Development of a BM7G(TKO/hCD46/hCD55/hTHBD/hEPCR) donor pig with endogenous promoter-driven transgenes for xenotransplantation

INTRODUCTION: Xenotransplantation holds promise for addressing the organ shortage crisis. Multi-genetic modification of pigs, such as knockout of three carbohydrate antigen-related genes and expression of immunoprotective proteins, can significantly improve xenograft survival. However, existing multi-gene modification strategies face challenges: transposon-based transgenic technology may lead to unstable expression, while exogenous promoters used in site-specific integration strategies are susceptible to epigenetic silencing, making it difficult to maintain long-term, stable expression levels. Therefore, developing a donor pig model capable of achieving stable and long-lasting multi-gene expression is a critical need in the field. METHODS: CRISPR-Cas9 technology was used to knockout three major glycan antigen genes (GGTA1, CMAH, β4GalNT2) to eliminate hyperacute rejection. Subsequently, four human protective genes (hCD55, hCD46, hTHBD, hEPCR) were site-specifically integrated into the porcine Rosa26 safe-harbor locus. Their expression was driven by the porcine endogenous Rosa26 promoter and the THBD core promoter, respectively, to ensure long-term stable and tissue-specific expression. Furthermore, the selection marker gene was efficiently removed using the Cre/loxP system. RESULTS: The three glycan antigens were completely absent at both cellular and tissue levels in BM7G genetically modified pigs. What's more, four protective proteins were stably expressed in vascular endothelial cells and major organs such as the heart, liver, and kidneys. Among them, hCD55 and hCD46 were widely expressed, while hTHBD and hEPCR were specifically expressed in the vascular region. In-vitro functional assays confirmed that BM7G porcine vascular endothelial cells significantly reduced the binding of human antibodies, effectively inhibited complement-dependent cytotoxicity, and decreased the formation of thrombin-antithrombin (TAT) complexes. CONCLUSION: In summary, by combining the knockout of xenoantigens with the use of endogenous promoters to drive the expression of multiple human protective genes, we successfully constructed a seven-gene modified pig model with low immunogenicity and synergistic protective functions. This provides an important donor resource for preclinical research in xenotransplantation.