Hepatic Non-viral Gene Therapy

摘要 许多遗传性和获得性肝脏疾病理论上可以用基因治疗来治疗。效率 除了流体动力学注射方法之外，非病毒载体的方法通常福尔斯落后于病毒载体。 然而，流体动力学程序的侵入性可能是不可接受的，超出了作为一种治疗方法的范围。 研究工具。Leaf Huang博士等人最近开发了一种纳米颗粒制剂， 无定形磷酸钙核心包裹着单脂双层膜。述纳米颗粒 命名为LCP（脂质/钙/磷酸盐）。半乳糖靶向LCP含有质粒DNA和半乳糖靶向LCP。 十肽CR 8 C在尾静脉注射后小鼠肝细胞中显示高水平的基因转染 以非流体动力学方式注入。CR 8 C的存在对于细胞核定位是必不可少的。 注射质粒DNA。初步数据表明，CR 8 C虽然对核进口至关重要， 浓缩的质粒DNA不能有效地从细胞核中的DNA解离。拟议目标1 一项研究将测试一系列组蛋白肽，添加和不添加寡聚天冬氨酸，这可以 通过核酶共价修饰以减少肽的阳离子电荷含量。的假设 是组蛋白肽将浓缩质粒DNA并将其带入细胞核，但从质粒DNA中解离。 原位共价修饰后的DNA。质粒DNA的这种受控释放应允许DNA 以增强转录。由于质粒DNA在肝细胞核中积累， 研究有效进口的内在机制。目的2旨在检验假设，即瞬时 由于LCP从内体释放其货物而导致的Ca浓度升高将刺激细胞核内的Ca-2+。 质粒DNA/CR 8 C复合物的导入。各种各样的细胞生物学实验，包括那些利用时间- 已经提出了lapsed活细胞共聚焦显微镜。超过20亿人感染了 B肝炎病毒（HBV）和全球3.5亿慢性HBV感染者，其中约25%死于 由感染引起的肝纤维化/肝硬化或肝癌（WHO：乙型肝炎-关键事实）。虽然 虽然预防性HBV疫苗是可用的，但对于3.5亿已经慢性感染的患者来说， 感染了Lishan Su博士开发了一种人源化小鼠模型（AFC 8-hu）， 肝脏中的人类肝细胞以及人类免疫系统。小鼠可被HBV感染， 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，特别是用于病毒性肝炎如HBV。在初步实验中，IFN-γ 2b可能是 在注射了含有该质粒的小鼠的肝脏中以临床相关浓度表达， 使用LCP作为载体的IFN-γ 2b的cDNA。在目的3中，我们将在AFC 8-hu小鼠中测试IFN基因治疗 慢性HBV感染。S/MAR序列将被掺入IFN-γ 2b质粒中，以延长IFN-γ 2b的表达。 基因表达。目的是开发一种每月一次的干扰素基因治疗乙型肝炎患者。

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.