Generation of the GGTA1/CMAH/hCD46 Genetically Modified Pigs for Xenotransplantation

The demand for organ transplantation has rapidly increased all over the world during the past decade. Genetically modified pigs provide a solution to the severe shortage of organs available for human transplantation. Porcine α-1,3-galactosyltransferase (GGTA1) gene is generates Gal-T epitopes that trigger hyperacute rejection in pig-to-human transplantation. Since production of GGTA1 knock-out pigs in 2002, non-gal antigens are considered to be the next xenoantigen involved in the rejection phenomenon. Here, we targeted the GGTA1 and CMP-Neu5Ac hydroxylase (CMAH) genes with CRISPR-Cas9 systems resulting in double knock-out pigs that no longer express α-Gal or Neu5Gc. Similar to GGTA1 gene, CMAH is widely expressed on the endothelial cells of many mammals except humans and this epitope is a potential porcine target for the antinon- gal antibody in humans. CMAH is responsible for the expression of Neu5Gc that key non-gal antigen. Additionally, hCD46 controls complement activation and when this gene expressed sufficiently as a transgene protects xenografts against complement-mediated rejection. This is report to describe generation of transgenic pigs that modify GGTA1, CMAH and hCD46. We expect to remove α-gal and Neu5Gc antigens and express hCD46 from pig for reducing human antibody mediated cytotoxicity.