Biodegradable Micelles for Liver-Targeted Gene Delivery

描述(申请人提供)：这项建议的总体目标是开发生物相容、可生物降解和自组装的共聚物/DNA胶束，以实现高效的肝靶向基因传递。肝脏是基因药物应用的重要靶点，因为转基因产物可以进入全身循环，而且它是许多代谢遗传疾病、病毒感染和恶性肿瘤的发源地。目前，由于缺乏安全有效的基因载体，肝靶向基因转移的全部潜力受到阻碍。具有DNA/聚阳离子复合体核心和亲水性冠层的聚合物胶束是一种很有前途的肝靶向基因传递载体。这些包裹DNA的胶束表现出几个有利于肝脏靶向基因传递的理想特征，如小尺寸(80-100 nm)、减少与生物成分的相互作用、延长血液循环和低毒性。新型聚乙二醇b-PPA/DNA胶束具有较高的DNA结合容量、体外转染率、较低的细胞毒性和良好的组织生物相容性。在本研究中，我们将(1)合成并表征一个具有更高的络合物稳定性、胶体稳定性、细胞内DNA释放、内体逃逸和肝细胞靶向性的聚乙二醇-b-PPA/DNA胶束的微型文库；(2)表征这一系列结构不同的共聚物/胶束对大鼠原代肝细胞、枯否细胞和正常大鼠胆管细胞的转染效率和细胞毒性，并与胶束结构相关联；(3)优化给药参数，以实现非分泌型蛋白(荧光素酶)和分泌型蛋白(干扰素-a2b)的高效肝基因表达。这将证明这种递送策略在表达全身性分布的蛋白质和局部肝特异性疾病方面具有广泛的实用性。将在体外和体内进行机制研究，以了解胶束的细胞外和细胞内运输。这项研究综合了聚合物化学、生物材料设计、分子/细胞生物学和临床科学方面的专业知识。它代表了基因递送策略在多个层面上的系统设计和优化。本研究为聚合物胶束在肝靶向基因治疗中的临床应用奠定了基础。公共卫生相关性这项建议的总体目标是开发安全和有效的非病毒纳米颗粒载体，用于将基因输送到肝脏。如果这项拟议的研究成功，这些新的基因载体将在治疗遗传性和代谢性肝病、肝癌、病毒性肝炎以及血友病A和B等系统性疾病方面获得广泛的临床应用。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to develop biocompatible, biodegradable and self- assembling copolymer/DNA micelles to achieve efficient liver-targeted gene delivery. Liver is a critically important target for gene medicine applications because of the access of the transgene product to systemic circulation, and because it is the site of many metabolic genetic disorders, viral infection and malignancies. At present, the full potential of liver-targeted gene transfer is hindered by a lack of safe and efficient gene carriers. Polymeric micelles with a DNA/polycation complex core and a hydrophilic corona represent a promising carrier for liver-targeted gene delivery. These DNA-encapsulating micelles exhibit several desirable features that favor liver- targeted gene delivery, such as small size (80-100 nm), reduced interaction with biological components, prolonged circulation in blood, and low toxicity. Novel PEG-b-PPA/DNA micelles exhibit high DNA binding capacity, in vitro transfection efficiency, low cytotoxicity and good tissue biocompatibility. In this study, we will (1) synthesize and characterize a mini-library of PEG-b-PPA/DNA micelles with improved complex stability, colloidal stability, intracellular DNA release, endosomal escape, and hepatocyte-targeting ability; (2) characterize the transfection efficiency and cytotoxicity in rat primary hepatocytes, Kupffer cells and normal rat cholangiocytes for this series of structurally distinct copolymers/micelles, and correlate with micelle structures; (3) optimize the administration parameters to achieve efficient liver gene expression for both a non-secretory protein (luciferase) and a secretory protein (interferon-a2b). This will demonstrate the broad utility of this delivery strategy for expression of proteins intended for systemic distribution and for localized liver-specific diseases. Mechanistic studies will be performed in vitro and in vivo to understand the extracellular and intracellular transport of the micelles. This study integrates expertise in polymer chemistry, biomaterial design, molecular/cell biology and clinical sciences. It represents a systematic design and optimization of gene delivery strategies at multiple levels. This study will lay the foundation for future clinical application of polymeric micelles for liver-targeted gene therapy. PUBLIC HEALTH RELEVANCE The overall objective of this proposal is to develop safe and efficient non-viral nanoparticle carriers for gene delivery to the liver. Should this proposed study be successful, these new gene carriers will find wide clinical applications for treating inherited and metabolic liver diseases, liver cancer, viral hepatitis, and systemic diseases, like hemophilia A and B.