Expression of Human CD46 Modulates Inflammation Associated With GalTKO Lung Xenograft Injury

Abstract

Evaluation of lungs from GalTKO.hCD46 pigs, genetically modified to lack the galactose-α(1,3)-galactose epitope (GalTKO) and to express human CD46, a complement regulatory protein, has not previously been described. Physiologic, hematologic and biochemical parameters during perfusion with heparinized fresh human blood were measured for 33 GalTKO.hCD46, GalTKO (n=16), and wild type pig lungs (n=16), and 12 pig lungs perfused with autologous pig blood. Median GalTKO.hCD46 lung survival was 171 minutes compared to 120 for GalTKO (p=0.27) and 10 for wild type lungs (p<0.001). Complement activation, platelet activation, and histamine elaboration were significantly reduced during the first 2h of perfusion in GalTKO.hCD46 lungs compared to GalTKO (ΔC3a at 120′ 812±230 vs. 1412±1047, p=0.02; ΔCD62P at 120′ 9.8±7.2 vs. 25.4±18.2, p<0.01; Δhistamine at 60′ 97±62 vs. 189±194, p=0.03). We conclude that, in addition to significant down-modulation of complement activation, hCD46 expression in GalTKO lungs diminished platelet and coagulation cascade activation, neutrophil sequestration and histamine release. Because GalTKO.hCD46 lung failure kinetics correlated directly with platelet and neutrophil sequestration, coagulation cascade activation, and a rise in histamine levels within the first hour of perfusion, further progress will likely depend upon improved control of these pathways, by rationally targeted additional modifications to pigs and pharmacologic interventions.

Lung xenotransplantation

“…For example human complementary regulatory proteins hCD46 (Membrane Cofactor Protein), hCD55 (Decay Accelerating Factor), and hCD59 (Membrane Attack Complex inhibitory protein) are associated with attenuated complement pathway amplification and prolonged lung survival. Human tissue factor pathway inhibitor (TFPI), thrombomodulin (TBM), and endothelial protein C receptor (EPCR) have been successfully introduced into pigs, generally with promising results (38, 43–51). In addition, hCD39 (an integral membrane protein expressed in endothelial and immune cell populations), and hHO-1 (Heme oxygenase-1, an anti-oxidant) have also been transfected into the pig genome; preliminary evidence with other organs and in the lung suggest that these modifications may reduce inflammation and improve function in ex vivo perfusion models (46–48).…”

Recent advances in understanding xenotransplantation: implications for the clinic

“…Increasing attention is being paid to an inflammatory response that develops after the transplantation of a pig organ into a NHP [48-50]. The inflammatory response can augment the immune response and the coagulation discrepancies [51].…”

The case for xenotransplantation

“…Liver and lung xenotransplantation are associated with more complex problems that have limited success to days rather than weeks or months (16, 17), but there is every prospect that these will be overcome through the introduction of pigs with an increasing number of genetic manipulations (18). In this respect, the recent introduction of new techniques of genetic engineering, e.g., zinc finger nucleases (ZFNs), TALENs (transcription activator-like effector nucleases), and the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system, are increasing the speed with which multiple gene manipulations can be introduced (19).…”