Alveolar Epithelial Cell-Specific Gene Therapy Plasmids

描述(申请人提供)：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，而不同时将它们转移到I型细胞。这是开发基因治疗方法来治疗包括表面活性物质缺乏和急性肺损伤在内的II型细胞参与的疾病的主要问题。我们已经找到了一种克服这个问题的方法。虽然正在解决载体设计的许多方面，但需要更多研究的一个关键领域是载体DNA的核进口。我们的目标是通过了解DNA和DNA-蛋白质复合体主动运输到细胞核的分子机制来设计更有效的用于肺的基因治疗载体。我们已经确定了一个DNA序列，它可以在肺泡上皮细胞中独特地增加核定位和随后的基因表达。DNA是表面活性蛋白C(SP-C)启动子的近端部分，包含几种细胞特异性转录因子的结合位点，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DNA的核输入。我们的工作假设是，含有核输入定位信号(NLSS)的转录因子与特定的SP-C DNA序列结合，从而用NLSS覆盖DNA，并允许DNA利用NLS介导的输入机制进行核进入。此外，我们开发了一种利用电场在体内传递基因的新技术，该电场可用于将这些非病毒II型细胞特异性载体靶向肺泡上皮。利用这种体内电穿孔，我们处于一个独特的位置来测试这种细胞选择性核输入序列在急性肺损伤动物模型中对II型细胞转染的影响。我们推测，SP-C DNA核靶向序列只会导致基因转移和表达在II型细胞中，而不是在活着的动物的其他肺细胞中。这项提议旨在检验这一假说，并将导致创建新的肺泡基因治疗载体，既具有细胞特异性，又能够提高基因转移效率。拟议的实验将从分子上表征肺泡上皮细胞DNA核输入的机制，并将研究结果扩展到体内模型系统，以转移Na+，K+-ATPase基因以增加受损肺的肺泡液清除。

DESCRIPTION (provided by applicant): 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 overcome this problem. While many aspects of vector design are being addressed, one critical area that needs more research is the nuclear import of vector DNA. Our goal is to design more effective gene therapy vectors for use in the lung by understanding the molecular mechanisms by which DNA 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 DNA sequences 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.