DELIVERY OF PROTEINS INTO CYTOSOL USING LLO-LIPOSOMES

描述（改编自研究者摘要）： 细胞是药物递送系统的重要靶点， 治疗剂的目的是干预或修饰特定的分子 发生在细胞质中的事件。 其中许多治疗 代理人，特别是近年来发现或设计的， 由于它们电荷或高分子量 重量. 这项建议的长期目标是制定战略， 以及可以将这类分子递送到细胞溶质中的方法 细胞的空间 这项提案的主要思想是将细胞生物学 李斯特菌的生物化学转化为药物，即，采取最低限度的 来自兼性胞内细菌李斯特菌的组分 单核细胞增多症，并构建基于脂质体的胞质递送载体。 李斯特菌溶血素O（LLO），这是必要的和足够的逃逸， 李斯特菌进入胞质溶胶后，将被纯化并掺入脂质体中。 含有LLO的脂质体，模仿李斯特菌如何传递自己， 进入它们生长的细胞质，将根据它们的 将大分子递送到胞质溶胶中的能力。 虽然拟议 战略应适用于其他类型的交付， 大分子，目前的建议将集中在交付 蛋白质大分子。 两种蛋白质在 将测试：（1）抗原蛋白， 诱导特异性细胞毒性T淋巴细胞（CTL）的活化;和（2）毒素 可以抑制蛋白质合成 胞质递送的量将 通过一种新开发的生物化学测定法来测量， 细胞溶质递送分子占总分子的百分比 细胞相关量。 还将进行体外抗原呈递测定。 用于测量递送到细胞溶质中的相对效率 抗原呈递途径。 这直接影响到疫苗 递送载体，因此将扩展至良好建立的小鼠 淋巴细胞性脉络丛脑膜炎病毒（LCMV）感染模型。 诱导 CTL，特异性针对LCMV的核蛋白（NP），通过含NP的 LLO-脂质体和随后的病毒攻击保护将是 在小鼠中监测。 除了递送抗原性蛋白质之外， 一种不透膜的毒素，白树毒素，将被包裹在里面 LLO-脂质体，并在细胞生长抑制试验中进行测试。 脂质体 循环时间较长的地层将采用LLO， 还将测试它们的胞质递送效率。 与此同时， 将研究LLO通过孔形成溶解内体的机制 使用生物物理学和分子生物学方法。

DESCRIPTION (Adapted from Investigator's Abstract): The cytosolic space of cells is an important target for drug delivery systems since many therapeutic agents are aimed at intervening or modifying specific molecular events occurring in the cytoplasm of cells. Many of these therapeutic agents, particularly the ones discovered or designed in recent years, are intrinsically membrane-permeant due to their charge or high molecular weight. The long term objective of this proposal is to develop strategies and methods that can deliver molecules in this category into the cytosolic space of cells. The main idea of this proposal is to import cell biology and biochemistry of Listeria into pharmaceutics, i.e., to take a minimal component from a facultative intracellular bacterium, Listeria monocytogenes, and construct a liposome-based cytosolic delivery vehicle. Listeriolysin O (LLO), which is necessary and sufficient in the escape of Listeria into cytosol, will be purified and incorporated into liposomes. The LLO-containing liposomes, which mimic how Listeria deliver themselves into the cytosol where they grow, will be characterized in terms of their ability to delivery macromolecules into the cytosol. Although the proposed strategy should be applicable to the delivery of other types of macromolecules, the current proposal will focus on the delivery of proteinaceous macromolecules. Two types of proteins that show effect when delivered into the cytosol will be tested: (1) an antigenic protein that induces activation of specific cytotoxic T lymphocytes (CTL); and (2) toxins that can inhibit protein synthesis. The amount of cytosolic delivery will be measured by a newly developed biochemical assay which monitors the percent of cytosolically delivered molecules out of the total cell-associated amount. In vitro antigen presentation assays will also be employed to measure relative efficiency of delivery into the cytosolic pathway of antigen presentation. This has direct implications as a vaccine delivery vehicle, and will therefore be extended to a well-established mouse model of lymphocytic choriomeningitis virus (LCMV) infection. Induction of CTL, specific against nucleoprotein (NP) of LCMV, by NP-containing LLO-liposomes and subsequent protection from viral challenge will be monitored in mice. In addition to delivering antigenic proteins, a membrane-impermeant toxin, gelonin, will be encapsulated inside LLO-liposomes and tested in the cell growth inhibition assays. The liposome formations that have a long circulation time will be made with LLO, and their efficiency of cytosolic delivery will also be tested. Concurrently, the mechanism of endosome lysis by LLO via pore formation will be studied using biophysical and molecular biological methods.