INTERFERON-b GENE THERAPY FOR VIRAL HEPATITIS

病毒性肝炎的干扰素-b 基因治疗

• 批准号: 05557033
• 负责人: SEO Hisao
• 金额: $ 4.48万
• 依托单位: NAGOYA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05557033/
• 关键词: HEPATITIS B; GENE THERAPY; LIPOSOME; INTERFERON; ASIARO-GLYCOPROTEIN; ウイルス性肝炎; 遺伝子療法; アシアロ糖蛋白; 肝細胞培養; リポゾーム

1. INHIBITION OF HEPATITIS beta VIRUS REPLICATION BY INTRODUCTION OF INTERFERON-BETA EXPRESSING PLASMIDIn developing gene therapy to treat the hepatitis B,we introduced plasmid expression human interferon-beta to a hepatoma cell line producing the hepatitis B virus. The cell line was established by transfecting a human hepatoma cell line, HepG2 using pHBV-3 containing the HBV genome. A liposome-mediated introduction of the interferon-beta expressing plasmid resulted in the secretion of interferon-beta into the medium at constnt levels for at least 6 days (30-40 IU/ml). However, exogenously added human interferon-beta (1,000 IU/ml) deteriorated rapidly and became undetectable after 3 days. Viral replication was assessed by determining the levels of the hepatitis B antigen in the culture medium. Introduction of the plasmid proved to markedly inhibit viral replication, while exogenously added human interferon-b caused almost no inhibition. It is thsu indicated that continuous productiono … More finterferon-beta by its gene delivery inhibits hepatitis beta viral replication much more efficiently than does an exogenous administration of linterferon-beta.2. HEPATOCYTE SPECIFIC GENE DELIVERYTo establish gene therapy for viral hepatitis by introducing interferon-beta expressing plasmid, it is mandatory to develop hepatocyte specific gene delivery. First we evaluated the usefulness of gene delivery via asialoglycoprotein receptor which is specifically expressed on the plasma membrane of the hepatocyte. Asialofetuin or asialo-alpha1-acid glycoprotein was conjugated with poly-L-lysine which binds with plasmid DNA due to its positive charge. A firefly luciferase-expressing plasmid was incubated with each of the conjugate and introduced into two forms of primary cultured rat hepatocytes or HepG2 cells. Efficiency of the gene transfer was assessed by the determination of luciferase activity. An inhibitor of the lysozomal enzyme chloroquine was required for efficient gene transfer. As a ligand for the receptor, asialo-alpha1-acid glycoprotein was more efficient than asialofetuin. Rapidly growing cells (HepG2 cells and monolayr cultured rat hepatocytes) were readily transfected while non-grwing rat hepatocytes in shperoid culture could not be transfected easily, suggesting that cell division in required for gene transfer. The transfected cell population was less than 10%. To increse the efficiency of transfection, luciferase reporter gene was introduced into replication defective adenovirus vector. The recombinat virus infected almost 100% of the hepatocytes either growing or not growing. When injected to the tail-vein fo the mouse, luciferase activity was detected in liver, kidney, lung and muscles, while the activity in liver was 10 times more than that in the rest of organs. It is thus demonstrated that adenovirus vector could be used for hepatocyte specific gene delivery when coupled with promoter region which define hepatocyte specific expression. Less

