Genetic modifications of the porcine genome regulate inflammatory and coagulation responses during ex-vivo porcine liver perfusion with human blood
Nikolaos Serifis1, Taylor M. Coe1, Danielle Detelich1, Charles G. Rickert1, Rudy Matheson1, Cailah Carroll1, Wenning Qin2, Yinan Kan2, Jacob Layer2, Gulcin Demirci2, Michele Youd2, William Westlin2, Luhan Yang2, Agnes M. Azimzadeh1, James F. Markmann1.
1Division of Transplant Surgery, Massachusetts General Hospital, Boston, MA, United States; 2eGenesis, Inc., Cambridge, MA, United States
Introduction: Xenotransplantation using pig organs provides a promising solution to the shortage of donor organs, but this is limited by human xenoreactive antibodies that cause early graft rejection. Genome editing can eliminate xenoantigens in donor pigs to minimize the impact of these xenoantibodies. Despite genome editing, profound thrombocytopenia, coagulopathy, and rejection are the main barriers remaining. Herein, we describe the effect of various genetic modifications of the pig donor on the inflammatory and coagulation responses of human blood after ex-vivo perfusion of porcine livers.
Materials and Methods: Ex-vivo liver perfusion of wild type (WT) (n=2), GTKO.CD55 (n=1) and two variations of triple xenoantigen knock out (TKO - GTKO, 4GalKO, CMAHKO) pigs with additional genetic modifications targeting complement, coagulation, and inflammation regulation (Pig 2.05; n=3, Pig 2.09 n=3) was completed using freshly collected, heparinized human whole blood and plasma on the Liver Assist (Organ Assist, Groningen, NL). Perfusion was continued until liver failure. EDTA plasma samples were collected for cytokine and coagulation marker measurement by Luminex assay. Targets of this study were human IL-1B, IL-6, IL-8, TNF-α, fibrinogen and D-dimer. Marker concentration was averaged at each time point within groups and presented with standard deviation.
Results: Liver perfusion lasted for 5-7 hours for WT, 3 hours for GTKO.CD55, 8-11 hours for 2.05 and 12-14 hours for 2.09. Elaboration of pro-inflammatory cytokines IL1-B, IL-6 and TNF-α and neutrophil recruiting cytokine IL-8 was lower in the 2.05 and 2.09 groups when compared to WT and GTKO.CD55 livers. Groups 2.05 and 2.09 showed decreased consumption of fibrinogen and decreased production of fibrin degradation product D-dimer compared to WT livers throughout ex-vivo perfusion.
Conclusion: Genetic modifications of pig donors has led to a marked decrease in pro-inflammatory cytokines and liver survival prolongation compared to WT pigs. Higher fibrinogen levels and lower D-dimer production in 2.05 and 2.09 livers suggest less coagulopathy and thrombosis in these groups. Additional studies will be conducted to evaluate other targets (i.e. anti-coagulant transgenes) implicated in xenograft injury.
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