Cytoplasmic trafficking of non-viral gene therapy vectors

描述（由申请人提供）：在使用任何方法的几乎所有条件下，基因转移到任何细胞的水平都很低，因为存在许多障碍来有效地将基因递送到细胞。更进一步，基因转移到活体动物体内的组织甚至更糟，至少部分是由于这些和额外的障碍，这些障碍来自组织的结构、这些组织内的机械力以及宿主对外源物质的反应。我们实验室的主要目标是识别和克服细胞内的障碍，以促进有效的基因转移在体外和体内。外源DNA，无论是病毒还是非病毒，必须穿过质膜进入细胞，穿过细胞质和细胞骨架网络，穿过核膜，定位于细胞核内的特定区域，并被转录，以便基因治疗成功。在2003年，我们证明了当多种细胞类型暴露于温和的等双轴拉伸时，它们吸收和表达外源DNA的能力比在静态条件下生长的细胞高10倍。我们已经证明，这种拉伸重组细胞骨架，并伴随着通过抑制HDAC 6（主要的细胞质α-微管蛋白脱乙酰酶）增加稳定的乙酰化微管的数量。我们利用这些信息，通过在细胞和动物中使用HDAC 6的药理学抑制剂来改善细胞内DNA移动和转染效率。最近，我们专注于质粒如何沿着沿着修饰和未修饰的微管移动，以便在转染和基因转移过程中将其运输到细胞核，并使用质谱和蛋白质组学鉴定了进入细胞质后立即形成的蛋白质-DNA复合物的成分。此外，通过比较质粒上的蛋白质复合物，这些蛋白质复合物通过细胞的生产性交通与质粒上的不移动，我们已经能够确定控制DNA运动的关键蛋白质，我们假设这些蛋白质的调制可以用于进一步改善基因传递。最后，我们也在研究质粒进入细胞核后是如何运输的，并发现质粒显示出高度动态的核内运动，可用于控制基因表达。质粒定位于细胞核内不同区域的基础上，他们的序列，我们假设，我们可以控制这种运动，以优化基因表达。在这个竞争性更新的实验将剖析用于细胞内运输的蛋白质和DNA-蛋白质复合物在细胞质和细胞核，以提高基因传递的途径。具体目的是（1）确定微管蛋白乙酰化在转染质粒胞质运输中的作用;（2）鉴定和表征活性DNA运输复合物的组成;（3）确定质粒如何在细胞核内移动以及如何调节基因表达。

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 due to these and additional barriers that arise from the architecture of th tissue, mechanical forces within these tissues, and the host's response to exogenous materials. The primary goal of our laboratory is to identify and overcome the intracellular barriers to promote effective gene transfer both in vitro and in vivo. Exogenous DNA, either viral or non-viral, must cross the plasma membrane into the cell, travel through the cytoplasm and the cytoskeletal networks, cross the nuclear envelope, localize to specific regions within the nucleus, and be transcribed in order for gene therapy to be successful. In 2003, we demonstrated that when multiple cell types were exposed to mild equibiaxial stretch, their ability to take up and express foreign DNA was 10-fold more efficient than cells grown under static conditions. We have since shown that such stretch reorganizes the cytoskeleton and concomitantly increases the numbers of stable, acetylated microtubules by inhibiting HDAC6, the major cytoplasmic a-tubulin deacetylase. We exploited this information to improve intracellular DNA movement and transfection efficiency by using pharmacologic inhibitors of HDAC6 in cells and animals. More recently we have focused on how plasmids move along modified and unmodified microtubules for their trafficking to the nucleus during transfection and gene transfer and have identified the constituents of the protein-DNA complexes that form immediately after entry into the cytoplasm and at various times afterward using mass spectrometry and proteomics. Further, by comparing the protein complexes on plasmids that productively traffic through the cell with those on plasmids that do not move, we have been able to identify key proteins that control DNA movement and we hypothesize that modulation of these proteins may be used to further improve gene delivery. Finally, we are also looking at how plasmids traffic once inside the nucleus and have found that plasmids show highly dynamic intranuclear movement that can be used to control gene expression. Plasmids localize to distinct regions within the nucleus based on their sequences and we hypothesize that we can control this movement to optimize gene expression. The experiments in this competitive renewal will dissect pathways used for intracellular trafficking of proteins and DNA-protein complexes in both the cytoplasm and nucleus to enhance gene delivery. The specific aims are to (1) Determine the role of tubulin acetylation in cytoplasmic trafficking of transfected plasmids; (2) Identify and characterize the composition of the active DNA trafficking complex; and (3) Determine how plasmids move within the nucleus and how this regulates gene expression.