Developing a CEST Reporter Gene (RMI)

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DESCRIPTION (provided by applicant): Non-invasive imaging of cell migration, trafficking, and homing is an emerging new field that can provide us with a deeper insight into the dynamics of cell-tissue interactions, as well as provide guidance to the development of novel cell therapies using stem cells and progenitors. Compared to other imaging modalities (i.e., PET, SPECT, and bioluminescent imaging), MR imaging has the highest spatial resolution and can provide both anatomical and functional information, but it suffers from a severely high signal-to noise threshold for the detection of cells using suitable labels/tracers. To a certain degree, this limitation has been resolved using intracellular endosomal tagging with super-paramagnetic nanoparticles. However, the magnetic susceptibility-based T2 (*) contrast induced this way has significant drawbacks, including the creation of hypointense "black holes" (obscuring tissue morphology), difficult differentiation between live and dead cells, the presence of hypointense imaging artifacts, uncertainty about long-term metal toxicity, and, most important, dilution of label following cell proliferation. We are proposing a new approach for the MR detection of labeled cells using a CEST (Chemical Exchange Saturation Transfer) reporter gene. The method is based on the expression of amide-enriched artificial proteins, i.e., lysine-rich protein (LRP) and argenine-rich protein (ARP) that can be detected by CEST imaging in the nanomolar range. The advantages of using these molecular probes are: 1) the gene product can be visualized directly without the need of a substrate (no tissue penetration needed); 2) detection sensitivity is not limited by cell proliferation; 3) only live cells should provide CEST contrast; 4) the contrast can be "switched-on" and "switched-off" repeatedly; and 5) double- or triple-cell labeling strategies may be pursued. We have initial data showing that a CEST reporter gene can be cloned, expressed in transfected cells, and specifically detected by MR CEST imaging in phantoms, without affecting cell viability or proliferation. We hypothesize that this detection is also possible in vivo. To achieve this goal, our aim is to synthesize novel, more efficient CEST reporter genes, and to detect double-labeled LRP/ARP transfected glioma cells and neural stem cells individually in live animals.