LIVER TRANSPLANTATION: WILL XENOTRANSPLANTATION BE THE ANSWER TO THE DONOR ORGAN SHORTAGE?

Abstract

Since the first report of a successful liver transplant in 1968, access to this operation has dramatically improved. In 2018, 8,250 patients underwent liver transplantation in the United States. Despite this remarkable advance, a persistent shortage of donor organs remains the primary obstacle to optimal utilization of this life-saving operation.

Over the past two decades, transplant professionals have pursued two broad strategies to overcome this roadblock: increasing the number of donor organs and decreasing the number of patients requiring transplantation through advances in medical interventions.

Despite these efforts, more than 13,500 patients remained on liver transplant waiting lists at the end of 2018. Almost 1,200 died while waiting, and 1,350 were removed from wait lists because they had become too sick to survive the operation. Clearly, a dramatic new approach to the donor organ shortage is needed.

One effort, first attempted by surgeons in the 1960s, was to utilize donor organs from other species (xenotransplantation). The major obstacle to xenotransplantation acceptance has been the fear of transmitting new infectious diseases from animals to humans. As the twentieth century came to a close, national moratoria on xenotransplantation ended both research and clinical activities in this field.

The recent discoveries that modern gene-editing techniques can be used to eliminate the retrovirus that is ubiquitous in pigs and that retrovirus-free pigs can be cloned has reopened the possibility that xenotransplantation may be a potentially game-changing approach to eliminating the donor shortage for liver and other solid organ transplant recipients.

In response to these advances, the FDA has released comprehensive industry guidelines regarding all aspects of xenotransplantation. This release has resulted in numerous preclinical studies in which organs from genetically modified pigs are transplanted into various nonhuman primates (NHPs). Use of a variety of gene-editing and immunosuppressive techniques has greatly increased the survival of recipient animals in the past few years.

Survival of NHP renal transplant recipients has been extended to 435 days, functional cardiac transplant recipients to 195 days, and liver transplant recipients to 29 days. Current research studies using various gene modification strategies combined with newer immunosuppressive protocols are attempting to further extend the survival of these experimental animals.

These encouraging results have raised the possibility that clinical xenotransplantation in humans is just beyond the horizon. The most likely candidates for initial clinical studies probably will be kidney transplant recipients who are difficult to crossmatch for human organs, neonates with severe congenital heart disease, and liver transplant candidates with acute liver failure.

INTRODUCTION

Liver disease accounts for an estimated 2 million deaths per year worldwide—one million from cirrhosis and another million from viral hepatitis and hepatocellular carcinoma (1). From 1999 to 2016, annual deaths in the United States from cirrhosis increased by 65%, to more than 34,000, while annual deaths from hepatocellular carcinoma doubled to greater than 11,000 (2). Liver disease accounts for an estimated 200,000 hospitalizations annually (3).

For patients with decompensated cirrhosis and most patients with hepatocellular carcinoma, liver transplantation remains the only effective treatment option.

Thomas Starzl reported the first extended survival following this operation in 1968 (4). Over the next 15 years, his team steadily improved both the surgical technique and immunosuppression related to the procedure. In 1983, the National Institutes of Health (NIH) held a Consensus Development Conference, in which the results of all transplants performed to that date were critically reviewed. Based on this evidence, the panel concluded that liver transplantation was a therapeutic modality for end-stage liver disease that deserved broader application (5). This led to a dramatic increase in U.S. liver transplant centers, and the number of liver transplants increased from 1,713 in 1988 to a record of 8,250 in 2018. Despite this remarkable improvement in access to the operation, almost 14,000 patients remained on waiting lists at U.S. liver transplant centers at the end of 2018.

The past two decades have shown that donor organ shortage is the greatest obstacle to optimal utilization of liver transplantation. In response to this challenge, transplant surgeons and physicians have sought to increase the availability of donor organs and to decrease the need for transplantation with advances in medical therapy.

