Cell and Developmental Biology Seminar Series: Strategies to produce multi-transgenic pigs for xenotransplantation

Allotransplantation is the only effective therapy for end-stage human organ failure. However, many patients in need do not receive a suitable organ owing to the ever growing shortage of human organ donors. More than 60,000 registered patients are currently waiting for a lifesaving organ in the European Union (http://ec.europa.eu). It is estimated that 4,100 patients died while officially placed on these waiting lists in 2013 within the 28 member states of the European Union. Thus, functional porcine xenografts and cells to treat patients with terminal organ failure would be of tremendous value for human medicine. It has been shown that most of the problems inherent within xenotransplantation stem from the independent evolutionary adaptation of pigs and primates. These difficulties affect a number of crucial biological processes, specifically those involved in the regulation of important molecular cascades, such as coagulation and complement. These issues need to be adequately addressed if long-term survival of pig organs transplanted into primates needs to be achieved.

The domestic pig shares many genetic, anatomical and physiological similarities to humans and the ability to genetically modify pigs significantly enhances their potential as organ donor. Moreover, the discovery of new molecular tools to modify complex mammalian genomes such as ZFNs, TALENs and CRISPR/Cas, and the improved genomic maps of human and pigs open new avenues towards efficient, precise and fast modification of the porcine genome. However, prior to clinical application of porcine xenografts, three major hurdles have to be overcome: (i) various immunological rejection responses, incl. HAR (hyperacute rejection response), AVR (acute vascular rejection), DXR (delayed cellular rejection), (ii) physiological incompatibilities between the porcine organ and the human recipient, incl. a severe dysfunction of the coagulation cascade, and (iii) the risk of transmitting zoonotic pathogens from pig to humans.

The generation of pigs with a genetic knockout of the a1.3-galactosyltransferase gene (GGTA1) was a milestone down the road towards clinical application of porcine xenografts. The HAR can now be reliably prevented and significantly extended survival times after pig-tobaboon xenotransplantation up to a maximum of 83 days for kidneys and more than two years for heterotopically transplanted hearts have been reported. After orthotopic (i.e. life supportive) heart transplants the average survival of the recipient is 30-50 days. Subsequently porcine xenografts are rejected due to inflammatory symptoms and severe perturbation of coagulation. Thus, the AVR remains the bottleneck to clinical xenotransplantation. Non-anti-Gal antibody binding activates the endothelium and results in cellular damage and thrombotic microangiopathy. The current view is that long-term survival of xenografts after transplantation into primates requires multiple modifications of the porcine genome and a specifically tailored immunosuppressive regimen compliant with current clinical standards. This requires the production and characterization of multi-transgenic pigs to control HAR, AVR and DXR. Several candidate genes, incl. hTM, hHO-1, hA20, CTLA4Ig,have been explored in their ability to improve long-term survival of porcine xenografts after transplantation into non-human primates. The presentation will provide an update on the current status in the production of multi-transgenic pigs for xenotransplantation, with emphasis on recent results from our laboratory, which could bring porcine xenografts closer to clinical application.