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 one 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 h 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 h 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.

Keywords: ex vivo lung perfusion; genetically engineered pig; immune response; lung xenotransplantation; thromboregulation.

Figure 1

Kaplan – Meier survival curve demonstrating association between cumulative genetic modifications and survival time. P < 0.01 for all inter-group comparisons. nb: 0 modifications group represents xenoperfusion of wild type pig lungs.

Figure 2

Association between cumulative genetic modifications and survival to 4 hours of perfusion. P < 0.0005. nb: 0 modifications group represents xenoperfusion of wild type pig lungs.

Figure 3

Perfusion parameters for parameters for all lungs, segregated by lung failure within four hours versus lungs surviving at four hours. X axis represents perfusion time in minutes. #: P < 0.05, *: P < 0.005

Figure 4

Perfusion parameters for lungs from all pigs including the background GalTKO.hCD46 genotype, segregated by lung failure within four hours versus lungs surviving at four hours. X axis represents perfusion time in minutes. #: P < 0.05, *: P < 0.005