Combined transgenic expression of alpha-galactosidase and alpha1,2-fucosyltransferase leads to optimal reduction in the major xenoepitope Galalpha(1,3)Gal.

Abstract

Hyperacute rejection of pig organs by humans involves the interaction of Galalpha(1,3)Gal with antibodies and complement. Strategies to reduce the amount of xenoantigen Galalpha(1,3)Gal were investigated by overexpression of human lysosomal alpha-galactosidase in cultured porcine cells and transgenic mice. The overexpression of human alpha-galactosidase in cultured porcine endothelial cells and COS cells resulted in a 30-fold reduction of cell surface Galalpha(1,3)Gal and a 10-fold reduction in cell reactivity with natural human antibodies. Splenocytes from transgenic mice overexpressing human alpha-galactosidase showed only a 15-25% reduction in binding to natural human anti-Galalpha(1,3)Gal antibodies; however, this decrease was functionally significant as demonstrated by reduced susceptibility to human antibody-mediated lysis. However, because there is residual Galalpha(1,3)Gal and degalactosylation results in the exposure of N-acetyllactosamine residues and potential new xenoepitopes, using alpha-galactosidase alone is unlikely to overcome hyperacute rejection. We previously reported that mice overexpressing human alpha1,2-fucosyltransferase as a transgene had approximately 90% reduced Galalpha(1,3)Gal levels due to masking of the xenoantigen by fucosylation; we evaluated the effect of overexpressing alpha-galactosidase and alpha1,2-fucosyltransferase on Galalpha(1,3)Gal levels. Galalpha(1, 3)Gal-positive COS cells expressing alpha1,3-galactosyltransferase, alpha1,2-fucosyltransferase, and alpha-galactosidase showed negligible cell surface staining and were not susceptible to lysis by human serum containing antibody and complement. Thus, alpha1, 2-fucosyltransferase and alpha-galactosidase effectively reduced the expression of Galalpha(1,3)Gal on the cell surface and could be used to produce transgenic pigs with negligible levels of cell surface Galalpha(1,3)Gal, thereby having no reactivity with human serum and improving graft survival.