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

基因枪介导 CTLA4-Ig 基因转移诱导免疫耐受

• 批准号: 11470269
• 负责人: MORI Yoshio
• 金额: $ 9.22万
• 依托单位: Gifu university
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470269/
• 关键词: organ transplantation; CTLA4-Ig; immunological tolerance

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

为了诱导供体抗原特异性免疫耐受，抑制辅助性T细胞活化所需的共刺激信号是一个重要的问题。我们研究了供体器官本身通过CTLA 4-IG基因转移抑制共刺激信号的可能性。为了避免病毒载体诱导应答，我们使用非病毒pCAGGS载体。另外，为了提高基因转移效率，我们采用基因枪系统代替逆转录病毒或腺病毒载体，将小鼠CTLA 4基因的胞外区与pBluescript载体中的人IgG 1或EGFP基因连接，再将mCTLA 4 hIgG 1或mCTLA 4 EGFP片段插入到改造后的pCAGGS载体中。用pCAGGS/mCTLA 4 hIgG 1或EGFP以5μg DNA/mg金颗粒包被金颗粒。用基因枪（Helios，BIORAD，USA）在400 psi的压力下转移BALB/c小鼠的皮肤、肌肉或肝脏。将pCAGGS/CTLA 4-EGFP基因轰击的同基因BALB/c小鼠皮肤移植， ...更多信息 通过荧光显微镜检测。将注射了pCAGGS/CTLA 4-IG的BALB/c小鼠同种异体皮肤移植到C57 BL/6小鼠的侧腹。以pCAGGS/2B 4 TCR-Ig基因转染的皮肤为对照，由于脂质体包裹的金颗粒在真皮层中被捕获，CTLA 4-EGFP在真皮中的表达难以检测。在表皮中，由于基因枪轰击引起的组织损伤，在基因枪轰击区域的中心部分没有EGFP表达，而在中心部分周围区域有强表达，即使在第1天，肝脏中也检测到CTLA 4-EGFP表达，几乎没有轰击引起的损伤。轰击肝脏未显示与轰击表皮相同的中心损伤。第5天，肌肉中检测到EGFP表达，但低于表皮和肝脏。在表皮和肌肉中观察到CTLA 4-EGFP表达1周，而在肝脏中观察到2至5周，表明基因枪介导的基因转移诱导肝脏中特别稳定的基因表达。移植皮肤中CTLA 4-EGFP表达的模式和持续时间没有改变，尽管基因枪介导的基因转移有效地进入表皮，但注射CTLA 4-IG的同种异体皮肤移植物的存活时间（MST±SD=10.8±0.9）与对照移植物（MST±SD=9.8±0.4）相比没有显著延长。在同基因皮肤移植物中观察到的有效基因枪介导的基因表达与天然表皮中的基因枪介导的基因表达相同。然而，与对照组相比，注射CTLA 4-IG的同种异体皮肤移植物的存活时间并没有延长。一些报道显示，CTLA 4-IG给药诱导了心脏、胰岛和肝移植中的免疫耐受，但在皮肤移植物中不诱导免疫耐受。除皮肤特异性抗原外，我们的研究结果表明，基因枪轰击诱导的炎症反应是导致同种异体皮肤移植物存活失败的另一个因素。应进行进一步的实验以尽量减少组织损伤。少

项目成果

Y.Umeda,: "Non-viral gene gun mediated CTLA4Ig-gene transfer for modification of donor organs"Transplantation Proceedings. 33. 243-245 (2001)

Y.Umeda，：“用于修饰供体器官的非病毒基因枪介导的 CTLA4Ig 基因转移”移植论文集。

Y.Umeda et.al: "Non-viral gene gun mediated CTLA4Ig-gene transfer for modification of donor organs"Transplantation Proceedings. 33. 243-245 (2001)

Y.Umeda 等人：“非病毒基因枪介导的 CTLA4Ig 基因转移用于修饰供体器官”移植论文集。