Hepatic Non-viral Gene Therapy

肝脏非病毒基因治疗

基本信息

批准号：
8731891
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/8731891
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 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

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治疗来治疗，但非病毒载体的效率通常落后于病毒载体，但水动力注射方法的侵入性除外。 Leaf Huang 博士等人最近开发了一种纳米颗粒制剂，该制剂由包裹着单个脂质双层膜的无定形磷酸钙核心组成。 LCP（脂质/钙/磷酸盐）。含有质粒 DNA 和十肽 CR8C 的半乳糖靶向 LCP 以非流体动力学方式注射后，在小鼠肝脏肝细胞中显示出高水平的基因转染。初步数据表明，CR8C 虽然对于浓缩质粒 DNA 的核导入至关重要，但其效率并不高。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，这些肽可以通过核酶进行共价修饰以减少肽的阳离子电荷含量。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，从而控制释放质粒DNA。由于质粒 DNA 在肝细胞核中积累，因此研究有效导入的机制非常重要，目标 2 旨在检验 LCP 导致 Ca 浓度短暂升高的假设。已经有超过 20 亿人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进行的实验。乙型肝炎病毒 (HBV) 感染者，全球有 3.5 亿人患有慢性乙型肝炎病毒感染，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）。乙肝疫苗已经问世，但对于 3.5 亿慢性感染患者来说，尚无治愈方法。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），其中不含乙肝疫苗。不仅是肝脏中的人类肝细胞，而且人类的免疫系统也可以被乙型肝炎病毒感染并出现肝炎症状，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，尤其是对于病毒性肝炎。在初步实验中，IFN-¿ 2b 可以在注射了含有 IFN-¿ cDNA 的质粒的小鼠肝脏中以临床相关浓度表达2b 使用 LCP 作为载体，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，将其纳入 IFN-¿ 2b质粒以延长基因表达，目标是为乙型肝炎患者开发每月一次的IFN基因疗法。