Reduction of α‐galactosyl xenoantigen by expression of endo‐β‐galactosidase C in pig endothelial cells

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Abstract
Background: Elimination of the Galα1–3Galβ1–4GlcNAc (αGal) epitope has been considered to be essential for successful pig-to-human xenotransplantation but, unfortunately, has not been achieved. Endo-β-galactosidase C (EndoGalC) is an endoglycosidase which cleaves the Galβ1–4GlcNAc linkage in the αGal epitope and digests out the Galα1–3Gal disaccharide. Because of its potent activity in physiological pH conditions, EndoGalC can remove αGal epitopes expressed on the cell surface of pig erythrocytes and vascular endothelial cells almost completely. In vivo or ex vivo administration of EndoGalC successfully reduced αGal expression in pig kidneys to an undetectable level, but αGal epitopes soon reappeared. Gene expression of EndoGalC in pig cells was attempted to solve this problem. As the terminal αGal is transferred in the trans-Golgi network by α-1,3-galactosyltransferase (α1,3GT), colocalization of the EndoGalC gene with the α1,3GT gene was expected to be one of the most reliable ways to eliminate the αGal epitope. Methods and results: The sequence of pig α1,3GT, including the cytoplasmic tail, transmembrane domain and stem region, was ligated upstream of EndoGalC, and the conjugated gene was expressed in pig aortic endothelial cells and COS7 cells. Following the introduction of the gene, the αGal epitope on pig aortic endothelial cells was effectively reduced. Transfection studies in COS7 cells using EndoGalC combined with α1,3GT showed that the expressed EndoGalC was localized not only inside, but also outside, the cells. The expression of EndoGalC conjugated with a murine immunoglobulin (Igκ)-chain signal sequence also showed a similar effect. Conclusions: These results suggest the effectiveness of gene transfer with EndoGalC into pig endothelial cells, and strongly encourage us to produce transgenic animals with the expressed enzyme.