Intracellular immunization strategy in inhibit HCV related liver cancer

描述（由申请人提供）：我们开发了一种细胞内免疫策略，使用基因工程人类抗体克隆作为一种新的抗病毒治疗丙型肝炎病毒。该抗体克隆靶向HCV NS3蛋白，该蛋白具有多种酶活性（蛋白酶、解旋酶和NTPase），对病毒基因组复制至关重要。我们已经证明重组人抗体克隆与HCV NS3解旋酶结构域发生反应并完全抑制解旋酶活性。在复制亚基因组RNA的稳定细胞系或瞬时全长HCV复制模型中，细胞内表达该抗体可降低HCV RNA和病毒蛋白的表达。在该应用程序的最后一个审查周期中，我们开发了用于HCV复制的小鼠异种移植肿瘤模型。我们已经证明HCV在SCID小鼠皮下肿瘤中的复制被干扰素抑制。这为使用该抗体检测HCV细胞内免疫提供了可靠的动物模型。我们还开发了使用可生物降解和FDA批准的聚合物将质粒DNA或纯化抗体封装成纳米颗粒的方法。在这项建议中，我们将在体外和体内使用丙型肝炎病毒的小动物模型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的技术，以抑制HCV在小鼠模型中的复制。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包封在可生物降解的纳米颗粒中，将抗体基因有效地递送到肝细胞，并可能为对干扰素无反应的慢性HCV患者提供一种新的治疗策略。在Specific Aim 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菌株的比例。在特异性目标2中，我们将研究在HCV细胞培养模型中出现的逃避抗体介导的病毒复制抑制的耐药病毒变体。在特异性目标3中，我们将使用非感染性丙型肝炎小动物模型确定重组抗体的细胞内表达是否有效地消除病毒RNA复制。如果本拨款申请中提出的实验成功，那么我们可能为慢性丙型肝炎感染患者提供替代抗病毒策略的基础，这些患者对当前干扰素治疗没有反应。

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.