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治疗。除流体动力注射法外，非病毒载体的效率通常低于病毒载体。然而，除了作为研究工具之外，水动力程序的侵入性可能是不可接受的。Leaf Huang博士等人最近开发了一种纳米颗粒配方，该配方由无定形磷酸钙核心包裹着单层脂质双层膜组成。这些纳米颗粒被命名为LCP（脂质/钙/磷酸盐）。含有质粒DNA和十肽CR8C的半乳糖靶向LCP经非水动力方式尾静脉注射后，在小鼠肝细胞中显示出高水平的基因转染。CR8C的存在对于注射的质粒DNA的核定位至关重要。初步数据表明，尽管CR8C对浓缩质粒DNA的细胞核输入至关重要，但它不能有效地与细胞核中的DNA分离。本研究的目的1将测试一系列组蛋白肽，无论是否添加低精氨酸，低精氨酸可以被核酶共价修饰以减少多肽的阳离子电荷含量。假设是组蛋白肽(s)将凝聚质粒DNA并将其带入细胞核，但在原位共价修饰后与DNA分离。这种质粒DNA的控制释放应该允许DNA增强转录。由于质粒DNA在肝细胞细胞核中积累，因此研究其有效输入的机制具有重要意义。目的2旨在验证一种假设，即LCP从核内体释放其货物导致Ca浓度短暂升高，从而刺激质粒DNA/CR8C复合物的核输入。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。全世界有超过20亿人感染了乙型肝炎病毒（HBV）， 3.5亿人患有慢性HBV感染，其中约25%的人死于感染引起的肝纤维化/肝硬化或肝癌（世卫组织：乙型肝炎关键事实）。虽然有预防性乙肝疫苗，但3.5亿已感染慢性乙肝的患者无法治愈。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），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。小鼠可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，特别是对HBV等病毒性肝炎。在初步实验中，以LCP为载体，注射含有IFN-¿2b cDNA的质粒后，IFN-¿2b可在小鼠肝脏中以临床相关浓度表达。在Aim 3中，我们将在慢性HBV感染的AFC8-hu小鼠中测试IFN基因治疗。将S/MAR序列整合到IFN-¿2b质粒中，延长基因表达。目标是为HBV肝炎患者开发一种每月一次的IFN基因疗法。

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.