Ксенотрансплантация сердца: кризис миновал или будущее уже наступило?


Постоянная нехватка донорских органов привела к возрождению интереса ксенотрансплантации (межвидовая трансплантация, в частности тканей свиньи человеку). Заметно возросло понимание причин отторжения ксенотрансплантата. В результате был разработан подход, который включает генетическую модификацию клеток свиньи и назначение иммуносупрессивной терапии реципиенту трансплантата. Применение (1) методов геномного редактирования для предотвращения повреждения вследствие врожденного иммунного ответа человека и (2) новых препаратов, ингибирующих костимуляцию Т-клеток CD40/CD154 - для предотвращения адаптивного иммунного ответа позволяет преодолеть многие известные барьеры на пути ксенотрансплантации. Трансплантация свиного сердца с множественными генетическими модификациями низшим приматам на фоне ингибирования у последних пути костимуляции Т-клеток CD40/CD154 привела к выживанию свиных сердец в течение многих лет после гетероскопических трансплантаций и в течение <9 мес после ортотопических трансплантаций. Это сделало возможным ортотопическую пересадку генетически модифицированного свиного сердца человеку. Впервые она была проведена в январе 2022 г. Реципиент умер от отторжения через 2 мес после операции, тем не менее, этот опыт позволил сделать важные выводы. В настоящее время существует большой интерес к проведению зарегистрированных клинических исследований ксенотрансплантации сердца в соответствии со строгими протоколами и нормативными указаниями.

Ключевые слова:редактирование генов; сердце; свинья; ксенотрансплантация

Финансирование. Исследование не имело спонсорской поддержки.
Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов.
Для цитирования: Азиз С., Азиз Д., Купер Д.К.С. Ксенотрансплантация сердца: кризис миновал или будущее уже наступило? // Клиническая и экспериментальная хирургия. Журнал имени академика Б.В. Петровского. 2023. Т. 11, № 1. С. 7-17. DOI: https://doi.org/10.33029/2308-1198-2023-11-1-7-17  (англ.)

The idea of changing body parts has fascinated humans since the beginning of time. Mythology includes creatures with body parts from different animal species, e.g., the chimera and lamassu.There is a story by Pien Ch’iao of a heart transplant between two Chinese soldiers, one of whom had a weak heart and a strong spirit and the other a strong heart and weak spirit. Then there is the legend of a leg transplant by the Saints Cosmas and Damian.

It was in the 20th century that contributions by many investigators, such as Alexis Carrel (Fig. 1), Charles Guthrie, Frank C. Mann, and Vladimir Demikhov (Fig. 2), began to make heart transplantation a possibility. Other great pioneers, such as Peter Medawar (Fig. 3), Billingham, Brent, Roy Calne (Fig. 4), an Thomas E. Starzl (Fig. 5), enhanced our understanding of the biological factors that resulted in rejection or tolerance, and of immunosuppressive therapy that made allotransplantation a clinical reality.

Success in the field of allotransplantation has been a stepwise process over many years. It was not until the late 1970s when Calne et al introduced the novel immunosuppressive agent, cyclosporine А (CsA), that ushered in the current era of widespread allotransplantation [1]. This amplified the problem of the chronic shortage of organs from deceased human donors, leading to a rekindled interest in xenotransplantation.

Cardiac allotransplantation

There has been a longstanding interest in transplanting the heart and lungs for end-stage heart and/or lung failure. Experimentally, Demikhov had shown the possibility of heart and heart-lung transplantation [2]. The modern era of cardiac surgery began under the leadership of Lillehei at the University of Minnesota. The first heart transplantation in man was a xenotransplant performed by James Hardy (Fig. 6) at the University of Mississippi using a chimpanzee heart [3]. Hardy also did the first allotransplant of the lung.

