Purpose: Expression of human complement pathway regulatory proteins (hCPRP’s) such as CD46 or CD55 has been associated with improved survival of pig organ xenografts in multiple different models.  Here we compare the results of ex vivo perfusion with human blood of lungs from GTKO swine heterozygous for hCD46 (with CD46 on one haplotype: GTKO.het-hCD46), homozygous for hCD46 (GTKO.hom-hCD46), or homozygous for hCD46 and heterozygous for hCD55 (GTKO.hom-hCD46.het-hCD55), to explore the hypothesis that an increased ‘hCPRP gene dose’ is associated with improved protection of the xenograft against rejection.

Methods: Lungs from 25 GTKO. het-hCD46 pigs, 9 GTKO.hom-hCD46 pigs, and 6 GTKO.hom-hCD46.het-hCD55 pigs were perfused with human blood for up to 4 hours in an ex vivo circuit.  Results from 5 GTKO pigs are shown as an additional historical control.

Results: Relative to GTKO.het-hCD46 lungs (139±83min, range 5-240; 6/25 surviving at 4 hrs), survival was significantly improved for GTKO.hom-hCD46 (200±72, range 45-240, p=0.023; 7/9 surviving at 4 hrs) or GTKO.hom-hCD46.het-hCD55 lungs (>240min, p=0.004; 6/6 surviving at 4 hrs). GTKO lungs had similar survival to the GTKO.het-hCD46 lungs (176±41-240, p=0.228; 3/5 surviving at 4 hrs). Surprisingly, GTKO.hom-hCD46.het-hCD55 lungs exhibited a relatively more rapid drop in neutrophils between minute 5 and 60 (p<0.001) and increased histamine and thromboxane B2 elaboration at all time-points (p<0.001). Otherwise, although coagulation pathway activation (F1+2) and platelet activation (CD62P) tended to be lower with increased hCD46 gene dose, there were no consistently significant differences between groups in hematocrit, monocyte or platelet counts, complement activation (C3a), or pulmonary vascular resistance in association with increasing hCPRP gene dose.

Conclusion: Genetic engineering approaches designed to augment hCPRP activity by increasing the expression of hCD46 through homozygosity or co-expressing hCD55 with hCD46 were associated with prolonged lung xenograft survival in this model. However, expected reductions in complement activation (C3a) and/or inflammation (neutrophil sequestration, histamine and thromboxane elaboration) were not consistently observed. If ongoing analysis of transgene expression in lung endothelium by immunohistochemistry and Western blot confirm that increased gene dose correlates with higher protein expression, we will conclude that the mechanism by which hCPRP expression provides a survival advantage for transgenic GalTKO swine lungs remains to be determined.