Cytoplasmic trafficking of non-viral gene therapy vectors

描述(申请人提供)：在几乎所有的条件下，使用任何方法，基因转移到任何细胞的水平都很低，因为有效地将基因传递到细胞存在许多障碍。更进一步，基因转移到活体动物体内的组织中更糟糕，至少部分是因为所有组织中的细胞都不断地暴露在机械应力下，如剪切、压缩和拉伸。因此，如果要提高基因治疗的有效性，就必须阐明静态条件下基因传递的途径和分子机制以及机械应变下的基因传递途径和分子机制。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，并被表达，才能使基因治疗成功。我们已经证明，在暴露于等轴拉伸的多种细胞类型中，基因传递和表达的效率是在静态条件下生长的细胞的10倍。我们还表明，这种循环拉伸重组细胞骨架，增加稳定的乙酰化微管的数量，其机制是通过抑制细胞质组蛋白脱乙酰酶HDAC6介导的，该酶的主要靶标是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，并增加结合货物的细胞质转运。我们假设，循环拉伸调节HDAC6的活性，导致乙酰化微管水平增加，并增加质粒DNA-蛋白质复合体向核的运输，以增强基因表达。尽管这不是一个生理上“正常”的过程，但对于科学家每天使用的方法和形成基因治疗的基础来说，质粒与宿主细胞的相互作用是至关重要的。这项应用中的实验将阐明拉伸增强在培养细胞和活动物中的基因传递的机制，重点是肺泡上皮，这是一种持续经历周期性拉伸的组织。最后，我们开发了一种高水平、安全、非病毒基因转移到肺的电穿孔方法，并将该方法用于治疗实验性动物模型的肺损伤。现在，我们将利用这种方法来探索体内基因转移的机制，并开发治疗受损肺的方法。其具体目的是(1)确定HDAC6和乙酰化微管在细胞内周期性拉伸增强的基因转移中的作用，(2)确定在拉伸和非拉伸细胞中促进质粒通过细胞质移动的DNA-蛋白质复合体的成分，以及(3)确定在活体小鼠肺组织中，机械通气是否通过HDAC6增加向肺泡上皮细胞的基因转移。 与公共卫生相关：基因疗法是一种令人兴奋的、潜在的非常有用的方法，可以在分子水平上治疗许多疾病。不幸的是，向细胞和动物传递基因存在许多障碍，必须在克服这些障碍之前对其进行表征，从而导致更高水平的基因转移和基因治疗。尽管大多数关于基因转移的研究都是在培养皿中原封不动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目标器官肺中的细胞，都在不断地经历各种形式的机械应变，包括循环拉伸。我们将在分离的细胞和小动物模型中确定细胞对周期性拉伸做出反应的分子机制，以重排细胞骨架，增加细胞内DNA的移动和基因治疗。

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.