Viral Immunoregulatory Genes and Hepatocyte Transplants

描述(申请人提供)：我们的目标是开发安全和有效的方法来对供体肝细胞进行基因修饰，以消除肝细胞移植后的同种异体排斥反应，而不需要使用长期的免疫抑制。腺病毒(Ad)早期转录区3(E3)编码免疫调节蛋白，我们以前曾用它在胰岛产生表达E3基因的转基因小鼠。在1型糖尿病模型小鼠系统中，这些胰岛没有经历β细胞的自身免疫破坏，当移植到同种异体受体中时也没有被排斥。我们的假设是，将部分或全部AdE3基因插入原代或永生化的肝细胞中，将防止它们移植到同种异体受体黄褐斑Gunn大鼠身上，这是Crigler-Najjar综合征1型(CN1)的动物模型。CN1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AdE3抑制免疫系统的各种潜在机制中，哪些对促进同种异体肝细胞移植的存活和功能最重要。可能的免疫靶点包括：Ad-gp19下调I类MHC；Ad E3-14.7K抑制肿瘤坏死因子的杀伤；Ad E3-10.4/14.5K复合体减少细胞表面促凋亡受体Fas、TRAIL和TNFR的表达；Ad E3-10.4/14.5K抑制NFkappaB和AP-1等信号转导通路；以及阻止趋化因子(MCP-1、IL-8、IP-10)的合成。此外，我们将试图剖析AdE3是通过内在机制仅作用于肝细胞，还是通过克隆删除或诱导抑制细胞来耐受而改变免疫系统的效应臂。建议的实验将使用SV40T抗原(Tag)有条件永生化的肝细胞和原代肝细胞。肝细胞带来了新的挑战和机遇，因为与胰岛不同，宿主肝脏可以通过控制破坏内源性肝细胞来操纵，从而允许移植细胞的后代在肝脏中广泛繁殖。我们将使用条件永生化的Fisher大鼠肝细胞系，通过转染法或慢病毒感染法快速将AdE3基因插入新的组合中。慢病毒技术还将使我们能够使用原代肝细胞来评估它们在肝脏中重建正常胆红素过程的效果。

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.