Gene transfer to the alveolar epithelium: effects of stretch

暴露于机械通气可引起肺泡上皮的深刻变化。我们建议开发非病毒基因治疗方法来治疗急性肺损伤。因此，我们必须了解在模拟肺损伤及其管理过程中发现的条件下肺泡上皮细胞（AEC）中的基因转移机制（即，通风）。机械牵张诱导AEC中的许多生物学反应，包括细胞骨架的改变、细胞信号传导通路的激活和转录因子的上调。这些反应与基因传递过程直接相关。外源DNA必须穿过质膜，穿过细胞质，进入细胞核，并表达，以使基因治疗成功。暴露于等双轴拉伸的原代大鼠肺泡II型细胞、人或小鼠AEC细胞系中的基因递送和表达比在静态条件下生长的细胞中的效率高10倍。我们假设细胞骨架 由机械拉伸诱导的重组和转录因子活化刺激外源DNA穿过细胞质并进入细胞核用于基因表达的能力。虽然不是一个生理学上的“正常”过程，但质粒与宿主细胞的相互作用对科学家每天使用的方法至关重要，并形成了基因治疗的基础。本实验旨在阐明牵张增强基因的作用机制 在肺泡上皮中递送。我们还将建立一种新开发的非病毒基因转移到肺部的电穿孔方法的功效，该方法产生高水平的表达，而没有任何与腺病毒介导的基因治疗相关的炎症反应或副作用。具体目的是（1）确定牵张诱导的细胞骨架组织变化是否改变AEC中质粒DNA的胞质迁移率;（2）确定周期性牵张是否激活AEC中的转录因子并导致增加的DNA核输入;（3）确定机械通气是否增加体内小鼠和大鼠肺中电穿孔介导的基因转移至肺泡上皮。

Exposure to mechanical ventilation can cause profound changes in the alveolar epithelium. We propose to develop non-viral gene therapy approaches to treat the acutely injured lung. As such, we must understand the mechanisms of gene transfer in alveolar epithelial cells (AEC) under conditions that mimic those found during lung injury and its management (i.e., ventilation). Mechanical stretch induces numerous biological responses in AEC, including alterations in the cytoskeleton, activation of cell signaling pathways, and upregulation of transcription factors. These responses are directly related to the process of gene delivery. Exogenous DNA must cross the plasma membrane, travel through the cytoplasm, enter the nucleus, and be expressed in order for gene therapy to be successful. Gene delivery and expression in primary rat alveolar type II ceils, human, or mouse AEC cell lines exposed to equibiaxial stretch is 10-fold more efficient than in cells grown under static conditions. We hypothesize that the cytoskeletal reorganization and transcription factor activation induced by mechanical stretch stimulates the ability of exogenous DNA to travel through the cytoplasm and into the nucleus for gene expression. Although not a physiologically "normal" process, the interactions of plasmids with the host cell are vital to methods scientists use everyday and form the basis of gene therapy. The experiments in this proposal will elucidate the mechanisms of stretch-enhanced gene delivery in the alveolar epithelium. We also will establish the efficacy of a newly developed electroporation method for non-viral gene transfer to the lung that yields high level expression without any of the inflammatory response or side effects associated with adenoviral mediated gene therapy. The specific aims are (1) To determine whether stretch-induced changes in cytoskeletal organization alter the cytoplasmic mobility of plasmid DNA in AEC; (2) To determine whether cyclic stretch activates Transcription Factors and leads to increased DNA nuclear import in AEC; and (3) To determine whether mechanical ventilation increases electroporation-mediated gene transfer to the alveolar epithelium in the mouse and rat lung in vivo.