Targeted Delivery, Trafficking, and Vector Design for Respiratory Gene Transfer

呼吸道基因转移的靶向递送、运输和载体设计

• 批准号: 8115076
• 负责人: Mark Eugene Hwang
• 金额: $ 4.43万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-16 至 2015-07-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8115076
• 关键词: A549; Accounting; Address; Affect; Arginine; Aspartic Acid; Behavior; Biochemical; Biological Assay; Buffers; Candidate Disease Gene; Caveolae; Caveolins; Cell Line; Cell model; Cell surface; Clathrin; Clathrin-Coated Vesicles; Clinical; Clinical Trials; Cystic Fibrosis; DNA; Data; Dendrimers; Dependence; Development; Disease; Effectiveness; Endocytic Vesicle; Endocytosis; Endosomes; Epithelial; Epithelial Cells; Face; Folate; Gene Delivery; Gene Expression; Gene Transfer; Glycine; Goals; Health; Hela Cells; Human; Immunofluorescence Immunologic; Integrins; Kinetics; Ligands; Literature; Lung; Lysosomes; Mediating; Methods; Modeling; Molecular Motors; Non-Viral Vector; Pathway interactions; Patients; Peptides; Phase; Polymers; Porifera; Process; Property; Protons; RGD (sequence); Relative (related person); Reporter Genes; Role; Safety; Series; Structure; Therapeutic Studies; Therapy Clinical Trials; Tissues; Transduction Gene; Transferrin; Transferrin Receptor; Transport Vesicles; Vesicle; airway epithelium; cell type; clinical application; coated pit; cytotoxic; design; folate-binding protein; gene delivery process; gene therapy; inhibitor/antagonist; particle; polycation; prevent; receptor; receptor internalization; respiratory; response; targeted delivery; therapeutic gene; trafficking; uptake; vector

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is an autosomal recessive disease and a prime candidate for gene therapy. CF represents a major target for gene therapy research as the airway epithelium is relatively isolated and, theoretically, readily accessible by nebulized vectors. However, CF gene therapy faces the same obstacles that prevent the widespread use of gene therapy for other diseases; of 1300gene therapy clinical trials conducted worldwide to-date, barely three percent of these have been approved for phase three clinical trials. Clinical application of gene therapy is hindered by lack of safe and efficient delivery methods. Non-viral vectors can provide the requisite safety, but design of efficient materials is limited by a lack of understanding of the intracellular mechanisms of gene delivery. Many approaches to vector design are empirical, as data provided in the literature do not definitively provide robust model criteria for the design of gene delivery vehicles. Therefore, the principal objective of this proposal is to elucidate design criteria for safe and efficient gene delivery vehicles that will be used to construct a model for epithelial airway gene delivery. Specifically, this study will examine the internalization mechanisms and intracellular behavior of integrin-mediated gene transfer vectors. The specific aims of this proposal are to determine (1) the effectiveness of vector-ligand conjugation for targeted, receptor-mediated internalization at the cell surface; (2) the roles of endocytic vesicle acidification, trafficking kinetics, and processing pathways- namely clathrin and caveolin mediated vesicle transport - in vector escape and subsequent DNA unpackaging from the delivery vehicle; and (3) the interplay of molecular motors and cytoskeletal structures in endocytic vesicle trafficking of vectors. Correlating targeted delivery efficiency and intracellular trafficking studies will produce insightful structure-property relationships that will aid in the rational design of gene delivery vehicles. This study will focus on the design of synthetic, targeted, non-viral vectors that are minimally cytotoxic and easily produced. Preliminary results have established generalizable principles for the design of targeted vectors to different endocytic pathways from an epithelial (HeLa) cell model using prototypical ligands transferrin and folate. The long term goal is to use these principles to affect targeted airway gene delivery in human lung and bronchial epithelial cell lines. The clinical goal of gene therapy is to treat disease by delivering therapeutic genes to a patient in a highly efficient, specific, and safe manner. PUBLICH HEALTH RELEVANCE: The aims described herein seek to elucidate design criteria for delivery approaches in human airway tissue, and ultimately, the development of cystic fibrosis treatment.

描述（由申请人提供）：囊性纤维化（CF）是一种常染色体隐性疾病，是基因治疗的主要候选者。 CF代表了基因治疗研究的主要靶标，因为气道上皮相对孤立，从理论上讲，雾化载体很容易访问。但是，CF基因疗法面临的障碍与阻止其他疾病的基因疗法广泛使用。在迄今为止在全球进行的1300种基因疗法临床试验中，其中只有3％已批准用于第三阶段临床试验。由于缺乏安全有效的递送方法，阻碍了基因治疗的临床应用。非病毒载体可以提供必要的安全性，但是有效材料的设计受到对基因递送的细胞内机制的了解的限制。许多矢量设计方法都是经验性的，因为文献中提供的数据并未确定为基因递送车设计设计的强大模型标准。因此，该提案的主要目的是阐明设计标准的安全有效基因输送车辆，该工具将用于构建上皮气道基因递送模型。具体而言，这项研究将检查整联蛋白介导的基因转移载体的内在化机制和细胞内行为。该提案的具体目的是确定（1）载体 - 配体共轭在细胞表面靶向的受体介导的内在化的有效性； （2）内吞囊泡酸化，运输动力学和加工途径的作用 - 即网状蛋白和小窝蛋白介导的囊泡转运 - 在载体逃生中以及随后从输送车中解开包装的DNA； （3）分子电动机和细胞骨架结构在载体的内吞囊泡运输中的相互作用。相关的有针对性的输送效率和细胞内贩运研究将产生洞察力的结构特性关系，这将有助于基因递送车的合理设计。这项研究将着重于最小细胞毒性且易于产生的合成，靶向，非病毒载体的设计。初步结果已经建立了使用典型的配体转运蛋白和叶酸，从而从上皮（HELA）细胞模型的不同内吞途径的靶向矢量设计到不同的内吞途径。长期目标是使用这些原理影响人肺和支气管上皮细胞系中靶向气道基因的递送。基因治疗的临床目标是通过以高效，特定和安全的方式向患者提供治疗基因来治疗疾病。 Publich Health相关性：本文所描述的目的旨在阐明人类气道组织输送方法的设计标准，并最终是囊性纤维化治疗的发展。