Norman Shumway (Fig. 7) and Richard Lower introduced the concept of using the left atrial anastomosis for avoiding the technically challenging individual four pulmonary vein anastomoses for implanting the donor heart [4]. Christiaan Barnard (Fig. 8) in Cape Town did the first successful human heart allotransplant in 1967 (using a deceased heart) [5]. Although many centers around the world followed suit, the long-term results were largely unsuccessful.

The introduction of CsA in experimental transplantation in Cambridge, UK, where Kostakis reported that CsA could prevent rejection of heterotopic heart transplants in rats [6] was soon followed by large animal solid organ transplants and eventually clinical abdominal organ transplantation [1]. Reitz et al at Stanford, using nonhuman primates (NHPs), demonstrated that CsA was able to provide long-term survival after cardiac and cardiopulmonary allotransplantation [7, 8]. This led to a rapid increase in clinical cardiac, pulmonary, and cardiopulmonary transplantation. Currently, cardiac allotransplantation provides excellent therapy for end-stage heart failure. Although we have not achieved the "holy grail" of tolerance, present immunosuppressive regimens have allowed excellent long-term survival [9].

Heart failure is currently a growing global epidemic [10]. Our ability to better treat acute coronary syndromes has led to an increasing number of patients who survive but eventually develop heart failure. Heart allotransplantation is effective therapy for end-stage heart failure. The shortage of donor organs has led to increasing disparity between supply and demand of hearts for transplantation. Although mechanical circulatory devices are available, they are still fraught with complications and have limitations [11]. This has led to interest anew in using animal organs (xenotransplantation) to fulfill the need for donor organs.

Some statements about xenotransplants from pioneers in transplantation

In 1907, Nobel Prizewinner Alexis Carrel wrote, “The ideal method would be to transplant in man organs of animals easy to secure and operate on, such as hogs (pigs), for instance. But it would in all probability be necessary to immunize organs of the hog against the human serum [12]. The future of transplantation of organs for therapeutic purposes depends on the feasibility of hetero (xeno) transplantation”. Carrel was correct as today, more than a 100 years later, we are genetically engineering the pig to protect its organs from the human immune response.

Another Nobelist, Sir Peter Medawar (1969), stated, “We should try to solve (the problem of organ transplantation) by using heterografts (xenografts) one day if we try hard enough, and maybe in less than 15 years” [13]. Clearly, even Nobel Prizewinners can get their predictions very wrong.

A more correct prediction was made by Sir Roy Calne (1995), who stated that, “Xenotransplantation is just around the corner, but it may be a very long corner” [14]. In contrast, Norman Shumway is reported to have pessimistically said, “Xenotransplantation is the future of transplantation, and always will be”.

Cardiac xenotransplantation in the modern era

In the cardiac arena, Hardy performed the first chimpanzee-to-human heart transplant in 1964 [3]. There was a significant size mismatch and the graft failed within 2 hours. In 1977 Barnard introduced the concept of heterotopic heart transplantation in the chest as a biological heart assist to support the failing heart [15]. In 1977, he used baboon and chimpanzee hearts to support two patients who could not be weaned from cardiopulmonary bypass after routine open heart operations, but without long-term success. Although some further cardiac xenotransplants were carried out, these were also unsuccessful. Bailey transplanted a baboon heart into Baby Fae with hypoplastic left heart syndrome and she survived for 20 days before succumbing to rejection [16].

Presently, the pig has emerged as the animal that will be the source of organs for transplantation. Over 100 million pigs are killed annually in the USA as sources of food. Although there are significant immunological barriers, there are also several advantages. These include sizeable litters, organ sizes that are compatible for humans, lack of widespread objection to the use of pigs by the public and society in general, and a large body of experimental work using pigs for cardiac xenotransplantation.

