Structures of Lipid-DNA Nonviral Gene Delivery Systems 3

脂质-DNA 非病毒基因传递系统的结构 3

• 批准号: 7822752
• 负责人: CYRUS R SAFINYA
• 金额: $ 27.96万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7822752
• 关键词: Acids; Address; Area; Award; Behavior; Biological; Caliber; Cell Communication; Cells; Charge; Chemical Engineering; Chemical Structure; Chemicals; Chemistry; Cleaved cell; Collaborations; Complex; Confocal Microscopy; Cryoelectron Microscopy; Custom; Cytoplasm; Cytosol; DNA; Dissociation; Distal; Double-Stranded RNA; Electrostatics; Encapsulated; Engineering; Gene Delivery; Gene Silencing; Goals; Head; Histidine; Human; In Vitro; Knowledge; Laboratories; Lead; Length; Ligands; Lipids; Liposomes; Measurement; Mediating; Membrane; Membrane Lipids; Methods; Micelles; Molecular; Nucleic Acids; Oligonucleotides; Peptides; Performance; Phase; Plasmids; Production; Property; RNA; Research; Research Activity; Research Institute; Science; Serum; Shapes; Solid; Structure; Synchrotrons; Techniques; Testing; Therapeutic; Toxic effect; Transfection; Tube; Viral Vector; Virus; base; chemical synthesis; density; design; disorder control; gene delivery system; gene therapy; gene therapy clinical trial; immunogenicity; improved; in vivo; liposome vector; liquid crystal; method development; non-viral gene delivery; public health relevance; retinal rods; synchrotron radiation; therapeutic gene; two-dimensional; vector

DESCRIPTION (provided by applicant): There is currently a very large research activity in developing lipid- based vectors for therapeutic applications because of their nonimmunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. Indeed cationic liposome (CL) based vectors are among the prevalent synthetic carriers of nucleic acids currently used in human clinical gene therapy trials worldwide. The vectors are studied both for gene delivery with CL-DNA complexes and gene silencing with CL-siRNA (short-interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. The low efficiencies are the result of poorly understood transfection-related mechanisms at the molecular and self-assembled levels, and a general lack of knowledge about interactions between membranes and double stranded nucleic acids resulting in stable complex formation, and between membrane-nucleic acid complexes and cellular components. The aims of this research application are (1) to use custom synthesized degradable and PEGylated lipids, and biophysical characterization, in order to clarify the interactions between lipid-nucleic acid complexes and cellular components for improved understanding of structure-function properties, and (2) to clarify structures and interactions between cationic membranes and siRNA in CL-siRNA complexes used in gene silencing. Modern methods of organic and solid phase chemistry will be employed to synthesize multivalent degradable lipids, peptide-PEG-lipids, and acid labile PEG-lipids. The structure of the lipid-nucleic acid complexes will be solved by using synchrotron x-ray diffraction techniques at the Stanford Synchrotron Radiation Laboratory and cryo-electron microscopy at UCSB and the Scripps Research Institute. Confocal microscopy will enable us to track the lipid-nucleic acid complexes and observe their interactions with cells. The structures will be correlated to the biological activity of complexes interacting with cells by quantitative measurements of transfection efficiency and silencing efficiency both in DMEM and in high-serum for in vivo applications. The broad long-range goal of the research is to develop a mechanistic understanding of the biophysical interactions between cationic membranes and biologically active double stranded nucleic acids and between CL-nucleic acid complexes and cells, which will generate custom lipid-carriers of nucleic acids ultimately for use in gene therapeutics and disease control. PUBLIC HEALTH RELEVANCE: The project proposes to use a mechanistic approach to further the understanding of lipid carriers of therapeutic DNA and RNA. This, in turn, will lead to new materials and methods and the development of efficient lipidic DNA and RNA carriers for disease control. The goals will be accomplished by applying biophysical scientific methods to custom designed lipids and therapeutic molecules, made available by advanced synthetic methods.

描述（由申请人提供）：目前在开发基于脂质的媒介的治疗应用中有非常大的研究活动，因为它们的非不发育性，低毒性，易于生产以及将大型DNA转移到细胞中的潜力。实际上，基于阳离子脂质体（CL）的载体是目前在全球人类临床基因治疗试验中使用的核酸的普遍合成载体之一。研究了载体，既可以用Cl-DNA复合物递送基因，又研究了用Cl-siRNA（短裂RNA）复合物的基因沉默。但是，与工程病毒载体相比，它们的转染效率和沉默效率仍然很低。低效率是在分子和自组装水平上与转染相关的机制知识较低的结果，并且普遍缺乏对膜之间相互作用和双链核酸之间的相互作用，从而导致稳定的复合形成，以及膜核酸络合物和细胞成分之间的稳定复合物。 The aims of this research application are (1) to use custom synthesized degradable and PEGylated lipids, and biophysical characterization, in order to clarify the interactions between lipid-nucleic acid complexes and cellular components for improved understanding of structure-function properties, and (2) to clarify structures and interactions between cationic membranes and siRNA in CL-siRNA complexes used in gene silencing.现代的有机和固相化学方法将用于合成多价降解脂质，肽-PEG-脂质和酸不稳定的PEG脂质。通过在Stanford Synchrotron辐射实验室和UCSB和Scripps Research Institute的Stanford Synchrotron辐射实验室和冷冻电子显微镜上，使用同步加速器X射线衍射技术来解决脂质核酸络合物的结构。共聚焦显微镜将使我们能够跟踪脂肪核酸复合物并观察它们与细胞的相互作用。结构将通过定量测量转染效率的定量测量和在DMEM中的沉默效率和在体内应用中的高核中进行沉默效率，与与细胞相互作用的复合物的生物学活性相关。该研究的广泛远距离目标是对阳离子膜与生物活性双链核酸以及Cl-核酸复合物和细胞之间的生物物理相互作用进行机械理解，这将最终产生核酸的定制脂质载体，以最终用于基因治疗和疾病控制。公共卫生相关性：该项目建议使用一种机械方法来进一步了解治疗性DNA和RNA的脂质载体。反过来，这将导致新的材料和方法以及有效的脂质DNA和RNA载体进行疾病控制。这些目标将通过将生物物理科学方法应用于定制设计的脂质和治疗分子（可通过高级合成方法提供）来实现。