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，它包括几个有利于体内基因传递到肝脏的关键特征。这种非病毒载体由一种新设计的双功能化合物组成，该化合物包含末端多胺基作为DNA结合段，末端半乳糖残基作为目标结合段，以及连接两个功能基团的脂肪连接体。当与DNA混合时，双功能化合物的DNA结合部分通过静电相互作用与DNA结合，允许形成中性复合物，其中DNA分子被双功能分子的鞘包裹，半乳糖残基暴露在表面。由于DNA被包裹在里面，它在从注射部位到靶细胞的过程中受到细胞外液酶降解的保护。这些静电中性颗粒体积小，表面带有靶向配体，可以避免与非靶向细胞、细胞外基质和血液成分的非特异性结合。此外，这些颗粒将能够穿过肝窗，通过asialal糖蛋白受体与肝细胞结合，并将转基因传递到肝细胞中。为了实现我们的目标，我们将优化双功能化合物中三个片段的结构，包括它们对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的影响。实验方法包括合成每个功能段的结构类似物，然后组装具有不同结构组合的双功能化合物池。然后通过生物化学、生物物理和生物学方法选择理想的双功能化合物结构。所选双功能化合物的靶特异性基因传递特性将在体外和体内通过细胞培养和动物模型进行评估。该目标的成功实现将为双功能化合物的合成建立新的方法，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.