Improved multi-transgenic pig lines for xenotransplantation
Final Report Abstract
During the funding period, three main goals were achieved: 1) Production of biallelic GalKO pigs using zinc finger nucleases: An important advantage of xenotransplantation is the opportunity to modify the genome of the donor animals. Two modifications are required for preventing the hyperacute rejection (HAR) in pig-to-human transplantation: 1) elimination of antigenic sugar residues via genetic knockout of the porcine alpha 1,3-galactosyltransferase (Gal) locus (homozygous Gal kO), and/or 2) suppression of the recipient's complement system by introducing one or more regulators of complement activation (RCA), such as CD55, CD46, or CD59. Zinc finger nucleases (ZFNs) are a class of engineered DNA-binding proteins that facilitate editing of the genome by creating double-strand breaks in DNA at targeted locations. Double-strand breaks are important for site-specific mutagenesis in that they stimulate the cell’s natural DNA-repair processes, such as homologous recombination and non-homologous end joining (NHEJ). We have pioneered ZFN application in porcine cells and produced viable female and male Gal-/- pigs via somatic cell nuclear transfer. ZFN technology allowed for a knockout of both alleles of the GGTA1 locus by a single transfection. Wild-type porcine fibroblasts were transiently transfected with plasmids coding for two Gal specific ZFNs. FACS analysis revealed ~1% Gal negative cells; these were selected with a magnetic bead sorting approach. This yielded a cell culture with 99% Gal-negative cells. Gal-negative cells served as donor cells for somatic cell nuclear transfer and lead to the birth of healthy offspring. These pigs were the first pigs worldwide carrying a biallelic knockout of an endogenous gene caused by zinc finger nucleases. 2) Production of pigs expressing functional proteins to suppress acute vascular rejection (AVR): Current view is that long term survival of xenografts after transplantation into primates would require a specifically tailored immunosuppressive regimen compliant with current clinical standards, and additional modifications of the pig genome. Several candidate genes, incl. hTM, hHO-1 and hA20 have been explored in their ability to improve long term survival of porcine xenografts after transplantation into nonhuman primates. During the funded Transregio research unit "Xenotransplantation", we produced the first pigs transgenic for either human thrombomodulin (hTM), human A20 or human heme oxygenase-1 (hHO-1). HHO-1 transgenic porcine aortic endothelial cells were significantly better protected against TNF-alpha mediated apoptosis as compared with their wild-type counterparts. HHO-1 transgenic kidneys maintained function in the ex vivo perfusion circuit with human blood while wild-type kidneys were destroyed within minutes. Additionally, we could show that after occlusion of the left anterior descending artery (LAD), hHO-1 transgenic hearts had significantly smaller infarct lesions and concomitantly a significantly improved global myocardial function than size-matched wild-type controls. 3) Production of pigs with a decreased PERV expression by shRNA approach: In collaboration with the group of Joachim Denner at RKI, we produced pigs carrying shRNA constructs directed against PERV expression. These animals showed significantly reduced PERV-expression for > 2 years. This approach could improve the safety of porcine xenografts.
