Viral Immunoregulatory Genes and Hepatocyte Transplants

描述（由申请人提供）：我们的目的是开发安全有效的方法来遗传修饰供体肝细胞，以消除肝细胞移植后的同种异体排斥反应，而无需使用长期免疫抑制剂。腺病毒（Ad）早期转录区3（E3）编码的免疫调节蛋白，我们以前使用产生转基因小鼠表达E3基因的胰岛。这些胰岛在1型糖尿病模型鼠系统中不经历β细胞的自身免疫破坏，并且当移植到同种异体受体中时不被排斥。我们的假设是，将部分或全部AdE 3基因插入原代或永生化肝细胞将防止其移植到异基因受体黄疸古恩大鼠（Crigler-Najjar综合征1型（CN 1）的动物模型）后的排斥反应。CN 1是一种潜在的致命性遗传性胆红素结合缺陷。我们的目标是确定AdE 3抑制免疫系统的各种潜在机制中哪一种在促进同种异体肝细胞移植的存活和功能方面是最重要的。Ad-gp 19下调I类MHC，AdE 3 -14.7K抑制TNF细胞溶解，AdE 3 - 10.4/14.5K复合物降低细胞表面促凋亡受体如FAS、TRAIL和TNFR的表达，AdE 3 - 10.4/14.5K复合物抑制TNF细胞溶解，AdE 3 - 10.4/14.5K复合物抑制Fas、TRAIL和TNFR的表达。通过Ad E3-10.4/14.5K抑制信号转导途径如NF κ B和AP-1;以及防止响应TNF α的趋化因子（MCP-1、IL-8、IP-10）合成。此外，我们将试图剖析是否广告E3的行为只对肝细胞的内在机制，也改变了免疫系统的效应臂通过克隆删除或诱导抑制细胞的耐受。拟定的实验将使用SV 40 T抗原（Tag）条件永生化的肝细胞和原代肝细胞。肝细胞提出了新的挑战和机遇，因为与胰岛不同，宿主肝脏可以通过内源性肝细胞的受控破坏来操纵，以允许肝脏与移植细胞的后代广泛再增殖。我们将使用条件永生化的Fisher大鼠肝细胞系，通过转染或慢病毒感染迅速地以新的组合插入Ad E3基因。慢病毒技术还将允许我们使用原代肝细胞来评估它们在重建肝脏中正常胆红素加工中的功效。

DESCRIPTION (provided by applicant): Our objective is to develop safe and efficient methods to genetically modify donor liver cells to abrogate allograft rejection after hepatocyte transplantation without the use of long-term immunosuppression. Adenovirus (Ad) early transcription region three (E3) encodes immunomodulatory proteins, which we have used previously to generate transgenic mice expressing the E3 genes in pancreatic islets. These islets did not undergo autoimmune destruction of beta-cells in model murine systems of type 1 diabetes and were not rejected when transplanted into allogeneic recipients. Our hypothesis is that insertion of some or all of the AdE3 genes into primary or immortalized hepatocytes will prevent their rejection upon transplantation into allogeneic recipient jaundiced Gunn rats, which are an animal model for Crigler-Najjar syndrome type 1 (CN1). CN1 is a potentially lethal inherited deficiency of bilirubin conjugation. Our goals are to determine which of the various potential mechanisms of AdE3 suppression of the immune system are most important in facilitating the survival and function of allogeneic hepatocyte transplants. Among the possible immunologic targets acting alone or together are the downregulation of class I MHC by Ad-gp19; inhibition of TNF cytolysis by Ad E3-14.7K; reduction of cell surface expression of pro-apoptotic receptors such as FAS, TRAIL and TNFR, by the complex of Ad E3- 10.4/14.5K; inhibition of signal transduction pathways such as NFkappaB and AP-1 by Ad E3-10.4/14.5K; and prevention of chemokine (MCP-1, IL-8, IP-10) synthesis in response to TNFalpha. In addition, we will attempt to dissect whether Ad E3 acts only on the hepatocyte by intrinsic mechanisms or also alters the effector arm of the immune system by tolerizing by clonal deletion or induction of suppressor cells. The experiments proposed will use both hepatocytes conditionally immortalized with SV40 T antigen (Tag) and primary hepatocytes. Hepatocytes present new challenges and opportunities, because unlike pancreatic islets, the host liver can be manipulated by controlled destruction of endogenous hepatocytes to permit extensive repopulation of the liver with the progeny of the transplanted cells. We will use a conditionally immortalized Fisher rat hepatocyte line to insert Ad E3 genes in new combinations expeditiously by transfection or lentivirus infection. The lentivirus technology also will allow us to use primary hepatocytes to assess their efficacy in reconstituting normal bilirubin processing in the liver.