Genetically-engineered pig-to-human organ transplantation: a new beginning

There has been a continuing inadequate number of human organs for the treatment of patients with terminal organ failure worldwide. The transplantation of organs from genetically engineered pigs may prove an alternative solution [1]. In recent years, great progress has been made in pig-to-nonhuman primate (NHP) organ transplantation models (Table S1 online), largely by (1) the availability of pigs with increasing numbers of genetic modifications aimed at protecting their organs from the human innate immune response, and (2) the introduction of novel immunosuppressive agents, particularly those that block the CD40/CD154 costimulation pathway, to control the human adaptive immune response. Furthermore, there has been increasing public awareness and acceptance of pig organ xenotransplantation as a potentially life-saving therapy and, in the USA, official regulatory approval of pigs with genomic alterations as sources of food and for potential therapeutic uses.

On the basis of these advances, there have recently been attempts to explore organ xenotransplantation in human subjects. Two such efforts, by surgical teams in New York (NY) and Birmingham, Alabama (Ala), investigated the transplantation of genetically-engineered pig kidneys into brain-dead human subjects, who were monitored for 2–3 d [2,3]. They contributed much significance to what was already known, on the basis of unperfect simulation between NHPs and humans. In the NY experience, the gene-edited pig selected was not optimal for a clinical trial. In spite of more gene modification may have more risks, β4GalNT2 knockout and/or hCD55 insertion seemed to be necessary as exemplified by the pig donors selected in many preclinical trials of renal xenotransplant (Table S1 online). However, they confirmed thymokidney could be an ideal treatment for overcoming xenogeneic acute rejection and an alternative to experimental anti-CD40 mAb administration, since the co-transplant of the donor-derived thymus has been proven to induce the selective development of recipient T cells to produce donor-specific immune tolerance. The pre-xenotransplant revascularization of thymus in the renal sub capsule theoretically facilitated the induction of immune tolerance. In this case, the pig kidneys quickly produced urine, and the volume of urine was about twice that of the native kidneys. It’s partly due to the fact that pigs had fewer nephrons and a lower percentage of long-looped nephrons, and thus had a reduced ability to concentrate urine [4]. Furthermore, the increase of kinetic estimated glomerular filtration rate (eGFR) and decline of creatinine level confirmed the potential alternative function of pig kidneys. In the Birmingham experience, the pig was more suitable for a clinical trial in theory (although some felt the Ala kidney with more genes inserted may have more risks than the less engineered NY choice) and the native kidneys were excised [2]. Administered clinically approved immunosuppressants, the right pig kidney quickly produced urine while there was a delay and inferior function recovery of the left pig kidney (partly because the left renal vein was damaged during kidney acquisition and additional repair caused prolonged ischemia and more renal injury). In addition, there were histopathological features suggesting either rejection or, more likely, a detrimental effect of the inflammatory response that follows brain death [2], which might account for the post-transplant increase in creatinine level. In spite of the avoidance of hyperacute rejection, “10-gene” manipulation still showed deficiencies in dealing with xenogeneic graft injury. Because the frequency of karyotype anomalies in multi-genetically engineered pigs raises the possibility of unforeseen genomic changes, including the exposure of neoantigens [5]. It is difficult to maintain graft function in brain-dead recipients who are hemodynamically and metabolically unstable, however, it’s likely that pig-to-brain-dead human model is a prudent and safe way to transition to clinical practice, and further experience in this model with extended period of observation will provide us more valuable data.

Of much greater interest and more scientific value was the transplantation of a gene-edited pig heart into a 57-year-old patient at the University of Maryland at Baltimore [6]. The patient was not an ideal candidate for the first clinical attempt, as he had been supported by extracorporeal membrane oxygenation (ECMO) for approximately 6 weeks. However, as no other life-saving therapy was available for him, approval was given on “compassionate” grounds by the US Food and Drug Administration (FDA) for the operation to proceed. The pig also expressed 10 genetic modifications and, although the operation was complicated by unanticipated problems that resulted in renal failure requiring dialysis, the pig heart initially functioned very well. On about post-operative day (POD) 40, the patient left the hospital bed and shook hands with the nursing staff. However, his condition deteriorates rapidly in the later period, with abnormal hypertrophy of the ventricular wall and reduced ventricular volume, and an increase of serum IgM and IgG antibodies, as well as troponin, all indicating a potential humoral rejection. The pig heart showed severe edema and nearly doubled in weight. There were myocardial necrosis, interstitial edema, and erythrocyte extravasation, but no microvascular thrombosis within the xenograft, which are inconsistent with typical rejection [6]. It was verified that the pig heart was affected by porcine cytomegalovirus (CMV), a preventable infection that is linked to devastating effects on transplants, and that this compromised cardiac function, contributing to the death of the patient 60 d after the transplant. Although the pig had been housed in a state-of-the-art designated pathogen-free facility, a blood test performed on POD 20 initially indicated the presence of porcine CMV, which was starting to multiply fast and setting off a possible “cytokine explosion”—a storm of immune-system molecules. As a result, greater attention will need to be directed in the future to the microorganisms carried by the organ-source pigs. Nevertheless, much valuable information might have been accumulated from this experience that might facilitate future attempts.

