Intracellular immunization strategy in inhibit HCV related liver cancer

抑制HCV相关肝癌的细胞内免疫策略

• 批准号: 7847457
• 负责人: Srikanta Dash
• 金额: $ 16.73万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-24 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7847457
• 关键词: A Mouse; Accounting; Amino Acid Sequence; Amino Acids; Animal Model; Antibodies; Antiviral Agents; Antiviral Therapy; Applications Grants; Cell Culture Techniques; Cell Line; Chronic Hepatitis; Chronic Hepatitis C; Cicatrix; Cirrhosis; Clinical; Collaborations; Development; Epitopes; Escape Mutant; FDA approved; Fluorescence; Genes; Genetic Polymorphism; Genomics; Goals; HCV Animal Models; Hepatitis C; Hepatitis C virus; Hepatocyte; Human; Human Engineering; IgG1; Immune response; Immunization; In Vitro; Individual; Infection; Infection prevention; Infectious hepatitides; Inflammation; Interferon-alpha; Interferons; Intravenous; Label; Lead; Length; Liver; Malignant neoplasm of liver; Mediating; Methods; Modeling; Mus; Pan Genus; Patients; Peptide Hydrolases; Pharmacy facility; Polymers; Primary carcinoma of the liver cells; Principal Investigator; RNA replication; Recombinant Antibody; Recombinants; Regimen; Relapse; Replicon; Research; Research Proposals; Resistance; Ribavirin; SCID Mice; Simulate; Technology; Testing; United States; Universities; Variant; Viral; Viral Genome; Viral Proteins; Virion; Virus; Virus Diseases; Virus Replication; base; expression vector; helicase; hepatitis C virus NS3 protein; high risk; improved; in vivo; interferon therapy; liver transplantation; mouse model; nanoparticle; novel; novel therapeutics; nucleoside triphosphatase; plasmid DNA; programs; protein expression; public health relevance; research study; stable cell line; subcutaneous; tumor; tumor xenograft; viral RNA

DESCRIPTION (provided by applicant): We have developed an intracellular immunization strategy with a genetically engineered human antibody clone as a novel antiviral therapy against the hepatitis C virus. This antibody clone targets the HCV NS3 protein, which has multiple enzymatic activities (protease, helicase and NTPase) that are crucial for viral genome replication. We have shown that a recombinant human antibody clone reacts with the helicase domain of HCV NS3 and completely inhibits the helicase activity. Intracellular expression of this antibody in either a stable cell line replicating subgenomic RNA, or a transient full-length HCV replication model, reduced both HCV RNA and viral protein expression. During the last review cycle of this application we have developed a mouse xenograft tumor model for HCV replication. We have shown that HCV replication in the subcutaneous tumors in the SCID mice is inhibited by interferon alpha. This provides a reliable animal model for testing intracellular immunization for HCV using the antibody. We also developed methods of encapsulation for plasmid DNA or purified antibodies into nanoparticles by using a biodegradable and FDA approved polymer. In this proposal we will be experimenting with the practical aspects of the intracellular immunization strategy in vitro as well as in vivo using a small animal model for hepatitis C virus. Our focus will be to improve technology for the systemic delivery of nanoparticles carrying the recombinant antibody to liver cells to inhibit HCV replication in a mouse model. Our hypothesis is that intracellular immunization with recombinant antibodies that block the NS3 helicase is an effective strategy for inhibiting hepatitis C virus replication and expression. We propose that encapsulation of the antibody gene into biodegradable nanoparticles will efficiently deliver the antibody gene to hepatocytes and may provide a novel therapeutic strategy for chronic HCV patients who are non-responders to interferon. In Specific Aim 1, we will define the epitope(s) of a recombinant human antibody clone and investigate what proportion of clinical HCV strains are inhibited by the recombinant human antibody clone. In Specific Aim 2, we will investigate the emergence of resistant virus variants that escapes from antibody-mediated inhibition of virus replication in HCV cell culture models. In Specific Aim 3, we will determine whether intracellular expression of recombinant antibody effectively eliminates virus RNA replication using a non-infectious small animal model for hepatitis C. If the experiments proposed in this grant application are successful, then we may have the basis for an alternative antiviral strategy for people with chronic HCV infections who do not respond to current interferon therapy. Public Health Relevance: Chronic hepatitis C virus infection accounts for 27% of liver cancer in the United States. This research proposal intends to develop an intracellular immunization strategy for inhibiting hepatitis C virus. If this project becomes successful then it can lead to a potential therapy to treat chronic hepatitis C infection and prevent liver cancer.

描述（由申请人提供）：我们开发了一种细胞内免疫策略，使用基因工程人抗体克隆作为抗丙型肝炎病毒的新型抗病毒疗法。该抗体克隆靶向HCV NS3蛋白，该蛋白具有对病毒基因组复制至关重要的多种酶活性（蛋白酶、解旋酶和NTR）。我们已经证明，重组人抗体克隆与HCV NS3的解旋酶结构域反应，并完全抑制解旋酶活性。这种抗体在稳定的细胞系复制亚基因组RNA或瞬时全长HCV复制模型中的细胞内表达降低了HCV RNA和病毒蛋白的表达。在本申请的最后一个审查周期中，我们开发了一种用于HCV复制的小鼠异种移植肿瘤模型。我们已经证明，在SCID小鼠皮下肿瘤中的HCV复制被干扰素α抑制。这为使用抗体测试HCV的细胞内免疫提供了可靠的动物模型。我们还开发了通过使用可生物降解和FDA批准的聚合物将质粒DNA或纯化的抗体封装到纳米颗粒中的方法。在本提案中，我们将使用丙型肝炎病毒的小动物模型在体外和体内试验细胞内免疫策略的实际方面。我们的重点将是改进携带重组抗体的纳米颗粒系统递送到肝细胞以抑制小鼠模型中HCV复制的技术。我们的假设是，用阻断NS3解旋酶的重组抗体进行细胞内免疫是抑制丙型肝炎病毒复制和表达的有效策略。我们建议将抗体基因封装到可生物降解的纳米颗粒中将有效地将抗体基因递送到肝细胞，并可能为对干扰素无应答的慢性HCV患者提供一种新的治疗策略。在具体目标1中，我们将定义重组人抗体克隆的表位，并研究重组人抗体克隆抑制临床HCV毒株的比例。在具体目标2中，我们将研究在HCV细胞培养模型中逃避抗体介导的病毒复制抑制的耐药病毒变体的出现。在特定目标3中，我们将使用丙型肝炎的非传染性小动物模型来确定重组抗体的细胞内表达是否有效地消除病毒RNA复制。如果本研究资助申请中提出的实验是成功的，那么我们可能有一个替代的抗病毒策略的基础，慢性HCV感染的人谁不响应目前的干扰素治疗。 公共卫生相关性：慢性丙型肝炎病毒感染占美国肝癌的27%。本研究计划旨在开发一种抑制丙型肝炎病毒的细胞内免疫策略。如果该项目取得成功，那么它可能会成为治疗慢性丙型肝炎感染和预防肝癌的潜在疗法。