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: 7595062
• 负责人: David A Dean
• 金额: $ 37.38万
• 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/7595062
• 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; Cell Nucleus; Cell physiology; Cells; Clinical; 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; Patients; Plasmids; Positioning Attribute; Proteins; Pulmonary Edema; Pulmonary Surfactant-Associated Protein C; Rattus; Receptor Gene; Reporter; Research; Research Personnel; Syndrome; System; Techniques; Testing; Transfection; Type I Epithelial Receptor Cell; Type II Epithelial Receptor Cell; Work; alternative treatment; alveolar epithelium; cell type; design; disorder prevention; efficacy testing; electric field; gene delivery system; gene therapy; in vivo; in vivo Model; injured; mortality; overexpression; plasmid DNA; programs; promoter; protein complex; research study; standard of care; 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，没有选择性地将基因转移到肺泡上皮II型细胞的方法。 将它们转移到I型细胞。这是基因治疗方法发展中的一个主要问题 治疗II型细胞受累的疾病，包括表面活性物质缺乏和急性肺损伤。我们有 开发了一种克服这个问题的方法。虽然矢量设计的许多方面都在讨论中，但其中一个 需要更多研究的关键领域是媒介DNA的核进口。我们的目标是设计更多 有效的基因治疗载体用于肺通过了解其分子机制 DNA和DNA-蛋白质复合体被积极地运输到细胞核中。我们已经确认了一种DNA 在肺泡上皮细胞中唯一增加核定位和随后基因表达的序列 细胞。DNA是表面活性蛋白C(SP-C)启动子的近端部分，包含结合 几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 因子介导了该启动子的细胞特异性转录，我们假设它们也介导了 核进口的DNA。我们的工作假设是，含有核的转录因子 核输入的定位信号(NLSS)与特定的SP-C DNA序列结合，从而“包被” 将DNA与NLSS结合，并允许DNA利用NLS介导的进口机制进行核进入。 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。 将这些非病毒II型细胞特异性载体靶向肺泡上皮细胞。在活体内使用这种方法 电穿孔，我们处于一个独特的位置来测试这种细胞选择性核进口序列的影响 急性肺损伤动物模型中II型细胞转染法的研究。我们假设SP-C DNA 核靶向序列只会导致基因在II型细胞中的转移和表达，而在其他类型的细胞中不会 活体动物的肺细胞。这项提议旨在检验这一假说，并将导致 新的肺泡基因治疗载体，既是细胞特异性的，又能进行更大的基因转移 效率。拟议的实验将从分子上表征肺泡上皮细胞的机制。 细胞DNA核输入，并将把这一发现扩展到体内模型系统，以转移用于 Na+，K+-ATPase促进肺损伤后肺泡液清除