to aggregate outcomes and safety signals across the small number of cases worldwide, and the creation of such registries should accompany any transition to formal trials[58,62].

Fifth, animal-welfare obligations accompany the clinical use of purpose-bred donor pigs. Source animals should be bred and maintained in DPF barrier facilities under the highest standards of husbandry, and research involving them should adhere to the principles of replacement, reduction, and refinement, with the number and type of genetic modifications justified by demonstrated need[57].

Finally, the regulatory framework remains provisional. All human cardiac XTx procedures performed to date have proceeded under single-patient expanded-access (“compassionate use”) authorization by the FDA rather than within formal clinical trials. The FDA guidance on source animal, product, preclinical, and clinical issues concerning XTx products provides the principal regulatory reference for trial development in the United States[59], while the position paper of the International Xenotransplantation Association Ethics Committee defines conditions-including independent oversight, informed consent, and surveillance-under which clinical application is considered ethically acceptable[57]. At the international level, the World Health Organization consultations on regulatory requirements, most recently summarized in the 2018 Changsha Communiqué, call for effective national regulatory oversight coupled with international cooperation, including data sharing and registries[62], and infectious-disease surveillance frameworks provide the corresponding public-health infrastructure[58,60]. The transition from case-by-case compassionate-use authorizations to regulated, prospectively designed trials is therefore a central near-term task for the field.

CURRENT DILEMMAS IN THE FIELD OF CARDIAC XTX

The optimal number and combination of genetic modifications in donor pigs remains unresolved. Although a xenograft from a 10-gene-edited pig functioned well for the first 47 days in the Maryland recipient[14], donor pigs carrying a minimal number of essential modifications may ultimately be preferable, for reasons that include the regulatory burden of demonstrating the efficacy and safety of each individual modification, time and cost constraints, unpredictable interactions among modifications, the increasing complexity of breeding strategies with each additional locus, and the possibility of unforeseen negative effects[33]. Experience from China illustrates this trade-off within the global evidence landscape. In 2023, a 5-gene-edited pig-to-rhesus monkey heterotopic heart XTx experiment; the donor pig carried GGTA1 knockout, β4GALNT2 knockout, and transgenic human CD46, CD55, and thrombomodulin, and the recipient survived for 40 days with satisfactory function of both hearts and no hyperacute or acute rejection[63,64]. In a subsequent orthotopic experiment, an 8-gene-edited Bama miniature pig heart (GGTA1KO/B4GALNT2KO/CMAHKO/hCD55/hCD 59/hTBM/hEPCR/hEPO) supported a rhesus macaque for 46 days, until the animal died of acute myocardial infarction[65]. These single-case NHP studies are preliminary and their survival durations remain shorter than leading results from GTKO.hCD46.hTBM-based programs[25]; nevertheless, they make a distinctive contribution by testing deliberately simplified editing combinations, complementing the more complex 10-edit strategy used clinically in the United States. Rational, effective, and parsimonious gene-editing strategies for donor pigs therefore still require systematic exploration.

A second dilemma concerns immunosuppression and biosecurity. A clinically applicable, non-nephrotoxic regimen anchored on CD40-CD154 co-stimulation blockade has been proposed as the maintenance backbone of cardiac XTx, yet some of the agents involved are not approved by national regulatory authorities and others have not been tested in humans[48]. Because intensive immunosuppression amplifies any residual infectious risk, the potential pathogenic consequences of xenozoonotic infections-for recipients and for the wider community-require continued investigation[64]. The fundamental principle of biosecurity must not be compromised: Heterologous infectious diseases should be prevented rather than treated after their occurrence.

A third dilemma is whether durable, rejection-free xenograft survival can be achieved with clinically tolerable immunosuppression alone, or whether tolerance induction will ultimately be required. Two approaches discussed by Sykes and Sachs[35] are mixed hematopoietic chimerism and porcine thymic transplantation. Mixed chimerism can, in principle, prevent hyperacute, acute vascular, T-cell-mediated, and chronic rejection of primarily vascularized xenografts by tolerizing donor-reactive T cells, preventing induced antibody responses, and inhibiting T-cell-independent natural antibody-producing B cells[66]. In the pig-to-baboon model, vascularized thymic grafts supported reconstitution of recipient-type T cells at early time points and induced donor-specific T-cell unresponsiveness in vitro[67]. Platt et al[65] proposed that, if neonatal tolerance could be safely and effectively exploited, XTx might meet the challenge of severe cardiac failure in the newborn infant. These strategies, however, remain distant from clinical translation. Mixed chimerism requires conditioning regimens whose toxicity and intensity are difficult to justify in patients with end-stage HF; thymic transplantation adds surgical complexity and has so far demonstrated only early, predominantly in vitro evidence of donor-specific unresponsiveness[66,67]; and neonatal tolerance, while conceptually attractive for pediatric bridging, is unproven beyond experimental models[68]. Because of the technical complexity of these approaches, progress toward their clinical application has been limited in recent years, and they should be regarded as long-term objectives rather than imminent clinical options.

CONCLUSION

Cardiac XTx has advanced from speculation to early clinical reality, yet the distance between proof of concept and routine practice remains substantial. The principal knowledge gaps concern the mechanisms of late xenograft failure-including the diastolic dysfunction observed in the first living recipient-the prevention, prediction, and monitoring of TM, and the standardization of PCMV/PRV surveillance in donor herds and recipients. The immediate methodological and clinical challenges are equally concrete: Securing regulatory approval of an immunosuppressive regimen that is currently anchored on an investigational anti-CD40 antibody; defining the minimal, sufficient combination of donor genetic modifications; and simplifying organ-preservation systems to permit broader clinical deployment. Over the next three to five years, the field would benefit most from (1) Carefully designed, formally regulated clinical trials rather than further isolated compassionate-use cases; (2) Coordinated international registries capturing outcomes, complications, and long-term surveillance data; (3) Incremental progress toward tolerance induction, which remains the most credible route to durable, low-toxicity graft acceptance; and (4) Rigorous evaluation of pediatric bridging, where the ethical and clinical case may be strongest. Cardiac XTx is best described as a promising but still investigational therapy whose further development must proceed with scientific discipline and regulatory transparency.

ACKNOWLEDGEMENTS

Thanks to Jie Yan and Dengke Pan for their unremitting efforts in gene editing work.