Hepatic Non-viral Gene Therapy

肝脏非病毒基因治疗

• 批准号: 8613538
• 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 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8613538
• 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; Genomics; Goals; 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; public health relevance; red fluorescent protein; research study; time use; tool; trafficking; vector

Abstract 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 for 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.

摘要 从理论上讲，许多遗传性和获得性肝脏疾病可以用基因疗法来治疗。效率 除流体动力注射方法外，非病毒载体的种类通常落后于病毒载体。 然而，流体动力程序的侵入性可能是不可接受的，除了作为一种 研究工具。黄叶博士等人。最近开发了一种纳米颗粒配方，由一种 无定形磷酸钙核心包裹着单一的脂质双层膜。这些纳米粒子是 命名为LCP(脂/钙/磷)。半乳糖靶向LCP同时含有一个质粒DNA和一个 十肽CR8C显示尾静脉注射后小鼠肝细胞基因转染率较高 以非流体动力方式注入。CR8C的存在对于核定位是必不可少的 注射质粒DNA。初步数据表明，CR8C虽然对核进口至关重要，但 浓缩的质粒DNA，不能有效地从细胞核中的DNA解离。建议的目标1 研究将测试一系列组蛋白多肽，在添加和不添加寡精氨酸的情况下，这可以是 用核酶共价修饰以降低多肽的阳离子电荷含量。假说 是组蛋白多肽(S)会凝聚质粒dna并将其带入细胞核，但会从 原位共价修饰后的DNA。这种受控释放的质粒DNA应该允许DNA 进行增强转录。由于质粒dna积聚在肝细胞的细胞核中，因此 研究高效进口背后的机制。目标2旨在检验这样一种假设，即瞬时 由于LCP从内吞体内释放其货物而导致的钙浓度升高将刺激细胞核 质粒DNA/CR8C复合体的导入。各种细胞生物学实验，包括利用时间的实验- 已经提出了失效的活细胞共聚焦显微镜。已有超过20亿人感染了这种病毒 全世界有3.5亿人感染了乙肝病毒，其中约25%的人死于 感染引起的肝纤维化/肝硬变或肝癌(世卫组织：乙肝--关键事实)。尽管一个 预防性乙肝疫苗问世，3.5亿慢性病患者无药可救 被感染了。北卡罗来纳大学的苏立山博士开发了一种人性化的小鼠模型(AFC8-HU)，它不仅包含 人体的肝细胞在肝脏中，也是人体的免疫系统。小鼠可以感染乙肝病毒和 出现肝炎症状，包括脂肪肝、纤维化和炎症。1型干扰素有效 抗病毒药物，特别是对乙肝病毒等病毒性肝炎。在初步实验中，干扰素-2b可能是 在小鼠肝脏中以临床相关浓度表达，注射含有 以LCP为载体的干扰素-2b基因的表达。在目标3中，我们将在AFC8-HU小鼠身上测试干扰素基因治疗 慢性感染乙肝病毒。S/MAR序列将被整合到干扰素-2b载体中，以延长 基因表达。目标是开发一种每月一次的干扰素基因疗法，用于治疗乙肝患者。