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

描述(申请人提供)：这项研究提案是一个实验性项目，将开发一种使用非病毒基因载体在肺部进行基因治疗的最佳配方方法。囊性纤维化是一种由单基因缺陷引起的疾病，该缺陷导致CFTR蛋白功能受损。虽然这种基因在20多年前就被分离出来了，但安全有效的递送策略的开发一直难以实现。发展非病毒基因载体的一个主要限制因素是缺乏对细胞外和细胞内屏障的基本了解。例如，以前的研究表明，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著降低。这是因为粘液是肺纤维化的主要屏障，在到达下面的呼吸道上皮之前有效地清除了基因携带者。为了解决这一问题，我们将确定通过人CF粘液和人支气管上皮细胞有效传递基因载体的限速屏障。这一系列研究的方法将是从简单系统到更复杂系统的系统化建设，并强调每一步的定量研究。这项建议的具体目的是：(1)制定生产在CF粘液中稳定的基因载体的方法。(2)测定和定量基因载体通过人CFs黏液的转运；(3)确定人支气管上皮细胞中基因载体有效转运到细胞核的细胞内屏障；(4)测定小鼠体内的基因转移效率和鼻腔电位差(NPD)。我们推测，重要的细胞外和细胞内屏障的识别将指导对非病毒基因载体的合理修饰，以实现成功的CF基因治疗。 公共卫生相关性：囊性纤维化(CF)是一种由单基因缺陷引起的疾病，该缺陷导致负责氯离子运输的CFTR蛋白功能受损。虽然这种基因在20多年前就被分离出来了，但安全有效的递送策略的开发一直难以实现。发展有效的非病毒基因治疗的一个主要限制因素是缺乏对细胞外和细胞内屏障的基本了解。我们推测，重要的限速屏障的识别将指导对非病毒基因载体的合理修饰，以实现成功的CF基因治疗。

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.