Kidneys From α1,3-Galactosyltransferase Knockout/Human Heme Oxygenase-1/Human A20 Transgenic Pigs Are Protected From Rejection During Ex Vivo Perfusion With Human Blood.

The hyperacute rejection (HAR) is the first immunological barrier occurring after pig-to-primate xenotransplantation. It can be reliably prevented by expressing human complement regulatory genes1-3 and/or by knocking out the α1,3-galactosyltransferase gene (GGTA1) in pigs.4 Current research efforts focus on the acute vascular rejection (AVR) which can be initiated by anti–non-Gal antibody-binding, followed by complement and endothelial activation, finally leading to microvascular thrombosis and graft rejection. Molecular incompatibilities between primate coagulation factors and porcine anticoagulant regulators are considered another causative factor in coagulation dysregulation after pig-to-primate xenotransplantation.5 Ischemia-reperfusion injury (IRI) is also an important factor in xenotransplantation, and the ischemic tissue is damaged after reperfusion by reactive oxygen species. The complement system, proinflammatory changes of the endothelium, and neutrophils are critically involved in this cascade of deteriorating events. Strategies to overcome AVR and IRI include the expression of human anticoagulant, anti-apoptotic, and/or anti-inflammatory transgenes on porcine tissues and organs. Previously, we had produced a human heme oxygenase-1 (hHO-1) transgenic pig line and demonstrated significant protection of hHO-1 transgenic porcine aortic endothelial cells against TNF-α–mediated apoptosis and prolonged survival of hHO-1 transgenic kidneys in ex vivo xenoperfusion experiments with human blood.6 The anti-apoptotic and anti-inflammatory properties of hHO-1 are mediated by catabolizing cytotoxic free heme to biliverdin, carbon monoxide (CO), and free iron. Biliverdin is reduced to bilirubin, which acts as a potent antioxidant.7 The CO provides anti-apoptotic and anti-inflammatory effects via activation of the p38 MAPK pathway.8 Free iron upregulates ferritin which protects cells from oxidative damage.9 This renders hHO-1 promising for attenuating the detrimental effects during xenograft rejection. Human TNF-α–induced protein 3, also known as human A20 (hA20), is a cytoplasmic zinc-finger protein that prevents inflammation by blockade of NF-κB activation10 and inhibition of TNF-α–mediated apoptosis.11 We had shown that transgenic hA20 expression in pigs also provided immune modulatory effects in porcine aortic endothelial cells by rendering them less susceptible to CD95(Fas)L-mediated cell death.12 Because of previous experience,12 we decided to construct a new hA20 expression vector based on the Sleeping Beauty (SB) transposon system13 to ensure high ubiquitous expression of the transgene. The SB transposons are less susceptible to rearrangements and not affected by reverse transcriptase-induced mutations,14 usually leading to stable gene expression profiles. Here, we report the production and characterisation of pigs that express cytoprotective hHO-1 and hA20 transgenes on a α1,3-galactosyltransferase knockout (GGTA1-KO) background. The GGTA1 gene was disrupted by targeting exon 9 of the GGTA1 gene coding for the catalytic domain of the α1,3 galactosyltransferase with zinc-finger nucleases (ZFN) as recently described.15 These genetic modifications were combined in 1 porcine genotype to evaluate if the triple genetic modifications would result in a better control of xenograft rejection. We studied the biological function of the transgenes in vitro in a 51Chromium (51Cr) release assay and performed ex vivo kidney xenoperfusions with human AB-pooled blood to investigate its protective effects on IRI.