Design of Gene Delivery System to Target Hepatocytes

描述（由申请人提供）： 非病毒（合成）核酸递送系统具有提供基于核酸的治疗剂的实际应用的潜力。我们已经设计并创建了一种可调的、自组装的、非病毒的核酸递送系统，该系统基于含环糊精的聚合物（CDP），并且该递送系统已被证明在动物中具有非常低的毒性，并且通过静脉内施用在靶组织中给予基因表达。 我们建议评估以下假设：如果粒径不超过70 nm且表面电荷在+/-10 mV范围内，则我们适当设计和工程化的、合成的、携带基于半乳糖的靶向部分的非病毒递送载体可以有效地将核酸递送至小鼠肝细胞。 具体目标： 1.配制含有siRNA的基于CDP的颗粒，其具有基于半乳糖的靶向配体，直径为70 nm或更小，表面电荷在+/-10 mV范围内，可被肝细胞上的去唾液酸糖蛋白受体识别和处理，并确定小鼠全身给药后肝基因最大下调时间和效率的最佳理化性质。 2.使用特定目标1中获得的结论配制含质粒DNA（pDNA）的CDP颗粒，以指导组装和给药方案，并使用标记物和治疗基因测定基因递送效率和小鼠肝细胞中基因表达的时程。 重要性：用于靶向肝细胞的有效的合成递送载体的开发将提供用于治疗多种疾病的通用方法。 长期目标：使用基于核酸的疗法（所谓的基因疗法）提供新的疾病治疗的潜力受到核酸的安全和有效递送的限制的限制。我们提案的长期目标是设计和工程化一种通用的合成系统，用于最终将基于核酸的治疗剂递送至人类肝细胞。

DESCRIPTION (provided by applicant): Non-viral (synthetic) nucleic acid delivery systems have the potential to provide for the practical application of nucleic acid-based therapeutics. We have designed and created a tunable, self-assembling, non-viral nucleic acid delivery system that is based on cyclodextrin-containing polymers (CDP) and this delivery system has been shown to have very low toxicity in animals and give gene expression in targeted tissues from an intravenous administration. We propose to evaluate the hypothesis that our properly designed and engineered, synthetic, non-viral delivery vehicle bearing galactose-based targeting moieties can effectively deliver nucleic acids to hepatocytes in mice if the particle diameters do not exceed 70 nm and the surface charges are in the range of +/-10 mV. Specific Aims: 1. Formulate siRNA-containing, CDP-based particles with galactose-based targeting ligands of ca. 70 nm diameter or less with surface charges within the range of +/-10mV that can be recognized and processed by the asialoglycoprotein receptor on hepatocytes and determine the optimal physicochemical properties for maximum downregulation time and efficiency of a hepatic gene following systemic administration to mice. 2. Formulate plasmid DNA (pDNA)-containing, CDP-based particles using the conclusions obtained in Specific Aim 1 to guide the assembly and administration protocols and determine the gene delivery efficiency and the time course of gene expression in hepatocytes of mice using both marker and therapeutic genes. Significance: The development of effective, synthetic delivery vehicles for targeting hepatocytes would provide a generalized methodology for treating numerous diseases. Long-term goals: The potential for providing new disease treatments using nucleic acid-based therapies, the so-called gene therapies, has been restricted by limitations in the safe and effective delivery of nucleic acids. The long-term objective of our proposal is to design and engineer a generalized, synthetic system for ultimately delivering nucleic acid-based therapeutics to hepatocytes in humans.