STRATEGIES TO INCREASE DONOR ORGAN AVAILABILITY

Three approaches have been taken to increase the availability of donor organs:

1. Expanding the criteria for brain-dead donors,

2. Increasing the use of organs from donors who die after cardiac death, and

3. Optimizing the use of living related transplants.

A clear definition of “brain death” was first proposed by a Harvard ad hoc committee in 1968 (6). “Brain death” subsequently was incorporated into the Uniform Determination of Death Act (7) and has been codified into the laws of every state. As a result, legally declared brain-dead individuals have become the primary source of liver donors in the United States.

A concerted effort has been made over the past 30 years to expand the criteria for brain-dead donors primarily by expanding the acceptable age of donors. In 1988, only 2.4% of liver donors were older than 50 years of age. In contrast, 33% of organ donors were older than 50 in 2018, and almost 10% were 65 years of age or older (8). Ongoing efforts are being taken to further expand the use of donors over 70 years of age by carefully matching them to ideal recipients; however, this is unlikely to have a major impact on the number of donor organs (9).

As the shortage of organs has become more acute, there also has been increased emphasis on donation after cardiac death (DCD) in individuals with severe brain injuries who do not meet the criteria for brain death (10). Formal guidelines for DCD were finalized by the United Network for Organ Sharing (UNOS) in 2007 (11). Despite the active pursuit of DCD by many transplant centers, DCD donors accounted for fewer than 10% of liver donors in 2018.

Living donor liver transplantation (LDLT) is another approach to expanding organ availability. This procedure has become standard practice in pediatric transplantation because of the low perioperative risk to the donor and excellent post-operation outcomes. Because adults require a larger functional mass of liver tissue for a successful outcome, most transplant centers perform adult-to-adult right lobe or, less commonly, left lobe LDLT. In contrast to pediatric LDLT, adult LDLT poses a clear risk of morbidity and mortality to the donor. The number of adult procedures performed in the United States peaked at 524 in 2001, declined thereafter, and accounted for only 402 transplants in 2018. These low numbers reflect continuing concerns over donor safety, as well as a very low acceptance rate among potential donors (12,13).

MEDICAL INTERVENTIONS TO REDUCE THE NEED FOR TRANSPLANTS

A complementary approach to transplantation has been to reduce the number of patients requiring transplantation through advances in medical therapy. The three causes of end-stage liver disease in which this has been most effective are primary biliary cholangitis (PBC), chronic hepatitis B, and chronic hepatitis C.

PBC was the leading indication for liver transplantation in the United States in the 1980s (14). Treatment with ursodeoxycholic acid, which was shown to improve survival even in patients with decompensated disease, was approved by the FDA in 1998 (15). With the widespread use of this agent, the need for liver transplantation for patients with PBC began to decline; by 2009, patients with PBC accounted for only 3% of U.S. transplants (16,17).

The advent of oral antiviral agents also has reduced the number of patients with chronic hepatitis B (HBV) who are referred for transplantation (18). In addition, the ability to control HBV in liver transplant candidates has virtually eliminated the need for transplantation in patients with decompensated cirrhosis. As a result, most patients with hepatitis B now have transplants for hepatocellular carcinoma rather than decompensated cirrhosis (19,20).

Chronic hepatitis C has been the leading indication for liver transplantation in the United States for the past two decades. The approval of numerous direct-acting antivirals (DAAs) has dramatically reduced the need for transplantation for patients with cirrhosis due to this chronic viral infection. The number of patients with decompensated cirrhosis listed for transplantation has decreased by more than 30% (21,22). Furthermore, the risk of dying while on the waiting list has decreased by more than 20% (23).

