Thursday, November 21, 2024
11/21/2024
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.
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