Inhibition of Xenogeneic Response in Porcine Endothelium Using RNA Interference

Abstract

Rejection mediated by antibody recognition of the alpha-Gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) is a major barrier in porcine-to-human xenotransplantation. Because the synthesis of alpha-Gal is dependent on alpha1,3 galactosyltransferase (alpha1,3GT), methods of blocking this enzyme are needed. RNA interference induced by small interfering RNA (siRNA) is a powerful technique for allowing the silencing of mammalian genes with great specificity and potency. In this study, we use siRNA for silencing of alpha1,3GT with the purpose of reducing expression of the alpha-Gal epitope and subsequently decreasing immunogenicity of porcine endothelial cells. alpha1,3GT-specific and control siRNAs were transfected into the porcine aortic endothelial cell line, PED. alpha-Gal expression was assessed by Western blotting, flow cytometry, and immunofluorescence. Protection from human-complement and natural killer (NK)-cell-mediated cytotoxicity was evaluated by Cr-release assays after incubation of PED with normal human serum (NHS) and NK92 cell, respectively. RNA interference was successfully achieved in PED as witnessed by the specific knock-down of alpha1,3GT mRNA levels. Flow cytometric analysis using the Griffonia simplicifolia isolectin B4 lectin confirmed the suppression of alpha1,3GT activity as evidenced by decreased alpha-Gal. Functional relevance of the knock-down phenotype was illustrated by the finding that silenced PED were protected from cytotoxicity of NHS. Protection from NK-mediated cytotoxicity was not observed. Our data are the first to demonstrate that RNA interference is a potent tool to down modulate alpha-Gal expression and to protect endothelial cells from complement-mediated cytotoxicity. Gene silencing by siRNA may represent a new approach for overcoming hyperacute and acute vascular rejection.