Transgenic pigs as organ donors in xenotransplantation

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Xenotransplantation is considered to be promising as an alternative method to solve the shortage of donor organs. As molecular biological technique has recently advanced, attempts to overcome the problem of antigen-antibody interaction has been directed towards the modification of the donors. Attention has been directed toward removal or replacement of alphaGal epitopes, since transgenic pigs expressing DAF (decay accelerating factor) could not suppress hyperacte rejection completely. alphaGal epitopes are synthesized by alphagalactosyltransferase (GT). Unfortunately, gene knockout is at present not achievable in the pig due to the anavailability of porcine embryonic stem cells. An alternative approach, which is called "enzymatic remodeling of the carbohydrate surface" has been attempted. That is to say, this concept is that the alphaGal molecules would be replaced with another oligosaccharides such as fucose by indtoruction of alpha1,2 fucosyltransferase (FT). We have produced alpha1, … More 2 FT transgenic pigs. However, this strain could not be established, since these pigs died of malignant tumor or incomplete development. We also showed the observation that such gene transfer decreased not only alphaGal expression but also sialylation in cultured endothelial cells and inhibited the capacity for tube formation. Our results suggests that extreme modification of carbohydrate antigens on cell surfaces may have a detrimental effect on developement or differentiation. We also demonstrated that alpha1,2 FT gene or GT ribozyme transfer using adenoviral vectors decreased alphaGal expression in vitro. We are now exploring direct gene replacement of alpha1,3 GT gene with human alpha1,2 FT gene, namely knock-in method. However, further experiment will be necessary to examine if modification of carbohydrate antigens in cell surface is valid for xenotransplantation.We analyzed the genomic structure of alpha1,3 GT in pigs and constructing a targeting vector with the aim of inducing alphaGal knockout pigs as soon as porcine embryonic stem cells or nuclear transplantation are available. We have shown several alternative splicing variants in pig alpha1,3 GT,which are related to functional differences in the enzyme activity. Some splicing variants, which are defective in exon 5,6 and 7, have been found to have less enzymatic activity, indicating the importance of the stem region in the enxyme activity. Furthermore, splicing varlants without exon 8 or mutant cDNA (two mutations in the catalytic region ; exon 9) were also devoid of enzymatic activity. These observations suggest new strategles for reducing alphaGal antigen expression in pigs, that is to say, the introduction of cDNA without exon 7,8 or of mutant cDNA that can inhibit alpha1,3 GT enzymatic activity in a dominant negative effect. Less