NOVEL MARKERS FOR IMAGING GENE EXPRESSION IN VIVO USING MRI

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The ability to non-invasively image the duration and location of gene expression in vivo is important for the future of biomedical research and molecular medicine. The goal of this application is to develop a general purpose MRI reporter gene cassette that can be used in a variety of molecular imaging applications. Recent results from our laboratory demonstrate that vectors encoding metalloprotein reporters delivered into the living mouse brain can elicit potent contrast in MR images. The reporter protein is made superparamagnetic as the cell sequesters endogenous iron from the organism. Our approach is unique because the cell constructs the MRI contrast agent in situ by genetic means. No bulky-metal complex is required, thereby simplifying intracellular delivery. Our proposal consists of three Specific Aims: (1) we will design second generation MRI reporters for improved sensitivity and minimal reporter sequence length. MRI reporters will be incorporated into replication-defective viral vectors including adenovirus, herpes simplex virus, and vesicular stomatitis virus, for subsequent cell culture and in vivo studies; (2) using the viral vectors we will perform extensive in vitro analyses of the chemical, biochemical, biophysical and toxicological properties of the reporters in established cell lines; (3) we will demonstrate proof-of-principle in vivo by longitudinally imaging the MRI reporters in the CNS of mice. We will quantitatively evaluate their contrasting effects in different tissues via focal injections and by targeting their expression using tissue-specific promoters. Finally, we will look for evidence of pathologic changes in reporter-transduced brain tissues using an array of neuropathological markers. Overall, this platform technology can be adapted to examine gene expression in many tissue types, and thus there are a large number of potential in vivo applications. Examples include preclinical testing of gene therapeutics, such as for the treatment of metastatic disease, and use in transgenic models for drug development. Thus, there is high relevance to the mission of the NIH. Lay Summary - Tools that enable one to visualize gene expression within the living organism are of fundamental importance to the future of medicine and biomedical research. The emerging field of genetic medicine requires non-invasive imaging methods that can indicate if the therapeutic genes have been delivered to the correct cells and if the desired proteins have been made. Towards this goal, our project will develop new technologies to visualize gene expression in vivo using conventional magnetic resonance imaging (MRI) scanners.