DESIGN OF TARGETED SYNTHETIC GENE DELIVERY VEHICLES

我们提出了一套基础研究，旨在了解通过靶向分子偶联物（复合物）载体提高基因递送到细胞的效率的机制基础。 我们计划分离涉及转基因表达整个过程的表达限制步骤：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，从而允许选择性地修饰其分子性质。 GFP将作为转基因，允许跨细胞群体分布的表达效率和水平的定量测量。 我们的实验方法的电池包括定量测定结合和内化，共聚焦和双光子显微镜可视化共轭定位在细胞内，定量测量的表达水平和详细的物理化学分析共轭结构和稳定性。

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.