title: Regulatory sequences of the porcine THBD gene facilitate endothelial-specific expression of bioactive human thrombomodulin in single- and multitransgenic pigs.
abstract: BACKGROUND
  Among other mismatches between human and pig, incompatibilities in the blood coagulation systems hamper the xenotransplantation of vascularized organs. The provision of the porcine endothelium with human thrombomodulin (hTM) is hypothesized to overcome the impaired activation of protein C by a heterodimer consisting of human thrombin and porcine TM.
  
  
  METHODS
  We evaluated regulatory regions of the THBD gene, optimized vectors for transgene expression, and generated hTM expressing pigs by somatic cell nuclear transfer. Genetically modified pigs were characterized at the molecular, cellular, histological, and physiological levels.
  
  
  RESULTS
  A 7.6-kb fragment containing the entire upstream region of the porcine THBD gene was found to drive a high expression in a porcine endothelial cell line and was therefore used to control hTM expression in transgenic pigs. The abundance of hTM was restricted to the endothelium, according to the predicted pattern, and the transgene expression of hTM was stably inherited to the offspring. When endothelial cells from pigs carrying the hTM transgene--either alone or in combination with an aGalTKO and a transgene encoding the human CD46-were tested in a coagulation assay with human whole blood, the clotting time was increased three- to four-fold (P<0.001) compared to wild-type and aGalTKO/CD46 transgenic endothelial cells. This, for the first time, demonstrated the anticoagulant properties of hTM on porcine endothelial cells in a human whole blood assay.
  
  
  CONCLUSIONS
  The biological efficacy of hTM suggests that the (multi-)transgenic donor pigs described here have the potential to overcome coagulation incompatibilities in pig-to-primate xenotransplantation.
authors.affiliation: 1 Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, LMU Munich, Germany. 2 Department of Clinical Research, and Graduate School of Cellular and Biomedical Sciences, University of Bern, Switzerland. 3 Institute of Veterinary Pathology, Center for Clinical Veterinary Medicine, LMU Munich, Germany. 4 Transplant Laboratory, Medizinische Hochschule Hannover, Germany. 5 Department for Veterinary Sciences, LMU Munich, Germany. 6 Meiji University International Institute for Bio-Resource Research, Kawasaki, Japan. 7 Institute of Pathology, LMU Munich, Germany. 8 Department of Anaesthesiology, LMU Munich, Germany. 9 Department of Cardiac Surgery, LMU Munich, Germany. 10 Revivicor, Inc., Blacksburg, Virginia. 11 Address correspondence to: Dr. Annegret Wuensch, Molecular Animal Breeding and Biotechnology, Hackerstr. 27, 85764 Oberschleissheim, Germany.
authors.forename: Annegret
authors.initials: A
authors.lastname: Wuensch
authors.forename: Andrea
authors.initials: A
authors.lastname: Baehr
authors.forename: Anjan K
authors.initials: AK
authors.lastname: Bongoni
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Kemter
authors.forename: Andreas
authors.initials: A
authors.lastname: Blutke
authors.forename: Wiebke
authors.initials: W
authors.lastname: Baars
authors.forename: Sonja
authors.initials: S
authors.lastname: Haertle
authors.forename: Valeri
authors.initials: V
authors.lastname: Zakhartchenko
authors.forename: Mayuko
authors.initials: M
authors.lastname: Kurome
authors.forename: Barbara
authors.initials: B
authors.lastname: Kessler
authors.forename: Claudius
authors.initials: C
authors.lastname: Faber
authors.forename: Jan-Michael
authors.initials: JM
authors.lastname: Abicht
authors.forename: Bruno
authors.initials: B
authors.lastname: Reichart
authors.forename: Ruediger
authors.initials: R
authors.lastname: Wanke
authors.forename: Reinhard
authors.initials: R
authors.lastname: Schwinzer
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nagashima
authors.forename: Robert
authors.initials: R
authors.lastname: Rieben
authors.forename: David
authors.initials: D
authors.lastname: Ayares
authors.forename: Eckhard
authors.initials: E
authors.lastname: Wolf
authors.forename: Nikolai
authors.initials: N
authors.lastname: Klymiuk
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D042783:Endothelial Cells / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D051916:Membrane Cofactor Protein / Q000032:analysis; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D012045:Regulatory Sequences, Nucleic Acid; D013552:Swine / Q000235:genetics*; D018180:Thrombomodulin / Q000235:genetics* / Q000502:physiology; D014183:Transplantation, Heterologous
chemical_list: D051916:Membrane Cofactor Protein; D019408:Platelet Endothelial Cell Adhesion Molecule-1; C546931:THBD protein, human; D018180:Thrombomodulin