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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.为了开发新的移植免疫抑制策略，我们利用携带CTLA4Ig(AdCTLA4Ig)或CD40Ig(AdCD40Ig)基因的腺病毒载体在急性脑梗塞大鼠至LEW大鼠器官移植中测试了阻断共刺激通路的效果。a)心脏移植：单独应用AdCTLA4Ig或AdCD40Ig可延长移植物存活。B)肝移植：单用AdCTLA4Ig或AdCD40Ig可延长移植物存活时间，并可诱导供者特异性免疫耐受。c)小肠移植：虽然联合基因治疗延长了移植物存活时间，但这一策略不足以诱导耐受和避免慢性排斥反应。D)异种心脏移植(仓鼠到大鼠)：当AdCTLA4Ig和AdCD40Ig WE…时，DXR和细胞排斥反应被成功控制，所有异种移植都接受了100天以上更多的患者在FK779诱导治疗下再次接受治疗。然而，在长期存活的异种移植物中，慢性排斥是不可避免的。为了避免腺病毒载体的毒性，建立高效、安全的基因转移，人们已经采取了多种策略。A)体外转移：在大鼠肝移植中，在4℃UW溶液中，1x10^9pfu AdCTLA4Ig的移植物在4℃UW溶液中作用8小时以上，可以获得高效的转染率和耐受性。B)改良的夹闭技术：使用短期的CT转导方法(在4 GLx冷保存10分钟的条件下)实现移植物的长期存活。c)Cre/loxP：在所需的时间给予含有Cre重组酶基因的腺病毒载体(AdexCACre)诱导Cre介导的重组从转导基因中切下CTLA4Ig基因，并在体内外终止CTLA4Ig的表达。CD40-CD154A阻断的免疫抑制机制)CD40Ig具有双向作用，即一方面通过CD40L与CD40L相互作用，促进IL-2的产生，另一方面向APC诱导产生CD4+CD25+T细胞。这些事件协同作用，导致产生功能性的CD4+CD25+T细胞，以抗原特异性的方式调节CTL活性。较少