Hepatic Non-viral Gene Therapy

肝脏非病毒基因治疗

• 批准号: 8882415
• 负责人: Leaf Huang
• 金额: $ 33.06万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-10 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8882415
• 关键词: Acetylation; Animals; Blood; CMV promoter; Cancer Etiology; Cell Nucleus; Cells; Cellular biology; Charge; Chronic Hepatitis B; Cirrhosis; Complementary DNA; Complex; Confocal Microscopy; Cyclic Peptides; Cytolysis; Cytoplasm; DNA; Data; Development; Differentiation Antigens; Dissociation; Dose; Drug Formulations; Encapsulated; Endosomes; Enzymes; Fatty Liver; Fibrosis; Fluorescence; Freezing; Galactose; Gene Delivery; Gene Expression; Gene Transfer; Genes; Genetic; Genetic Transcription; Genomic DNA; Goals; Health; Hepatic; Hepatitis; Hepatitis B; Hepatitis B Vaccines; Hepatitis B Virus; Hepatocyte; Histone H3; Histones; Human; Immune system; In Situ; In Vitro; Infection; Inflammation; Injection of therapeutic agent; Interferons; Intravenous Bolus; Label; Life; Lipid Bilayers; Lipids; Liver; Liver Fibrosis; Liver diseases; Luciferases; Malignant neoplasm of liver; Measures; Mediating; Mental Depression; Methods; Methylation; Modeling; Modification; Moods; Mus; N-terminal; Names; Non-Viral Vector; Nuclear; Nuclear Import; Nucleic Acids; Patients; Peptides; Phosphorylation; Plant Leaves; Plasma; Plasmids; Play; Post-Translational Protein Processing; Preventive; Procedures; Proteins; Recombinants; Research; Research Personnel; Role; Schedule; Series; Symptoms; Tail; Testing; Toxic effect; Transfection; Transgenes; Veins; Viral; Viral Vector; Viral hepatitis; Virus Diseases; Work; anti-hepatitis B; calcium phosphate; clinically relevant; controlled release; design; falls; gene therapy; hepatoma cell; histone modification; immunopathology; in vivo; intein; interferon therapy; light scattering; mouse model; nano; nanoparticle; non-viral gene therapy; plasmid DNA; promoter; prototype; psychologic; red fluorescent protein; research study; time use; tool; trafficking; vector

DESCRIPTION (provided by applicant): Many genetic and acquired diseases of the liver can be theoretically treated with gene therapy. The efficiency of non-viral vectors typically falls behind that of viral vectors, except the hydrodynamic injection method. However, the invasiveness of the hydrodynamic procedure is probably not acceptable beyond serving as a research tool. Dr. Leaf Huang et al. have recently developed a nanoparticle formulation consisting of an amorphous calcium phosphate core wrapped with a single lipid bilayer membrane. The nanoparticles are named LCP (lipid/calcium/phosphate). Galactose targeted LCP containing both a plasmid DNA and a decapeptide CR8C showed a high level of gene transfection in the liver hepatocytes of mice after tail vein injection in a non-hydrodynamic manner. The presence of CR8C was essential for nuclear localization of the injected plasmid DNA. Preliminary data suggest that CR8C, although essential for the nuclear import of the condensed plasmid DNA, dose not efficiently dissociate from the DNA in the nucleus. Aim 1 of the proposed study will test a series of histone peptides, with and without the addition of oligoarginines, which can be covalently modified by nuclear enzymes to reduce the cationic charge content of the peptides. The hypothesis is that the histone peptide(s) will condense the plasmid DNA and bring it into the nucleus, but dissociate from the DNA after covalent modification in situ. Such controlled release of the plasmid DNA should allow the DNA for enhanced transcription. Since the plasmid DNA accumulated in the nuclei of the hepatocytes, it is important to study the mechanism underlying the efficient import. Aim 2 is designed to test the hypothesis that transiently elevated Ca concentration as a result of LCP releasing its cargo from the endosome will stimulate the nuclear import of the plasmid DNA/CR8C complex. Various cell biology experiments, including the ones using time- lapsed live-cell confocal microscopy, have been proposed. Over 2 billion people have been infected with the hepatitis B virus (HBV) and 350 million live with chronic HBV infection worldwide, about 25% of whom die from liver fibrosis/cirrhosis or liver cancer caused by the infection (WHO: Hepatitis B-Key facts). Although a preventive HBV vaccine is available, there is no cure for the 350 million patients who are already chronically infected. Dr. Lishan Su at UNC has developed a humanized mouse model (AFC8-hu) which contains not only the human hepatocytes in the liver but also the human immune system. The mice can be infected by HBV and develop hepatitis symptoms, including fatty liver, fibrosis and inflammation. Type-1 interferons are effective anti-virals, especially fo the viral hepatitis such as HBV. In preliminary experiments, IFN-�2b could be expressed at the clinically relevant concentration in the liver of mouse injected with a plasmid containing the cDNA for IFN-�2b using LCP as a vector. In Aim 3, we will test the IFN gene therapy in the AFC8-hu mice chronically infected with HBV. S/MAR sequence will be incorporated into the IFN-�2b plasmid to prolong the gene expression. The goal is to develop a once-a-month IFN gene therapy for the HBV hepatitis patients.

