Hepatic Non-viral Gene Therapy

描述（由申请人提供）：许多遗传性和获得性肝脏疾病理论上可以用基因疗法治疗。除了流体动力学注射方法之外，非病毒载体的效率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除了作为研究工具之外可能是不可接受的。Leaf Huang博士等人最近开发了一种纳米颗粒制剂，其由包裹有单个脂质双层膜的无定形磷酸钙核心组成。这种纳米颗粒被命名为LCP（脂质/钙/磷酸盐）。含有质粒DNA和十肽CR 8 C的半乳糖靶向LCP在以非流体动力学方式尾静脉注射后在小鼠肝细胞中显示高水平的基因转染。CR 8 C的存在对于注射的质粒DNA的核定位是必不可少的。初步的数据表明，CR 8 C，虽然必不可少的浓缩质粒DNA的核输入，不有效地从细胞核中的DNA解离。目的1的拟议研究将测试一系列组蛋白肽，添加和不添加寡聚天冬氨酸，它可以被核酶共价修饰，以减少肽的阳离子电荷含量。假设组蛋白肽将使质粒DNA凝聚并将其带入细胞核，但在原位共价修饰后从DNA解离。质粒DNA的这种受控释放应允许DNA增强转录。由于质粒DNA在肝细胞核中积累，因此研究有效输入的机制是重要的。目的2旨在检验以下假设：由于LCP从内体释放其货物而导致的瞬时升高的Ca浓度将刺激质粒DNA/CR 8 C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。全世界有超过20亿人感染了B型肝炎病毒（HBV），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（WHO：乙型肝炎-关键事实）。虽然预防性乙肝疫苗是可用的，但没有治愈3.5亿已经慢性感染的患者。Lishan Su博士开发了一种人源化小鼠模型（AFC 8-hu），该模型不仅包含肝脏中的人类肝细胞，还包含人类免疫系统。这些小鼠可以被HBV感染并出现肝炎症状，包括脂肪肝，纤维化和炎症。1型干扰素是一种有效的抗病毒药物，特别是对病毒性肝炎如HBV。在初步实验中，使用LCP作为载体，用含有IFN-<$2b的cDNA的质粒注射小鼠，IFN-<$2b可以在小鼠肝脏中以临床相关浓度表达。目的3：在慢性HBV感染的AFC 8-hu小鼠中进行IFN基因治疗试验。将S/MAR序列掺入IFN-γ 2b质粒中以延长基因表达。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。

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.