Biodegradable Micelles for Liver-Targeted Gene Delivery

描述（由申请人提供）：本提案的总体目标是开发生物相容性、生物可降解性和自组装共聚物/DNA胶束，以实现有效的肝脏靶向基因递送。肝脏是基因医学应用的一个至关重要的靶点，因为转基因产物可以进入体循环，并且因为它是许多代谢遗传疾病、病毒感染和恶性肿瘤的发生部位。目前，肝靶向基因转移的全部潜力受到缺乏安全有效的基因载体的阻碍。具有DNA/聚阳离子复合物核心和亲水性冠的聚合物胶束代表了用于肝靶向基因递送的有前途的载体。这些DNA包封胶束表现出有利于肝靶向基因递送的几个期望的特征，例如小尺寸（80-100 nm）、与生物组分的相互作用减少、血液循环延长和低毒性。新型PEG-b-PPA/DNA胶束具有较高的DNA结合能力、体外转染效率、较低的细胞毒性和良好的组织相容性。本论文的主要工作是：（1）合成和表征一个具有较好的复合稳定性、胶体稳定性、细胞内DNA释放、内体逃逸和肝细胞靶向能力的PEG-b-PPA/DNA胶束微型库;（2）表征该系列结构不同的共聚物/胶束在大鼠原代肝细胞、枯否细胞和正常大鼠胆管细胞中的转染效率和细胞毒性，并与胶束结构相关;（3）优化给药参数以实现非分泌性蛋白（荧光素酶）和分泌性蛋白（干扰素-α 2b）的有效肝脏基因表达。这将证明这种递送策略对于表达旨在用于全身分布和局部肝脏特异性疾病的蛋白质的广泛用途。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转运。这项研究整合了聚合物化学，生物材料设计，分子/细胞生物学和临床科学的专业知识。它代表了在多个水平上对基因递送策略的系统设计和优化。本研究为聚合物胶束用于肝靶向基因治疗的临床应用奠定了基础。本提案的总体目标是开发安全有效的非病毒纳米颗粒载体，用于将基因递送至肝脏。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、病毒性肝炎和系统性疾病（如血友病A和B）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.