Immunological barriers

The major initial impediment to clinical pig-to-human xenotransplantation was hyperacute rejection, i.e., antibody-mediated rejection occurring within 24 hours [17]. Wild-type (genetically-unmodified) pig hearts transplanted into NHPs were rejected, usually within a matter of minutes, from hyperacute rejection [18]. This was due to the presence in humans and NHPs of preformed antibodies against the pig. It is believed that humans develop these antibodies as infants in the first year of life as a defensive response to colonization of the gastrointestinal tract to bacteria and viruses that express carbohydrates that are also expressed on pig cells [19]. Investigators have identified three carbohydrate antigens that are known to be targets for human anti-pig antibodies (see Table) [20]. Immunosuppressive agents used to prevent rejection of allotransplants do not prevent hyperacute rejection.

Although hyperacute rejection has largely been prevented by judicious gene-editing of the organ-source pig, if the immunosuppressive therapy administered to the recipient is not entirely effective, delayed antibody-mediated rejection, developing against other pig antigens, remains a barrier to successful xenotransplantation. Early techniques to prevent hyperacute rejection in NHPs utilized pre-transplant absorption of anti-pig antibodies or plasmapheresis, but resulted in xenograft survival of only a few days [21, 22]. These studies, however, established that antibody-dependent, complement-mediated cytotoxicity played an important role in xenograft rejection.

Our understanding of xenotransplantation rejection continues to increase. It is now apparent that this phenomenon is complex and involves a cycle of immune reactions involving both innate and adaptive immune systems, in which antibody, complement, macrophages, natural killer cells, and T-cells all play significant roles. T-cell activation is mediated via direct and indirect pathways. Through the indirect pathway, presentation of xenogeneic antigens by recipient cells leads to activation of CD4+ T-cells, leading to B-cell activation and the production of new antibodies [18].

Genetic engineering of pigs

Initially, it only proved possible to introduce a ‘protective’ human transgene into pig cells, but not to delete expression of a pig xenoantigen. Using this technique, David White (Fig. 9) and his colleagues [23] reported an orthotopic cardiac xenotransplant - between a pig expressing a human complement-regulatory protein, CD55 (decay accelerating factor; hDAF) and an adult baboon treated with a short course of cyclophosphamide and maintenance therapy with CsA. The baboon lived for 39 days.

In 1996, cloning became possible in large mammals [24]. With advances in genetic engineering, it became possible to “knockout” the gene for the enzyme that leads to the expression of Gal on the vascular endothelium and other cells of pigs, producing α-1,3-galactosyltransferase gene-knockout [GTKO] pigs (see Table). These GTKO pigs were born towards the end of 2003 [25, 26]. Investigators have since identified the presence of two other pig glycan antigens (see Table) [27, 28]. Estrada et al [29] showed that all three antigens can be eliminated by using CRISPR-Cas9 technology.

The use of genetically engineered pigs represents a major shift in transplantation whereby the ‘donor’ is modified prior to transplantation to make it more ‘compatible’ with the recipient. This is the first time in the 70 years or organ transplantation that the donor can be modified, rather than just the recipient being treated, a major conceptual change.

This excitement was soon dampened by a publication on the potential risks of the transplantation of pig organs containing porcine endogenous retroviruses (PERVs), which are, in fact, expressed in all pig cells [30].

Recent investigations have used pigs with 10 genes edits, all aimed at protecting the pig organ from the human immune response. Improving results in the pig-to-NHP model have led to a resurgence of interest in clinical xenotransplantation, exemplified by the recent compassionate use of a clinical pig heart transplant at the University of Maryland (Fig. 10) at Baltimore in 2022.

The role of CD40/154 costimulation pathway blockade

Buhler et al in 2000 in a model of pig hematopoietic progenitor cell transplantation in NHPs reported that calcineurin-based immunosuppressive therapy did not prevent the adaptive immune response [31, 39] but that blockade of the CD40/CD154 T-cell costimulation pathway was much more effective. This was recently confirmed by Yamamoto et al. [40], who reported a beneficial effect of anti-CD154 monoclonal antibody (mAb) therapy to the NHP recipient, usually in combination with rapamycin or MMF (mycophenolate mofetil). More recently, preliminary evidence has been reported suggesting that maintenance therapy with costimulation blockade alone may suffice [32].