描述（申请人提供）：理论上，许多遗传性和后天性肝脏疾病都可以通过基因疗法来治疗。除水动力注射方法外，非病毒载体的效率通常落后于病毒载体。然而，流体动力学程序的侵入性除了作为研究工具之外可能是不可接受的。黄叶博士等。最近开发了一种纳米颗粒制剂，由包裹着单个脂质双层膜的无定形磷酸钙核心组成。该纳米粒子被命名为LCP（脂质/钙/磷酸盐）。以非流体动力学方式尾静脉注射后，含有质粒DNA和十肽CR8C的半乳糖靶向LCP在小鼠肝脏肝细胞中显示出高水平的基因转染。 CR8C 的存在对于注射质粒 DNA 的核定位至关重要。初步数据表明，CR8C 虽然对于浓缩质粒 DNA 的核输入至关重要，但不能有效地与细胞核中的 DNA 解离。拟议研究的目标 1 将测试一系列添加或不添加寡精氨酸的组蛋白肽，寡精氨酸可以通过核酶共价修饰以减少肽的阳离子电荷含量。假设组蛋白肽会压缩质粒 DNA 并将其带入细胞核，但在原位共价修饰后与 DNA 分离。质粒DNA的这种受控释放应该允许DNA增强转录。由于质粒DNA在肝细胞核中积累，因此研究其有效导入的机制具有重要意义。目标 2 旨在测试以下假设：由于 LCP 从内体释放其货物而导致 Ca 浓度短暂升高，将刺激质粒 DNA/CR8C 复合物的核输入。已经提出了各种细胞生物学实验，包括使用延时活细胞共聚焦显微镜的实验。全球有超过 20 亿人感染乙型肝炎病毒 (HBV)，其中 3.5 亿人患有慢性 HBV 感染，其中约 25% 死于感染引起的肝纤维化/肝硬化或肝癌（世界卫生组织：乙型肝炎-关键事实）。尽管有预防性乙肝疫苗，但对于 3.5 亿已经慢性感染的患者来说，尚无法治愈。北卡罗来纳大学的苏立山博士开发了一种人源化小鼠模型（AFC8-hu），该模型的肝脏中不仅含有人类肝细胞，还含有人类免疫系统。小鼠可能会​​感染乙型肝炎病毒并出现肝炎症状，包括脂肪肝、纤维化和炎症。 1型干扰素是有效的抗病毒药物，尤其是针对乙肝病毒等病毒性肝炎。在初步实验中，使用 LCP 作为载体，注射含有 IFN-α2b cDNA 的质粒的小鼠肝脏中，IFN-α2b 可以以临床相关浓度表达。在目标 3 中，我们将在慢性感染 HBV 的 AFC8-hu 小鼠中测试 IFN 基因治疗。 S/MAR 序列将被整合到 IFN-α2b 质粒中以延长基因表达。目标是为乙型肝炎患者开发每月一次的干扰素基因疗法。