Meta‐analysis of the independent and cumulative effects of multiple genetic modifications on pig lung xenograft performance during ex vivo perfusion with human blood

Abstract

Background Genetically modified pigs are a promising potential source of lung xenografts. Ex-vivo xenoperfusion is an effective platform for testing the effect of new modifications, but typical experiments are limited by testing of a single genetic intervention and small sample sizes. The purpose of this study was to analyze the individual and aggregate effects of donor genetic modifications on porcine lung xenograft survival and injury in an extensive pig lung xenoperfusion series. Methods Data from 157 porcine lung xenoperfusion experiments using otherwise unmodified heparinized human blood were aggregated as either continuous or dichotomous variables. Lungs were wild type in 17 perfusions (11% of the study group), while 31 lungs (20% of the study group) had 1 genetic modification, 40 lungs (39%) had 2, and 47 lungs (30%) had 3 or more modifications. The primary endpoint was functional lung survival to 4 hours of perfusion. Secondary analyses evaluated previously identified markers associated with known lung xenograft injury mechanisms. In addition to comparison among all xenografts grouped by survival status, a subgroup analysis was performed of lungs incorporating the GalTKO.hCD46 genotype. Results Each increase in the number of genetic modifications was associated with additional prolongation of lung xenograft survival. Lungs that exhibited survival to 4 hours generally had reduced platelet activation and thrombin generation. GalTKO and the expression of hCD46, HO-1, hCD55 or hEPCR were associated with improved survival. hTBM, HLA-E, and hCD39 were associated with no significant effect on the primary outcome. Conclusion This meta-analysis of an extensive lung xenotransplantation series demonstrates that increasing the number of genetic modifications targeting known xenogeneic lung injury mechanisms is associated with incremental improvements in lung survival. While more detailed mechanistic studies are needed to explore the relationship between gene expression and pathway-specific injury, and explore why some genes apparently exhibit neutral (hTBM, HLA-E) or inconclusive (CD39) effects, GalTKO, hCD46, HO-1, hCD55, and hEPCR modifications were associated with significant lung xenograft protection. This analysis supports the hypothesis that multiple genetic modifications targeting different known mechanisms of xenograft injury will be required to optimize lung xenograft survival.

Platelet sequestration and activation during GalTKO.hCD46 pig lung perfusion by human blood is primarily mediated by GPIb, GPIIb/IIIa, and von Willebrand Factor

“…hEPCR, hTBM, hCD39) have been created, and are currently being evaluated in lung perfusion studies to determine the extent to which each intervention successfully controls the targeted pathway and modulates organ injury. Our findings to date using these pigs (, L. Burdorf unpublished observations, 2015) show that, despite high expression of these transgenes – and in some instances associated with significant reduction of coagulation cascade activation parameters – platelet activation and sequestration remain prominent features associated with ex vivo lung perfusion .…”

Lung xenotransplantation

“…As deletion of vWF results in a pig with high risk of bleeding (54), current efforts seek to replace key segments of pvWF with the analogous human vWF epitope (38). We monitor platelet and neutrophil sequestration and activation, and thrombin, thromboxane, and histamine generation, as well as lung survival or pulmonary vascular resistance; we find that each of the genetic modifications discussed here is associated with some improvement in one or more of these parameters (47). …”

Immunobiological barriers to xenotransplantation

“…The immunobiological problems associated with transplantation of pig livers [46] and lungs [47] are more complex, and to date have limited graft survival to days rather than weeks, months or years (reviewed in [48]). Humans are known to have natural antibodies to at least two other antigens on pig cells, namely N-glycolylneuraminic acid (NeuGc) (reviewed in [49]) and β1,4 N-acetylgalactosaminyltransferase [50].…”