Unfortunately, the initial anti-CD154mAbs that were used were thrombogenic [33, 34] were withdrawn. The development of Fc-modified anti-CD154 agents that are non-thrombogenic has recently been reported [35, 36]. These latter developments in costimulation agents are important as they form the cornerstone of immunosuppressive regimens currently used in xenotransplantation [37, 38].

Muhammad Mohiuddin (Fig. 11) et al. used an anti-CD40mAb that was not thrombogenic in a pig-to-baboon cardiac transplantation model. With additional genetic manipulation to prevent thrombo-dysregulation, they reported heterotopic heart xenograft survival of nearly 3 years [39, 40]. The same group used a humanized form of this anti-CD40 agent in their first xenograft implant in a patient [41].

Accumulating evidence suggests that anti-CD154 agents are more efficient than anti-CD40 agents in xenotransplantation [42, 43]. Blockade of the CD28/B7 costimulation pathway with the available agents seems to be less effective [44]. However, geneediting techniques have been used to enable the pig to produce CTLA4-Ig itself [45]. This was highly successful, but unfortunately resulted in excessive production of CTLA4-Ig, causing immunosuppression in the pig and leading to a high occurrence of infectious complications. Nevertheless, it is an approach that requires further exploration.

Coagulation abnormalities in NHPs with pig organs

Because of differences in the coagulation systems between pigs and primates, it was predicted that coagulation dysfunction would be seen in recipient NHPs with pig organs [18, 46]. These fears were confirmed in pig-to-NHP models by Kozlowski [47], and coagulation abnormalities still impact the success of xenotransplantation [48, 49]. The accumulation of platelets and fibrin in the pig graft results in a thrombotic microangiopathy (Fig. 12), resulting in a consumptive coagulopathy in the recipient [50, 51]. Importantly, this was still seen in regimens where anti-CD154mAb therapy was substituted with anti-CD40mAb therapy [52].

Therefore, one of the goals of genetic manipulation has been to regulate the coagulation system by creating source animals that express at least one human coagulation pathway regulatory protein, e.g., thrombomodulin (hTBM), tissue factor inhibitor (hTFPI), and/or endothelial protein C receptor (hEPCR) [53]. When such pigs were available, pig kidneys transplanted into NHPs showed an increased survival to approximately 8 months [52].

Systemic inflammation

A systemic inflammatory response to the pig graft in xenograft recipients (SIXR) may occur before activation of coagulation [88]. Indeed, SIXR may be an important factor in the development of dysregulation of coagulation and may also contribute to resistance to immunosuppressive therapy. Recipient activated innate immune cells that express tissue factor are increased irrespective of the of immunosuppressive therapy administered. In NHP recipients of pig organs, increased levels of C-reactive protein and other markers of inflammation are seen before the development of features of consumptive coagulopathy. The insertion of a human anti-inflammatory or “apoptotic” transgene into the pig has been reported [54, 55].

Other aspects of the donor pig heart during the xenotransplant process

The pig heart is sensitive to cold ischemia following explantation whilst it is being transported and transplanted into the recipient. Early investigators reported a phenomenon they termed ‘perioperative cardiac xenograft dysfunction’ (PCXD) that may be seen in the first 48 hours after orthotopic implantation of a pig heart in a NHP [56, 57]. This process may or may not be reversible within the first 2 weeks after transplantation. Histologic PCXD differs from what is seen with hyperacute rejection and includes a preserved myocardium but with some deposition of antibody. It has been suggested it is related to cardiac stunning and ischemia-reperfusion injury. The team at Munich used continuous non-ischemic perfusion of the heart to resolve this problem [56-59]. The team at the University of Maryland used this system in their clinical cardiac xenotransplant [41]. However, PCXD may be prevented if the cold ischemic period is short [60].

