Xenotransplantation—A Basic Science Perspective

Introduction

The gene-edited pig heart transplant carried out on compassionate grounds in a patient at the University of Maryland earlier this year, who lived for 60 days before developing graft failure,^1,2 indicated that xenotransplantation is close to formal clinical trials. This initial clinical experiment was based on almost 40 years of basic and translational scientific research by groups around the world. A brief summary of progress in xenotransplantation science is given here.

The first important point to stress is that, unlike allotransplantation, our ability to genetically engineer the organ-source pig (by techniques such as CRISPR-cas9) provides us with the first real opportunity in >70 years of clinical organ transplantation to modify the donor and not just treat the recipient. This opens immense possibilities, and it is largely gene editing that has resulted in the progress that has been made.

Most research has been directed to the immunobiological hurdles that have been faced, which include (1) the immediate innate immune response (which is similar to that seen after some patients of ABO-incompatible organ allotransplantation or in human leukocyte antigens [HLA]–sensitized patients) and (2) the subsequent adaptive immune response (as seen in uncomplicated allotransplantation).

The Innate Immune Response

The transplantation of an organ from a wild-type (i.e., a genetically unmodified) pig into a nonhuman primate (NHP) recipient (or into a human recipient, which has been carried out in the past) results in hyperacute rejection of the graft, even when full conventional pharmacologic immunosuppressive therapy is administered to the recipient.³

This is initiated by binding of anti-pig antibodies to carbohydrate antigens expressed on pig vascular endothelial cells, which activates the complement and coagulation cascades, initiates an inflammatory response, and activates innate immune cells, e.g., neutrophils, natural killer cells, and macrophage. It is believed that these natural anti-pig antibodies develop during infancy as a defensive mechanism when the human gastrointestinal tract is colonized by microorganisms that express the same carbohydrate molecules as pigs (Figure 1, top). To overcome the innate immune response, the organ-source pigs have been genetically engineered to (1) delete expression of the three known pig xenoantigens (Table 1), resulting in triple-knockout (TKO) pigs, and (2) introduce transgenes for human protective proteins, e.g., complement-regulatory and coagulation-regulatory proteins and anti-inflammatory proteins.

Figure 1.: Correlation of human serum IgM and IgG binding to WT and TKO pig RBCs with age. (Top) GM binding and age correlation of human serum IgM (A) and IgG (B) antibodies with WT pig RBCs. 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 (A) and IgG (B) antibodies with TKO pig RBCs. There is virtually no increase in IgM or IgG antibodies during the first year of life and a very low level of antibodies in adults. Note the great difference in the scale on the Y axis between top and bottom. The dotted lines indicate no IgM or IgG binding. GM, geometric mean; RBC, red blood cells; TKO, triple-knockout; WT, wild-type. Reproduced with permission from Li Q, et al. Ann Thorac Surg. 2020;109:1268–1273.

Table 1. - Carbohydrate xenoantigens that have been deleted in gene-edited pigs

Carbohydrate (Abbreviation)	Responsible Enzyme	Gene-Knockout Pig
1. Galactose-α1,3-galactose (Gal)	α1,3-galactosyltransferase	GTKO
2. N-glycolylneuraminic acid (Neu5Gc)	Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH)	CMAH-KO
3. Sda	β-1,4N-acetylgalactosaminyltransferase	β4GalNT2-KO

Human serum antibody binding to TKO pig cells is greatly reduced (Figure 1, bottom) and, indeed, is absent in many humans. Whatever serum cytotoxicity remains is largely neutralized by the expression of human complement regulatory proteins, e.g., CD46, CD55, on the pig cells.⁴ There are significant molecular incompatibilities between the pig and primate coagulation systems, resulting in the development of a thrombotic microangiopathy within the pig graft and a consumptive coagulopathy in the recipient. These have largely been prevented by judicious gene editing of the pig, e.g., by the transgenic expression of human thrombomodulin (TBM) and/or endothelial cell protein C receptor (EPCR). Similarly, the graft can be protected to some extent from the primate inflammatory response by the expression of one or more anti-inflammatory proteins, e.g., hemoxygenase-1, and the introduction of human CD47 helps regulate innate cells, i.e., macrophages.

