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

1. 为了开发移植免疫抑制的创新策略，我们利用ACI中编码CTLA4Ig（AdCTLA4Ig）或CD40Ig（AdCD40Ig）基因的腺病毒载体，测试了阻断共刺激通路对LEW大鼠器官移植的影响。A)心脏移植：AdCTLA4Ig或AdCD40Ig单次治疗可延长移植物存活时间。虽然联合基因治疗可以使移植物长期存活，但这种策略不足以诱导耐受性和避免慢性排斥反应。B)肝移植：AdCTLA4Ig或AdCD40Ig单次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：虽然联合基因治疗可延长移植物存活，但该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）：在FK779诱导治疗下，AdCTLA4Ig和AdCD40Ig重新给予AdCTLA4Ig和AdCD40Ig，成功控制了DXR和细胞排斥反应，所有异种移植均接受100天以上。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副作用，建立高效、安全的基因转移途径，本文提出了几种策略。A)离体移植：在大鼠肝移植中，1 × 10^9pfu AdCTLA4Ig在4℃UW溶液中放置8hrs以上的移植物可以获得高效的转染率和耐受性。B)改良钳夹技术：采用短期CT转导法（在4℃低温保存10分钟的条件下）实现移植物长期存活。在需要的时间给药含有Cre重组酶基因的腺病毒载体（adexacre），诱导Cre介导的重组在Adex1CALoxCTLA4IgGLox载体衍生的基因内，并从转导的基因中切除CTLA4IgG的cDNA，终止CTLA4IgG在体外和体内的表达。cd40 - cd154a) CD40Ig阻断的免疫抑制机制具有双向作用，即一个方向是针对CD4 + T细胞，通过CD40L交联增强IL-2的产生，另一个方向是针对apc，导致CD4 + CD25 + T细胞的产生。这些事件共同导致产生功能性CD4 + CD25 + T细胞，以抗原特异性方式调节CTL活性。少

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