Design of Non-viral Gene Carriers that Overcome Extra- and Intracellular Barriers

克服细胞外和细胞内屏障的非病毒基因载体的设计

• 批准号: 8128093
• 负责人: Anthony J. Kim
• 金额: $ 5.84万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-29 至 2013-08-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8128093
• 关键词: Address; Cell Nucleus; Chloride Ion; Complex; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Development; Disease; Drug Formulations; Epithelial Cells; Gene Delivery; Gene Transfer; Genes; Human; Ion Transport; Lung; Methods; Modification; Mucous body substance; Mus; Nose; Research Proposals; Single-Gene Defect; System; Tissues; Viral Genes; airway epithelium; design; extracellular; gene therapy; in vivo; non-viral gene therapy

DESCRIPTION (provided by applicant): This research proposal is an experimental project that will develop an optimal formulation method for gene therapy in the lungs using non-viral gene carriers. Cystic Fibrosis (CF) is a disease caused by a single-gene defect, which leads to impaired function of the CFTR protein. Although this gene was isolated more than 20 years ago, the development of safe and effective delivery strategies have been difficult to realize. A major limitation in the development of non-viral gene carriers has been the lack of basic understanding of the extracellular and intracellular barriers. For example, it was previously shown that the gene transfer efficiency of gene carriers exposed to mucus lining was dramatically reduced, when compared to mucus-depleted tissues. This is because mucus acts as the primary barrier in the CF lungs, and efficiently removes gene carriers prior to reaching the underlying airway epithelium. To address this, we will determine the rate limiting barriers through human CF mucus and human bronchial epithelial cells for efficient delivery of gene carriers. The approach to this range of studies will be one of systematic building from simple to more complex systems, with an emphasis on quantitative studies at each step. The specific aims of this proposal are: (1) to formulate methods that produce gene carriers that are stable in CF mucus. (2) to determine and quantify gene carrier transport through human CF mucus; (3) to determine intracellular barriers to efficient gene carrier transport to the nucleus in human bronchial epithelial cells; and (4) to determine in vivo gene transfer efficiencies and nasal potential differences (NPD) in mice. We hypothesize that the identification of important extra- and intracellular barriers will guide the rational modification of the non-viral gene carriers for successful CF gene therapy. PUBLIC HEALTH RELEVANCE: Cystic Fibrosis (CF) is a disease caused by a single-gene defect, which leads to impaired function of the CFTR protein responsible for chloride ion transport. Although this gene was isolated more than 20 years ago, the development of safe and effective delivery strategies have been difficult to realize. A major limitation in the development of effective non-viral gene therapy has been the lack of basic understanding of the extracellular and intracellular barriers. We hypothesize that the identification of important rate-limiting barriers will guide the rational modification of the non-viral gene carriers for successful CF gene therapy.

描述（由申请人提供）：该研究计划是一个实验项目，将开发一种使用非病毒基因载体进行肺部基因治疗的最佳配方方法。囊性纤维化（CF）是一种由单基因缺陷引起的疾病，导致 CFTR 蛋白功能受损。尽管该基因在20多年前就被分离出来，但安全有效的递送策略的开发一直难以实现。非病毒基因载体发展的一个主要限制是缺乏对细胞外和细胞内屏障的基本了解。例如，之前的研究表明，与粘液耗尽的组织相比，暴露于粘液衬里的基因载体的基因转移效率显着降低。这是因为粘液充当 CF 肺部的主要屏障，并在到达下面的气道上皮之前有效地去除基因载体。 为了解决这个问题，我们将确定通过人 CF 粘液和人支气管上皮细胞的限速障碍，以有效递送基因载体。这一系列研究的方法将是从简单到更复杂的系统的系统构建，重点是每一步的定量研究。该提案的具体目标是：（1）制定产生在CF粘液中稳定的基因载体的方法。 (2) 确定和量化通过人类 CF 粘液的基因载体转运； (3) 确定人支气管上皮细胞中基因载体有效转运至细胞核的细胞内障碍； (4) 确定小鼠体内基因转移效率和鼻电位差 (NPD)。我们假设重要的细胞外和细胞内屏障的识别将指导非病毒基因载体的合理修饰以实现成功的 CF 基因治疗。 公共健康相关性：囊性纤维化 (CF) 是一种由单基因缺陷引起的疾病，会导致负责氯离子转运的 CFTR 蛋白功能受损。尽管该基因在20多年前就被分离出来，但安全有效的递送策略的开发一直难以实现。开发有效的非病毒基因疗法的一个主要限制是缺乏对细胞外和细胞内屏障的基本了解。我们假设，重要限速障碍的识别将指导非病毒基因载体的合理修饰，以实现成功的 CF 基因治疗。