Targeting Airway Smooth Muscle for Asthma Gene Therapy

描述(申请人提供)：哮喘是影响全球超过3亿人的发病率和死亡率的主要原因。虽然有多种治疗方法可以对症治疗呼吸道炎症和支气管痉挛，但基因治疗可以改变驱动疾病发病机制的分子功能障碍。不幸的是，这种治疗方法因无法选择性地将基因转移到未分裂的血管和呼吸道平滑肌细胞而不造成损害而受到阻碍。我们正在进行的目标是通过阐明主动和选择性地将DNA和DNA-蛋白质复合体运输到特定细胞类型的细胞核的分子机制来设计更有效的基因治疗载体。我们已经确定了一个来自于平滑肌γ肌动蛋白(SMGA)启动子的DNA序列，该序列增加了平滑肌细胞的核定位和随后的基因表达，而平滑肌细胞是哮喘基因治疗的关键靶点。我们已经证明，SMGA DNA核靶向序列(或DTS)的细胞选择性核导入是由转录因子SRF和Nkx3.1/3.2介导的，这两个转录因子唯一地在平滑肌细胞中共表达，而不是在呼吸道的其他细胞中共表达。在最近的基于蛋白质组学的研究中，我们确定了一些可能参与DNA蛋白复合体在平滑肌细胞中运输的候选蛋白质。此外，我们已经开发了一种利用电场在体内传递基因的新技术，该电场可以用于将这些非病毒的平滑肌特异性载体靶向呼吸道。利用体内电穿孔，我们计划测试我们的平滑肌特异性核输入序列在哮喘动物模型中用于基因转移的有效性。我们假设SMGA DNA核靶向序列将导致核输入和随后DNA的转录，仅在活动物的呼吸道平滑肌中，而不是在其他类型的肺细胞中。本研究的具体目的是(1)确定最小SMGA DNA核靶向序列及其细胞特异性DNA核导入的机制，(2)测试最小SMGA DNA核靶向序列在驱动肺内气道平滑肌特异性基因转移方面的效率，以及(3)测试使用平滑肌特异性核靶向构建的2-肾上腺素能受体基因传递是否以及如何在体内抑制气道反应性和平滑肌增殖。 与公共卫生相关：基因疗法是一种令人兴奋的、潜在的非常有用的方法，可以在分子水平上治疗许多疾病。不幸的是，向细胞和动物传递基因存在许多障碍，必须在克服这些障碍之前对其进行表征，从而导致更高水平的基因转移和基因治疗。一个需要更多关注的领域是将基因传递到特定类型的细胞。我们已经确定了一种DNA序列，它只能促进质粒在平滑肌细胞中的核进入，在我们正在进行的研究中，我们将确定导致这种DNA运动的分子机制。我们还开发了一种新技术，这是现有的唯一将基因输送到哮喘基因治疗的关键靶点--气道平滑肌的方法。我们将利用所获得的信息来开发更好的基因转移载体，并测试它们是否可以用于治疗哮喘的几种动物模型的呼吸道。我们将使用分离的细胞和小动物模型以及药理学和遗传学方法来回答这些问题。

DESCRIPTION (provided by applicant): Asthma is a leading cause of morbidity and mortality affecting more that 300 million people worldwide. While numerous therapies exist to treat airway inflammation and bronchospasm symptomatically, gene therapy can alter the molecular dysfunctions driving disease pathogenesis. Unfortunately, this therapeutic approach has been hindered by the inability to selectively transfer genes to non-dividing vascular and airway smooth muscle cells without inducing damage. Our ongoing goal is to design more effective gene therapy vectors by elucidating the molecular mechanisms that transport DNA and DNA-protein complexes actively and selectively into the nucleus of specific cell types. We have identified a DNA sequence derived from the smooth muscle gamma actin (SMGA) promoter that increases nuclear localization and subsequent gene expression uniquely in smooth muscle cells, a critical target in asthma gene therapy. We have shown that the cell-selective nuclear import of the SMGA DNA nuclear targeting sequence (or DTS) is mediated by the transcription factors SRF and Nkx3.1/3.2 that are uniquely co-expressed in smooth muscle cells but not other cells of the airway. In recent proteomics-based studies we identified a number of candidate proteins that may be involved in trafficking of the DNA protein complexes in smooth muscle cells. Further, we have developed a new technique for in vivo gene delivery using electric fields that can be used to target these non-viral smooth muscle specific vectors to the airways. Using in vivo electroporation, we plan to test the efficacy of our smooth muscle-specific nuclear import sequence for gene transfer in animal models of asthma. We hypothesize that the SMGA DNA nuclear targeting sequence will lead to nuclear import and subsequent transcription of the DNA only in airway smooth muscle of living animals and not in other cells types of the lung. The specific aims of this proposal are to (1) determine the minimal SMGA DNA nuclear targeting sequence active in airway smooth muscle and the mechanisms of its cell specific DNA nuclear import, (2) test the efficiency of minimal SMGA DNA nuclear targeting sequences for driving airway smooth muscle-specific gene transfer in the lung, and (3) Test whether and how gene delivery of the ¿2-adrenergic receptor using smooth muscle-specific nuclear targeting constructs inhibits airway reactivity and smooth muscle proliferation in vivo. PUBLIC HEALTH RELEVANCE: Gene therapy is an exciting and potentially very useful approach to treat a number of diseases at the molecular level. Unfortunately, many barriers for gene delivery to cells and animals exist that must be characterized before they can be overcome, leading to greater levels of gene transfer and gene therapy. One area that requires more attention is that of gene delivery to specific cell types. We have identified a DNA sequence that can promote nuclear entry of plasmids in smooth muscle cells only, and in our ongoing studies we will determine the molecular mechanisms responsible for this DNA movement. We have also developed a new technique that is the only existing approach to deliver genes to airway smooth muscle, a critical target for asthma gene therapy. We will use the information obtained to develop better gene transfer vectors and test whether they can be used in the airways to treat asthma in several animal models of the disease. We will use isolated cell and small animal models along with pharmacological and genetic approaches to answer these questions.