CENTRAL MESSAGE
Gene-edited pigs could eventually provide organs that are safely and effectively protected from the human immune response without exogenous immunosuppression.
Keywords: gene editing, heart, kidney, pig, xenotransplantation
Graphical Abstract
Hyperacute rejection of a wild-type pig heart after transplantation into a baboon.
Feature Editor's Introduction—
Genome editing technology has revolutionized heart xenotransplantation and made transplantation of bioengineered pig hearts into humans a possibility. This first clinical application resulted from a tremendous amount of research. Dramatic early attempts of clinical cardiac xenotransplantation during the last century paved the way to modern xenotransplantation using bioengineered pig hearts. It appears that such genome-edited hearts will be most suitable for neonates and infants because of their immature immune system. The bioengineered pig heart may also be used as a bridge to human heart transplantation, avoiding the risk of thromboembolic events of durable ventricular assist devises in these young children. It is also intriguing to think that bioengineered hearts using pigs as a host may result in a new source of donor hearts that would not evoke the human immune response and minimize, if not eliminate, the need for immunosuppression.
It this issue of the Journal, a group of experts led by Dr Cooper, whose personal work spans over 50 years of heart transplantation research, outline the current state of the genome editing of bioengineered hearts and discuss the prospects of clinical application.
“That’s one small step for man, one giant leap for mankind.”
Neil Armstrong, as he stepped onto the surface of the moon in 1969.
Two-month survival of the first clinical heart transplant from a gene-edited pig is a milestone in the development of xenotransplantation.1,2 This “small step for man” was based on 35 years of incremental advances in pig-to-nonhuman primate (NHP) models contributed by multiple research groups worldwide.
Genetic engineering using CRISPR-Cas9 has enabled production of multiple lines of pigs with multiple gene edits aimed at protecting their organs from the human innate immune response. When coupled with novel immunosuppressive agents that efficiently block the CD40/CD154 T-cell costimulation pathway and thus prevent human-anti-pig adaptive immune injury,3 orthotopically transplanted hearts from gene-edited pigs have survived for several months in NHPs.4–6
A case can be made for pig cardiac xenotransplantation in patients with terminal heart failure who are unable to receive an allograft or are unsuitable for mechanical circulatory support.7
UNDERSTANDING THE PROBLEM
Initially, in the mid-1980s, the transplantation of organs from wild-type (ie, genetically unmodified) pigs was followed by rapid antibody-dependent, complement-mediated destruction of the graft, usually within minutes or hours (although occasionally longer).8 The antibodies responsible for hyperacute rejection are directed at carbohydrates. The production of “natural” (or “preformed”) anti-ABO blood group and anti-pig antibodies in infant humans (and infant NHPs) is thought to be a response to microbial colonization of the gastrointestinal tract and environmental exposure to viruses (Figure 1, top). To date, 3 carbohydrate xenoantigens have been identified in pigs against which humans have natural (preformed) antibodies (Table 1). The most important is galactose-α1,3-galactose (Gal).
FIGURE 1.
Top: Geometric mean (GM) binding and age correlation of human serum immunoglobulin (Ig)M (A) and IgG (B) antibodies to wild-type pig red blood cells. There is a steady increase in IgM and IgG during the first year of life. Bottom: GM binding and age correlation of human serum IgM (C) and IgG (D) antibodies to TKO pig red blood cells. There is virtually no increase in IgM or IgG antibodies during the first year of life. Note the great difference in the scale on the Yaxis between top and bottom. The dotted red lines indicate no IgM or IgG binding. Reproduced with permission from Li Q, Hara H, Banks CA, Yamamoto T, Ayares D, Mauchley DC, et al. Anti-pig antibody in infants: can a genetically engineered pig heart bridge to allotransplantation? Ann Thorac Surg. 2020;109:1268–73. TKO, Triple knockout; RBC, red blood cell.
TABLE 1.
