Cell-specific gene delivery methods for expression and silencing in the lung

描述(申请人提供)：阻碍基因治疗方法发展的主要问题之一，包括使用RNAi进行基因沉默，是相对缺乏将基因/RNAi传递到特定细胞类型的方法。此应用程序解决了这一限制。我们的主要兴趣是开发基因治疗方法来治疗急性肺损伤，在美国，每年有超过5万人死于急性肺损伤。我们已经开发出一种在小鼠和猪模型上高度有效的治疗这种疾病的方法，它使用普遍存在的Na，K-ATPase过度表达来增加先前受伤肺的肺泡液清除。我们的实验表明，这种治疗方法不仅可以改善水肿消退(以及肺功能和存活率)，还可以通过上调紧密连接复合体来改善肺泡上皮/内皮细胞屏障功能。然而，我们不知道这种保护和治疗需要在什么细胞中转移和表达Na，K-ATPase基因，也不知道是否需要上调紧密连接复合体才能发挥最大作用。为了了解这种治疗方法的机制，我们必须能够将基因特异性地输送到肺泡中的单个细胞类型，并以类似的细胞特异性方式抑制基因的表达。不幸的是，目前还没有统一有效的方法将RNAi传递到体内特定类型的细胞。我们已经找到了一种克服这个问题的方法。我们已经证明，在没有细胞分裂的情况下，质粒的核定位是序列特异性的，并且需要转录因子来进行核输入。我们已经确定了一些DNA序列，这些序列显示了核进口的细胞特异性，因为它们与细胞特异性转录因子结合。这些序列包括在体外和活体动物中作用于内皮细胞、肺泡1型和肺泡2型上皮细胞的序列。我们已经在体内使用这些序列在这些细胞中过度表达基因，现在将使用它们通过shRNA传递来沉默基因。这是一种全新的定向细胞特异性RNAi传递的方式。我们的目标是：(1)确定哪种类型的肺泡细胞需要Na，K-ATPase基因转移来改善肺泡液清除和诱导紧密连接；(2)通过建立细胞特异性RNAi传递方法，测试Na，K-ATPase基因转移介导的急性肺损伤的保护和治疗是否需要紧密连接；(3)确定单独转移紧密连接复合蛋白是否可以治疗肺损伤，或者进一步加强Na，K-ATPase基因转移介导的急性肺损伤的治疗。

DESCRIPTION (provided by applicant): One of the major problems hindering development of gene therapy approaches, including the use of RNAi for gene silencing, is the relative lack of methods to deliver genes/RNAi to specific cell types. This application addresses this limitation. Our major interest is to develop gene therapy approaches to treat acute lung injury, which accounts for over 50,000 deaths each year in the US. We have developed a highly effective treatment for this disease in mouse and pig models that uses the ubiquitous overexperssion of the Na+,K+-ATPase to increase alveolar fluid clearance from the previously injured lung. Our experiments show that this treatment not only improves edema resolution (and lung function and survival), but also improves alveolar epithelial/endothelial barrier function by upregulating tight junction complexes. However, we do not know in what cells transfer and expression of the Na+,K+-ATPase gene is needed for this protection and treatment, nor whether upregulation of tight junction complexes is needed for maximal effect. In order to understand the mechanisms responsible for this treatment approach, we must be able to deliver genes specifically to the individual cell types in the alveolus and silence gene expression in a similar cell-specific manner. Unfortunately, there are no uniformly effective ways to deliver RNAi to specific cell types in vivo. We have developed a way to overcome this problem. We have shown that the nuclear localization of plasmids in the absence of cell division is sequence-specific and requires transcription factors for nuclear import. We have identified a number of DNA sequences that show cell-specificity of nuclear import because they bind to cell- specific transcription factors. These include sequences that act in endothelial cells, alveolar type 1, and alveolar type 2 epithelial cells in vitro and in living animals. We have used these sequences to overexpress genes in these cells in vivo and will now use them to silence genes by shRNA delivery. This is an entirely new way to direct cell-specific RNAi delivery. Our aims are to (1) Determine in which alveolar cell types gene transfer of the Na+,K+-ATPase is needed for improved alveolar fluid clearance and induction of tight junctions, (2) Test whether tight junctions are needed for Na+,K+-ATPase gene transfer-mediated protection and treatment of acute lung injury by developing a method for cell-specific RNAi delivery, and (3) Determine whether gene transfer of tight junction complex proteins alone can treat lung injury or further enhance Na+,K+- ATPase gene transfer-mediated treatment of acute lung injury.