TRANSPLANT IMMUNOLOGY OF THE GENETICALLY MODULATED LIVER

这一建议的中心假设如下：肝脏感染 带有能够抑制血管生成的基因表达的移植物 同种异体反应性免疫反应会影响局部免疫。几个 要检验这一假设，必须满足基本前提条件。第一，一个制度 有效地将基因传递到感兴趣的细胞和器官必须是 精制，基因表达的功能蛋白必须得到保证。 最后，插入基因的蛋白质产物必须能够 抑制免疫反应。 因此，第一个目标是定义有效的病毒介导的基因方法。 转移到老鼠的肝脏里。我们将进行系统的定性和 定量分析方法、效率、感染细胞亚型、 腺病毒感染的肝移植中基因转移的稳定性 向量。我们的初步结果表明，当腺病毒载体 与逆转录病毒载体相比，它的用途更广，并实现了 有效的基因传递的必要先决条件。 第二个目标是量化肝脏中的蛋白质产量。 在不同条件下进行基因转移后，确认生物活性 并研究启动子在基因表达中的调控作用。 蛋白质生产。因此，重组人的基因表达范围 蛋白质将被定义。 第三个目的是研究肝脏局部免疫反应。 移植感染转化生长因子-β的移植物。转化生长因子-β将被用作 一种具有代表性的有效免疫调节蛋白。体外和体内 化验将被用来确定当地是否过度生产 这种抑制因子改变同种异体反应性T细胞的反应，并且 同种异体移植排斥反应的模式。 总之，这项建议探索了使用基因疗法的方法。 解决肝移植机制问题的技术 免疫生物学。最终目标是通过以下方式来定义生物学原理 哪些基因治疗可以应用于实验和临床 肝脏移植。

The central hypothesis in this proposal is as follows: Infection of liver grafts with genes expressing factors capable of inhibition of the alloreactive immune response will influence regional immunity. Several basic prerequisites must be met to test this hypothesis. First, a system of efficient gene delivery to cells and organs of interest must be refined, and gene expression of functional protein must be assured. Finally, the protein products of inserted genes must be capable of inhibiting the immune response. The first aim, then, is to define efficient methods of viral mediated gene transfer into rat livers. We will perform a systematic qualitative and quantitative analysis of the methods, efficiency, infected cell subtypes, and stability of gene transfer in liver grafts infected with adenoviral vectors. Our preliminary results demonstrate that adenoviral vectors, when compared to retroviral vectors, are more versatile and fulfill the necessary prerequisites for efficient gene delivery. The second aim is the quantification of protein production in the liver after gene transfer under various conditions, to confirm biologic activity of expressed protein, and to examine the regulatory role of promoters in protein production. Thus, the range of gene expression for recombinant proteins will be defined. The third aim is to study the regional hepatic immune response after transplantation of grafts infected with TGF-beta. TGF-beta will be used as a representative potent immune regulatory protein. In vitro and in vivo assays will be used to determine whether excessive local production of this inhibitory factor modifies the response of alloreactive T cells, and patterns of allograft rejection. In summary, this proposal explores methods of using gene therapy techniques to address mechanistic issues of liver transplantation immunobiology. The ultimate goal is to define biological principles by which gene therapy efforts may be applied to experimental and clinical liver transplantation.