Development of a BM7G(TKO/hCD46/hCD55/hTHBD/hEPCR) Donor Pig with Endogenous Promoter-Driven Transgenes for Xenotransplantation

Xenotransplantation represents a critical strategy for alleviating the global organ shortage crisis; however, traditional multi-gene modification strategies often struggle to achieve long-term, stable transgene expression due to instability associated with transposon systems or the susceptibility of exogenous promoters to epigenetic silencing. In this study, we utilized CRISPR-Cas9 technology to knock out the three major glycan antigen genes (GGTA1, CMAH, and β4GalNT2) to eliminate hyperacute rejection.

Subsequently, we site-specifically integrated four protective genes (hCD55, hCD46, hTHBD, and hEPCR) into the porcine Rosa26safe-harbor locus. Expression was driven by the endogenous porcine Rosa26promoter, and selection markers were efficiently removed using the Cre/loxP system. The results demonstrated that the BM7G genetically modified pigs achieved complete ablation of the three glycan antigens and stable, specific expression of the four human protective proteins in vascular endothelial cells and major organs.