Structures of Lipid-DNA Nonviral Gene Delivery Systems 4

脂质-DNA 非病毒基因传递系统的结构 4

基本信息

批准号：
8502078
负责人：
CYRUS R SAFINYA
金额：
$ 31.1万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA BARBARA
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-05-01 至 2017-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8502078
关键词：
Acids Adenovirus Vector Affinity Biological Biological Assay Capsid Cell membrane Cells Chemicals Chemistry Clinical Trials Communities Complex Cryoelectron Microscopy Custom Cytosol DNA Data Development Electrostatics Elements Endosomes Engineering Equilibrium Ethylene Glycols Event Exhibits Fluorescence Gene Delivery Gene Silencing Genes Goals Human Immune Immune response In Vitro Incidence Insertional Mutagenesis Knowledge Laboratories Lead Life Ligands Lipids Liposomes Measurement Measures Mediating Medical Research Membrane Methods Microscopy Minority Molecular Non-Viral Vector Nucleic Acid Regulatory Sequences Nucleic Acids Optics Pathway interactions Peptides Phase Population Property Proteins Publishing RGD (sequence)RNA Radial Reaction Reporting Research Research Activity Research Institute Resources Retroviral Vector Roentgen Rays Science Series Serum Shapes Small Interfering RNA Stimulus Structure Surface Synchrotrons Tail Techniques Testing Therapeutic Time Toxic effect Transfection Viral Viral Vector Virus X ray diffraction analysis X-Ray Diffraction base cellular imaging design disorder control endosome membrane ethylene glycol fluorescence microscope gene delivery system gene therapy gene therapy clinical trial immunogenic improved in vivo liposome vector membrane assembly non-viral gene delivery novel particle prevent public health relevance success synchrotron radiation therapeutic gene uptake vector

项目摘要

DESCRIPTION (provided by applicant): The current level of research activity involving gene therapy with either synthetic vectors (carriers) or engineered viruses is unprecedented. Cationic liposomes (CLs) have emerged worldwide as among the most prevalent synthetic vectors employed in human clinical trials, due to their near lack of immune response and low toxicity. CL-vectors are also able to carry large pieces of DNA (consisting of entire genes and regulatory regions) into cells, which is not feasible with engineered viral vectors due to the limited size of virus capsids. CL_nucleic acid (CL_NA) complexes are employed both as DNA carriers and as siRNA (short interfering RNA; for gene silencing) carriers. However, the transfection efficiency and silencing efficiency of CL-vectors compared to viral vectors remains low for in vivo applications. Improvement of the efficiencies of synthetic vectors intended for in vivo applications requires a knowledge of the structures of CL-DNA and CL-siRNA complexes (in particular, the electrostatic interactions stabilizing assemblies of membranes and double-stranded NAs) as well as a mechanistic understanding of their interactions with cell membranes and the events leading to release of DNA and siRNA inside the cell. The aims of this research application are (1) to develop a new class of multi-component surface-functionalized CL-NA complexes, which will enable a mechanistic understanding of the initial pathway of complex uptake by the cell and the subsequent release of the NA-containing complex into the cell interior (cytosol), and (2) to understand the influence of lipid shape and membrane elastic properties on the formation of a new class of CL-DNA complexes possessing the membrane shape desired for interactions with endosome membranes inside the cell that optimally facilitate endosomal escape. Modern methods of organic and peptide chemistry will be employed to synthesize distinct PEG-lipids for cell targeting and endosome escape properties. These will be strategically combined in order to prepare high efficiency PEGylated CL-DNA complexes. The structures of CL-NA complexes will be solved using modern synchrotron x-ray diffraction techniques at the Stanford Synchrotron Radiation Lightsource and cryo-electron microscopy at the National Resource for Automated Molecular Microscopy at the Scripps Research Institute. Live-cell imaging with state-of-the-art optical fluorescence microscopes will allow us to visualize the interactions between CL-NA complexes and cellular components. Their structures will be correlated to their biological activity by quantitative measurements of transfection efficiency and silencing efficiency. The broad, long-term objective of our research is to develop a fundamental science base (via mechanistic studies of interactions of CL-NA complexes and cells) that will lead to the design and synthesis of synthetic carriers of DNA and siRNA optimized for gene therapeutics and disease control.

描述（由申请人提供）：目前涉及使用合成载体（载体）或工程病毒进行基因治疗的研究活动水平是前所未有的。阳离子脂质体（CL）由于几乎缺乏免疫反应且毒性低，已成为全球人类临床试验中最常用的合成载体之一。 CL载体还能够将大片段DNA（由整个基因和调控区域组成）携带到细胞中，这对于工程化病毒载体来说是不可行的，因为DNA片段的大小有限。病毒衣壳。 CL_核酸 (CL_NA) 复合物既可用作 DNA 载体，也可用作 siRNA（短干扰 RNA；用于基因沉默）载体。然而，与病毒载体相比，CL 载体的转染效率和沉默效率在体内应用中仍然较低。提高用于体内应用的合成载体的效率需要了解 CL-DNA 和 CL-siRNA 复合物的结构（特别是稳定膜和双链 NA 组件的静电相互作用）以及机械理解它们与细胞膜的相互作用以及导致细胞内 DNA 和 siRNA 释放的事件。本研究应用的目的是 (1) 开发一类新型多组分表面功能化 CL-NA 复合物，这将使人们能够从机理上理解细胞摄取复合物的初始途径以及随后释放 NA - 包含复合物进入细胞内部（细胞质），以及（2）了解脂质形状和膜弹性特性对新型 CL-DNA 复合物形成的影响，该复合物具有与细胞内内体膜相互作用所需的膜形状细胞那个最佳地促进内体逃逸。现代有机和肽化学方法将用于合成不同的 PEG-脂质，以实现细胞靶向和内体逃逸特性。这些将被战略性地组合以制备高效的聚乙二醇化 CL-DNA 复合物。 CL-NA 复合物的结构将使用斯坦福同步辐射光源的现代同步辐射 X 射线衍射技术和斯克里普斯研究所国家自动分子显微镜资源中心的冷冻电子显微镜来解析。使用最先进的光学荧光显微镜进行活细胞成像将使我们能够可视化 CL-NA 复合物与细胞成分之间的相互作用。通过定量测量转染效率和它们的结构将与其生物活性相关联消音效率。我们研究的广泛、长期目标是开发一个基础科学基础（通过 CL-NA 复合物和细胞相互作用的机制研究），这将导致设计和合成针对基因治疗优化的 DNA 和 siRNA 合成载体和疾病控制。