Aptamers for Imaging and Therapy

描述（由申请人提供）： 一期 目前的提议描述了识别药物PD 173955的适体的选择，所述药物PD 173955是酪氨酸蛋白激酶的取代的吡啶并[2，3-d]嘧啶抑制剂的大家族之一。适体是一种单链核酸，其作用类似于抗体，但可以在细胞内表达和发挥功能。PD 173955 RNA适体将被选中，为开发一种新的成像技术奠定基础，该技术也可用于增强药物治疗。其概念是存在于细胞内或靶向细胞表面的适体可以将药物集中在细胞内或周围。该药物的放射性类似物可用于成像。 提出的成像基础涉及放射性标记药物通过适体的累积。细胞内或细胞周药物浓度的增加应增加药物的有效性。然而，预期适体的动力学参数对于最佳成像和治疗增强非常重要。因此，我们正在开发一种计算模型，该模型基于适体、药物和药物靶向酶的动力学和扩散常数的数学渲染。数学模型的开发将与aptarner选择并行进行，并指导选择协议。 提出了以下具体目标来开发用于成像和治疗应用的PD 173955适体：1）制备连接至固体支持物的PD 173955以用于适体选择，2）选择识别PD 173955的RNA适体，和3）开发用于适体在成像和增强药物治疗中的使用的数学模型。 二期 目前的提议描述了一种基于适体的新型核酸结构，该适体将在细胞中表达以标记它们，因此它们可以通过可用的成像技术在体内检测。我们将这些标记物称为细胞内多适体遗传标签（IMAGEtags）。IMAGEtag设计的基本创新是在活细胞中表达适体，该适体可用于通过非侵入性程序在体内追踪细胞。表达IMAGE标签的细胞将通过浓缩的放射性标记的配体来检测，所述配体通过IMAGE标签的适体组分结合并捕获在细胞中。表达IMAGE标签的启动子将决定哪些细胞可以成像。例如，许多导致增殖增加的基因在癌细胞中表达，而在大多数正常细胞中不表达。如果使用这些启动子或仅在癌细胞中高度表达的“合成”启动子来驱动IMAGEtag表达，则癌细胞将被成像。 我们还建议IMAGE标签也可以应用于增强药物治疗。由于成像的机制涉及配体的积累，识别特定药物的IMAGE可以将更多的药物吸入细胞并增加药物的有效性。 IMAGE标签及其靶配体的设计和选择将通过计算建模来指导，以优化适体及其配体的动力学特征，用于成像和增强的治疗应用。 用于成像和治疗应用的MAGE标签的开发将通过以下具体目标实现：1）改进PD 173955适体以在细胞内发挥作用并制备稳定表达适体的细胞，2）改进用于体内成像的数学模型，3）使用培养的细胞验证用适体进行体内成像的能力，和4）使用PD 173955适体在动物中成像。

DESCRIPTION (provided by applicant): Phase I The current proposal describes the selection of an aptamer that recognizes the drug PD173955, which is one of large family of substituted pyrido[2,3-d]pyrimidine inhibitors of tyrosine protein kinases. An aptamer is a single stranded nucleic acid that acts like an antibody but that can be expressed and function inside cells. The PD173955 RNA aptamer will be selected to lay the groundwork for developing a novel imaging technology that could also be used for enhancing drug therapy. The Concept is that the aptamer, present inside the cell or targeted to the cell surface, could concentrate drug in or around the cell. A radioactive analogue of the drug could be used for imaging. The proposed basis of imaging involves accumulation of radiolabeled drug by aptamers. The increased intracellular or pericellular drug concentration in the cell should increase the effectiveness of the drug. However, the kinetic parameters of the aptamer are expected to be very important for optimal imaging and therapeutic enhancement. Therefore, we are developing a computational model that is based on a mathematical rendering of the kinetic and diffusion constants of the aptamer, drug and the enzyme targeted by the drug. Development of the mathematical model will proceed in parallel with the aptarner selection and guide the choice of selection protocols. The following specific aims are proposed for development of an aptamer to PD173955 for imaging and therapeutic applications: 1) prepare PD173955 linked to a solid support for aptamer selection, 2) select an RNA aptamer that recognizes PD173955, and 3) develop a mathematical model for the use of aptamers in imaging and enhancing drug therapy. Phase II The current proposal describes a novel nucleic acid structure based on aptamers that will be expressed in cells to mark them so they can be detected in viva by available imaging technology. We call the markers intracellular Multi-Aptamer Genetic Tags" (IMAGEtags). The basic innovation in the proposed IMAGEtag design is the expression, in living cells, of aptamers that can be used to track the cells in vivo by a noninvasive procedure. Cells that express IMAGEtags will be detected by virtue of the concentrated radiolabeled ligands that are bound and trapped in the cells by the aptarner components of IMAGEtags. The promoter from which the IMAGEtags are expressed will determine which cells can be imaged. For example, many genes that are responsible for increased proliferation are expressed in cancer cells and not in most normal cells. If these promoters, or a "synthetic" promoter that was highly expressed only in cancer cells, were used to drive IMAGEtag expression then cancer cells would be imaged. We also propose that the IMAGEtags could also be applied to enhancing drug therapy. Because the mechanism of imaging involves accumulation of ligand, IMAGEtas that recognize a particular drug could draw more drug into the cell and increase the effectiveness of the drug. The design and selection of IMAGEtags and their target ligands will be guided by computational modeling to optimize the kinetic characteristics of the aptamers and their ligands for imaging and enhanced therapeutic applications. Development of MAGEtags for imaging and therapeutic applications will be achieved by the following specific aims: 1) refine the PD173955 aptamer to function inside cells and prepare cells that stably express the aptamer, 2) refine the mathematical model for in vivo imaging, 3) validate the ability to image with aptamers in vivo using cultured cells, and 4) Use the PD173955 aptamer to image in animals.