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缀合物（多聚体）载体提高基因递送至细胞的效率的机制基础。 我们计划分离转基因表达整个过程中涉及的表达限制步骤：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缀合物的分子特性。 这一目标将通过构建模块化缀合物来实现，允许选择性修改其分子特性。 GFP 将作为转基因，允许定量测量整个细胞群分布的表达效率和水平。 我们的一系列实验方法包括结合和内化的定量测定、细胞内缀合物定位的共焦和双光子显微镜可视化、表达水平的定量测量以及缀合物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.