Tools for Targeted Delivery of DNA into Mammalian Cells

描述（由申请方提供）：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，由于将感兴趣的DNA掺入病毒颗粒的方案相当冗长和繁琐，这些载体尚未广泛用于细胞生物学领域。在基因治疗领域，这些载体仍然是不受挑战的工具，但它们生产昂贵，不能仅靶向选定类型的细胞，并且它们的应用可能与相当大的健康风险相关。相反，噬菌体噬菌体衍生的载体使用起来容易且安全。它们具有相当大的容量，并且可以大量重复获得。多年来，基于噬菌体噬菌体的载体已被用作将外源DNA递送到细菌中而不是哺乳动物细胞中的通用工具。当前提案的目的是确定将λ噬菌体从分子生物学中的通用工具转换为细胞生物学中的通用工具的方法。我们打算证明，提供的手段，以识别哺乳动物细胞表面上的特异性受体，并穿透细胞膜，λ噬菌体颗粒将能够提供延长的DNA序列到哺乳动物细胞。在第一阶段期间，将通过将能够指导绿色荧光蛋白合成的DNA递送到哺乳动物细胞中来实现概念的证明。在第二阶段的努力将针对优化这样的重要过程，如受体识别，膜渗透，从噬菌体衣壳释放的DNA和运输的DNA进入细胞核。作为这些努力的结果，我们期望创造一种新的工具，将允许有效穿梭的DNA构建体（包括组合文库）从大肠杆菌。卷曲成哺乳动物细胞。此外，我们期望为构建适合基因治疗应用的定制的基于噬菌体的DNA递送系统奠定基础。

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.