Carbohydrate xenoantigens that have been deleted in gene-edited pigs
| Carbohydrate (abbreviation) | Responsible enzyme | Gene-knockout pig |
|---|---|---|
| Galactose-α1,3-galactose (Gal)9–11 | α1,3-galactosyltransferase | GTKO |
| N-glycolylneuraminic acid (Neu5Gc)12,13 | Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) | CMAH-KO |
| SID antigen14 | β–1,4N-acetylgalactosaminyltransferase | β4GalNT2-KO |
Efforts to remove anti-pig antibodies from the blood, for example, by (1) plasmapheresis or by perfusion of blood or plasma through (2) a donor-specific pig organ or (3) an immunoaffinity column of a synthetic glycan, extended graft survival, but the return of antibody inevitably resulted in graft failure.8
GENE EDITING OF PIGS TO PROTECT AGAINST THE HUMAN ANTI-PIG INNATE IMMUNE RESPONSE
Xenotransplantation of pig organs is the first opportunity we have had in more than 70 years of clinical organ transplantation to modify the donor, rather than just treat the recipient. The potential this provides is immense, and it is our prediction that eventually the pig will be so successfully engineered that, after transplantation, the organ will be completely protected from the human immune response and that no exogenous immunosuppressive therapy will be required.
Introduction of Human Complement-Regulatory Transgenes
When human complement is activated, for example, in response to an invading infectious microorganism, the native cells are to a large extent protected from complement injury by the expression of human complement-regulatory proteins (CRPs) on the vascular endothelium. Pigs have similar CRPs, but these are less protective against human complement. Studies in the 1990s incorporated a transgenic human CRP, CD55 (decay-accelerating factor), or CD46 (membrane cofactor protein), by microinjection of DNA directly into the pronucleus of a fertilized pig egg.15 When cyclosporine-based immunosuppressive therapy was administered, this resulted in a significant prolongation of pig heart survival after heterotopic transplantation into NHP recipients, extending to approximately 3 months. One baboon with a pig orthotopic heart transplant survived for 1 month.
Deletion of Expression of Pig Xenoantigens
The key initiating factor in complement activation is binding of anti-pig antibodies to the pig cells, for example, binding of anti-Gal antibodies to Gal antigens. Once Gal had been identified as the major xenoantigen involved in hyperacute rejection of a pig organ graft, the proposal was made in 1993 to knock out the gene for α1,3-galactosyltransferase and thus delete expression of Gal in the pig (from an α1,3-galactosyltransferase gene-knockout pig) (Table 1), although this was not possible at that time.
With the introduction of cloning by somatic cell nuclear transfer in large animals, it became possible to propagate pigs using cells in which a pig gene had been “knocked out,” and thus create pigs lacking expression of the associated xenoantigen. In 2003, the first pigs that did not express Gal were produced (Table 1).16 Survival of heterotopic heart transplants from α1,3-galactosyltransferase gene-knockout pigs in NHPs was maximally extended to 6 months.17
Subsequent gene editing technologies, like TALENS and CRISPR, enabled more precise and complex gene modification, facilitating additional deletion of expression of the remaining known carbohydrate xenoantigens, yielding double knockout and triple knockout (TKO) pigs18 (Table 1). There is minimal development of antibodies to TKO pig cells in infants and children (Figure 1, bottom), which greatly increases the likelihood of success when xenotransplants are carried out in infants, who also have weaker complement systems. The combination of deletion of expression of the 3 xenoantigens and expression of 1 of more human CRPs protected pig cells completely from complement injury in vitro.
Complicating preclinical assessment of TKO pig organs is the observation that deletion of expression of N-glycolylneuraminic acid (Neu5Gc) in the pig appears to expose another carbohydrate xenoantigen (sometimes called the “fourth xenoantigen”) against which NHPs (but not humans) have a positive crossmatch, presumably due to anti-pig antibodies.18 Deletion of expression of Neu5Gc may not be the entire explanation because even when Neu5Gc expression is present, many NHPs demonstrate a high level of complement-mediated cytotoxicity of these pig cells19 (Figure 2). This problem currently presents a substantial translational challenge in preparing for clinical trials of xenotransplantation, and we suspect that the pigto-NHP model may no longer be accurately predictive of TKO pig organ transplantation into human recipients.
FIGURE 2.
