(2017), IXA 2017- Abstracts of the 14th Congress of the International Xenotransplantation Association, Baltimore, USA. Xenotransplantation, 24: n/a, e12328. doi:10.1111/xen.12328
ImmuImmunogenicity of Genetically-Engineering Porcine Liver Sinusoidal Endothelial Cells
Zheng-Yu Wang1, Luz M Reyes1, Thomas A Tector1, Juan Li1, Mathew F Tector1, Joseph A Tector1.
1Surgery, University of Alabama at Birmingham, School of Medicine, Birmingham, AL, United States
Introduction: Xenotransplantation has the potential to solve the shortage of human organs needed for transplantation. Disrupting the porcine GGTA1, CMAH and b4GalNT2 genes eliminates the gal-α(1,3)-gal, N-glycolylneuraminic acid, and Sd(a) carbohydrate xenoantigens recognized by human antibodies on red blood cells, renal endothelial cells and peripheral blood mononuclear cells. It is important to examine rejection pathways at an organ-specific level. The object of this study is to evaluate the human preformed antibody reactivity against porcine liver sinusoidal endothelial cells (LSEC) deficient in these carbohydrate-modifying genes in vitro.
Methods: Primary LSEC were isolated from wild type pigs. The immortalized cell lines were generated using Lenti-SV40 T. The Cas9 endonuclease and gRNA were used to create lacking GGTA1, GGTA1/b4GalNT2, GGTA1/CMAH, or GGTA1/CMAH/b4GalNT2 genes. Characteristics of these cell lines were analyzed using flow cytometry. Human IgG/IgM binding to porcine cell lines was examined using flow cytometric crossmatch assay. Complement-dependent cytotoxicity was performed using MTS cell proliferation assay.
Results: Immortalized porcine LSEC exhibited endothelial morphologic features and expression of SV40 T and endothelial marker CD31. Carbohydrate expression for GGTA1, b4GalNT2 and CMAH genes were not detectable on these genetically-engineering cell lines using IB4 lectin/DBA lectin/Neu5Gc antibody. Simultaneously removing these xenoantigen genes reduced human IgG and IgM binding toward the porcine LSEC. Furthermore, complement-dependent cytotoxicity was reduced in the porcine LSEC deficient in GGTA/b4GalNT2/CMAH genes.
Conclusions: Effective genetic editing can silence the porcine GGTA1, CMAH and B4GalNT2 genes in LSEC, and reduce human preformed antibody binding to LSEC. Porcine LSEC will be a useful reagent for the further study of xenoimmunology
United Therapeutics .