Alveolar Epithelial Cell-Specific Gene Therapy Plasmids

肺泡上皮细胞特异性基因治疗质粒

• 批准号: 7529330
• 负责人: David A Dean
• 金额: $ 29.45万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7529330
• 关键词: Acute; Acute Lung Injury; Address; Adrenergic Receptor; Adult; Affect; Alveolar; Animal Model; Animals; Area; Automobile Driving; Binding; Binding Sites; Biological Models; Capsid Proteins; Caring; Cell Nucleus; Cell physiology; Cells; Clinical; Complex; Condition; DNA; DNA Sequence; Development; Disease; Distress; Electroporation; Epithelial; Epithelial Cells; Gene Delivery; Gene Expression; Gene Transduction Agent; Gene Transfer; Genetic Transcription; Goals; Human; Importins; In Vitro; Injury; Lead; Life; Liquid substance; Lung; Mediating; Methods; Mitosis; Molecular; Mus; Na(+)-K(+)-Exchanging ATPase; Nuclear; Nuclear Import; Nuclear Localization Signal; Nuclear Pore Complex; Numbers; Patients; Plasmids; Positioning Attribute; Protein C; Protein Import; Protein Overexpression; Proteins; Pulmonary Edema; Rattus; Receptor Gene; Reporter; Research; Research Personnel; Standards of Weights and Measures; Syndrome; System; Techniques; Testing; Transfection; Type I Epithelial Receptor Cell; Type II Epithelial Receptor Cell; Work; alveolar epithelium; cell type; design; disorder prevention; electric field; gene delivery system; gene therapy; in vivo; in vivo Model; injured; mortality; plasmid DNA; programs; promoter; research study; surfactant; surfactant deficiency; therapeutic gene; transcription factor; uptake; vector

At present, there are no methods to selectively transfer genes to alveolar epithelial type II cells without also transfering them to type I cells. This is a major problem in the development of gene therapy approaches to treat diseases with type II cell involvement, including surfactant deficiencies, and acute lung injury. We have developed a way to overcomethis problem. While many aspects of vector design are being addressed, one critical area that needs more research is the nuclear import of vector DMA. Our goal is to design more effective gene therapy vectors for use in the lung by understanding the molecular mechanisms by which DMA and DNA-protein complexes are actively transported into the nucleus. We have identified a DNA sequence that increases nuclear localization and subsequent gene expression uniquely in alveolar epithelial cells. The DNA is the proximal portion of the surfactant protein C (SP-C) promoter which contains binding sites for several cell-specific transcription factors, including TTF-1, GATA-6, and TAZ. These transcription factors mediate the cell-specific transcription of this promoter, and we hypothesize that they also mediate the nuclear import of the DNA. Our working hypothesis is that transcription factors, containing nuclear localization signals (NLSs) for their nuclear import, bind to specific SP-C DNAsequences thereby 'coating' the DNA with NLSs, and allowing the DNA to utilize the NLS-mediated import machinery for nuclear entry. Further, we have developed a new technique for in vivo gene delivery using electric fields that can be used to target these nonviral type II cell specific vectors to the alveolar epithelium. Using this in vivo electroporation, we are in a unique position to test the effects of this cell-selective nuclear import sequence on type II cell transfection in animal models for acute lung injury. We hypothesize that the SP-C DNA nuclear targeting sequence will lead to gene transfer and expression only in type II cells, and not in other lung cells of living animals. This proposal is designed to test this hypothesis and will lead to the creation of new alveolar gene therapy vectors that are both cell-specific and capable of greater gene transfer efficiencies. The proposed experiments will molecularly characterize the mechanisms of alveolar epithelial cell DNA nuclear import and will extend the findings to an in vivo model system to transfer the genes for the Na+,K+-ATPase to increase alveolar fluid clearance in injured lungs.

目前还没有方法可以将基因选择性转移到II型肺泡上皮细胞中 将它们转移至 I 型细胞。这是基因治疗方法发展中的一个主要问题 治疗涉及 II 型细胞的疾病，包括表面活性剂缺乏和急性肺损伤。我们有 开发了一种方法来克服这个问题。虽然矢量设计的许多方面正在得到解决，但其中一个 需要更多研究的关键领域是矢量 DMA 的核心导入。我们的目标是设计更多 通过了解肺部有效基因治疗载体的分子机制 DMA 和 DNA-蛋白质复合物被主动转运到细胞核中。我们已经鉴定出DNA 增加肺泡上皮细胞核定位和随后基因表达的序列 细胞。 DNA 是表面活性剂蛋白 C (SP-C) 启动子的近端部分，其中包含结合 多种细胞特异性转录因子的位点，包括 TTF-1、GATA-6 和 TAZ。这些转录 因子介导该启动子的细胞特异性转录，我们假设它们也介导 DNA 的核输入。我们的工作假设是，含有核的转录因子 其核输入的定位信号 (NLS) 与特定的 SP-C DNA 序列结合，从而“包被” DNA 与 NLS，并允许 DNA 利用 NLS 介导的输入机制进入核。 此外，我们还开发了一种利用电场进行体内基因传递的新技术，可用于 将这些非病毒 II 型细胞特异性载体靶向肺泡上皮。在体内使用这个 电穿孔，我们处于独特的地位来测试这种细胞选择性核输入序列的效果 急性肺损伤动物模型中 II 型细胞转染的研究。我们假设 SP-C DNA 核靶向序列只会在 II 型细胞中导致基因转移和表达，而不会在其他细胞中导致基因转移和表达。 活体动物的肺细胞。该提案旨在检验这一假设，并将导致创建 新的肺泡基因治疗载体具有细胞特异性并能够进行更大的基因转移 效率。所提出的实验将从分子角度表征肺泡上皮细胞的机制 细胞 DNA 核输入，并将研究结果扩展到体内模型系统，以转移基因 Na+,K+-ATP 酶可增加受损肺部的肺泡液清除率。