Cytoplasmic trafficking of non-viral gene therapy vectors

非病毒基因治疗载体的细胞质运输

• 批准号: 8930146
• 负责人: David A Dean
• 金额: $ 33.85万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8930146
• 关键词: Acetylation; Actins; Animal Model; Animals; Architecture; Cell Nucleolus; Cell Nucleus; Cell membrane; Cells; Complex; Cytoplasm; Cytoskeleton; DNA; DNA Polymerase I; DNA Polymerase II; Deacetylase; Disease; Environmental air flow; Funding; Gene Delivery; Gene Expression; Gene Transduction Agent; Gene Transfer; Genes; Genetic Materials; Genetic Transcription; Goals; Grant; HDAC6 gene; Health; Histone Deacetylase; Immune response; In Vitro; Laboratories; Length; Life; Lung; Mass Spectrum Analysis; Mechanics; Messenger RNA; Methods; Microtubule-Associated Proteins; Microtubules; Molecular; Molecular Chaperones; Motor; Movement; Mus; Nuclear Envelope; Nuclear Export; Nuclear Import; One-Step dentin bonding system; Organ; Pathway interactions; Plasmids; Play; Production; Protein Binding; Proteins; Proteomics; Rattus; Role; Running; Stretching; Time; Tissues; Transfection; Travel; Tubulin; Viral; Work; base; cell type; depolymerization; gene delivery process; gene therapy; genetic approach; improved; in vivo; inhibitor/antagonist; non-viral gene therapy; promoter; protein complex; protein transport; rRNA Promoters; research study; tool; trafficking; transcription factor

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.

描述（由申请人提供）：在几乎所有条件下使用任何方法，基因转移的水平都较低，因为存在许多障碍，以有效地递送基因向细胞传递。进一步迈出了一步，至少部分是由于这些组织的结构，这些组织中的机械力以及宿主对外源物质的反应而产生的，至少部分是由于这些基因转移到活动物中的组织更糟。我们实验室的主要目的是识别和克服细胞内屏障，以促进体外和体内有效基因转移。外源性DNA，无论是病毒还是非病毒，都必须跨越质膜进入细胞，穿过细胞质和细胞骨架网络，越过核膜，位于原子核内的特定区域，并进行转录以使基因治疗成功。在2003年，我们证明，当多种细胞类型暴露于轻度的倍轴延伸时，它们的摄取和表达外源DNA的能力比在静态条件下生长的细胞高10倍。此后，我们已经表明，这种拉伸重新组织了细胞骨架，并通过抑制HDAC6（主要的细胞质A-微管脱乙酰基酶）来增加稳定的乙酰化微管的数量。我们利用这些信息来通过在细胞和动物中使用HDAC6的药理抑制剂来提高细胞内DNA运动和转染效率。最近，我们专注于质粒在转染和基因转移过程中如何沿着修饰和未修饰的微管移动到核的运输，并确定了蛋白-DNA复合物的成分，这些蛋白-DNA复合物在进入细胞质后立即形成，然后在后来使用质谱和蛋白质组学。此外，通过比较质粒的蛋白质复合物，这些质粒在细胞中有效地流动的质粒与质粒上的质量络合物与不动的质粒的蛋白质复合物进行比较，我们已经能够鉴定控制DNA运动的关键蛋白质，并且我们假设对这些蛋白质的调节可以用于进一步改善基因递送。最后，我们还在研究质粒如何在细胞核内进行的流量，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。质粒基于其序列在核内的不同区域定位，我们假设我们可以控制这种运动以优化基因表达。该竞争性更新的实验将剖析用于细胞质和细胞核中蛋白质和DNA-蛋白质复合物的细胞内运输途径，以增强基因递送。具体目的是（1）确定微管蛋白乙酰化在转染质粒的细胞质运输中的作用； （2）识别并表征活性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该如何调节基因表达。