Sulfhydryl Cross-linked Targeted Gene Delivery

描述（由申请人提供）：本研究将研究一种基于肽DNA缩合物内巯基交联形成的新型基因递送系统。所研究的基因递送系统是多组分肽浓缩DNA制剂。关键成分包括质粒DNA，质粒DNA由肽缀合物的混合物凝聚而成，每个肽缀合物含有多个巯基，设计为当与DNA结合时自发地交联和交联。静脉内给药后，建议使用交联的DNA缩合物在循环期间和靶细胞内瞬时稳定质粒DNA。交联肽将用单个聚乙二醇（PEG）链共价衍生化，以在DNA缩合物表面上形成空间层，其阻断蛋白质结合并掩蔽网状内皮系统对DNA缩合物的识别。靶向特异性将通过用单个N-聚糖衍生交联肽来实现，从而产生直接靶向肝细胞上的脱唾液酸糖蛋白受体或枯否细胞上的甘露糖受体的糖肽。将使用交联糖肽和PEG-肽的二元混合物制备DNA缩合物。交联肽将被修饰以缓冲内体并允许DNA浓缩物释放到靶细胞的胞质溶胶中。一旦在胞质溶胶中，交联的DNA缩合物被提议在二硫键还原后缓慢释放质粒DNA。要测试的中心假设是，增加DNA凝聚物的稳定性和控制DNA释放到胞质溶胶中的速率将延长DNA的半衰期，并在体内产生更高水平的基因表达。这些研究的目的是系统地优化瞬时基因表达的水平在体内使用一种新的化学机制，以克服目前限制非病毒基因传递系统的效率的障碍。

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.