A, Correlation of human (n = 9) and baboon (n = 72) serum IgM antibody binding with serum complement-dependent cytotoxicity (CDC) (at 50% serum concentration) to TKO pig peripheral blood mononuclear cells. In both humans and baboons, there was a significant increase in cytotoxicity as IgM and IgG antibody binding to TKO pig peripheral blood mononuclear cells increased. In baboons, cytotoxicity was high whether IgM binding was high (eg, 80% cytotoxicity at a relative GM of 8) or relatively lower (eg, 75% at a relative GM of 2) (**P<.01). B, Human and Old World monkey IgM (left) and IgG (middle) binding and complement-dependent cytotoxicity (at 25% serum concentration) (right) to wild-type, α1,3-galactosyltransferase gene-knockout, and TKO pig peripheral blood mononuclear cells. Results are expressed as mean ± standard error of the mean. (*P<.05, **P<.01). On the y axis, the dotted line represents cutoff value of binding (relative GM: IgM 1.2, IgG 1.1). For complement-dependent cytotoxicity on the y axis, the dotted line represents cutoff value of cytotoxicity (6.4%). Note the difference in scale on the y axis between IgM and IgG. In Old World monkeys, serum IgM binding to and complement-dependent cytotoxicity of TKO peripheral blood mononuclear cells are greater than of human serum. Reproduced with permission from Yamamoto T, Iwase H, Patel D, Jagdale A, Ayares D, Anderson D, et al. Old World monkeys are less than ideal transplantation models for testing pig organs lacking three carbohydrate antigens (triple-knockout). Sci Rep. 2020;10:9771. Ig, Immunoglobulin; WT, wild-type; GTKO, α1,3-galactosyltransferase gene-knockout; TKO, triple knockout; N.S., not significant; OWM, Old World monkey.
Introduction of Human Coagulation-Regulatory Transgenes
Antibody or complement or inflammatory activation of the vascular endothelial cells of the pig graft may result in a change from a local anticoagulant state to a procoagulant state, resulting in the development of thrombotic microangiopathy in the graft and consumptive coagulopathy in the recipient.20 As with complement regulation, pig coagulation-regulatory proteins are inefficient in maintaining a state of vascular endothelial anticoagulation in the pig organ after its transplantation into a human or NHP.
This complication was largely overcome by the introduction into the pig of human coagulation-regulatory proteins, for example, thrombomodulin, endothelial protein C receptor, tissue factor pathway inhibitor, or CD39. Transgenic expression of human thrombomodulin or endothelial protein C receptor works to provide an anticoagulant (and anti-inflammatory) state that has been demonstrated to reduce the development of thrombotic microangiopathy and consumptive coagulopathy, and extended survival of pig heart and kidney transplants.
Introduction of Human Anti-Inflammatory (Apoptotic) Transgenes
A sustained systemic inflammatory response develops to the presence of a pig organ in an NHP host. Although this may be reduced to some extent by drug therapy, for example, by blockade of interleukin-6 (although the efficacy of this therapy has been questioned), transgenic expression of a human anti-inflammatory (antiapoptotic) protein, for example, hemeoxygenase-1, A20,21 may provide local protection of the graft by blunting inflammation associated with ischemia-reperfusion and innate immune activation.
Gene Editing to Suppress Macrophage or Natural Killer Cell Activation
Macrophages serve as a bridge for the innate immune response to institute T-cell activation that can mediate cellular xenograft rejection through direct cytotoxicity. Thus, a method to limit the activation of recipient macrophages may prolong graft survival.22 Human CD47 is recognized by macrophage signal-regulatory protein-α, which inhibits macrophage activation. However, pig CD47 is recognized by primate macrophages as foreign, and thus macrophage activity is not inhibited. The introduction of a transgene for human CD47 into the pig may significantly reduce phagocytosis by human/NHP macrophages, suppress inflammatory cytokine production, and decrease T-cell infiltration of pig xenografts.
Likewise, an important role for natural killer cells in xenograft injury has been demonstrated. Natural killer cells identify nonself by lack of expression of human leukocyte antigen (HLA)-E (or HLA-G). Accordingly, expression of HLA-E on pig cells protects them from natural killer cell–mediated cell killing.
Deletion of Expression of Pig Growth Hormone Receptors
With these in vitro and in vivo background data, it was suggested that the optimal organ-source pig for clinical xenotransplantation at the present time would be one with 9 gene edits, that is, TKO.CD46.CD55.TBM.EPCR.HO-1.CD47.23 However, rapid growth of a pig organ graft in an NHP continues for several weeks or months after transplantation and could be problematic (particularly after orthotopic heart transplantation in the confined space within the chest). This led to a proposal for adding knockout of the gene for growth hormone receptors.24 Therefore, a pig with 10 gene edits was the source of the heart in the recent first clinical study.
