Bifunctional Compounds for Targeted Gene Delivery

描述（由申请人提供）：我们计划开发一种新的合成载体，其包括有利于体内基因递送至肝脏的几个关键特征。 这种类型的非病毒载体由双功能化合物的新设计组成，所述双功能化合物含有作为DNA结合区段的末端多胺基团、作为靶结合区段的末端半乳糖残基和将两个官能团桥接在一起的脂肪族接头。 当与DNA混合时，双官能化合物的DNA结合区段通过静电相互作用结合DNA，允许形成中性复合物，其中DNA分子被双官能分子的鞘包被，并且半乳糖残基暴露在表面上。 由于DNA被包裹在里面，当它从注射部位到达靶细胞时，它被保护免受细胞外液中的酶降解。 由于尺寸小并且在表面上携带靶向配体，这些静电中性颗粒将避免与非靶向细胞、细胞外基质和血液组分的非特异性结合。 此外，颗粒将能够穿过肝窗，通过脱唾液酸糖蛋白受体与肝细胞结合，并将转基因递送到肝细胞中。 为了实现这一目标，我们将从DNA结合、DNA复合物的形成、复合物在血清中的稳定性、靶向特异性结合和基因传递等方面对双功能化合物中的三个片段的结构进行优化。 实验方法包括合成每个功能片段的结构类似物，然后组装具有不同结构组合的双功能化合物的池。 然后利用生物化学、生物物理学和生物学方法选择双功能化合物的理想结构。 将采用细胞培养和动物模型在体外和体内评价所选双功能化合物的靶特异性基因递送的性质。 该目标的成功实现将为双功能化合物的合成提供新的方法学基础，为基因治疗提供新的合成载体。

DESCRIPTION (provided by applicant): We plan to develop a novel synthetic vector, which includes several key features that favor in vivo gene delivery to the liver. This type of non-viral vector consists of a new design of bifunctional compounds that contain a terminal polyamine group as the DNA binding segment, terminal galactose residues as the target binding segment and an aliphatic linker to bridge the two functional groups together. When mixed with DNA, the DNA binding segment of the bifunctional compounds binds DNA through electrostatic interaction, allowing the formation of a neutral complex in which the DNA molecule is coated with a sheath of bifunctional molecules and the galactose residues exposed on the surface. As DNA is wrapped inside, it is protected against enzyme degradation in extracellular fluid as it travels from the site of injection to the target cells. Being small in size and bearing targeting ligands on surface, these electrostatically neutral particles will avoid nonspecific binding to non-targeted cells, the extracellular matrix and blood components. In addition the particles will be able to pass through liver fenestrae, bind to hepatocytes through asialoglycoprotein receptors and deliver transgene into hepatocytes. To achieve our goal, we will optimize the structure of each of the three segments in the bifunctional compounds with respect to their effect on DNA binding, formation of DNA complex, the stability of complexes in serum, target specific binding and gene delivery. The experimental approach includes the synthesis of structural analogues of each functional segment followed by assembling a pool of bifunctional compounds with different structural combinations. An ideal structure of bifunctional compound will then be selected using biochemical, biophysical and biological methods. The properties of target specific gene delivery of the selected bifunctional compounds will be evaluated in vitro and in vivo employing cell culture and animal models. The successful accomplishment of the proposed objective will establish new methodologies for synthesis of bifunctional compounds that will provide a novel synthetic vector for the gene therapy.