Regulatory sequences of the porcine THBD gene facilitate endothelial-specific expression of bioactive human thrombomodulin in single- and multitransgenic pigs.

Wuensch, Annegret; Baehr, Andrea; Bongoni, Anjan; Kemter, Elisabeth; Blutke, Andreas; Baars, Wiebke; Haertle, Sonja; Zakhartchenko, Valeri; Kurome, Mayuko; Kessler, Barbara; Faber, Claudius; Abicht, Jan-Michael; Reichart, Bruno; Wanke, Ruediger; Schwinzer, Reinhard; Nagashima, Hiroshi; Rieben, Robert; Ayares, David; Wolf, Eckhard and Klymiuk, Nikolai (2014). Regulatory sequences of the porcine THBD gene facilitate endothelial-specific expression of bioactive human thrombomodulin in single- and multitransgenic pigs. Transplantation, 97(2), pp. 138-147. Lippincott Williams & Wilkins 10.1097/TP.0b013e3182a95cbc

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > Forschungsbereich Mu50 > Forschungsgruppe Herz und Gefässe
09 Interdisciplinary Units > Microscopy Imaging Center (MIC)

Graduate School:

Graduate School for Cellular and Biomedical Sciences (GCB)

UniBE Contributor:

Bongoni, Anjan and Rieben, Robert

Subjects:

600 Technology > 610 Medicine & health

ISSN:

0041-1337

Publisher:

Lippincott Williams & Wilkins

Language:

English

Submitter:

Verena de Serra Frazao-Bill

Date Deposited:

13 Mar 2015 08:49

Last Modified:

20 Feb 2019 09:43

Publisher DOI:

10.1097/TP.0b013e3182a95cbc

PubMed ID:

24150517

BORIS DOI:

10.7892/boris.64347

URI:

https://boris.unibe.ch/id/eprint/64347

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