COMBINED TRANSGENIC EXPRESSION OF ALPHA-GALACTOSIDASE AND ALPHA-1,2-FUCOSYL-TRANSFERASE LEADS TO OPTIMAL REDUCTION IN THE MAJOR XENOEPITOPEGAL-ALPHA(1,3)GAL

Hyperacute rejection of pig organs by humans involves the interaction of Gal alpha(1,3)Gal with antibodies and complement, Strategies to red uce the amount of xenoantigen Gal alpha(1,3)Gal were investigated by o verexpression of human lysosomal cy-galactosidase in cultured porcine cells and transgenic mice. The overexpression of human alpha-galactosi dase in cultured porcine endothelial cells and COS cells resulted in a 30-fold reduction of cell surface Gal alpha(1,3)Gal and a 10-fold red uction in cell reactivity with natural human antibodies, Splenocytes f rom transgenic mice overexpressing human alpha-galactosidase showed on ly a 15-25% reduction in binding to natural human anti-Gal alpha(1,3)G al antibodies; however, this decrease was functionally significant as demonstrated by reduced susceptibility to human antibody-mediated lysi s, However, because there is residual Gal alpha(1,3)Gal and degalactos ylation results in the exposure of N-acetyllactosamine residues and po tential new xenoepitopes, using alpha-galactosidase alone is unlikely to overcome hyperacute rejection, We previously reported that mice ove rexpressing human alpha 1,2-fucosyltransferase as a transgene had appr oximate to 90% reduced Gal alpha(1,3)Gal levels due to masking of the xenoantigen by fucosylation; we evaluated the effect of overexpressing a-galactosidase and alpha 1,2-fucosyltransferase on Gal alpha(1,3)Gal levels, Gal alpha(1,3)Gal-positive COS cells expressing alpha 1,3-gal actosyltransferase, alpha 1,2-fucosyltransferase, and alpha- galactosi dase showed negligible cell surface staining and were not susceptible to lysis by human serum containing antibody and complement, Thus, alph a 1,2-fucosyltransferase and alpha-galactosidase effectively reduced t he expression of Gal alpha(1,3)Gal on the cell surface and could be us ed to produce transgenic pigs with negligible levels of cell surface G al alpha(1,3) Gal, thereby having no reactivity with human serum and i mproving graft survival.