Pig heart function after xenotransplantation

Although considerable work has been carried out to successfully overcome the immunological barriers, only time will tell whether pig hearts will function well in the different physiological environment of humans. For example, differences in blood pressure between pigs and humans could impact xenograft hypertrophy or growth. Additionally, serum levels of cholesterol in humans are three times higher than in pigs and could theoretically impact the development of xenograft vasculopathy. Other uncertainties include the ability of the porcine heart to adapt to cardiometabolic stress during exercise and the effect of being in an upright versus horizontal position. It is encouraging to note that in NHP models, orthotopic pig cardiac xenotransplants have functioned well for almost 9 months until antibody-mediated rejection developed [61].

Growth of pig heart after transplantation of NHP

Many genetically-engineered pig lines for xenotransplantation use pigs that naturally experience rapid body and organ growth. This intrinsic growth pattern can adversely impact long-term xenograft function after orthotopic transplantation in NHPs [62]. Because growth hormone is a major stimulator of postnatal growth, growth hormone receptors have been knocked out in the pig (GHR-KO) to reduce the growth rate of the pig and its organs [63]. The pigs had normal birth weight with subsequent impairment of growth at 5 weeks and a 60% reduction in body weight by 6 months.

This approach reduced the growth of the pig organ after transplantation in NHPs [64]. Clinically, this strategy may prevent compression of the heart after orthotopic transplantation, particularly in pediatric patients [65, 66]. Rapid growth of a cardiac xenograft may result in diastolic cardiac failure, compressing the surrounding lungs, and causing pulmonary edema and hypoxia. Attempts to prevent this have included steroid therapy and temsirolimus, which blocks the mechanistic target of Rapamycin signaling pathway and inhibits growth hormone [62, 66].

Monitoring for xenograft rejection

There remains a lack of standardization among methods to diagnose and predict xenograft rejection. Measures to monitor rejection in cardiac allografts have included echocardiography, measurement of troponin, endomyocardial biopsies, flow crossmatching and use of genomic markers, etc. Similar methods are being explored in experimental cardiac xenotransplantation. A rise in serum anti-pig antibodies may not always be seen, as the antibody may be adsorbed on to the pig xenograft.

It is important to develop noninvasive markers of rejection in experimental and clinical xenotransplantation. Circulating DNA is released upon cell death or apoptosis, and may indicate rejection in xenotransplantation [67]. The release of circulating pig-specific DNA (cpsDNA) reflects the infiltration of immune cells in the graft and precedes the production of anti-pig IgM/IgG antibodies in pig-to-mouse models. Likewise, cell-free DNA (cfDNA) levels also correlate with tissue injury in xenograft models [68]. While data regarding organ-specific microRNAs (miRNA) in xenotransplantation remain limited, they have shown promising usage as biomarkers of rejection [69].

Hopefully commercial kits will be developed using multiplex detection of porcine markers using xMAP technology to simultaneously detect porcine cytokines, chemokines, complement activation markers, growth factors, and markers of cardiac injury.

Potential infectious complications

The current opinion of experts in the infectious complications that occur in immunosuppressed patients with allografts is that the incidence and nature of these complications is likely to be similar after xenotransplantation [70]. The organ-source pigs will be bred and housed in biosecure environmentally-controlled conditions, and there will be frequent screening for infectious agents and archiving of blood and tissue samples prior to organ excision and transplantation. Organ-source pigs should be known to be free of all relevant pathogenic microorganisms, e.g., cytomegalovirus, before the organ is transplanted (although the inadvertent presence of porcine CMV in the graft may have contributed to graft failure in the University of Maryland patient [71, 72]. Indeed, from an infection perspective, designated pathogen-free pigs should be greatly preferable sources of organs than most deceased human donors.

