Proteosome Inhibitor Enhanced Non-Viral Gene Delivery

蛋白酶体抑制剂增强非病毒基因传递

• 批准号: 7039140
• 负责人: KEVIN G RICE
• 金额: $ 21.61万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7039140
• 关键词: Kupffer' s cell; crosslink; gene delivery system; laboratory mouse; liver cells; nucleic acid denaturation; nucleic acid metabolism; plasmids; protease inhibitor; proteasome; receptor mediated endocytosis; technology /technique development; vesicle /vacuole

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.

描述（由申请人提供）： 本研究将探讨蛋白酶体抑制作为一种新的机制，以增加在体内的非病毒基因传递系统的表达。所研究的基因递送系统是一种多组分肽浓缩靶向基因递送系统。关键成分是由聚乙二醇（PEG）-肽、靶向糖肽和融合肽组成的巯基交联聚合物凝聚的质粒DNA。使用与肝细胞上的去唾液酸糖蛋白受体或枯否细胞上的甘露糖受体结合的糖肽实现特异性靶向。在通过受体介导的内吞作用内化之后，巯基交联的DNA缩合物在内体的还原环境中脱乙酰化。释放的融合肽促进内体裂解和DNA逃逸到胞质溶胶中。在冷凝物解包之后，DNA靶向细胞核。基因转移的效率受到DNA浓缩物逃逸内体和避免在胞质溶胶中降解的能力的限制。初步研究表明，蛋白酶体代谢细胞质中的肽-DNA缩合物，导致质粒DNA被细胞质DNA酶过早降解。本研究将探讨蛋白酶体抑制剂在细胞质中阻断DNA缩合物代谢和提高基因转移效率的应用。待检验的中心假设是，蛋白酶体抑制将通过稳定质粒DNA使其免于代谢而增强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肽的内在蛋白酶体抑制剂纳入靶向肝细胞或枯否细胞的巯基交联非病毒基因递送系统中。拟议研究的总体目标是使用蛋白酶体抑制的新机制来阻断DNA缩合物代谢，以提高非病毒基因递送的效率。这些研究的成功结果应该提供一个合理的方法来开发更有效的基因传递系统，可用于治疗各种人类疾病。