1.通过引入干扰素-β表达质粒抑制乙型肝炎病毒复制在开发治疗B型肝炎的基因疗法中，我们将表达人干扰素-β的质粒引入产生B型肝炎病毒的肝癌细胞系。该细胞系通过使用含有HBV基因组的pHBV-3转染人肝癌细胞系HepG2来建立。脂质体介导的干扰素-β表达质粒的引入导致干扰素-β以恒定水平分泌到培养基中至少6天（30 - 40 IU/ml）。然而，外源性添加的人干扰素-β（1，000 IU/ml）迅速恶化，3天后无法检测到。通过测定培养基中乙型肝炎B抗原的水平来评估病毒复制。质粒的引入被证明能显著抑制病毒复制，而外源性人干扰素-b几乎没有抑制作用。这表明，连续化生产 ...更多信息 干扰素-β通过其基因传递抑制乙型肝炎病毒复制比外源性给予干扰素-β 2有效得多。肝细胞特异性基因递送为了通过引入干扰素-β表达质粒来建立病毒性肝炎的基因治疗，必须开发肝细胞特异性基因递送。首先，我们评估了通过特异性表达于肝细胞质膜上的去唾液酸糖蛋白受体进行基因递送的有用性。脱唾液酸胎球蛋白或脱唾液酸-α 1-酸性糖蛋白与聚-L-赖氨酸缀合，聚-L-赖氨酸由于其正电荷而与质粒DNA结合。将萤火虫转移酶表达质粒与每种缀合物一起孵育，并引入两种形式的原代培养大鼠肝细胞或HepG2细胞中。通过测定荧光素酶活性来评估基因转移的效率。有效的基因转移需要溶菌酶的抑制剂氯喹。作为受体的配体，去唾液酸-α 1-酸性糖蛋白比去唾液酸胎球蛋白更有效。快速生长的细胞（HepG2细胞和单层培养的大鼠肝细胞）容易转染，而在类球形培养中的非生长的大鼠肝细胞不能容易地转染，这表明细胞分裂是基因转移所必需的。转染的细胞群体小于10%。为了提高转染效率，将荧光素酶报告基因导入复制缺陷型腺病毒载体。重组病毒几乎100%感染生长或不生长的肝细胞。小鼠尾静脉注射荧光素酶后，在肝、肾、肺和肌肉中检测到荧光素酶活性，其中肝中的荧光素酶活性是其它器官的10倍。因此证明，当与限定肝细胞特异性表达的启动子区偶联时，腺病毒载体可用于肝细胞特异性基因递送。少

项目成果

M.Menjo: "Mechanism involved in the different responsiveness to thyroid hormonebetween monolayer and spheroid cultures of rat hepatocytes." Environmental Medicine. 38. 107-110 (1994)

M.Menjo：“大鼠肝细胞单层和球状培养物对甲状腺激素的不同反应性的机制。”

Masafumi Menjo: "Effects of Thyroid and Glucocorticoid Hormones on the levels of messenger ribonucleic acid for lodothyronine Type l 5′-Deiodinase in Rat Primary Hepatocyte Cultures Grown as Spheroids" Endocrinology. 133. 2984-2990 (1993)

Masafumi Menjo：“甲状腺和糖皮质激素对大鼠原代肝细胞球体培养物中洛多腺原氨酸 5′-脱碘酶信使核糖核酸水平的影响”内分泌学 133。2984-2990 (1993)

Masafumi Menjo: "Mechanism involved in the different responsiveness to thyroid hormone between monolayer and spheroid cultures of hepatocytes." Environmental Medicine. 38. 107-110 (1994)

Masafumi Menjo：“肝细胞单层和球状培养物对甲状腺激素的不同反应性的机制。”

Yoshio Nomura et al.: "Targeted gene delivery to the hepatocytes by asialoglycoprotein receptor-mediated endocytosis" Environmental Medicine. 39. 11-14 (1995)

Yoshio Nomura 等人：“通过脱唾液酸糖蛋白受体介导的内吞作用将靶向基因递送至肝细胞”环境医学。

長屋敬: "スフェロイド肝細胞培養における単層培養と異なる甲状腺ホルモンレセプター補助因子の誘導:ゲルシフトアッセイを用いた検討" 名古屋大学環境医学研究所年報. 45. 169-171 (1994)

Takashi Nagaya：“单层培养中球状肝细胞培养物中不同甲状腺激素受体辅助因子的诱导：使用凝胶转移测定法进行的研究”名古屋大学环境医学研究所年度报告45。169-171（1994）。