TECHNIQUES OF PIG GENETIC ENGINEERING
Gene editing of pigs has evolved markedly since the early days and has been summarized by Eyestone and colleagues.25 The development of somatic cell nuclear transfer (cloning) was critical to its success (Figure 3). Today, CRISPR/Cas9 technology allows for efficient gene targeting for creating knockouts and, with homology-directed repair, transgenes can be inserted into predetermined “landing pad” sites in the genome. The use of bicistronic or multicistronic vectors enables 2 or more transgenes to be inserted under the control of 1 or more promoter. Incorporating several transgenes into a single vector incurs benefits for mitigating the innate immune response to the pig organ. Therefore, judicious genetic engineering of the organ-source pigs has resulted in significant prolongation of pig heart function in immunosuppressed NHP recipients.4–6
FIGURE 3.
Steps involved in somatic cell nuclear transfer. The nuclei of porcine oocytes are replaced by genetically modified nuclei, typically from donor pig fibroblasts. The newly constructed oocytes develop into pigs that express the genetic modifications made in the donor fibroblast nuclei. Using somatic cell nuclear transfer technology and homologous recombination, knockout of genes can be made in the pig fibroblasts and incorporated into the oocytes. The pigs derived from these oocytes will lack the gene and phenotypically lack expression of the specific pig antigen, for example, Gal (Table 1). Genes can also be inserted into the oocytes, for example, into a site vacated after knockout of a xenoantigen. The choice of a promoter can determine whether the transgenic protein is expressed ubiquitously in all tissues throughout the pig or expressed only in the endothelial cells. For example, human CRPs are more effective if widely expressed, whereas human coagulation-regulatory proteins may be associated with bleeding tendencies in the pig, and therefore are preferably expressed only in endothelial cells. Once the initial pigs have been produced by cloning technology, future generations can be bred naturally. Reprinted with permission from Eyestone and colleagues.25
Gene Editing of Pigs to Protect Against the Human Anti-Pig Adaptive Immune Response
The adaptive immune response causes or contributes to xenograft loss unless effective immunosuppressive therapy is administered. Thrombotic microangiopathy and other features characteristic of antibody-mediated rejection are typically prominent in failed or failing xenografts and, once detected, have proved extremely difficult or impossible to reverse. Today, most preclinical protocols include peritransplant induction using agents that deplete T and B cells combined with a monoclonal antibody that blocks the CD40/CD154 T-cell costimulation pathway.26
Deletion or Reduction of Expression of Swine Leukocyte Antigens
In pigs, swine leukocyte antigens (SLAs) correspond to HLAs and are expressed by all nucleated cells. Like HLAs, the SLAs can be divided into class I and II. After pig organ transplantation in humans/NHPs, expression of SLA class I is associated with the presentation of intracellular peptides to host CD8+T cells, whereas SLA class II presents extracellular peptides to CD4+T cells. Depletion of CD4+T cells, but not CD8+T cells, in NHP recipients of pig grafts is particularly important in extending graft survival.
SLAs have been confirmed to be xenoantigens,27,28 but only less than 5% of HLA-nonsensitized persons have detectable levels of anti-SLA antibodies (possibly associated with prior viral infection). Martens and colleagues29 studied sera from 820 patients waitlisted for kidney allotransplantation, of whom 119 had panel-reactive antibodies of more than 80%. Only 13 samples contained SLA class I-specific immunoglobulin-G, thus demonstrating that only a relatively few patients highly sensitized against HLA (~11%) demonstrate cross-reactivity with SLA. This represents perhaps less than 2% of all waitlisted patients.
This suggests that, in many patients, prior sensitization to HLA will not be detrimental to the outcome of a pig xenograft. Nevertheless, there is a need to screen all potential recipients for the presence of anti-HLA antibodies that cross-react with SLA.
Organ xenotransplants from pigs that, through gene editing, do not express any SLA would stimulate a much weaker human/NHP immune response, but may render the organ-source pig more susceptible to infection. Recent approaches have been focused on deleting expression of SLA class I antigens (by gene knockout) or reducing the expression of SLA class II antigens (because knockout of class II results in immunodeficiency and may be lethal).
When anti-HLA antibodies are present that cross-react with a particular SLA, it should be possible by genetic engineering of the pig to delete the target antigen(s). In this respect, Ladowski and colleagues30 have demonstrated that genetic modification of the amino acids on the surface of a pig cell, for example, by mutating an arginine to pro-line, can result in a reduction in antibody binding to the cell.
