Intracellular Trafficking of Antisense and siRNA Oligonucleotides in Cancer Cells

描述（由申请人提供）：该提案的主旨是获得关于反义和siRNA寡核苷酸的细胞摄取和细胞内运输及其对癌症药理学有效性的影响的新颖和基本见解。我们已经合成了对整合素，g蛋白偶联受体和其他受体具有高亲和力配体的寡核苷酸偶联物。我们可以改变配体的类型、亲和力和配价。使用这些试剂，我们将阐明受体介导的细胞摄取和运输途径。我们还将利用化学抑制剂和分子工具（如参与贩运的蛋白质的激活或显性阴性版本）来操纵这些途径。我们将通过敏感的报告基因检测来评估这些操作对反义rna和siRNA的药理学效果的影响。我们将寻求确定摄取和运输途径，提供最有效的递送到细胞质和细胞核，从而允许最大的药理作用。这些研究将在三种不同的背景下进行。标准二维（2-D）培养癌细胞系将提供一个设置，分子操作和定量共聚焦荧光显微镜可以相对容易地进行。三维（3-D）癌细胞培养将被用作一种重要的中间物，它可能更接近地反映肿瘤的情况，但这仍然是可以操纵的。最后，通过使用活体显微镜和其他工具，我们将检查反义核苷酸和siRNA寡核苷酸在异种移植肿瘤中的摄取、分布和作用。2-D， 3-D和体内系统的协同使用将提供丰富的相关信息流，这将对设计有效的受体介导的癌症治疗性寡核苷酸递送策略具有重要意义。

DESCRIPTION (provided by applicant): The thrust of this proposal is to attain novel and fundamental insights into the cellular uptake and intracellular trafficking of antisense and siRNA oligonucleotides and the implications for pharmacological effectiveness in cancer. We have synthesized oligonucleotide conjugates bearing high affinity ligands for Integrins, G-Protein Coupled Receptors, and other receptors. We can vary the ligand type, affinity, and valency. Using these reagents we will elucidate pathways of receptor- mediated cellular uptake and trafficking. We will also manipulate those pathways utilizing both chemical inhibitors and molecular tools such as activated or dominant-negative versions of proteins involved in trafficking. We will evaluate the impact of such manipulations on the pharmacologic effectiveness of antisense and siRNA via sensitive reporter assays. We will seek to identify the uptake and trafficking pathway(s) that provide the most effective delivery to the cytosol, and nucleus, and thus allow the greatest pharmacologic effect. These studies will be performed in three different contexts. Standard two-dimensional (2-D) culture of cancer cell lines will provide a setting where molecular manipulations and quantitative confocal fluorescence microscopy can be performed with relative ease. Three-dimensional (3-D) cancer cell cultures will be used as an important intermediate that may more closely reflect the situation in tumors, but that is still amenable to manipulation. Finally, via use of intravital microscopy and other tools, we will examine the uptake, distribution, and effect of antisense and siRNA oligonucleotides in xenograft tumors. The concerted use of 2-D, 3-D and in vivo systems will provide a rich stream of correlated information that will be of importance in the design of effective strategies for receptor-mediated delivery of therapeutic oligonucleotides in cancer. PUBLIC HEALTH RELEVANCE: Antisense and siRNA oligonucleotides have great potential for cancer therapeutics. However our ability to use these molecules effectively is limited by lack of detailed molecular understanding of their cellular uptake and intracellular trafficking. The current proposal will address these issues using a variety of pharmacological, molecular and imaging techniques. These studies will be pursued in single cells, multi-cellular assemblies, and tumors in vivo. This integrated approach will provide a rich stream of novel information that will enhance and expedite the development of oligonucleotides as therapeutic agents in cancer.