Alveolar Epithelial Cell-Specific Gene Therapy Plasmids

描述（由申请人提供）：目前，还没有选择性地将基因转移到肺泡上皮II型细胞而不将它们转移到I型细胞的方法。这是发展基因治疗方法以治疗II型细胞参与的疾病（包括表面活性剂缺乏和急性肺损伤）中的主要问题。我们已经找到了解决这个问题的方法。虽然载体设计的许多方面正在得到解决，但需要更多研究的一个关键领域是载体DNA的核输入。我们的目标是通过了解DNA和DNA-蛋白质复合物被主动转运到细胞核中的分子机制，设计更有效的基因治疗载体用于肺。我们已经确定了一个DNA序列，增加核定位和随后的基因表达独特的肺泡上皮细胞。DNA是表面活性蛋白C（SP-C）启动子的近端部分，其含有几种细胞特异性转录因子（包括TTF-1、加塔-6和TAZ）的结合位点。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，含有核定位信号（NLS）的核进口，结合到特定的SP-C DNA序列，从而'涂层'的DNA与NLS，并允许DNA利用NLS介导的进口机制进入核。此外，我们已经开发了一种新的技术，在体内基因传递使用电场，可用于针对这些非病毒II型细胞特异性载体的肺泡上皮细胞。使用这种体内电穿孔，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对急性肺损伤动物模型中II型细胞转染的影响。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，而不是在活动物的其他肺细胞中。该提议旨在验证这一假设，并将导致创建新的肺泡基因治疗载体，这些载体既具有细胞特异性，又具有更高的基因转移效率。所提出的实验将从分子水平表征肺泡上皮细胞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+-ATP酶的基因，从而增加受损肺的肺泡液体清除率。

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.