CURRENT STATUS OF LIVER TRANSPLANTATION

The number of potential liver transplant recipients has continued to grow at an alarming rate in the United States. As a result, the demand for donor organs has never been higher. Almost 14,000 patients remained on liver transplant waiting lists at the end of 2018, a total of 1,187 died during the year while on waiting lists, and an additional 1,343 were removed from waiting lists because they had progressed to the point of being “too sick for a transplant.”Indications for the operation have significantly shifted as well. Since 2015, alcoholic liver disease and nonalcoholic steatohepatitis (NASH) have replaced hepatitis C as the two leading indications for transplantation in the United States (22,24). The prevalence of alcohol use, high-risk drinking, and alcohol use disorders are on the rise across all U.S. sociodemographic groups, and treatment for patients with these disorders is vastly underutilized (25). NASH also has become one of the leading causes of cirrhosis and hepatocellular carcinoma in the United States. Other than lifestyle modification through diet and exercise, no approved treatments are available for this condition (26). Because of the limited treatment options for these conditions, the number of U.S. patients requiring transplantation can be expected to increase in the decades to come.

THE POTENTIAL ROLE OF XENOTRANSPLANTATION

If patients with end-stage liver disease or hepatocellular carcinoma are to have a reasonable chance of undergoing liver transplantation, there must be a dramatic increase in donor organs.

One possible source for these organs could from other species (xenotransplantation). This is not a new idea. Between 1966 and 1993, a total of 12 hepatic xenografts were performed. The donor animals included baboons (7), chimpanzees (4), and one pig. The longest survival following these operations was 70 days (27).

Xenotransplantation has many advantages. The supply of donor organs would be unlimited, the organs would be available electively, and genetic modification of the donor to enhance acceptance of the graft could be planned (28).

The major concern regarding xenotransplantation has been the potential for transmission of asymptomatic xenogenic infections to another species. This problematic outcome caused the FDA to prohibit human transplantation using primate organs in 1999 (29).

Because of the many disadvantages of using nonhuman primates as organ donors, pigs have become the animals most likely to solve the donor organ shortage (30). They have a short gestation period, grow rapidly, and reach a size suitable for adult transplantation in three to six months (31). Furthermore, pigs are plentiful; their heart, kidneys, and liver are anatomically similar to those of humans; and they can be cloned (32).

Two major concerns have emerged regarding the use of pigs as organ donors: the risk of rapid rejection of the graft and the potential for transmitting xenogeneic infections.

Transplantation of organs from wild type pigs into nonhuman primates or humans results in a rapid antibody-dependent, complement-mediated hyperacute rejection similar to that seen when ABO-incompatible allografts are transplanted between humans. The clinical evolution and histologic features of this type of rejection suggest that it is mediated by the action of preformed antibodies and complement on unique epitopes found on the vascular endothelium of pig. The major donor epitopes responsible for this type of rejection have been found to be carbohydrate moieties on porcine cells that have been lost with evolution from the pig to primates and humans (33).

A much bigger concern has been the fear of transplanting endogenous retroviruses from pigs to humans. Porcine endogenous retroviruses (PERVs) have been transmitted from pig cell lines to human cells (34). The immunosuppressed condition of the xenotransplant recipient could allow rapid and persistent replication of these retroviruses that might lead to lethal immunosuppression and tumorigenesis and even spread to the normal population. These concerns led to a call for a moratorium on xenotransplantation using pigs as organ donors at the end of the twentieth century (35).

This moratorium effectively ended research and clinical activities in xenotransplantation for over 15 years.

THE IMPACT OF MODERN GENE-EDITING TECHNIQUES

The development of modern gene-editing techniques has revitalized the field of xenotransplantation. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has become the platform of choice because it is efficient, versatile, easy to use, and relatively inexpensive (36).

In a landmark publication, George Church's team at Harvard, in collaboration with scientists in China, was able to completely eradicate PERVs from an epithelial kidney cell line using two CRISPR-Cas9 guide RNAs that targeted the catalytic center of the pol gene on PERVs. The product of this gene functions as a reverse transcriptase that is essential for PERV replication and infection (37).

In a follow-up publication two years later, his group reported the cloning of the first PERV free pigs using somatic cell nuclear transfer. This was accomplished by inactivating PERV activities in a porcine fetal fibroblast cell line. These cells were used to produce PERV-inactivated embryos. These embryos were then transferred into surrogate sows to produce PERV-inactivated fetuses and live pigs (38).

Although the risk posed to humans by PERVs remains controversial (39), these two publications eliminated the major public obstacle to xenotransplantation.

