Viral Immunoregulatory Genes and Hepatocyte Transplants

病毒免疫调节基因与肝细胞移植

• 批准号: 6873970
• 负责人: JAYANTA ROY-CHOWDHURY
• 金额: $ 36.74万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6873970
• 关键词: Adenoviridae; Lentivirus; RNase protection assay; biotechnology; cell line; cell transplantation; enzyme linked immunosorbent assay; gene expression; genetic manipulation; green fluorescent proteins; homologous transplantation; immune tolerance /unresponsiveness; immunocytochemistry; immunoregulation; immunosuppressive; laboratory rat; liver cells; liver transplantation; polymerase chain reaction; regulatory gene; transfection; transfection /expression vector; transplant rejection; transplantation immunology; virus genetics; western blottings

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.

描述（由申请人提供）：我们的目标是开发安全有效的方法来对供体肝细胞进行基因改造，以消除肝细胞移植后的同种异体移植排斥反应，而无需使用长期免疫抑制。腺病毒 (Ad) 早期转录区三 (E3) 编码免疫调节蛋白，我们之前已使用它来产生在胰岛中表达 E3 基因的转基因小鼠。在 1 型糖尿病小鼠模型系统中，这些胰岛没有经历 β 细胞的自身免疫破坏，并且在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肝细胞中，将可防止移植到同种异体受体黄疸 Gunn 大鼠（1 型 Crigler-Najjar 综合征（CN1）的动物模型）后发生排斥反应。 CN1 是一种潜在致命的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的哪一种对于促进同种异体肝细胞移植物的存活和功能最重要。单独或共同作用的可能免疫靶标包括 Ad-gp19 下调 I 类 MHC； Ad E3-14.7K 抑制 TNF 细胞溶解； Ad E3-10.4/14.5K复合物减少细胞表面促凋亡受体如FAS、TRAIL和TNFR的表达； Ad E3-10.4/14.5K抑制NFkappaB和AP-1等信号转导途径；以及防止响应 TNFα 的趋化因子（MCP-1、IL-8、IP-10）合成。此外，我们将尝试剖析 Ad E3 是否通过内在机制仅作用于肝细胞，或者还通过克隆删除或诱导抑制细胞的耐受来改变免疫系统的效应臂。拟议的实验将使用用 SV40 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，从而允许移植细胞的后代在肝脏中广泛重新繁殖。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快速将Ad E3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其在肝脏中重建正常胆红素处理的功效。