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

描述（由申请人提供）：阻碍基因治疗方法（包括使用RNAi进行基因沉默）发展的主要问题之一是相对缺乏将基因/RNAi递送至特定细胞类型的方法。本申请解决了该限制。我们的主要兴趣是开发治疗急性肺损伤的基因治疗方法，在美国每年有超过50，000人死亡。我们已经在小鼠和猪模型中开发了一种针对这种疾病的高效治疗方法，该方法使用普遍存在的Na+，K+-ATP酶过度表达来增加肺泡液从先前受损的肺中的清除。我们的实验表明，这种治疗不仅改善水肿的解决（和肺功能和生存），而且还改善肺泡上皮/内皮屏障功能，通过上调紧密连接复合物。然而，我们不知道在什么细胞中转移和表达的Na+，K+-ATP酶基因是需要这种保护和治疗，也不知道是否需要上调紧密连接复合物的最大效果。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传递到肺泡中的单个细胞类型，并以类似的细胞特异性方式沉默基因表达。不幸的是，没有统一有效的方法将RNAi递送到体内特定的细胞类型。我们已经找到了解决这个问题的方法。我们已经表明，在细胞分裂的情况下，质粒的核定位是序列特异性的，需要转录因子的核输入。我们已经鉴定了许多显示细胞特异性核输入的DNA序列，因为它们结合细胞特异性转录因子。这些包括在体外和活动物中在内皮细胞、肺泡1型和肺泡2型上皮细胞中起作用的序列。我们已经使用这些序列在体内这些细胞中过表达基因，现在将使用它们通过shRNA递送沉默基因。这是一种指导细胞特异性RNAi递送的全新方式。我们的目的是（1）确定哪种肺泡细胞类型需要Na+，K+-ATP酶的基因转移来改善肺泡液体清除和诱导紧密连接，（2）通过开发细胞特异性RNAi递送方法来测试Na+，K+-ATP酶基因转移介导的保护和治疗急性肺损伤是否需要紧密连接，和（3）确定单独的紧密连接复合物蛋白的基因转移是否可以治疗肺损伤或进一步增强Na+，K+-ATP酶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.