As the ultimate goal of xenotransplantation, necessary clinical trials would further help to answer a slew of questions, including (1) the suitable gene editing, (2) the physiological compatibility of transplanted organs, (3) the optimal immunosuppressive regimen, (4) the management of interspecies pathogen transmission, (5) the best pig breed suited for growing transplant organs, and (6) the impact on transplant success of co-occurring health conditions.

Techniques of gene editing have steadily evolved and improved over the past 30 years. The current CRISPR-Cas9 technique enables multiple edits to be made quickly and more cheaply [7]. However, gene editing may not always prove as beneficial as anticipated, as exemplified by the knockout of the genes for the enzymes that add the 3 known pig xenoantigens (Gal, Neu5Gc, and Sda) to the underlying glycans on pig cells. Deletion of expression of Neu5Gc appears to expose another glycan (often known as the “4th pig xenoantigen”) that is antigenic in NHPs, but not in humans [5]. The NHP antibody response to the 4th xenoantigen has proved problematic and is inhibiting progress in this model which is arguably no longer representative of the pig-to-human model. Nevertheless, for clinical trials, deletion of expression of all 3 xenoantigens (triple-knockout [TKO] pigs) will be advantageous, as many humans do not have antibodies against cells from these pigs. However, there is convincing evidence that further protection from the human innate immune response can be provided by the additional transgenic expression of one or more human complement-regulatory proteins (e.g., CD46 and CD55) [8] and one or more human coagulation-regulatory proteins (e.g., thrombomodulin and endothelial protein C receptor). Expression of an anti-inflammatory protein (e.g., hemoxygenase-1 and A20) and the “anti-macrophage” protein, CD47, may also be beneficial [8]. The heart transplanted into the recipient at the University of Mayland had all of the above gene modifications, and in addition, had undergone deletion of expression of growth hormone receptors (GHR-KO) to prevent the reported rapid post-transplant growth of the pig heart in the restricted confines of the patient’s chest. Furthermore, different donor organs may need diverse gene knockouts and/or knockins. As an alternative, expression in the pig of soluble complement-receptor-1 has been proposed, and key segments of pig vWF have been suggested to be replaced by analogous human vWF segments, coupled with ASGR1 knockout. Pigs with transgenic expression of human human leukocyte antigens (HLA)-E/β2m and CD178 (FasL) have been produced to inhibit natural killer cell cytotoxicity [1]. Transgenic human CTLA4-Ig (CD152) and CD253 (TRAIL) expression interrupt T-cell activation and induce T-cell apoptosis, respectively [1]. According to our experience and others, human CD200, and IL-18BP are also potential knockin genes in protecting pig grafts from xenogeneic injury [1].

Acute rejection of an allograft is predominantly represented by a cell-mediated immune response, whereas acute xenograft rejection is principally antibody-mediated. There is some evidence that the immune response to a pig xenograft is stronger than to an allograft. After xenotransplantation, effective immunosuppressive therapy is required to suppress the immune response to other xenoantigens expressed on pig cells, e.g., swine leukocyte antigens (SLA) I and/or II. Although the expression of these xenoantigens can be deleted by gene editing, this has the potential to render the pigs immunocompromised, which may be detrimental to their health. Conventional immunosuppressive therapy (e.g., tacrolimus-based), as administered to patients with allografts, has not proven fully effective in preventing the immune response to a pig xenograft [9]. The optimal regimen in xenotransplantation appears to be (1) induction therapy with anti-thymocyte globulin (ATG), an anti-B cell monoclonal antibody (e.g., rituximab), and an agent that inhibits systemic complement activity (e.g., a C1-esterase inhibitor), and (2) maintenance therapy based on blockade of the CD40/CD154 costimulation pathway, perhaps combined with an agent such as rapamycin or mycophenolate mofetil. Blockade of the CD28-CD80/86 costimulation pathway (e.g., with belatacept) has proven less effective. Rapamycin has the additional benefit of being an inhibitor of the growth of the graft. There is increasing evidence of a systemic inflammatory response to a pig xenograft [10], and the administration of an anti-inflammatory agent may therefore be beneficial. Tocilizumab and/or etanercept have been added by some groups, but evidence for their efficacy remains unproven. Adjunctive agents that may contribute to the prevention of platelet aggregation include low-dose aspirin and/or low-molecular-weight heparin.

