Targeting Airway Smooth Muscle for Asthma Gene Therapy

描述（由申请人提供）：哮喘是全球超过3亿人发病和死亡的主要原因。虽然有许多治疗方法可以对症治疗气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，这种治疗方法由于无法选择性地将基因转移到非分裂血管和气道平滑肌细胞而不引起损伤而受到阻碍。我们正在进行的目标是通过阐明将DNA和DNA-蛋白复合物主动和选择性地转运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平滑肌γ肌动蛋白（SMGA）启动子的DNA序列，该序列在平滑肌细胞中独特地增加核定位和随后的基因表达，这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）的细胞选择性核输入是由转录因子SRF和Nkx3.1/3.2介导的，这两种转录因子在平滑肌细胞中共同表达，而在气道的其他细胞中不表达。在最近基于蛋白质组学的研究中，我们发现了一些可能参与平滑肌细胞中DNA蛋白质复合物运输的候选蛋白质。此外，我们还开发了一种利用电场进行体内基因递送的新技术，该技术可用于将这些非病毒平滑肌特异性载体靶向到气道。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的基因转移效果。我们假设SMGA DNA核靶向序列仅在活体动物的气道平滑肌中导致核输入和随后的DNA转录，而在其他类型的肺细胞中则不会。本研究的具体目的是：(1)确定在气道平滑肌中活性的最小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核靶向序列在肺内驱动气道平滑肌特异性基因转移的效率。(3)在体内测试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抑制气道反应性和平滑肌增殖。

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.