Aptamers for Imaging and Therapy

描述(由申请人提供)： 第一阶段 目前的建议描述了一种识别药物PD173955的适体的选择，PD173955是酪氨酸蛋白激酶的取代吡啶[2，3-d]嘧啶抑制剂大家族中的一种。适配子是一种单链核酸，其作用类似于抗体，但可以在细胞内表达和发挥作用。PD173955 RNA适配子将被选中，为开发一种新的成像技术奠定基础，这种成像技术也可以用于加强药物治疗。其概念是，存在于细胞内或靶向细胞表面的适体可以在细胞内或细胞周围浓缩药物。这种药物的放射性类似物可以用于成像。 所提出的成像基础涉及适体对放射性标记药物的累积。细胞内或细胞外药物浓度的增加应该会增加药物的有效性。然而，适体的动力学参数对于最佳的成像和治疗增强是非常重要的。因此，我们正在开发一种计算模型，该模型基于适体、药物和药物靶向的酶的动力学和扩散常数的数学呈现。数学模型的开发将与分配人选择并行进行，并指导选择选择协议。 为开发PD173955的适体用于成像和治疗应用，提出了以下具体目标：1)制备连接到用于适体选择的固体载体的PD173955，2)选择识别PD173955的RNA适体，以及3)为适体在成像和增强药物治疗中的使用开发数学模型。 第二阶段 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，从而可以通过现有的成像技术在体内检测到它们。我们称这些标记为“细胞内多适配子遗传标记”(IMAGE标记)。拟议中的IMAGE标签设计的基本创新是在活细胞中表达适体，这种适体可以通过非侵入性程序在体内跟踪细胞。表达IMAGE标签的细胞将通过IMAGE标签的配基成分结合并捕获在细胞中的浓缩型放射性标记配体而被检测到。表达IMAGE标签的启动子将决定哪些细胞可以被成像。例如，许多促进增殖的基因在癌细胞中表达，而在大多数正常细胞中不表达。如果这些启动子，或者只在癌细胞中高表达的“合成”启动子，被用来驱动IMAGE标签的表达，那么癌细胞将被成像。 我们还建议IMAGE标签也可以应用于加强药物治疗。由于成像的机制涉及配体的积累，识别特定药物的IMAGEtas可以将更多的药物吸引到细胞内，从而提高药物的有效性。 IMAGE标签及其目标配体的设计和选择将以计算建模为指导，以优化适体及其配体的动力学特性，用于成像和增强治疗应用。 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PD173955适配子以在细胞内发挥作用并制备稳定表达该适配子的细胞；2)改进体内成像的数学模型；3)利用培养细胞验证体内用适配子成像的能力；以及4)使用PD173955适配子在动物体内成像。

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.