Adenovirus-mediated expression of anti-sense pig α (1,3) galactosyltransferase or human secretor type α (1,2) fucosyltransferase reduces level of Gal α (1,3) Gal epitope on NIH3T3 cells
One of major obstacles to xenotransplantation of pig organs into humans is the occurrence of hyperacute rejection (HAR) caused by the binding of natural human IgG and IgM antibodies to the antigenic molecules on the membrane of pig cells, particularly endothelial cells in vascularized organs, followed by the complement cascade reaction. The main antigenic molecule is carbohydrate epitope Galα(1,3)Gal (gal epitope) synthesized by α(1,3) galactosyltransferase [α(1,3) GT]. Gal epitope is present in pigs, mice, and New World monkeys, but is replaced with H blood group antigen in humans, apes, and Old World monkeys due to lack of a functional α(1,3) GT. H blood group antigen is synthesized by α(1,2) fucosyltransferase [α(1,2) FT]. For down-regulation of gal epitope expression in xenotransplantation, we constructed two recombinant adenoviruses (rAdv) expressing different regions of anti-sense pig α(1,3) galactosyltransferase (Ad5anti-sGT600 and Ad5anti-sGT1100) and one rAdv expressing human secretor type α(1,2) fucosyltransferase (Ad5hSeFT), and infected NIH3T3 cells. The expression levels of H blood group antigen and Gal epitope were analyzed by flow cytometry using FITC-UEA-I and FITC-GS-IB4 lectins, respectively. We found that there was about 30% less expression of gal epitope on the surface of NIH3T3 cells infected by Ad5anti-sGT600 and Ad5anti-sGT1100. And H blood antigen was detected on NIH3T3 cells after Ad5hSeFT infection and resulted in more than 40% reduction in the level of gal epitope on the cell surface. This reduction increased the resistance of infected NIH3T3 cells to lysis by normal human serum. In addition, coexpression of anti-sense pig α(1,3) galactosyltransferase and human secretor type α(1,2) fucosyltransferase led to a further reduction of Gal epitope. Inhibition of Gal epitope in our studies would be of petentiation for prevention of HAR in Xenotransplantation.