CURRENT STATUS OF XENOTRANSPLANTATION

Efforts at bringing xenotransplantation to clinical practice now are focused on two broad areas: clarification of regulatory guidelines and scientific studies of transplants from pigs to nonhuman primates.

REGULATORY GUIDELINES

In December 2016, the FDA published detailed industry guidance entitled “Issues Concerning the Use of Xenotransplantation Products in Humans.” This very progressive document outlined the details of donor animal welfare, screening of potential donor animals for pathogens, surveillance of donors and recipients, and the preclinical efficacy studies required prior to approval of clinical trials (40).

PIG TO NONHUMAN PRIMATE TRANSPLANTS

The classical preclinical model of xenotransplantation consists of transplanting organs from genetically modified pigs into nonhuman primates (NHP). Before the development of modern gene-editing techniques, breeding pigs with a single-gene knockout could take up to three years in expensive and often wasteful breeding programs (41). Gene editing using CRISPR has revolutionized this process.

Using CRISPR, it is possible to simultaneously modify multiple genes in a process that takes only a few months (41). The CRISPR components can be transfected into cultured porcine cells that are subsequently used to clone pigs by somatic cell nuclear transfer, or they can be injected as mRNA into zygotes that can be subsequently transferred into foster mothers (42).

The first step in the development of modern pig to nonhuman primate models was to eliminate the porcine genes that code for the glycoproteins and glycolipids associated with early antibody mediated rejection. The three moieties most clearly associated with this process included galactose-α1,3-galactose (αGal), Nglycolylneuraminic acid (Neu5Gc), and an Sd_a-like structure. CRISPR-Cas9 has been essential to the generation of pigs with single, double, or even triple knockout (KO) pigs in which the genes encoding these moieties have been eliminated (43,44). Use of organs from these genetically modified pigs has dramatically prolonged the survival of various NHP transplant recipients.

Subsequent studies in NHP models revealed the importance of the complement pathway in rapid rejection of xenografts. When the complement pathway is activated in humans, it does not result in tissue injury because of the presence of complement regulatory proteins (CRPS) on the surface of vascular endothelial cells. Pigs have similar CRPS, but these are limited in their efficiency in protecting pig cells from human complement-mediated injury. This knowledge led to the concept that pigs in which human CRPS have been genetically introduced (knockins) could enhance the survival of NHP transplant recipients. Other genetic modifications have included the insertion of immunomodulatory and thromboregulatory genes and deletion of growth hormone to slow the excessive growth of transplanted organs (45-47).

There have been major breakthroughs in the survival of pig to NHP renal and cardiac transplants recently. By combining genetic modification of donor pigs, screening recipients for preformed antibodies, and providing adequate immunosuppression, the survival of pig to NHP kidney recipients has been extended to 435 days (48). In similar fashion, using hearts from pigs that have been genetically modified to eliminate expression of galactose-α1,3-galactose and to express the human complement regulatory protein CD46 and the coagulation-regulatory protein thrombomodulin has been the key to avoiding early xenograft rejection. Baboons with transplanted hearts from these animals have now survived up to 195 days (49).

From 1968 to 2012, pig to NHP liver transplant survival was limited to nine days. In addition to hyperacute rejection, the obstacles unique to liver transplantation have included severe thrombocytopenia, profound coagulopathy, thrombotic microangiopathy, and lethal bleeding. These problems have been overcome recently by the use of α1,3-galactosyltransferase gene knockout pig donors, continuous infusion of a human prothrombin concentrate complex, and immunosuppression including costimulation blockade. This regimen has resulted in control of the often fatal coagulation disturbances and survival of recipient baboons for almost a month (50).

POTENTIAL CLINICAL APPLICATIONS OF XENOTRANSPLANTATION

The clinical application of xenotransplantation in the near future will most likely focus on three areas: renal transplantation, cardiac transplantation for neonates with congenital heart disease, and hepatic xenotransplants as a bridge to transplantation for patients with acute liver failure.

