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The induction of tolerance using the brand new gene therapy in transplantation setting and clarification of the mechanism.

在移植环境中使用全新基因疗法诱导耐受并阐明其机制。

基本信息

批准号：
13470248
负责人：
FURUKAWA Hiroyuki
金额：
$ 9.15万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

1. To develop innovative strategies for transplant immunosuppression, we tested the effect of the blockade of costimulatory pathways by using adenovirus vector coding CTLA4Ig(AdCTLA4Ig) or CD40Ig(AdCD40Ig)genes in ACI to LEW rat organ transplantations.A) Heart transplantation : A single treatment with AdCTLA4Ig or AdCD40Ig prolonged graft survival. Although the combined gene therapy allowed long term graft survival, this strategy was not enough to induce tolerance and to avoid chronic rejection.B) Liver transplantation : A single treatment with AdCTLA4Ig or AdCD40Ig prolonged graft survival, and could induce donor-specific tolerance.C) Small bowel transplantation : Although the combined gene therapy prolonged graft survival, this strategy was not enough to induce tolerance and to avoid chronic rejection.D) Xeno heart transplantation(Hamster to Rat) : DXR and cellular rejection were controlled successfully and all xenografts were accepted for over 100 days when AdCTLA4Ig and AdCD40Ig we … More re administered under FK779 induction therapy. However, chronic rejection was inevitable in the long-term surviving xenografts.2. Several strategies have been made in order to avoid the toxicity of adenoviral vector and to establish an efficient and safety gene transfer.A) Ex-vivo transfer : In rat liver transplantation, the graft with 1x10^9pfu AdCTLA4Ig in 4℃ UW solution for more than 8hrs could get efficient transfection rate and tolerance.B) Modified Clamp Technique : Using the short term CT transduction method (under the condition of 4℃ cold preservation for 10 minutes) long term graft survival are achieved.C) Cre/loxP : The administration of adenovirus vector containing Cre recombinase gene(AdexCACre) at the desired time induced Cre-mediated recombination within a gene derived from Adex1CALoxCTLA4IgGLox vector, and the cDNA of CTLA4IgG was excised from the transduced gene and terminated the expression of CTLA4IgG in vitro and in vivo.3. Immunosuppressive mechanism of blockade of CD40-CD 154A) CD40Ig has bi-directional effects, i.e., one direction towards CD4 + T cells that enhances IL-2 production by cross-linking CD40L, and the other to APCs, which results in generation of CD4 + CD25 + T cells. These events, in concert, lead to generation of functional CD4 + CD25 + T cells that regulate CTL activity in an antigen-specific manner. Less

1.为了开发移植免疫抑制的创新策略，我们在ACI至LEW大鼠器官移植中测试了使用编码CTLA4Ig（AdCTLA4Ig）或CD40Ig（AdCD40Ig）基因的腺病毒载体阻断共刺激途径的效果。A）心脏移植：AdCTLA4Ig或AdCD40Ig单次治疗延长移植物存活。虽然联合基因治疗允许长期移植物存活，但该策略不足以诱导耐受并避免慢性排斥。B) 肝移植：AdCTLA4Ig 或 AdCD40Ig 单一治疗可延长移植物存活，并可诱导供者特异性耐受。C) 小肠移植：虽然联合基因治疗延长移植物存活，但该策略不足以诱导 D) 异种心脏移植（仓鼠至大鼠）：当我们在 FK779 诱导治疗下重新施用 AdCTLA4Ig 和 AdCD40Ig 时，成功控制了 DXR 和细胞排斥，并且所有异种移植物均已接受超过 100 天。然而，长期存活的异种移植物不可避免地会出现慢性排斥反应。2．为了避免腺病毒载体的毒性，建立高效、安全的基因转移，已经采取了多种策略。A) 离体转移：在大鼠肝移植中，移植物在4℃ UW溶液中加入1x10^9pfu AdCTLA4Ig 8小时以上，可以获得高效的转染率和耐受性。B) 改良的Clamp技术：使用短期CT转导方法（下） C) Cre/loxP：在所需时间给予含有Cre重组酶基因的腺病毒载体(AdexCACre)，诱导来自Adex1CALoxCTLA4IgGLox载体的基因内发生Cre介导的重组，并从转导物中切除CTLA4IgG的cDNA 基因并终止CTLA4IgG的体外和体内表达。 3．阻断 CD40-CD 154A) CD40Ig 的免疫抑制机制具有双向作用，即一个方向朝向 CD4 + T 细胞，通过交联 CD40L 增强 IL-2 的产生，另一个方向朝向 APC，从而产生 CD4 + CD25 + T 细胞。这些事件共同导致功能性 CD4 + CD25 + T 细胞的产生，以抗原特异性方式调节 CTL 活性。较少的