Justification of specific genetic modifications in pigs for clinical organ xenotransplantation

Abstract

Xenotransplantation research has made considerable progress in recent years, largely through the increasing availability of pigs with multiple genetic modifications. We suggest that a pig with nine genetic modifications (ie, currently available) will provide organs (initially kidneys and hearts) that would function for a clinically valuable period of time, for example, >12 months, after transplantation into patients with end-stage organ failure. The national regulatory authorities, however, will likely require evidence, based on in vitro and/or in vivo experimental data, to justify the inclusion of each individual genetic modification in the pig. We provide data both from our own experience and that of others on the advantages of pigs in which (a) all three known carbohydrate xenoantigens have been deleted (triple-knockout pigs), (b) two human complement-regulatory proteins (CD46, CD55) and two human coagulation-regulatory proteins (thrombomodulin, endothelial cell protein C receptor) are expressed, (c) the anti-apoptotic and "anti-inflammatory" molecule, human hemeoxygenase-1 is expressed, and (d) human CD47 is expressed to suppress elements of the macrophage and T-cell responses. Although many alternative genetic modifications could be made to an organ-source pig, we suggest that the genetic manipulations we identify above will all contribute to the success of the initial clinical pig kidney or heart transplants, and that the beneficial contribution of each individual manipulation is supported by considerable experimental evidence.

Keywords: clinical; genetically engineered; heart; human; kidney; pig; transgenes; triple-knockout; xenotransplantation.

FIGURE 1

A, Human IgM (left) and IgG (right) antibody binding to wild-type (WT), GTKO, double-knockout (DKO, ie, KO of Gal and Sda), and TKO pig red blood cells (RBCs). Human serum antibody binding to pRBCs (n = 14) was measured by flow cytometry using the relative geometric mean (GM), which was calculated by dividing the GM value for each sample by the negative control, as previously described (refs^,). Negative controls were obtained by incubating the cells with secondary anti-human antibodies only (with no serum). Binding to TKO pig RBCs was not significantly different from human IgM and IgG binding to human RBCs of blood type O. (Note that our data support the observation that the deletion of β4GalNT2 has little effect in reducing antigenicity to humans, as opposed to baboons (Figure 2A) or New World monkeys (Figure 2B)). B, Pooled human serum complement-dependent cytotoxicity (hemolysis) to WT, GTKO, and TKO (GTKO/β4GalKO/CMAHKO) pig RBCs was performed, as previously described (ref). Briefly, RBCs were incubated with diluted serum for 30 min at 37°C. After washing, RBCs were incubated with rabbit complement (Sigma; final concentration 20%) for 150 min at 37°C. After centrifugation, supernatant was collected, and hemolysis was evaluated using a Multi-Label Microplate Reader (Perkin Elmer Victor3). The absorbance of each sample at 541 nm was measured. Cytotoxicity of the same serum to autologous human O RBCs was tested as a control. C, Human T-cell proliferative response to WT, GTKO.hCD46, and GTKO.hCD46.CMAHKO (Neu5GcKO) pig peripheral blood mononuclear cells (PBMCs) in mixed leukocyte reaction (MLR; ref). (TKO pig PBMCs were not available to us.)

FIGURE 2

(A) Baboon (n = 14) and (B) capuchin monkey (a New World monkey) (n = 10) IgM and IgG antibody binding to WT, GTKO, DKO (KO of Gal and Sda), and TKO pig RBCs. Serum antibody binding to pig RBCs was performed, as previously described (ref). (Note that CMAHKO in pig cells appears to expose a fourth xenoantigen against which Old World NHPs, but not New World monkeys, have natural antibodies. TKO pig organ transplantation in Old World NHPs may therefore be problematic and disadvantageous.)

FIGURE 3

Human serum cytotoxicity to WT, GTKO, GTKO. hCD46, and GTKO.hCD46.hCD55 pig corneal endothelial cells both before (left) and after (right) activation with pig IFN-γ (ref). Before activation, there was no serum cytotoxicity to GTKO, GTKO/CD46, or GTKO/CD46/CD55 pig cells. After activation, serum cytotoxicity was significantly increased, but the expression of two human complement-regulatory proteins (CD46 and CD55) almost completely prevented cytotoxicity

FIGURE 4

A, Rapid development of thrombocytopenia (consumptive coagulopathy) in two baboons with life-supporting GTKO.hCD46 pig kidney grafts (indicated in red), and maintenance of normal platelet counts in two baboons (treated identically) with life-supporting GKTO.hCD46.hTBM pig kidney grafts (indicated in black). (Modified from Iwase H et al, ref). B, Results of human platelet aggregation assay when human platelets were co-incubated with WT, GTKO.hCD46, GTKO.hCD46.hCD55, GTKO.hCD46. hTBM, and GTKO.hCD46.hEPCR pig aortic endothelial cells (AECs). Human platelet aggregation when exposed to human AECs is shown as a control. (Reproduced with permission from Iwase et al, ref)

FIGURE5

Human hemeoxygenase-1 (hHO-1) transgenic pig aortic endothelial cells (PAEC) are protected against TNF-α–mediated apoptosis, measured by a caspase 3/7 assay. PAECs from hHO-1 transgenic pigs were better protected against TNF-α–mediated apoptosis compared to WT PAEC. (Modified from, and with the courtesy of, Petersen et al, ref)

FIGURE 6

A, Schematic representation of CD47-SIRP-α interaction in relation to natural expression of SIRP-α on human macrophages. Left: After transplantation of an unmodified pig lung into a human, the expression of pig CD47 on the endothelial cells of the pulmonary blood vessels will not be recognized by human SIRP-α–expressing macrophages, which will therefore not be inhibited but will become activated; inflammatory cytokines will be produced and graft injury will occur. Right: When a lung from a pig transgenic for human CD47 is transplanted, the human SIRP-α–expressing macrophages will recognize the pig tissues as “self,” and activation will be inhibited; cytokine production and graft injury will not occur. (Reproduced with permission from Cooper et al, 2012, ref). B, Human (h) CD47 surface expression on pig aortic endothelial cells reduces phagocytosis. Labeled control (GTKO. CD46) pig endothelial cells (white bar) or hCD47-expressing pig endothelial cells (black bar) isolated from transgenic pigs were co-incubated with human macrophages derived from freshly isolated monocytes. High surface expression of CD47 was associated with a significant reduction in phagocytosis (measured by flow cytometry). Statistical comparisons were calculated using a paired T test (*P < 0.05)