Targeting Airway Smooth Muscle for Asthma Gene Therapy

描述（由申请人提供）：哮喘是发病率和死亡率的主要原因，影响全球3亿多人。虽然存在许多治疗气道炎症和支气管痉挛的药物，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，这种治疗方法受到了阻碍，不能选择性地将基因转移到非分裂的血管和气道平滑肌细胞而不诱导损伤。我们正在进行的目标是通过阐明将DNA和DNA-蛋白质复合物主动和选择性地转运到特定细胞类型的细胞核中的分子机制来设计更有效的基因治疗载体。我们已经确定了来自平滑肌γ肌动蛋白（SMGA）启动子，增加核定位和随后的基因表达独特的平滑肌细胞，在哮喘基因治疗的关键目标的DNA序列。我们已经表明，SMGA DNA核靶向序列（或SMGA）的细胞选择性核输入是由转录因子SRF和Nkx3.1/3.2介导的，它们在平滑肌细胞中而不是气道的其他细胞中独特地共表达。在最近基于蛋白质组学的研究中，我们确定了一些可能参与平滑肌细胞中DNA蛋白复合物运输的候选蛋白质。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，可用于靶向这些非病毒平滑肌特异性载体的气道。使用体内电穿孔，我们计划在哮喘动物模型中测试我们的平滑肌特异性核输入序列用于基因转移的功效。我们假设SMGA DNA核靶向序列将导致核输入和随后的DNA转录仅在活体动物的气道平滑肌中，而不是在肺的其他细胞类型中。本提案的具体目的是（1）确定在气道平滑肌中有活性的最小SMGA DNA核靶向序列及其细胞特异性DNA核输入的机制，（2）测试最小SMGA DNA核靶向序列驱动肺中气道平滑肌特异性基因转移的效率，以及（3）测试是否以及如何将SMGA DNA核靶向序列的基因递送到肺中。2-肾上腺素能受体使用平滑肌特异性核靶向结构抑制气道反应性和平滑肌增殖在体内。

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.