Sulfhydryl Cross-linked Targeted Gene Delivery

描述(由申请人提供)：这项研究将研究一种新的基因传递系统，该系统基于多肽DNA缩合物中巯基交联物的形成。正在研究的基因递送系统是一种多组分肽缩合DNA制剂。主要成分是由多肽结合物缩合而成的质粒DNA，每个多肽结合物都含有多个巯基，当结合到DNA上时，这些巯基可以自发聚合和交联。跟随静脉注射。剂量，交联的DNA缩合物被建议在循环期间和靶细胞内瞬时稳定质粒DNA。交联肽将与单个聚乙二醇链共价衍生，在DNA凝聚物表面形成空间层，阻止蛋白质结合，并掩盖网状内皮系统对DNA凝聚物的识别。通过将交联肽与单个N-糖衍生物进行衍生化，得到直接靶向肝细胞上的去唾液酸糖蛋白受体或库普弗细胞上的甘露糖受体的糖多肽，从而实现靶向性。DNA缩合物将使用交联糖肽和聚乙二醇肽的二元混合物来制备。交联肽将被修饰以缓冲内体，并允许DNA凝聚体释放到靶细胞的胞浆中。一旦进入胞浆，交联的DNA缩合物被认为是在二硫键还原后缓慢释放质粒DNA。需要检验的中心假设是，增加DNA凝聚体的稳定性并控制DNA释放到胞浆中的速度将延长DNA的半衰期，并在体内产生更高水平的基因表达。这些研究旨在利用一种新的化学机制系统地优化体内瞬时基因表达水平，以克服目前限制非病毒基因递送系统效率的障碍。

DESCRIPTION (provided by applicant): This study will investigate a novel gene delivery system that is based on the formation of sulfhydryl crosslinks within peptide DNA condensates. The gene delivery system under investigation is a multi-component peptide condensed DNA formulation. The key components consist of plasmid DNA condensed by an addmixture of peptide conjugates each containing multiple sulfhydryl groups designed to spontaneously polymerize and cross-link when bound to DNA. Following i.v. dosing, cross-linked DNA condensates are proposed to transiently stabilize plasmid DNA during circulation and inside the target cells. Cross-linking peptides will be covalently derivatized with a single polyethylene glycol (PEG) chain to form a steric layer on the surface of DNA condensates that blocks protein binding and masks DNA condensate recognition by the reticuloendothelial system. Targeting specificity will be achieved by derivatizing a cross-linking peptide with a single N-glycan resulting in glycopeptides that direct targeting to either the asialoglycoprotein receptor on hepatocytes or the mannose receptor on Kupffer cells. DNA condensates will be prepared using binary addmixtures of cross-linking glycopeptide and PEG-peptide. Cross-linking peptides will be modified to buffer endosomes and allow DNA condensates to release into the cytosol of target cells. Once in the cytosol, crosslinked DNA condensates are proposed to slowly release plasmid DNA following disulfide bond reduction. The central hypothesis to be tested is that increasing DNA condensate stability and controlling the rate of DNA release into the cytosol will prolong the half-life of DNA and produce higher levels of gene expression in vivo. These studies aim to systematically optimize the level of transient gene expression in vivo using a novel chemical mechanism to overcome barriers that currently limit the efficiency of nonviral gene delivery systems.