Other Gene-Editing Methods of Immunomodulation
The role of gene editing will increasingly be directed to control of the adaptive immune response to pig grafts, thus minimizing the need for exogenous immunosuppressive therapy, with its potential complications.
Some of the potential approaches have been mentioned in this article, but others include the transgenic expression of programmed death-ligand 1 (PD-L1). The PD-1/PD-L1 pathway plays a vital role in the maintenance of the balance between immune activation and induction of tolerance through the generation of inhibitory signals. The expression of PD-L1 is associated with reduced immunogenicity and renders cells and tissues into an immune-privileged/tolerogenic state. Other approaches may include cytotoxic lymphocyte-associated molecule-4 ligand production by the graft. Drug-responsive promoters that turn off (or turn on) the immunomodulator would be an important safety feature.
Gene Editing to Prevent Potential Complications Associated With Porcine Endogenous Retroviruses
There is one other area in which gene editing of the pig could play a role, and that is in minimizing or preventing a potential complication related to the presence of porcine endogenous retroviruses (PERVs) that are present in the nucleus of every pig cell. These have been present in pigs for thousands of years and do not appear to do any harm to the pig. Human cells contain similar human endogenous retroviruses, and these are believed to be equally innocuous. However, concern was raised as to whether PERVs might be harmful after the transplantation of a pig organ into a human, for example, by possibly causing immunodeficiency or malignant change, or might combine with fragments of their human counterparts to form a hybrid virus. The potential risk to the recipient of a pig xenograft (and possibly more importantly to his/her close contacts) will be unknown until clinical trials are initiated.
However, gene-editing techniques have been developed to reduce or abrogate any potential risk. Activation of PERVs can be prevented, and multiple copies of PERV have been knocked out, thus rendering the pig PERV-free.31 Whether the national regulatory authorities will consider this to be necessary remains uncertain. Although considered unlikely, if a PERV-related infection develops, drugs are available that are likely to successfully treat such an infection.
DISCUSSION
In addition to these approaches, gene editing can be used to address any physiologic incompatibilities that are identified between the pig organ and the needs of the human recipient. To date, however, pig heart function in NHPs appears to be satisfactory, although a persistent tachycardia is common.
Patients who might benefit from pig heart transplantation include (1) neonates and infants with complex congenital heart disease (eg, single-ventricle physiology, in whom mechanical circulatory support devices are associated with poor results) who will be bridged to allotransplantation by a xenograft; (2) adults with restrictive or hypertrophic cardiomyopathy, a dysfunctional valve prosthesis, or an atrial or ventricular septal defect; (3) those with a high level of sensitization to HLAs but who have no antibodies that cross-react with pig antigens; and (4) those with chronic rejection in need of retransplantation.7 We suggest that, initially, further patients will be offered pig heart transplantation on a compassionate basis, followed by formal clinical trials. Within the next 5 to 10 years, we anticipate that cardiac xenotransplantation will become an accepted form of bridging, and subsequently, with improved gene-edited pigs, of destination therapy.2
CONCLUSIONS
Further advances in genetic engineering are likely to make this “moonshot” possible and enable a second “giant leap for mankind” by providing access to lifesaving organ transplantation.
CENTRAL MESSAGE.
Gene-edited pigs could eventually provide organs that are safely and effectively protected from the human immune response without exogenous immunosuppression.
Acknowledgments
D.K.C.C. and R.N.P. received grant support from the National Institutes of Health (NIH NIAID U19 Grant AI090959 and UO1 Grant AI153612) and the Department of Defense (Grant W81XWH2010559; D.K.C.C.), and previously received research funding from Revivicor, a subsidiary of United Therapeutics. R.N.P. has received research support from eGenesis and Tonix. R.C. is supported by the Benjamin Walter Research Fellowship from the German Research Foundation (DFG).
Footnotes
Conflict of Interest Statement
D.K.C.C. is a consultant to eGenesis Bio of Cambridge, Massachusetts, but the opinions expressed in this article are his own and do not necessarily reflect those of eBiogenesis. All other authors reported no conflicts of interest.
The Journal policy requires editors and reviewers to disclose conflicts of interest and to decline handling or reviewing manuscripts for which they may have a conflict of interest. The editors and reviewers of this article have no conflicts of interest.
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