Hepatic Non-viral Gene Therapy

肝脏非病毒基因治疗

基本信息

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
项目类别：
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

项目摘要

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.

抽象的理论上可以通过基因疗法治疗许多肝脏的遗传和获得性疾病。效率除流体动力注射方法外，非病毒载体通常落后于病毒载体的载体。但是，流体动力学程序的侵入性除了作为一个研究工具。 Leaf Huang等人。最近开发了一个纳米颗粒公式，由用单个脂质双层膜包裹的无定形磷酸钙核心。纳米颗粒是命名为LCP（脂质/钙/磷酸盐）。半乳糖靶向的LCP含有质粒DNA和A 二肽CR8C在尾静脉后的肝脏肝细胞中显示出高水平的基因转染以非流动力学方式注射。 CR8C的存在对于核定位置至关重要注射的质粒DNA。初步数据表明，CR8C虽然对于核进口至关重要凝结的质粒DNA，剂量不能有效地从核中的DNA中分离出来。拟议中的目标1 研究将测试一系列的组蛋白辣椒，有或不加入寡素金素，这可以是通过核酶重新修饰，以减少宠物的阳离子电荷含量。假设是Hisstone肽（S）会凝结质粒DNA并将其带入细胞核，但与共价修饰原位后的DNA。质粒DNA的这种受控释放应允许DNA 用于增强的转录。由于质粒DNA积聚在肝细胞的核中，这很重要研究有效进口的基础机制。 AIM 2旨在测试瞬时的假设由于LCP从内体释放其货物，CA浓度升高将刺激核导入质粒DNA/CR8C复合物。各种细胞生物学实验，包括使用时间的实验已经提出了失去的活细胞共聚焦显微镜。超过20亿人感染了丙型肝炎病毒（HBV）和3.5亿个全球慢性HBV感染，其中约25％死于肝纤维化/肝硬化或肝癌是由感染引起的（WHO：乙型肝炎事实）。虽然可预防性HBV疫苗可用，对于已经长期已经长期已经存在的3.5亿患者无法治愈已感染。 UNC的Lishan Su博士开发了人源化的小鼠模型（AFC8-HU），不仅包含肝脏中的人类肝细胞，也是人类免疫系统。小鼠可以被HBV感染，并且发展肝炎症状，包括脂肪肝，纤维化和炎症。 1型干扰素有效抗病毒，特别是对于病毒肝炎，例如HBV。在初步实验中，IFN-€2b可能是在注射含有质粒的小鼠肝脏的临床相关浓度下表达使用LCP作为载体的IFN- - 2B的cDNA。在AIM 3中，我们将测试AFC8-HU小鼠的IFN基因治疗长期感染HBV。 S/MAR序列将纳入IFN-€2B质粒以延长基因表达。目的是为HBV肝炎患者开发一个月一次的IFN基因治疗。