hEPCR.hTBM.hCD47.hHO-1 with donor clodronate and DDAVP treatment improves perfusion and function of GalTKO.hCD46 porcine livers perfused with human blood

Abstract

Introduction: Platelet sequestration, inflammation, and inappropriate coagulation cascade activation are prominent in liver xenotransplant models and are associated with poor outcomes. Here, we evaluate a cassette of six additional genetic modifications to reduce anti-pig antibody binding (α-1,3-galactosyl transferase knockout [GalTKO]) and target coagulation dysregulation (human endothelial protein C receptor [hEPRC] and thrombomodulin [hTBM]), complement pathway regulation (human membrane cofactor protein, hCD46), inflammation heme oxygenase 1 [hHO-1]), and a self-recognition receptor (integrin-associated protein [hCD47]), as well as donor pharmacologic treatments designed to blunt these phenomena.

Methods: Livers from GaltKO.hCD46 pigs ("2-gene," n = 3) and GalTKO.hCD46 pigs also transgenic for hEPRC, hTBM, hCD47, and hHO-1 ("6-gene," n = 4) were perfused ex vivo with whole human blood. Six-gene pigs were additionally pretreated with desmopressin (DDAVP) and clodronate liposomes to deplete vWF and kupffer cells, respectively.

Results: The average perfusion times increased from 304 (±148) min in the 2-gene group to 856 (±61) min in the 6-gene group (p = .010). The average heparin administration was decreased from 8837 U/h in the 2-gene to 1354 U/h in the 6-gene group (p = .047). Platelet sequestration tended to be delayed in the 6-gene group (p = .070), while thromboxane B2 (TXB2, a platelet activation marker) levels were lower over the first hour (p = .044) (401 ± 124 vs. 2048 ± 712 at 60 min). Thrombin production as measured by F1+2 levels tended to be lower in the 6-gene group (p = .058).

Conclusions: The combination of the hEPCR.hTBM.hCD47.hHO-1 cassette along with donor pig DDAVP and clodronate liposome pretreatment was associated with prolonged function of xenoperfused livers, reduced coagulation pathway perturbations, and decreased TXB2 elaboration, and reflects significant progress to modulate liver xenograft injury in a pig to human model.

Keywords: ex-vivo perfusion; liver xenotransplantation; transgenic pigs.

FIGURE 1.

Protein expression by capillary western and immunohistochemistry. (A) hTBM, hEPCR, hHO-1, and hCD46 expressed at detectable levels in preperfusion liver samples. hCD47 expression is below the assay detection limit. (B) Protein expression is normalized to actin and mean values from two different experiments are represented in arbitrary units. (C) Representative IHC shows detection of hTBM (black arrows), hEPCR, hHO-1, and hCD46. Expression of hCD47 falls below the level of detection. *indicated positive control for hCD47 detection

FIGURE 2.

Function of transgene protein expression. (A) The 2-gene group had significantly lower levels of APC production compared to the 6-gene group and human samples (p = .023, .002, respectively). The difference in APC production between the 6-gene group and human samples was not statistically significant. (B) There was significantly less apoptosis in the 6-gene group compared to the 2-gene group (p = .007) (*p < .05, **p < .005)

FIGURE 3

Graft viability. Kaplan–Meier curves showing significantly longer perfusion duration of the 6-gene group compared to the 2-gene group (p = .010)

FIGURE 4

Transaminase levels. Measured ALT (A) and AST (B) values comparing anhepatic, allogenic, and xenogeneic groups. ALT levels in all experiments remained within physiologic ranges. AST levels rose to supraphysiologic levels in the allogenic and the 2- and 6-gene xenogeneic groups. There was no significant difference between the 2- and 6-gene groups

FIGURE 5.

Platelet conservation. (A) Platelet levels measured by commercial lab (hemocytometer) during the first 15 min of perfusion in anhepatic, allogenic, and xenogeneic groups. Platelet counts were significantly different between the allogenic and 2-gene groups (p = .021). There was no significant difference between the allogeneic and 6-gene groups (p = .25) or the 2- and 6-gene groups (p = .097). (B) Platelet counts measured by flow cytometry over 4 h of perfusion. Platelet levels were lower in the 2-gene compared to 6-gene group with a trend toward significance (p = .07)

FIGURE 6

Thromboxane generation. Both the anhepatic and 6-gene experiments saw a steady rise in TXB2 over the course of perfusion. The 2-gene group had an initial sharp rise in TXB2 levels within 30 min of perfusion which peaked at 1 h and then declined. The difference in TXB2 levels between the 2- and 6-gene groups at 1 h was statistically significant (p = .044)

FIGURE 7

β-TG elaboration. Β-TG elaboration increased over time in the anhepatic, 2-gene, and 6-gene groups, though there were no significant differences

FIGURE 8

Heparin requirements. Heparin rate and bolus dosing (arrows) for allogenic and xenogeneic experiments. After initial blood pretreatment, a low rate continuous heparin infusion was started by protocol but was quickly titrated off based on therapeutic (>400) ACT values for allogenic experiments. In contrast, the xenogeneic experiments required continued heparin administration to maintain a therapeutic ACT, with the 2-gene group requiring escalating rates and boluses particularly around the time of graft failure. The cumulative heparin dose administered over the course of each xenogeneic experiment was significantly greater for 2-gene than 6-gene livers (p = .047)

FIGURE 9

Thrombin generation as measured by F1 + 2 levels. The anhepatic experiments had stable very low levels of F1 + 2 throughout the duration of perfusion. The 2-gene and 6-gene groups had similar levels of F1 + 2 production through the first hour of perfusion and then diverged with the levels in the 2-gene group being approximately double that of the 6-gene group. These results trended toward statistical significance (p = .058)

FIGURE 10

Histology of perfusion samples. Biopsies taken after 1, 4, and 8 hours of perfusion demonstrated preserved liver architecture (Figure 10). Liver tissues appeared viable without evidence of arterial thrombi. Increasing sinusoidal congestion was noted over time