Hemolysis-induced hepatic ferroptosis following xenotransfusion of genetically modified pig red blood cells.

To overcome cross-species immunological barriers, researchers utilize gene editing to remove the expression of three major carbohydrate xenoantigens (α-Gal, Neu5Gc, and Sd(a)) and insert genes, such as human protective genes (hCD55 and hCD39). This study aimed to investigate the mechanisms underlying long-term hepatic injury following xenotransfusion of genetically modified pig red blood cells (pRBCs). We xenotransfused pRBCs genetically engineered with GGTA1(-/-)(α-Gal (-)); CMAH(-/-)(Neu5Gc(-)); β4GALNT2(-/-)(Sd(a) (-)); hCD55; hCD39 into a non-human primate (NHP) model of acute hemorrhage hemolysis and evaluated the long-term immune response to xenogeneic RBCs in the liver, a key organ for RBC metabolism. Immediately after xenotransfusion (D + 1), significant elevations of liver enzymes (AST, ALT) and iron-related factors were observed in the serum levels of the recipients, which were normalized by D + 21. However, long-term analysis revealed excessive accumulation of iron ions in the liver and decreased expression of antioxidant enzymes. The resulting endoplasmic reticulum stress (increased GRP78/BiP expression), lipid peroxide accumulation, and iron-dependent cell death (ferroptosis) persisted in the liver of the recipient. These results suggest that, beyond gene editing, additional hurdles must be overcome to completely block oxidative stress damage caused by impaired RBC lysis. Notably, transcriptomic and immunohistochemical analyses in the NHP model identified the direct role of ferroptosis in xenotransfusion-induced liver injury. Therefore, we propose that a multilayered approach, including blocking the ROS-Iron axis and administering ferroptosis inhibitors, is needed to ensure long-term safety in the clinical development of xenotransfusion.