Proteasome Inhibitor Enhanced Non-Viral Gene Delivery

描述(由申请人提供)： 本研究将研究蛋白酶体抑制作为一种新的机制来增强体内非病毒基因传递系统的表达。正在研究的基因递送系统是一种多组分肽缩合靶向基因递送系统。其关键成分是由聚乙二醇肽、靶向糖肽和融合肽组成的巯基交联聚合物缩合的质粒DNA。通过结合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肽来实现特异性靶向。通过受体介导的内吞作用进行内化后，硫基交联的DNA凝聚体在内体的还原环境中解聚。释放的融合肽促进内体裂解和DNA逃逸到胞浆中。在凝聚物去涂层后，DNA以细胞核为靶标。基因转移的效率受到DNA凝聚体逃逸内体和避免在胞浆中降解的能力的限制。初步研究表明，蛋白酶体代谢胞浆中的肽-DNA缩合物，导致胞质DNA酶过早降解质粒DNA。本提案将研究使用蛋白酶体抑制剂来阻断胞浆中DNA凝集物的代谢，并提高基因转移效率。需要检验的中心假设是，抑制蛋白酶体将通过稳定代谢中的质粒DNA来提高体内瞬时基因表达的水平和持续时间。拟议的研究旨在将基于内源性多肽的蛋白酶体抑制剂整合到以肝细胞或Kupffer细胞为靶点的巯基交联非病毒基因递送系统中。这项拟议研究的总体目标是利用一种新的蛋白酶体抑制机制来阻断DNA凝集物代谢，从而提高非病毒基因的传递效率。这些研究的成功结果应该为开发更有效的基因输送系统提供一种合理的方法，这些基因输送系统可以用于治疗各种人类疾病。

DESCRIPTION (provided by applicant): This study will investigate proteasome inhibition as a novel mechanism to augment the expression of nonviral gene delivery systems in vivo. The gene delivery system under investigation is a multi-component peptide condensed targeted gene delivery system. The key components consist of plasmid DNA condensed by a sulfhydryl cross-linking polymer composed of polyethylene glycol (PEG)-peptide, targeting glycopeptide, and fusogenic peptide. Specific targeting is achieved using glycopeptides that bind to either the asialoglycoprotein receptor on hepatocytes or the mannose receptor on Kupffer cells. Following internalization via receptor mediated endocytosis, sulfhydryl cross-linked DNA condensates depolymerize in the reducing environment of the endosome. Released fusogenic peptides facilitate endosomal lysis and DNA escape into the cytosol. Following condensate un-coating, DNA targets the nucleus. The efficiency of gene transfer is limited by the ability of DNA condensates to escape endosomes and avoid degradation in the cytosol. Preliminary studies indicate the proteasome metabolizes peptide-DNA condensates in the cytosol, resulting in premature degradation of plasmid DNA by cytosolic DNAse. The present proposal will investigate the use of proteasome inhibitors to block DNA condensate metabolism in the cytosol and enhance gene transfer efficiency. The central hypothesis to be tested is that proteasome inhibition will enhance the level and duration of transient gene expression in vivo by stabilizing plasmid DNA from metabolism. The proposed studies aim to incorporate intrinsic peptide-based proteasome inhibitors into sulfhydryl cross-linked non-viral gene delivery systems that target either hepatocytes or Kupffer cells. The overall objective of the proposed study is to increase the efficiency of non-viral gene delivery using a novel mechanism of proteasome inhibition to block DNA condensate metabolism. The successful outcome of these studies should provide a rational approach to develop more efficient gene delivery systems that can be used to treat a variety of human diseases.