Expression of human thrombomodulin gene on genetically engineered pig hearts prolong cardiac xenograft survivals in non-human primates
Avneesh Singh1, Corbin E. Goerlich1, Gheorghe Braileanu1, Alena Hershfeld1, Tianshu Zhang1, Ivan Tatarov1, Billeta Lewis1, Faith Sentz1, David Ayares2, David Kaczorowski1, Bartley P. Griffith1, Muhammad Mohiuddin1.
1Surgery, SOM, University of Maryland Baltimore, Baltimore, MD, United States; 2Revivicor Inc, Blacksburg, VA, United States
Introduction: Thrombomodulin (TBM) is a coagulation regulatory protein that has shown a protective role against coagulation dysregulation and prevented thrombotic microangiopathy and premature loss of cardiac xenografts. We have previously reported long-term cardiac xenograft survival with human TBM gene expression on genetically engineered (GE) pig hearts along with immunosuppression that includes anti-CD40 antibody. In this study, we have investigated the correlation of hTBM expression level on GE donor pig hearts with xenograft survival.
Materials and Methods: Heterotopic and orthotopic cardiac xenotransplantations (XTx) were performed in SPF baboons from 3-gene (i.e. GTKO.CD46.hTBM; (n=7), multigene (i.e.GTKO.CD46.hTBM.EPCR.CD47.HO1; (n=2) and GTKO.CMAH-KO.TBM.EPCR.CD46.DAF; (n=2) GE pigs at NHLBI/NIH and University of Maryland, Baltimore. Recipients were treated with anti-CD20 mAb, CVF, ATG, anti-CD40 mAb (clone 2C10R4) i.e. low dose for short term and high dose, MMF and tapering dose of steroids. Recipients also received continuous intravenous. heparin infusion. Xenograft survival was monitored with telemetry, echocardiography, and manual palpation. The level of hTBM gene expression on porcine endothelial cells (PAEC) and explanted heart samples were examined by quantitative real-time PCR using sybr green kit from Biorad.
Results: Cardiac XTx were performed without any difficulty and baboons were extubated immediately following surgery and were active, eating, and generally well soon thereafter. Orthotopic cardiac xenograft survived ranging from few hours to 30 days and whereas heterotopic cardiac xenograft survived up to 945 days. Quantitative real-time PCR demonstrated a variable hTBM expression (range 2 to 38 folds) on PAEC and explanted xenografts. The level of hTBM expression directly correlated with xenograft survival in both heterotopic and orthotopic cardiac XTx when 3 genes (GTKO.CD46.hTBM) porcine hearts were used. However, a strong correlation was not found between hTBM expression and graft survival when these xenografts also expressed other genes on them, or recipients received a low dose of anti CD40 antibody for the short term.
Conclusion: These results suggest the importance of hTBM expression on graft survival and their high expression on donor pig along with high dose of anti CD40 mAb treatment is required to prolong cardiac xenograft survival.
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