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

描述（由申请人提供）：本研究计划是一项实验项目，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。虽然该基因早在20多年前就被分离出来，但开发安全有效的给药策略一直很难实现。发展非病毒基因载体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，先前的研究表明，与没有黏液的组织相比，暴露于黏液衬里的基因携带者的基因转移效率大大降低。这是因为黏液在CF肺中起主要屏障作用，并在到达下层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屏障，以便有效地递送基因携带者。这一系列研究的方法将是一个从简单到更复杂系统的系统构建，每一步都强调定量研究。本课题的具体目的是：(1)研究制备在CF粘液中稳定的基因载体的方法。(2)测定并量化人CF黏液中基因载体的转运；(3)确定人支气管上皮细胞内基因载体向细胞核有效转运的胞内屏障；(4)测定小鼠体内基因转移效率和鼻电位差异（NPD）。我们假设，识别重要的细胞外和细胞内屏障将指导非病毒基因载体的合理修饰，从而成功治疗CF基因。

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.