Intracellular immunization strategy in inhibit HCV related liver cancer

描述(由申请人提供)：我们已经开发了一种细胞内免疫策略，使用基因工程的人类抗体克隆作为对抗丙型肝炎病毒的新的抗病毒疗法。该抗体克隆以丙型肝炎病毒NS3蛋白为靶标，该蛋白具有多种对病毒基因组复制至关重要的酶活性(蛋白酶、解旋酶和NTPase)。我们已经证明，重组人抗体克隆与丙型肝炎病毒NS3的解旋酶结构域发生反应，并完全抑制解旋酶的活性。这种抗体在复制亚基因组RNA的稳定细胞系或瞬时的全长丙型肝炎病毒复制模型中的细胞内表达，降低了丙型肝炎病毒RNA和病毒蛋白的表达。在本申请的最后一次审查周期中，我们开发了一个用于复制丙型肝炎病毒的小鼠异种移植瘤模型。我们已经证明，干扰素α可以抑制丙型肝炎病毒在SCID小鼠皮下肿瘤中的复制。这为利用该抗体检测丙型肝炎病毒细胞内免疫提供了可靠的动物模型。我们还开发了一种方法，通过使用FDA批准的可生物降解的聚合物将质粒DNA或纯化的抗体包裹到纳米颗粒中。在这项提案中，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用方面。我们的重点将是改进系统地将携带重组抗体的纳米颗粒输送到肝细胞以抑制小鼠模型中的丙型肝炎病毒复制的技术。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。我们认为，将抗体基因包裹到可生物降解的纳米颗粒中，可以有效地将抗体基因传递到肝细胞，并可能为干扰素无效的慢性丙型肝炎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(S)，并研究重组人抗体克隆对临床丙型肝炎病毒株的抑制比例。在特定的目标2中，我们将研究在丙型肝炎病毒细胞培养模型中，逃避抗体介导的病毒复制抑制的耐药病毒变体的出现。在具体目标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。如果这项拨款申请中提出的实验成功，那么我们可能有基础为对当前干扰素治疗无效的慢性丙型肝炎患者提供替代的抗病毒策略。 公共卫生相关性：慢性丙型肝炎病毒感染占美国肝癌的27%。这项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。如果这个项目取得成功，那么它可能会带来一种治疗慢性丙型肝炎感染和预防肝癌的潜在疗法。

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.