DESIGN OF TARGETED SYNTHETIC GENE DELIVERY VEHICLES

我们提出了一系列基础研究，旨在了解通过靶向分子偶联（polyplex）载体提高基因传递到细胞效率的机制基础。我们计划分离整个转基因表达过程中的表达限制步骤：细胞表面结合、内化、内体逃逸、核定位和转录，并阐明控制每个步骤的偶联物的分子特性。这一目标将通过构建模块化共轭物来实现，允许对其分子性质进行选择性修饰。GFP将作为转基因，允许在细胞群分布中定量测量表达效率和水平。我们的一系列实验方法包括结合和内化的定量分析，细胞内共轭定位的共聚焦和双光子显微镜可视化，表达水平的定量测量以及共轭结构和稳定性的详细物理化学分析。

We propose a set of fundamental studies aimed at understanding the mechanistic basis for improving efficiency of gene delivery to cells via targeted molecular conjugate (polyplex) vectors. We plan to isolate the expression-limiting steps involved in the overall process of transgene expression: cell surface binding, internalization, endosomal escape, nuclear localization and transcription, and to elucidate the molecular properties of the conjugate governing each step. This goal will be achieved by constructing a modular conjugate, permitting selective modification of its molecular properties. GFP will serve as the transgene, allowing quantitative measurement of both expression efficiency and level across a cell population distribution. Our battery of experimental approaches includes quantitative assays for binding and internalization, confocal and two-photon microscopy visualization of conjugate localization within cells, quantitative measurements of expression levels and detailed physico-chemical analyses of conjugate structure and stability.