Bioengineering for In Vivo-Viral Gene Delivery

体内病毒基因传递的生物工程

• 批准号: 6917836
• 负责人: KENNETH J LONGMUIR
• 金额: $ 49.61万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-15 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6917836
• 关键词: bioengineering /biomedical engineering; biomedical automation; biomimetics; biotechnology; cell nucleus; gene delivery system; gene expression; gene targeting; laboratory mouse; membrane fusion; microdialysis; molecular dynamics; nanotechnology; polyethylene glycols; serum; technology /technique development; transfection; vesicle /vacuole

DESCRIPTION (provided by applicant): Non-viral gene delivery systems must overcome the following sequential "barriers" in order to effectively deliver genetic material. 1) Effective self-assembly of the delivery system with the genetic material, 2) stability of the delivery system in the extracellular (serum and blood) environment, 3) escape from the endosomal/lysosomal compartments of the cell, and 4) delivery into the nucleus without the requirement for cell division. We have found that no single barrier is predominant. Instead, effective non-viral gene delivery systems will become therapeutic realities only with a multidisciplinary research effort that addresses all four of these barriers in an integrated fashion, and only with an integrated delivery system with chemical and biophysical properties that allow all these barriers to be overcome. In this regard, we have developed a delivery system that has several extraordinary features, making it a highly attractive candidate for systemic administration in mammals. These features include resistance to inactivation by serum, releasable serum protection components, multiple endosomal escape mechanisms with low cytotoxicity, and most uniquely, the ability to transfect cells equally well with or without cell division. For this project, we have assembled a multidisciplinary team to address the sequential barriers to non-viral gene delivery in an integrated fashion. 1) Effective self-assembly will be achieved by developing fully automated microfluidic technology to carry out reconstitution, mixing, dialysis, and concentration. Stability in serum will be achieved by polyethyleneglycol (PEG) stabilization, with triggered release of the PEG chains upon entry into endosomal compartments. Endosomal escape will be achieved with multiple membrane fusion mechanisms, including fusogenic peptide mimetics designed to eliminate protease inactivation and immune response. Nuclear entry, without cell division, will be achieved with nuclear targeting signals that by-pass classical nuclear targeting pathways, and instead bind directly to the nuclear pore - a mechanism used by those viruses that are the most effective at nuclear entry without cell division. These signals will also be in the form of peptide mimetics to eliminate protease degradation and immune response. The development of this technology will have broad implications for the controlled self-assembly of a wide range of nanoparticle systems, particularly those intended for in vivo drug delivery.

描述（由申请人提供）：非病毒基因输送系统必须克服以下顺序“屏障”，以有效地传递遗传物质。 1）使用遗传物质的递送系统的有效自组装，2）在细胞外（血清和血液）环境中递送系统的稳定性，3）逃离细胞的内体/溶酶体隔室，而4）递送到细胞核中，无需细胞分裂。 我们发现没有一个障碍是主要的。 取而代之的是，只有通过多学科研究工作，有效的非病毒基因输送系统才能成为治疗现实，该研究以综合方式解决所有这些障碍，并且只有具有具有化学和生物物理特性的集成输送系统，从而允许所有这些障碍都可以克服。 在这方面，我们开发了一个具有多个非凡功能的交付系统，使其成为哺乳动物系统性管理的高度吸引人的候选人。 这些特征包括血清抗活化，可释放的血清保护成分，具有低细胞毒性的多个内体逃生机制，以及最独特的，可以在或没有细胞分裂的情况下同样良好地转染细胞的能力。 对于这个项目，我们组建了一个多学科团队，以综合方式解决非病毒基因传递的顺序障碍。 1）将通过开发全自动微流体技术来进行重组，混合，透析和集中度来实现有效的自组装。 血清中的稳定性将通过聚乙二醇（PEG）稳定来实现，并在进入内体隔室后触发了PEG链的释放。 将通过多种膜融合机制来实现内体逃逸，包括旨在消除蛋白酶失活和免疫反应的融合肽模拟物。 核靶向信号将实现核进入，而没有细胞分裂，该信号将经典的经典核靶向途径直接与核孔结合 - 那些在没有细胞分裂的核进入核进入的病毒中使用的机制。 这些信号还将以肽模拟物的形式消除蛋白酶降解和免疫反应。 该技术的开发将对广泛的纳米颗粒系统的受控自组装有广泛的影响，尤其是那些用于体内药物输送的纳米颗粒系统。