Hepatic Non-viral Gene Therapy

描述(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来治疗。除流体注射方法外，非病毒载体的效率通常落后于病毒载体。然而，除了作为一种研究工具之外，流体动力程序的侵入性可能是不可接受的。黄叶博士等人。最近开发了一种纳米颗粒配方，由包裹着单一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/钙/磷)。半乳糖靶向LCP同时含有质粒DNA和十肽CR8C，尾静脉注射后，小鼠肝细胞中以非流体动力学方式获得了高水平的基因转移。CR8C的存在对于注射的质粒DNA的核定位是必不可少的。初步数据表明，尽管CR8C对于浓缩的质粒DNA的核输入是必不可少的，但它并不能有效地与细胞核中的DNA解离。拟议研究的目标1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，这些寡精氨酸可以被核酶共价修饰，以降低多肽的阳离子电荷含量。假说是组蛋白多肽(S)在原位共价修饰后与DNA解离，使其凝聚进入细胞核。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，因此研究高效导入的机制非常重要。目的2旨在验证一种假说，即由于LCP从内体释放货物而导致的短暂的钙浓度升高，将刺激质粒DNA/CR8C复合体的核输入。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，已经被提出。全世界已有超过20亿人感染了乙肝病毒，3.5亿人患有慢性乙肝病毒感染，其中约25%死于由感染引起的肝纤维化/肝硬化或肝癌(世卫组织：乙肝-关键事实)。尽管有预防性的乙肝疫苗可用，但对于已经慢性感染的3.5亿患者来说，没有治愈方法。北卡罗来纳大学的苏立山博士开发了一种人源化的小鼠模型(AFC8-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。这些小鼠可能会感染乙肝病毒，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，尤其对乙肝病毒等病毒性肝炎有效。在初步实验中，以液晶蛋白为载体，注射含有干扰素-�2b基因的质粒，可在小鼠肝脏中以临床相关浓度表达干扰素-�2b。在目的3中，我们将在慢性感染乙肝病毒的AFC8-Hu小鼠中测试干扰素基因治疗。将S/MAR序列导入干扰素-�2b载体，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。

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.