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GalT-KO/CD46/hTM triple-transgenic donor animals for pig-to-baboon heart transplantation

Nikolai Klymiuk¹, Annegret Wuensch¹, Mayuko Kurome¹, Barbara Kessler¹, Andrea Baehr¹, Hiroshi Nagashima², David Ayares³, Eckhard Wolf¹

¹Molecular Animal Breeding, LMU Munich, Munich, Germany; ²Laboratory of Developmental Engineering, Meiji University, Kawasaki, Japan; ³Revivicor Inc., Blacksburg, VA, United States

Complement mediated rejection of xenografts is commonly assumed to be alleviated by using transgenic pigs carrying a knockout of the a1,3 galactosyl-galactose transferase (GalT-KO) combined with the expression of human CD46 complement regulator. Beside these humorally mediated mechanisms, other restrictions prevent the long-time acceptance of xenografts and, thus, will require the production of multi-transgenic donor pigs.

In this study we focused on the incompatibilities of the human and porcine coagulation system. The inability of porcine thrombomodulin (TM) to activate human protein C prompted the equipment of donor organs with its human orthologue. Initial experiments revealed that a genomic fragment from the porcine TM locus drives strong and endothelial specific expression of the human TM (hTM) gene in transgenic pigs. Consequently, the same construct was transfected into primary pig cells of GalT-KO/CD46 background which were then used for somatic cell nuclear transfer. Three founder animals were generated with two of them exhibiting strong expression of hTM on the endothelium of all examined organs. Primary cells from one line were chosen for reproduction by another round of nuclear transfer. In total, we generated 34 triple-transgenic animals by re-cloning and supplied 10 of them for pig-to-baboon orthotopic heart transplantation.

This, for the first time, reports the production multi-transgenic pigs with endothelial specific human thrombomodulin.