184 INHIBITION OF HUMAN COMPLEMENT-MEDIATED CYTOTOXICITY IN MINIPIG CELLS BY EXPRESSION OF hCD59

Abstract

Xenotransplantation of a pig organ to a human is a possible solution for the shortage of donor organs for transplantation. However, hyperacute rejection (HAR) due to natural antibodies (Nab) present major obstacle in pig-to-human xenotransplantation. To overcome this, much effort has been dedicated to preparing transgenic pigs that express human complement regulatory proteins (CRPs). One of CRPs, the human CD59 gene, can prevent the terminal polymerization of the membrane attack complex by complement. In this study, we investigated the inhibitory effect of hCD59 on complement-mediated cytotoxicity in hCD59-transfected minipig cells. To generate cell lines expressing hCD59, we transfected the hCD59 gene into minipig fetal fibroblasts and established seven transgenic clonal cell lines. The integration of hCD59 gene was confirmed by PCR and expression levels were measured by RT-PCR, fluorescence-activated cell sorting (FACS), Western blot, and immunohistochemistry. FACS analysis of hCD59 clonal cell lines demonstrated a substantial increment of hCD59 expression. The level (82% to 95%) of hCD59 protein expression was increased relative to that of the control. Human complement-mediated cytotoxicity was measured using a CytoTox96^® (Promega Corp., Sydney, Australia) non-radioactive cytotoxicity (LDH) assay using normal minipig cells as a negative control. In the LDH release assay, human complement-mediated cytotoxicity was also significantly reduced to 39.6 ± 17.8% in comparison to that of the control group (73.6 ± 19.1%; P < 0.05; n = 6). These results indicate that the expression of hCD59 gene in minipig cells can efficiently control complement-mediated cytotoxicity.