Sulfhydryl Cross-linked Targeted Gene Delivery

描述（由申请人提供）：本研究将研究一种新的基因传递系统，该系统基于肽DNA凝聚物中巯基交联的形成。所研究的基因传递系统是一种多组分肽浓缩DNA制剂。关键成分包括质粒DNA，由肽偶联物的混合物凝聚而成，每个肽偶联物含有多个巯基，这些巯基被设计成在与DNA结合时自发聚合和交联。在静脉给药后，交联DNA凝聚物被提议在循环和靶细胞内暂时稳定质粒DNA。交联肽将与单个聚乙二醇（PEG）链共价衍生，在DNA凝聚物表面形成一个空间层，阻止蛋白质结合并掩盖网状内皮系统对DNA凝聚物的识别。靶向特异性将通过衍生具有单个n聚糖的交联肽来实现，从而产生直接靶向肝细胞上的asialal糖蛋白受体或Kupffer细胞上的甘露糖受体的糖肽。用交联糖肽和聚乙二醇肽的二元混合物制备DNA凝聚物。交联肽将被修饰以缓冲内体，并允许DNA凝聚物释放到靶细胞的细胞质中。一旦进入细胞质溶胶，交联DNA凝聚物被认为可以在二硫键还原后缓慢释放质粒DNA。要验证的中心假设是，增加DNA凝聚稳定性和控制DNA释放到细胞质中的速度将延长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瞬时基因在体内的表达水平，以克服目前限制非病毒基因传递系统效率的障碍。

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.