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）：目前涉及合成载体（载体）或工程病毒基因治疗的研究水平是前所未有的。阳离子脂质体（CLS）由于几乎缺乏免疫反应和低毒性而在全球范围内出现为人类临床试验中最普遍的合成媒介之一。 CL-向量还能够将大块DNA（由整个基因和调节区域组成）携带到细胞中，由于工程病毒向量的大小有限，因此不可行病毒衣壳。 CL_N核酸（CL_NA）复合物既用作DNA载体，又用作siRNA（短干扰RNA；用于基因沉默）载体。然而，与病毒载体相比，Cl-量的转染效率和沉默效率在体内应用中仍然很低。 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需要与细胞内部内体膜相互作用的膜形状，从而最佳地促进内体逃逸。现代的有机和肽化学方法将采用用于细胞靶向和内体逃生特性的不同PEG脂质的不同PEG脂质。这些将在战略上合并，以制备高效率的pegyated Cl-DNA复合物。 CL-NA复合物的结构将使用斯坦福同步辐射照明仪和冰冻电子显微镜的现代同步加速器X射线衍射技术解决。具有最先进的光学荧光显微镜的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。它们的结构将通过定量转染效率的定量测量和沉默效率。我们研究的广泛长期目标是开发基本的科学基础（通过CL-NA复合物和细胞的相互作用的机械研究），这将导致DNA和SIRNA的合成载体的设计和合成，以优化基因疗法和疾病控制。