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，因为存在许多将基因有效递送到细胞的障碍。更进一步说，基因转移到活体动物体内的组织甚至更糟，至少部分原因是所有组织中的细胞都不断暴露于机械应力，如剪切，压缩和拉伸。因此，我们必须阐明的途径和分子机制的基因传递在静态条件下和那些机械应变，如果我们要提高基因治疗的疗效。机械拉伸在细胞中诱导许多与基因递送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，在多种细胞类型中暴露于等双轴拉伸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。我们还表明，这种循环拉伸重组细胞骨架，并增加稳定的，乙酰化微管的数量通过抑制细胞质组蛋白去乙酰化酶HDAC 6介导的机制，其主要目标是1-微管蛋白。已经表明，微管乙酰化引起动力蛋白马达的募集并增加结合货物的细胞质运输。我们推测，周期性拉伸调节HDAC 6活性，导致乙酰化微管水平增加，质粒DNA-蛋白质复合物向细胞核的细胞质运输增加，以增强基因表达。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，并构成基因治疗的基础。本申请中的实验将阐明培养细胞和活体动物中拉伸增强基因递送的机制，重点是肺泡上皮细胞，一种持续经历周期性拉伸的组织。最后，我们开发了一种电穿孔方法，用于高水平，安全，非病毒基因转移到肺，并使用这种方法来治疗实验动物模型中的肺损伤。我们现在将利用这种方法来探索体内基因转移的机制，并为受伤的肺开发治疗方法。具体目的是（1）确定HDAC 6和乙酰化微管在细胞中循环拉伸增强基因转移中的作用，（2）鉴定DNA-蛋白质复合物的组分，其促进质粒在拉伸和未拉伸细胞中通过细胞质的移动，以及（3）确定机械通气是否通过HDAC 6增加体内小鼠肺中肺泡上皮的基因转移。 公共卫生相关性：基因治疗是一种令人兴奋的和潜在的非常有用的方法，在分子水平上治疗许多疾病。不幸的是，基因传递到细胞和动物的许多障碍存在，必须在克服它们之前进行表征，从而导致更高水平的基因转移和基因治疗。虽然大多数基因转移的研究都是在培养皿中不受干扰地生长的细胞中进行的，但体内的大多数细胞，特别是我们感兴趣的靶器官肺中的细胞，都在不断地经历各种形式的机械应变，包括周期性拉伸。我们将确定细胞响应周期性拉伸的分子机制，以重新排列其细胞骨架，并在分离的细胞和小动物模型中增加细胞内DNA运动和基因治疗。

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.