NEW CATIONIC PHOSPHOLIPIDS FOR TRANSFECTION

拟议调查的总体目标是综合 一种新的带正电荷的脂质类的各种成员， 它们是基于天然存在的 磷脂，其中许多有望成为异常有效的药剂， 基因转染并阐明它们相互作用的机制 并促进其进入细胞。目前，阳离子脂质 通常用于转染培养物中的真核细胞， 成为分子生物学研究中不可或缺的工具。最近， 该技术的医学用途已经通过应用 在整个动物中，基因转染已经被显著地 成功地将功能性基因导入大部分的 许多组织中的细胞。这些动物模型的成功表明， 阳离子脂质可能成为基因的主要递送系统之一 治疗和基因治疗，尤其是传染病， 癌 虽然阳离子脂质作为基因转移有相当大的希望， 试剂，迄今为止最流行的阳离子脂质不适合 分子剪裁以优化其效率。因此， 已知脂质如何与DNA相互作用， 如何进入细胞以及DNA如何被释放以最终转移到 在细胞核中表达。我们提出了一个研究的结构和 与一类新的DNA形成的DNA复合物的转染效率 衍生自天然磷脂的阳离子脂质 以使转染效率最大化，并且作为变体， 由天然分子组成，可生物降解，以排除毒性 在有机体中积累。第一个目标要求有系统地 这些脂质的结构的变化，这是由 各种可用的前体和这些前体的容易改性 前体组织转染效率的测定 培养细胞将揭示分子结构的哪些方面 关键的转染和是否有特定的相互作用， 特定类型的细胞。第二个目的是阐明 复杂的。 选择在转染中差异很大的化合物 为了提高效率，我们将研究它们与DNA复合物的结构， 确定复合体的哪些特征对于 转染 特别地，将测试复合物的稳定性 以确定它在细胞条件下是否不稳定， 可以部分解释为什么这些复合物能有效地转导 剂.第三个目标是了解复合物如何进入细胞， 融合在细胞表面或通过内吞作用，以及鉴定 细胞中复合物解离的区域。最后，化合物 将评估细胞毒性和代谢结局。

The overall objective of the proposed investigation is to synthesize a variety of members of a new class of positively-charged lipids, all of which are based on the backbone structure of naturally occurring phospholipids many of which promise to be unusually effective agents for gene transfection and to elucidate the mechanism by which they interact with DNA and facilitate its entry into cells. Presently, cationic lipids are commonly used for transfection of eukaryotic cells in culture and are becoming indispensable tools in molecular biology research. Recently, medical uses of the technique have been indicated by application of the technique to whole animals in which gene transfection has been remarkably successful in introducing functional genes into a large proportion of cells in many tissues. These successes with animal models indicate that cationic lipids could become one of the primary delivery systems for gene therapy and gene therapeutics of, among others, infectious diseases and cancer. Although cationic lipids hold considerable promise as gene transfer agents, the cationic lipids most popular to date are not amenable to molecular tailoring for optimizing their efficiency. Hence, little is known about how the lipid interacts with DNA, how the resulting complex enters the cell and how the DNA is released for eventual transfer to and expression in the nucleus. We propose a study of the structure and transfection efficiency of DNA complexes formed with a new class of cationic lipids derived from natural phospholipids which can be altered synthetically to maximize transfection efficiency and which, as variants of naturally-occurring molecules, are biodegradable to preclude toxic accumulation in the organism. The first aim calls for the systematic variation of the structure of these lipids, which is facilitated by the variety of precursors available and the ease of modification of these precursors. Determination of the efficiency of transfection of tissue culture cells will reveal which aspects of molecular structure are critical for transfection and whether there are specific interactions with particular types of cells. The second aim is to elucidate the structure of the complex. Choosing compounds that vary widely in transfection efficiency, we will examine the structure of their complexes with DNA to determine what characteristics of the complex are critical for transfection. In particular, the stability of the complex will be tested to determine if it is unstable under cellular conditions, a property which would partially explain why these complexes are effective transducing agents. The third aim is to understand how the complex enters the cell, by fusion at the cell surface or by endocytosis, as well as to identify the region in the cell where the complex dissociates. Finally, the compounds will be assessed with respect to cellular toxicity and metabolic fate.