About the prophylaxis against infectious complications, as reactivation of human CMV and/or pneumocystis can occur in immunosuppressed patients, prophylactic ganciclovir and trimethoprim-sulfamethoxazole are often included in the post-transplant regimen. It should be noted that ganciclovir is relatively ineffective against porcine CMV, and so it is essential to breed pigs that do not carry CMV (which can be achieved by early weaning of the piglet from the sow). Some scholars recommend the use of orphan drug Maribavir to treat porcine CMV infection. In addition, whether the presence of porcine endogenous retroviruses (PERVs) in every pig cell will prove problematic after organ transplantation into an immunosuppressed recipient will probably not be known until clinical trials are undertaken [11]. Although PERVs can be inactivated by gene editing, which would clearly be advantageous, the potential risks associated with PERVs may not be thought sufficient by the regulatory authorities to insist on this genetic modification. Furthermore, there are anti-retroviral drugs that are likely to be active against PERV infection.

The selection of recipients for the initial clinical trials will also be critical [12]. Candidates for pig kidney transplantation include (1) elderly patients (with no significant comorbidities) who are likely to die or be withdrawn from the waiting list before a deceased human donor organ becomes available; (2) those with a high level of HLA-sensitization, but without antibodies that cross-react with SLA; and possibly (3) those with loss of vascular access, or (4) rapidly recurrent kidney disease. Cardiac candidates include (1) infants with complex congenital heart disease, e.g., single ventricle; those with (2) complex anatomic or pathologic considerations preventing ventricular assist device (VAD) implantation; (3) restrictive cardiomyopathy; or (4) in need of re-transplantation; as well as (5) those with high sensitization to HLA. Progress in experimental pig liver and lung transplantation has not progressed sufficiently for clinical trials to be realistically considered, except possibly when a pig liver is used as a bridge to allotransplantation (e.g., in fulminant liver failure, after primary liver allograft failure, acute decompensation of chronic liver disease), or until the native liver has recovered normal function (e.g., after drug-induced acute hepatic failure). Although there is evidence that a small number of allosensitized patients have antibodies that cross-react with SLA [13], which may be detrimental to the outcome of a xenograft, there is no evidence to date that suggests that sensitization to pig antigens will be detrimental to a sub-sequent allotransplant [14], indicating the safety of bridging with a pig organ in this respect.

In the regulation aspect of clinical organ xenotransplantation, there have been several revisions to the guidelines for xenotransplantation issued by the US FDA. The 2018 Changsha Communique, drafted at the Third World Health Organization Global Consultation on Regulatory Requirements for Xenotransplantation Clinical Trials, suggested that the minimum preclinical experimental requirement for a clinical trial was the relatively consistent survival of an NHP with a life-supporting pig organ for periods of at least six months in a series of at least six experiments. However, given the encouraging advances being made in the field of xenotransplantation, we suggest that the time has surely come when we need to consider moving from the laboratory to the clinic. Progress is much more likely to be made from small clinical trials than if we persist in carrying out experiments in an animal model that no longer mimics the clinical situation. Recently, the US regulatory agency has signaled willingness to allow the first xenotransplant trials [15].

The modest partial success of the recent pig heart transplant at the University of Maryland attracted worldwide attention, and has possibly increased the public’s acceptance of xenotransplantation as a potential life-saving strategy. Before this, the attitudes of patients, healthcare professionals, and the public were ascertained. This indicated that pig organ xenotransplantation is generally acceptable to the major religious groups, as the maintenance of human life takes precedence over other factors [16]. However, public support was in part dependent on the results of xenotransplantation being comparable to those of allotransplantation, which, of course, cannot be guaranteed as yet, and indeed, is unlikely to be the case until clinical experience has been gained.

In China, there are >300,000 patients on the waiting list for an organ transplant. Since the implementation of voluntary organ donation on January 1, 2015, a total of 4.53 million people have registered as potential donors, and 119,053 organs have been donated to date. Nevertheless, in no country does the availability of deceased human organs match the needs of patients with terminal organ failure. We believe that the initial clinical trials of gene-edited pig organ xenotransplantation will represent a new beginning for organ transplantation that will in time solve the problem of the shortage of human donor organs worldwide.

Biographies

Xuan Zhang is an M.D. and an attending surgeon at the Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University. His research interest lies primarily in organ transplantation, ischemia-reperfusion injury, and liver cancer, with a focus on pig-to-nonhuman primate liver xenotransplantation at present.

Kefeng Dou is the Director of the Organ Transplant Institute, Xijing Hospital, Fourth Military Medical University, Vice President of Surgery Society of Chinese Medical Association (CMA) and Organ Transplant Society of Chinese Medical Doctor Association (CMDA). His research interest includes allo- and xeno- liver transplantation, non-alcoholic fatty liver disease, liver fibrosis, and liver cancer.