Reducing Immunogenicity of Porcine Bioprosthetic Heart Valves via GGTA1/β4GalNT2/CMAH Gene Targeting

Background Bioprosthetic heart valves (BHVs) originated from pigs are extensively used for diseased heart valve replacement. However, BHVs undergo chronic calcification which is associated with recipient immune responses, leading to structural valve deterioration (SVD). Three major xenoantigens including Galactose a1,3 galactose (αGal), N-glycolylneuraminic acid (Neu5Gc), and glycan products of β-1,4-N-acetyl-galactosaminyl transferase 2 (β4GalNT2) have been identified to be implicated in SVD. The use of valves derived from genetically modified pigs avoid of these pig antigens may improve BHV durability. Methods: CRISPR/Cas9 mediated gene targeting was employed to disrupt porcine GGTA1/CMAH/β4GalNT2 genes. Peripheral blood mononuclear cells (PBMC) and pericardial tissue of GGTA1/CMAH/β4GalNT2 triple gene knockout (TKO) pigs were examined for the targeted xenoantigen expression and human IgG/IgM binding. The pericardial collagen composition was analyzed by Trichrome Masson staining and immunohistochemistry. The total collagen content and the mechanical properties of pericardium of pericardium were evaluated by hydroxyproline estimation method and uniaxial tensile testing, respectively. Results: αGal, Neu5Gc and β4GalNT2 expression was high on WT pig PBMC and pericardium but undetectable on TKO pigs. PBMC and pericardium from GGTA1/CMAH/β4GalNT2 deficient pigs exhibited decreased human IgM/IgG binding compared to those from WT pigs. Collagen expression and content of pericardial tissue showed no significant difference between TKO and WT pigs. Mechanical proprieties of TKO pericardium were comparable with those of WT pigs. Conclusion Elimination of αGal, Neu5Gc and β4GalNT2 xenoantigens had no significant impact on the physical proprieties of porcine pericardium but dramatically reduced its immunogenicity, which highlighted the potential of TKO pigs as preferable sources of BHVs. A grant from the National Natural Science Foundation of China (No. 81570402). a grant from the National Key R&D Program of China (2017YFC1103701). a grant from the National Key R&D Program of China (2017YFC1103702). a grant from the Jiangsu Key Laboratory of Xenotransplantation (BM2012116). grants from the Shenzhen Foundation of Science and Technology (No. JCYJ20160229204849975 and GCZX2015043017281705). a grant from the Sanming Project of Medicine in Shenzhen, the Fund for High Level Medical Discipline Construction of Shenzhen (No. 2016031638).