RENAL TRANSPLANTATION

Kidney transplantation offers better survival and quality of life for patients with end-stage renal disease (ESRD) than chronic dialysis (51). However, more than 95,000 Americans are on waiting lists for kidney transplantation, and over 18,000 of these have pre-formed antibodies against human donor HLA antigens that place them at a high risk for developing hyperacute or accelerated acute antibody-mediated rejection. Finding a kidney donor with an acceptable crossmatch for these patients is very difficult. Because of extended waiting periods, many of these patients die before an acceptable donor organ becomes available.

The prospects for xenotransplantation in these patients have improved dramatically in the past few years. In a recent study, 30% of sera from patients on a transplant waiting list showed negative crossmatches against tissues from triple KO pigs (52). Additional gene editing will be required for most patients; however, increased understanding of the xeroantigens involved and existing gene-editing techniques raises the possibility that immunologically acceptable donor pigs could be created for most, if not all, patients on renal transplant waiting lists (53).

CARDIAC TRANSPLANTATION

Hypoplastic left heart syndrome (HLHS), which is characterized by critical underdevelopment of the left heart structures, is uniformly fatal without medical intervention. In the 1980s, survival after heart transplantation was significantly better than after staged surgical procedures. As a result, HLHS was the leading indication for infant heart transplantation. However, by the mid-1990s, fewer than 100 neonatal cardiac donor offers were occurring annually and wait list mortality had become prohibitive at most transplant centers. The high wait list mortality, combined with improved results from staged surgical procedures, resulted in HLHS being the indication for fewer than 20% of infants listed for heart transplantation by 2006 (54).

Despite tremendous progress over the past 30 years, the Fontan procedure, the final stage of surgical palliation for HLHS, leaves the patient with a systemic right ventricle and a number of complications including arrhythmias, pulmonary and systemic thrombi, hepatic dysfunction that can progress to cirrhosis and hepatocellular carcinoma, and protein-losing enteropathy (55,56).

In comparison, neonates under 30 days old who undergo cardiac transplantation for HLHS have a 59% 25-year survival rate; however, these remarkable results exclude wait list mortality, which accounts for up to 63% of first-year mortality in HLHS infants selected for transplantation (57,58). The lack of donor availability clearly has been the overriding factor in the preference for staged palliation as primary therapy for HLHS over the past 20 years.

In many ways, infants with HLHS are the most attractive candidates for xenotransplantation because they typically do not develop antibodies to pig glycoproteins during the first three months of life. Sera from neonates have minimal reactions to tissues from triple knockout pigs (57). By including pig thymus transplantation after removal of the recipient's thymus, which is typically performed at the time of cardiac transplantation, immunosuppression could be discontinued within the first few years after xenotransplantation for HLHS in neonates (57).

LIVER TRANSPLANTATION

Acute liver failure (ALF) is a dramatic clinical syndrome marked by the sudden loss of hepatic function in individuals with no prior history of liver disease. There are no treatments of proven benefit except for emergency liver transplantation. Given the rapid disease progression in these patients, more than half of those listed for transplantation die before a donor organ can be found (59). Those who die waiting for transplantation spend an average of three days on the transplant wait list prior to their death, highlighting the critical shortage of organs as a major factor in their failure to survive (60).

In contrast to patients with renal or cardiac failure, where dialysis and cardiac assist devices can prolong survival in those waiting for transplantation, there is no evidence that any artificial support system reduces mortality in ALF patients (61).

Because of these various factors, the clearest indication for clinical trials of hepatic xenotransplantation is as a bridge to transplantation in critically ill ALF patients (62).

Due to the relatively short survival time in current hepatic pig to nonhuman primate models, liver xenotransplantation will not likely become a destination therapy for patients with chronic liver disease in the foreseeable future. Whether a pig liver can provide sufficient metabolic support for patients in the long term remains to be seen (63). The problems of thrombocytopenia and coagulopathy will require further genetic engineering of donor pigs before any clinical trial can be contemplated (64).

