Cytoplasmic trafficking of non-viral gene therapy vectors

描述（由申请人提供）：在几乎所有条件下，使用任何方法，基因转移到任何细胞的水平都很低，因为存在许多障碍，无法有效地将基因传递到细胞中。再进一步说，基因转移到活体动物的组织中就更糟糕了，至少在某种程度上，因为所有组织中的细胞都不断地暴露在机械压力下，比如剪切、压缩和拉伸。因此，为了提高基因治疗的有效性，必须阐明基因在静态条件和机械应变条件下传递的途径和分子机制。机械拉伸在细胞中诱导了许多与基因传递过程直接相关的生物反应。外源DNA必须进入细胞，穿过细胞质，进入细胞核，并表达，基因治疗才能成功。我们已经证明，暴露于等双轴拉伸的多种细胞类型中的基因传递和表达比在静态条件下生长的细胞效率高10倍。我们还表明，这种循环拉伸通过抑制细胞质组蛋白去乙酰化酶HDAC6介导的机制重组了细胞骨架，增加了稳定的乙酰化微管的数量，其主要靶点是1-微管蛋白。已有研究表明，微管乙酰化导致动力蛋白马达的募集，并增加细胞质中结合物的运输。我们假设循环拉伸调节HDAC6活性，导致乙酰化微管水平增加，质粒dna -蛋白复合物向细胞核的细胞质运输增加，从而增强基因表达。虽然不是生理上的“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，并构成了基因治疗的基础。本应用的实验将阐明拉伸增强基因在培养细胞和活体动物中的传递机制，重点是肺泡上皮，一个持续经历循环拉伸的组织。最后，我们开发了一种电穿孔方法，用于高水平、安全、非病毒的基因转移到肺部，并在实验动物模型中使用这种方法治疗肺损伤。我们现在将利用这种方法来探索体内基因转移的机制，并开发损伤肺的治疗方法。具体目的是：(1)确定HDAC6和乙酰化微管在细胞内循环拉伸增强基因转移中的作用，(2)鉴定拉伸和未拉伸细胞中促进质粒通过细胞质运动的dna -蛋白复合物的成分，以及(3)确定机械通气是否通过HDAC6增加基因在小鼠肺内肺泡上皮的转移。

DESCRIPTION (provided by applicant): Under almost all conditions, using any method, the levels of gene transfer to any cell are low because many barriers exist for the efficient delivery of genes to cells. Taken one step further, gene transfer to tissues within living animals is even worse, at least in part since cells in all tissues are constantly exposed to mechanical stresses such as shear, compression, and stretch. Thus, we must elucidate the pathways and molecular mechanisms of gene delivery under static conditions and those of mechanical strain if we are to increase the efficacy of gene therapy. Mechanical stretch induces numerous biological responses in cells that are directly related to the process of gene delivery. Exogenous DNA must enter the cell, cross the cytoplasm, enter the nucleus, and be expressed for gene therapy to be successful. We have shown that gene delivery and expression in multiple cell types exposed to equibiaxial stretch is 10-fold more efficient than in cells grown under static conditions. We have also shown that this cyclic stretch reorganizes the cytoskeleton and increases the numbers of stable, acetylated microtubules by a mechanism mediated by inhibition of the cytoplasmic histone deacetylase HDAC6, whose main target is 1-tubulin. It has been shown that microtubule acetylation causes the recruitment of dynein motors and increases cytoplasmic trafficking of bound cargoes. We hypothesize that cyclic stretch modulates HDAC6 activity resulting in increased levels of acetylated microtubules and increased cytoplasmic trafficking of plasmid DNA-protein complexes toward the nucleus for enhanced gene expression. Although not a physiologically "normal" process, the interactions of plasmids with the host cell are vital to methods that scientists use every day and form the basis of gene therapy. The experiments in this application will elucidate the mechanisms of stretch-enhanced gene delivery in cultured cells and living animals, with a focus on the alveolar epithelium, a tissue that continuously undergoes cyclic stretch. Finally, we have developed an electroporation method for high-level, safe, non-viral gene transfer to the lung and have used this approach to treat lung injury in experimental animal models. We will now utilize this approach to explore the mechanisms of in vivo gene transfer and develop treatment approaches for the injured lung. The specific aims are to (1) determine the role of HDAC6 and acetylated microtubules in cyclic stretch-enhanced gene transfer in cells, (2) identify the components of the DNA-protein complex that facilitate movement of plasmids through the cytoplasm in stretched and unstretched cells, and (3) determine whether mechanical ventilation increases gene transfer to the alveolar epithelium in the mouse lung in vivo through HDAC6. 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. Although most work on gene transfer has been studied in cells growing undisturbed in dishes, most cells in the body, especially those in the lung, our target organ of interest, are constantly undergoing various forms of mechanical strain including cyclic stretch. We will determine the molecular mechanisms by which the cells respond to cyclic stretch to rearrange their cytoskeleton and increase intracellular DNA movement and gene therapy in isolated cells and small animal models.