New transgenic pigs for xenotransplantation, part 1

Abstract

The hyperacute rejection response (HAR) after porcine-to-human xenotransplantation can now be reliably overcome. The next immunological hurdle is the acute vascular rejection (AVR) primarily caused by endothelial cell activation followed by disseminated intravascular coagulopathy, increased apoptosis and inflammatory symptoms. Several genes have been proposed to show protective effects against AVR, including human heme oxygenase-I (hHO-1) and human A20 (hA20) gene. HHO-1 has primarily anti-apoptotic and cell protective properties. The hA20 molecule possesses protective features against inflammatory and apoptotic stimuli in endothelial cells. Thus transgenic expression of these genes in pigs may be promising to prolong survival of porcine xenografts. We used somatic cell nuclear transfer (SCNT) for production of transgenic pigs. We produced pigs transgenic for human heme oxygenase 1 (hHO-1) to evaluate the protective effects of that molecule and to compare it with other transgenes used to control of the hyperacute rejection response (HAR), e.g. the DAF transgenes which gave HAR protection in in vitro cell death assays. Importantly, hHO-1 transgenic porcine aortic endothelial cells were significantly better protected against TNF-α mediated apoptosis. In close collaboration with partners at the LMU Munich (Prof. Kupatt et al.) the transgenic pig lines were tested in an ischemia/reperfusion (I/R) circuit. After occlusion of the left anterior descending artery (LAD), hHO-1 transgenic hearts had significantly smaller infarct lesions and concomitantly significantly better global myocardial function than size-matched wild-type controls. In close collaboration with partners at Hannover Medical University (Prof. Winkler et al.), hHO-1 transgenic porcine kidneys were perfused with pooled human blood for the maximum period of 240 min without addition of C1-Inhibitor in an ex vivo perfusion circuit. In parallel, we produced and characterized pigs that express hHO-1 on a Gal–/– background. Gal–/–/hHO-1 pig hearts were tested in the I/R circuit and preliminary results indicate a protective effect shown by decreased infarct size, less inflammation and improved global and regional myocardial function after LAD occlusion. Expression of hA20 from the CAGGS promoter was found in skeletal muscle, heart and PAECs. Cultured human A20-transgenic PAECs showed significantly reduced apoptosis when compared to their wild type counterparts. Only partial protection of hA20-transgenic pig hearts was observed after I/R. While infarct size was not different between the two groups after ischemic assault, hA20-transgenic pig hearts showed a significantly better hemodynamic performance (determined as SES) than the wild type porcine hearts. MPO activity was reduced in transgenic vs. wild type hearts. We also produced pigs carrying shRNA constructs directed against PERV expression. These animals showed significantly reduced PERV-expression for over 6 months compared to wild-type and sham controls. This approach could improve the safety of porcine xenografts. We will now produce pigs carrying hHO-1 on the Gal–/–/hCD46 background. Tissues and organs from these animals will be tested in the previously established in vitro systems, and when positive results are obtained, hearts and kidneys will be transplanted into baboons. A second line of multi-transgenic pigs will have both hA20 and shRNA against PERV expression on the Gal–/–/hCD46/hHO-1 background. The new somatic cloning protocol developed recently will allow rapid screening of promising transgene combinations and will ensure that we achieve our ambitious goals and move xenotransplantation closer to clinical application. This study was funded by grants from the Deutsche Forschungsgemeinschaft Ni 256/ 22-1, -2, -3,-4.