Alveolar Epithelial Cell-Specific Gene Therapy Plasmids

目前，还没有方法选择性地将基因转移到肺泡上皮II型细胞，而同时又不需要额外的细胞因子。 将它们转化为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型细胞参与的疾病，包括表面活性剂缺乏和急性肺损伤。我们有 developed发达a way to overcome克服this problem问题.虽然载体设计的许多方面正在解决， 需要更多研究的关键领域是载体DMA的核输入。我们的目标是设计更多 通过了解肺中使用的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。我们已经鉴定出一个DNA 在肺泡上皮细胞中独特地增加核定位和随后的基因表达的序列 细胞DNA是表面活性蛋白C（SP-C）启动子的近端部分， 几种细胞特异性转录因子的位点，包括TTF-1、加塔-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，转录因子，包含核 定位信号（NLS）用于其核输入，与特异性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，我们已经开发了一种使用电场的体内基因递送新技术， 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，我们在一个独特的位置，以测试这种细胞选择性核输入序列的影响， II型细胞转染在急性肺损伤动物模型中的作用。我们假设SP-C DNA 核靶向序列将导致基因转移和表达仅在II型细胞中，而不是在其他细胞中。 活体动物的肺细胞。这项建议旨在检验这一假设，并将导致建立 新的肺泡基因治疗载体，既有细胞特异性，又能更好地转移基因 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核导入，并将研究结果扩展到体内模型系统，以转移基因， Na+，K+-ATP酶增加损伤肺的肺泡液体清除。

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.