As discouraging as this may be to hepatologists, we should continue to hope as Thomas Starzl did when he wrote: “History tells us that procedures which were inconceivable yesterday, and are barely achievable today, often become routine tomorrow” (65).

DISCUSSION

Vierling, Houston: Bob, that was terrific, and I applaud what you have summarized for us. I have one comment and one question. The comment is that in the backdrop of those dying on the waiting list, we had the luxury of transplanting record numbers of livers over the past three years. Never before have over 8,000 liver transplants been performed in a year, but we did so on the back of the increase in anoxic brain death due to the ongoing opioid epidemic. This is something we, in society, must solve. When we do, we are going to be in a deep hole of disparity between those waiting for a transplant and the available donors. So this is a very timely subject. The question I have relates to the issues of immunology and rejection. You mentioned the high porcine reactive antibodies (PRAs) in patients needing renal transplants that may be theoretically overcome. We don't have HLA equivalent here, there is a major dis-compatibility, and we probably don't have the direct LO-reaction against a pig, but if we did, we could probably engineer it out. This leaves us with perhaps a different paradigm and I'm wondering whether you've thought ahead or looked at what has been done in primates to understand what will be the concept of immunosuppression for long-term survival of the allograft.

Carithers, Seattle: Potential renal transplant recipients with high PRAs can wait for 5 to 10 years for a transplant. In reality, most die while on dialysis waiting for a transplant. Clearly, there is an urgent need for a new source of donor organs. The development of triple knockout (TKO) pigs in which the major porcine glycan epitopes have been eliminated by gene editing has been a major step in this direction. Recent studies showing that 30% of high-PRA patients awaiting renal transplantation have no cross-reactivity to these pigs are very encouraging. For the remaining 70% of high-PRA patients, additional genetic modification of the pigs will almost certainly be required. The epitopes of greatest interest currently are the swine leukocyte antigens (SLAs), which are structurally similar to human leukocyte antigens (HLAs) and have 70% sequence identities with HLAs. Studies in which human serum is mixed with TKO pig peripheral blood mononuclear cells and then bound to HLA beads have shown that patients with anti-HLA-A antibodies are likely to cross-react with class I SLA and that anti-HLA class II antibodies are likely to cross-react with class II SLA. Work is in progress to genetically modify both the SLA class I and class II antigens in donor pigs to further reduce the risk of graft rejection. Thus, the three primary approaches to renal xenotransplantation now include: (1) eliminating xenoreactive pig antigens through genetic modification, (2) carefully screening potential human transplant recipients with high PRAs for antibodies to porcine antigens, and (3) finding the optimal immunosuppressive regimen—one that minimizes the risk of graft loss in nonhuman primates but could be tolerated by humans.

Another exciting area is the potential for xenotransplantation for neonates with severe congenital heart disease. Children who are fortunate enough to have a cardiac transplant within the first 30 days of life do much better long term than children who undergo staged procedures resulting in the Fontan procedure; however, transplantation has largely been abandoned because of the shortage of donor organs. These neonates also are in a very unique spot because they don't develop antibodies against pig antigens until they are about three months old. Furthermore, with thymus implantation from the pig, there is a possibility these children can stop immunosuppression within a few years after the transplant.

Sacher, Cincinnati: Since the liver cell has such a tremendous capacity for regeneration, what is the status of using induced pluripotent stem cells and maybe nano-platforms or scaffolds to regenerate the liver with either of those techniques?

Carithers, Seattle: There is considerable excitement around stem cell transplantation, which holds promise as an alternative to xenotransplantation. A number of clinical trials have shown improvement in liver function tests in patients with cirrhosis. However, long-term efficacy has not yet been proven. Organ bioengineering also represents a promising approach to the creation of organs for transplantation. This requires a decellularized scaffold with an intact extracellular matrix as a backbone on which to create a tissue-engineered organ. Efforts aimed at selecting an ideal scaffold and cell types for human application are ongoing. However, clinical trials are some ways off. Thank you.

Note: Numbers regarding liver transplant statistics were obtained from: https://optn.transplant.hrsa.gov/data/view-data-reports/national-data/.