NEW CATIONIC PHOSPHOLIPIDS FOR TRANSFECTION

拟议调查的总体目标是综合一个 一类新的带正电荷的脂类的各种成员，所有的 它们是基于自然产生的 磷脂，其中许多有望成为非同寻常的有效药物 基因导入并阐明它们相互作用的机制 与DNA结合，并促进其进入细胞。目前，阳离子脂类 通常用于培养中的真核细胞的转染，并且是 成为分子生物学研究中不可或缺的工具。最近， 这项技术的医学用途已经通过应用 转基因技术在整个动物中的应用已经非常显著 成功地将功能基因引入到很大比例的 许多组织中的细胞。这些动物模型的成功表明 阳离子脂类可能成为基因的主要传递系统之一 传染病和其他疾病的治疗和基因治疗 癌症。 尽管阳离子脂类在基因转移方面有很大的希望 试剂，迄今为止最流行的阳离子脂类不服从于 分子剪裁以优化它们的效率。因此，很少有 知道脂质是如何与DNA相互作用的，产生的复合体是如何 以及DNA如何被释放，最终转移到和 在细胞核中的表达。我们建议对该结构和 一类新基因形成的DNA复合体的转染率 天然磷脂衍生的阳离子脂是可以改变的 综合最大限度地提高转基因效率，作为变种 自然产生的分子，可生物降解以排除有毒物质 在生物体中的积累。第一个目标是系统化的 这些脂类的结构变化，这是由 可获得的前体的种类以及这些前体的易变性 先驱物。组织转染率的测定 培养细胞将揭示分子结构的哪些方面 对转染率至关重要，以及是否存在与 特定类型的细胞。第二个目的是阐明 这个建筑群。选择在转染过程中变化很大的化合物 效率，我们将研究它们与DNA的络合物的结构，以 确定复合体的哪些特征对 转染法。特别是，将测试该复合体的稳定性 以确定它在蜂窝条件下是否不稳定，该属性 部分解释了为什么这些复合体是有效的转导 探员们。第三个目标是了解复合体是如何进入细胞的，通过 在细胞表面或通过内吞作用进行融合，以及鉴定 细胞中复合体解离的区域。最后，化合物 将根据细胞毒性和代谢命运进行评估。

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.