Pigs are now available with ten or more gene edits, which has been facilitated by the introduction of the CRISPR-Cas9 technology. The ideal organ-source pig to protect from the innate immune response in clinical kidney transplantation has been suggested to be one with the following gene edits—TKO, CD46.CD55.TBM.EPCR.HO-1.CD47⁵ (it was from one of these pigs that the heart was transplanted in Maryland).

The Adaptive Immune Response

Even if the innate immune response is successfully controlled, the recipient's immune system is then exposed to other xeoantigens, e.g., swine leukocyte antigens, the pig equivalent to HLA, against which humans do not have natural antibodies. Exposure to these antigens, however, can initiate T-cell and B-cell responses, but, as in allotransplantation, these can be prevented by effective immunosuppressive therapy. Conventional immunosuppressive therapy, e.g., tacrolimus-based, is relatively ineffective in xenotransplantation, whereas agents that block the CD40/CD154 T-cell costimulation pathway (but not those that block the CD28/B7 pathway, e.g., belatacept) are associated with a high degree of success.⁶

There is growing evidence that anti-CD154mAb therapy has advantages over anti-CD40mAb therapy, but in both cases, these agents have been combined with at least one other immunosuppressive agent (e.g., rapamycin or mycophenolate mofetil),⁷ although there is very preliminary evidence that costimulation pathway blockade alone may be sufficient in some patients.

Rapid Growth of the Pig Organ after Transplantation

There has been one further problem that required resolution and that relates to the observed rapid growth of the pig organ after transplantation. When the organ is obtained from a domestic pig that grows rapidly to a large size, e.g., a Yorkshire or Landrace pig, for several months the organ grows as if it is still in a rapidly growing young pig. In our experience, this has generally not proved to be problematic after kidney transplantation, but it has proved to be a problem after heart transplantation into the more restricted confines of the chest. This rapid growth can be reduced by further gene editing, i.e., by knock-out of growth hormone receptors, in the pig.⁸ Although beneficial in heart transplantation, regarding kidney transplantation, this was initially associated with ureteric complications (Cooper DKC, unpublished data), the cause of which has not yet been fully clarified. An alternative is the transplantation of organs from one of the many breeds of miniature pigs, e.g., Yucatan, which grow more slowly.

Other Roles for Genetic Engineering of the Organ-Source Pig

The function of pig kidneys in NHPs has only recently begun to be investigated. The evidence from the literature⁹ and from recent studies by our own group (Hansen-Estruch C, et al., submitted for publication) indicates that function will be adequate. This conclusion is supported by the observation that several NHPs remain healthy >1 year after pig kidney transplantation with no evidence of renal dysfunction. However, if functional deficits are observed, further gene editing may resolve the problem. For example, although the evidence is that erythropoietin produced by the pig kidney is sufficient to prevent anemia, the gene for human erythropoietin could be introduced into the pig.

For some years, there has been some concern that porcine endogenous retroviruses, which are present within the genome of every pig cell and thus inevitably transferred with the organ, might be pathogenic in immunosuppressed human recipients. There is little evidence for this, but expression of these viruses has been deleted by genetic engineering, thus definitively negating any potential risk.¹⁰

However, careful screening (by a combination of sophisticated PCR-based and immunological assays, e.g., Western blot) and selection of pigs free from potential zoonotic microorganisms, e.g., porcine cytomegalovirus, will be required.

Comment

It is possible that some of the transgenes introduced in the 10-gene pig will eventually be proven not to be essential and that alternatives may be beneficial in protecting from the innate immune response, e.g., HLA-E or HLA-G expression. It is more likely that future gene editing will be aimed toward providing protection against the adaptive immune response, e.g., by expression of PD-LI or reduction in expression of swine leukocyte antigens class I or II, thus enabling a reduction in exogenous immunosuppressive therapy (although care will have to be taken to ensure that the pig is not rendered immunodeficient).

With our increasing ability to genetically engineer the organ-source pig, combined with the efficacy of the novel costimulation blockade agents, we are surely poised to initiate a new era in organ transplantation.