Selection of target genes and production of transgenic pigs for xenotransplantation

Abstract

Xenotransplantation is considered as a promising strategy to surmount donor organ shortage in transplantation. However, there are several hurdles for successful xenotransplantation such as immunological graft rejection, safety of donor organ, and ethical issues. To surmount the immunological graft rejection, generation of transgenic pigs is considered as one of strategies. Actually, hyperacute rejection (HAR) was overcome by the generation of α1,3-galactosyltransferase (GalT) KO pigs and/or human complement regulatory proteins (hCRPs). However, acute humoral xenograft rejection (AHXR) is still remained. To surmount the AHXR, new transgenic pigs to reduce immunological responses were needed using anti-inflammatory, anti-apoptotic, and cytoprotective genes. In this study, to effective selection of target genes which have anti-inflammatory, anti-apoptotic, and cytoprotective effects, in vitro function tests were performed. Anti-inflammatory gene (shTNFRI-Fc), anti-apoptotic and cytoprotective gene (heme oxygenase1

HO1), and combination of two genes were validated. In human TNF-α stimulation, expressions of adhesion molecules and chemokines were reduced in shTNFRI-Fc expressing porcine endothelial cells. In addition, combination of shTNFRI-Fc and HO1 showed better cell viability in TNF-α with CHX or H2O2 treated NPCCs than that of single gene expressing NPCCs. Based on the in vitro function tests, shTNFRI-Fc transgenic pig was generated by somatic cell nuclear transfer and performed validation of transgene expression in transgenic pig tissue and function in hTNF-α stimulation. shTNFRI-Fc transgenic pig serum showed effective inhibition in human TNF-α treated porcine endothelial cells. In addition, F1 generation of shTNFRI-Fc transgenic pig was generated successfully by natural breeding with wild-type pig. Three of seven piglets showed the expression of shTNFRI-Fc. In addition, double transgenic pigs expressing shTNFRI-Fc and HAHO1 were also generated by SCNT. Transgene insertion and copy number was confirmed by southern blot. Expression of each transgenes was confirmed by western blot and immunohistochemistry (IHC) in various tissues derived from transgenic pigs. The function of transgenes was confirmed by the measurement of cell viability and Caspase-3/-7 activity in hTNF-α with CHX treatment. Double transgenic pig cells showed more protective effects than wild-type and single transgenic pigs. On the other hands, another target molecules related with thrombin generation was validated. Fibrinogen-like protein2 (FGL2) is known as direct prothrombinase which produce thrombin from prothrombin without classical prothrombinase complex (Factor Xa/Factor Va). FGL2 up-regulation through CD40 stimulation induced an increase of thrombin generation. Knock-down of FGL2 or CD40 showed decrease of thrombin generation. These results indicate that CD40-CD40L signaling may up-regulate the expression of FGL2 and increase thrombin generation. Therefore, blocking the CD40 signal or lacking FGL2 might be helpful to surmount AHXR. In summary, selected target genes were validated their function in inflammation or apoptosis condition. shTNFRI-Fc or combination of shTNFRI-Fc and HAHO1 showed better effect. The expression and function of target genes in shTNFRI-Fc transgenic pig and shTNFRI-Fc-2A-HAHO1 double transgenic pigs were confirmed by measuring cell viability in cytokine-mediated apoptotic circumstances. Furthermore, FGL2, another target gene, up-regulation by CD40 signal was confirmed and increase of thrombin was also confirmed. In conclusion, to overcome the AHXR, validations of the function of target genes were performed and transgenic pigs with anti-inflammatory, anti-apoptotic, and cytoprotective genes were successfully generated in this study. Transgenic pigs generated in this study showed effective anti-inflammatory, anti-apoptotic and cytoprotective function. All things taken together, transgenic pigs produced in this study might be helpful to surmount the AHXR and contribute to successful xenotransplantation through the combination with other genetic modification to overcome the hypaeracute rejection (i.e. GalT knockout and transgenic of complement regulatory proteins).