The single topic that has given concern over the past 30 years is the presence of porcine endogenous retroviruses (PERVs) within the genome of every pig cell, and which therefore will inevitably be transferred to the recipient with the organ [73]. Although humans have similar species-specific viruses and virus particles in every cell (which do not appear to be pathogenic in the hosts), the question was raised of whether PERVs (which are not pathogenic in pigs) will be pathogenic and detrimental to the health of the human recipient. In the 1990s, many ethical questions were raised, and it was suggested that a moratorium should be placed on xenotransplantation. As a result, the British bioengineering company, Imutran (a subsidiary of the Swiss pharmaceutical company, Novartis) closed its xenotransplantation research program.

Since then there has been no evidence that humans exposed to pig tissues, e.g., spleens, skin, etc., or immunosuppressed NHPs with pig organ grafts have experienced any complications from PERV. However, although expert opinion is that the risk is low, a conclusive answer will not be known until clinical trials with long-term follow-up take place. To resolve this potential risk, genetic engineering has been used to inactivate the PERVs [74], and the development of CRISPR technology enabled PERV to be deleted from the pig genome [75], thus negating these fears [76].

Selection of patients for initial clinical trials of cardiac xenotransplantation

It was transplant pioneer, Thomas Starzl, who stated, “History tells us that procedures that were inconceivable yesterday, and are barely achievable today, often become routine tomorrow”. In the coming months, if US Food and Drug Administration approval can be obtained (on compassionate grounds), further xenotransplants using pig hearts are likely to be carried out in patients for whom a cardiac allograft cannot be obtained and in whom a mechanical ventricular assist device is contraindicated [77]. This could include patients with a restrictive or hypertrophic cardiomyopathy, patients with a mechanical cardiac valve prosthesis, or with refractory ventricular dysrhythmias, and patients in whom an allotransplant fails and a retransplant is needed.

There are data suggesting that a small number of highly-allosensitized patients (<5%) might be at a disadvantage if undergoing pig organ transplantation [78, 79], because there is some cross-reactivity between anti-HLA antibodies and swine leukocyte antigens. However, if a crossmatch between human serum and pig cells is negative, a pig graft should be successful. Ultimately, patients highly sensitized to HLA are likely to be among those who benefit most from the opportunities offered by xenotransplantation.

Another area of intense interest is infants with complex congenital heart disease, e.g., single ventricle pathology, where mechanical support devices have a poor record. Here, a pig xenograft may act as a bridge until a suitable human heart becomes available [60, 80].

The less mature infant immune system, as evidenced by excellent results in neonates of cardiac allografts across the ABO blood group barrier [81], might be more accepting of the xenograft, particularly as a total thymectomy is carried out at the time of heart transplantation in the age group.

The future of clinical xenotransplantation

Studies of pig organ transplantation into brain-dead human subjects have provided evidence for short term survival (<3 days) of pig organ transplants without the development of hyperacute rejection, but these short-term models cannot provide information on longer-term follow-up [82, 83].

The ultimate goal of transplantation, be it allo or xeno, is to achieve the holy grail of donor-specific tolerance. There is limited experimental evidence to suggest that the induction of mixed hematopoietic chimerism or thymic transplantation may eventually be successful, though not in the immediate future [84-87].

We have come a long way since the first successful cardiac allotransplantation by Barnard in 1967. Better management of acute coronary syndromes has increased the numbers of patients with heart failure. Xenotransplantation is a solution for the growing shortage of donor allografts for end-stage heart failure. Advances in genetic engineering and newer immunomodulatory agents that impact costimulatory pathways have opened the door to a new era in xenotransplantation.

We still have hurdles to overcome, but the future looks bright for xenotransplantation. It is wise to recall Tom Starzl who stated “History tells us that procedures that were inconceivable yesterday, and are barely achievable today, often become routine tomorrow”. It is likely that more pig cardiac xenotransplants will be carried out on compassionate grounds in 2023 as we “turn the very long corner” predicted by the great transplant pioneer, Sir Roy Calne.


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Доктор медицинских наук, профессор (Москва, Россия)

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