Tools for Targeted Delivery of DNA into Mammalian Cells

将 DNA 靶向递送至哺乳动物细胞的工具

• 批准号: 6742277
• 负责人: Alexey G Zdanovsky
• 金额: $ 14.49万
• 依托单位: ALPHA UNIVERSE, LLC
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-15 至 2005-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6742277
• 关键词: Escherichia coli; Mammalia; bacteriophage lambda; biotechnology; capsid; cell surface receptors; cytolysins; gene delivery system; genetic library; green fluorescent proteins; reporter genes; technology /technique development; tissue /cell culture; transfection /expression vector

DESCRIPTION (provided by applicant): Viral vectors are the most efficient tools currently available for genetic modification of mammalian cells in vitro and in vivo. However, because protocols for the incorporation of DNA of interest into viral particles are rather lengthy and cumbersome, these vectors have not been extensively used in the area of cell biology. In the area of genetic therapy these vectors remain unchallenged tools, but they are expensive to produce, suffer from the inability to target only chosen types of cells and their application could be associated with substantial health risks. On the contrary, bacteriophage lambda-derived vectors are easy and safe to use. They have a substantial capacity and could be reproducibly obtained in high quantities. For years bacteriophage lambda-based vectors have been used as versatile tools for the delivery of foreign DNA into bacterial, but not into mammalian cells. The purpose of the current proposal is to identify ways to convert bacteriophage lambda from being a general toot in molecular biology to a general toot for cell biology. We intend to prove that provided with means to recognize specific receptors on the surface of mammalian cells and penetrate through the cell membrane, bacteriophage lambda particles will be able to deliver extended DNA sequences into mammalian cells. During Phase I proof of the concept will be achieved through the delivery of DNA capable of directing the synthesis of green fluorescent protein into mammalian cells. During Phase II efforts will be directed towards optimization of such important processes as receptor recognition, membrane penetration, release of DNA from a phage capsid and transport of DNA into the cell nucleus. As a result of these efforts we expect to create a new tool that will allow for efficient shuttling of DNA constructs (including combinatorial libraries) from E. coil into mammalian cells. Also, we expect to lay a foundation for construction of customized bacteriophage-based DNA delivery systems suitable for genetic therapy applications.

描述（由申请人提供）：病毒载体是目前可用于体外和体内哺乳动物细胞遗传修饰的最有效工具。然而，由于将目的 DNA 掺入病毒颗粒的方案相当冗长且繁琐，因此这些载体尚未在细胞生物学领域广泛使用。在基因治疗领域，这些载体仍然是无可争议的工具，但它们的生产成本昂贵，无法仅针对选定类型的细胞，而且它们的应用可能会带来重大的健康风险。相反，源自 lambda 噬菌体的载体使用起来简单且安全。它们具有巨大的容量，并且可以大量重复获得。多年来，基于噬菌体 lambda 的载体一直被用作将外源 DNA 递送到细菌中的多功能工具，但不能递送到哺乳动物细胞中。当前提案的目的是确定将噬菌体 lambda 从分子生物学中的通用工具转变为细胞生物学中的通用工具的方法。我们打算证明，如果具备识别哺乳动物细胞表面特定受体并穿透细胞膜的方法，噬菌体 lambda 颗粒将能够将延伸的 DNA 序列传递到哺乳动物细胞中。在第一阶段，这一概念的证明将通过将能够引导绿色荧光蛋白合成的DNA传递到哺乳动物细胞中来实现。在第二阶段，我们将致力于优化受体识别、膜穿透、噬菌体衣壳释放 DNA 以及将 DNA 转运到细胞核等重要过程。作为这些努力的结果，我们期望创建一种新工具，允许将 DNA 构建体（包括组合文库）从大肠杆菌有效穿梭到哺乳动物细胞中。此外，我们期望为构建适合基因治疗应用的基于噬菌体的定制 DNA 传递系统奠定基础。