Abstract
Abstract BACKGROUND Genetic engineering pig models of pig-to-human xenotransplantation may overcome a growing shortage of available solid organs. Development of pigs deficient in galactose α1,3 galactose and N-glycolylneuraminic acid have significantly improved cell survival when challenged by human antibody and complement in vitro. The b4GalNT2 glycosyltransferase may also synthesize xenoantigens in pigs. In this study, we used RBC, as a cell model, to assess human antibody-mediated response to non-SLA xenoantigens of pigs deficient in these carbohydrate-modifying genes. MATHODS AND MATERIALS RBC were isolated from wild type pigs and pigs deficient in GGTA1/CMAH knockout (DKO) and GGTA1/CMAH/β4GalNT2 knockout (TKO). Human ABO sera were purchased from Valley Biomedical. Human ABO RBC were isolated from healthy people. Total antibody binding was assessed by hemagglutination assay; human IgG or IgM binding to RBC was characterized by flow cytometry; opsonized RBC phagocytosis by THP-1 macrophages was analyzed using confocal microscope. RESULTS Phenotype of TKO RBC was characterized by flow cytometry. Silencing the three genes reduced hemagglutination levels of TKO RBC compared with that of DKO RBC by all three human blood-type A, O, and AB sera but except blood-type B sera, and decreased both of IgG and IgM binding to TKO RBC compared with DKO RBC. Confocal image data showed that phagocytosis of opsonized TKO RBC was lower than that of opsonized DKO RBC by THP-1 cells. DISCUSSION Non-SLA xenoantigenicity of pig RBC can be disrupted using gene engineering tools, which greatly reduced the humoral barrier to xenotransplantation
Published Version
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