Gene gun mediated CTLA4-Ig gene transfer for immunological tolerance induction

Research Project

Project/Area Number	11470269
Research Category	Grant-in-Aid for Scientific Research (B).
Allocation Type	Single-year Grants
Section	一般
Research Field	Thoracic surgery
Research Institution	Gifu university
Principal Investigator	MORI Yoshio Gifu Univ.Hospital, Associate Professor, 医学部・附属病院, 助教授 (40220032)
Co-Investigator(Kenkyū-buntansha)	TAKAGI Hisato Gifu Univ. Assistant Professor, 医学部, 講師 (50262770) HIROSE Hajime Gifu Univ. Professor, 医学部, 教授 (20101272) IWATA Hisashi Gifu Univ.Hospital, Associate Professor, 医学部・附属病院, 講師 (90303495)
Project Period (FY)	1999 – 2000
Project Status	Completed (Fiscal Year 2000)
Budget Amount *help	¥14,400,000 (Direct Cost: ¥14,400,000) Fiscal Year 2000: ¥3,200,000 (Direct Cost: ¥3,200,000) Fiscal Year 1999: ¥11,200,000 (Direct Cost: ¥11,200,000)
Keywords	organ transplantation / CTLA4-Ig / immunological tolerance
Research Abstract	To induce the donor antigen specific immunological tolerance, it is an important issue to inhibit the costimulatory signal required for helper T cell activation. We examined the possibility that donor organs themselves had the performance to inhibit the costimulatory signal by CTLA4-Ig gene transfer. To avoid viral vector inducing responses, we used non-viral pCAGGS vector. In addition, we used gene gun system instead of retro-or adeno-viral vector to achieve higher gene transfer frequency. The extra-cellular domain of mouse CTLA4 gene was ligated to human IgG1 or EGFP gene in pBluescript vector, and then mCTLA4hIgGI or mCTLA4EGFP fragment was inserted to the modified pCAGGS vector. Gold particles were coated with pCAGGS/mCTLA4hIgG1 or EGFP at 5μg DNA/mg gold particles. The skin, muscle, or liver of BALB/c mouse was transferred with the gene gun (Helios, BIORAD, USA) at the pressure of 400 psi. Syngeneic BALB/c skin bombarded with pCAGGS/CTLA4-EGFP was transplanted, and EGFP expression … More was detected by fluorescence microscopy. C57BL/6 mice were engrafted on the flank with the pCAGGS/CTLA4-Ig injected allogeneic skin of BALB/c mice. Skin bombarded with pCAGGS/2B4TCR-Ig was transplanted as control graft. In vivo expression of CTLA4-EGFP was hard to detect in dermis due to the trapment of the plasmid coated gold particles in its fatty layer. In epidermis, there was no expression of EGFP in the central portion of gene gun bombarded-area, because of tissue damage by bombardment, whereas there was strong expression in the area around the central portion, even on day 1, CTLA4-EGFP expression in liver was also detected on day 1 with little bombardment-induced damage. Bombarded liver showed no central damage as per bombarded epidermis. EGFP expression in muscle was detected on day 5, but less than in epidermis and liver. CTLA4-EGFP expression in epidermis and muscle were observed for 1 week, while it was observed in liver for 2 to 5 weeks, suggesting that gene gun mediated gene transfer induces particularly stable gene expression in the liver. Pattern and duration of CTLA4-EGFP expression in epidermis was not changed in the grafted skin. Despite efficient gene gun mediated gene transfer into epidermis, survival of the CTLA4-Ig injected allogeneic skin graft (MST±SD=10.8±0.9) was not significantly prolonged compared with the control grafts (MST±SD=9.8±0.4). The efficient gene gun mediated gene expression observed in the syngeneic skin graft was identical to that in the native epidermis. However, survival of allogeneic skin grafts injected with CTLA4-Ig was not prolonged compared with the controls. Several reports showed that CTLA4-Ig administration induced immunological tolerance in cardiac, islet, and liver transplantation, but not in skin grafts. In addition to the skin specific antigen, our results suggested that the inflammatory response induced by gene gun bombardment was another factor that contributed to the failure of allogeneic skin graft survival. Further experiments to minimize tissue damage should be carried out. Less