New Class of Reporter Genes for MRI Based on Chemical Exchange Saturation Transfer (CEST)
Among the clinically applied 3D imaging modalities, magnetic resonance (MR) imaging has the highest resolution and is therefore the preferred method for detection of cellular gene expression. However, because of the low sensitivity of MR, no suitable reporter genes exist. This invention comprises a new class of reporter genes for MR imaging which do not employ paramagnetic metal substrates. The genes provide MR contrast when the sample / subject is irradiated at a specifc off resonance radio-fequency (RF). The contrast mechanism relies on chemical exchange saturation transfer (CEST). The contrast is highly dependent on the protein chemical exchange rate, which is dependent on the amino acid sequence of the protein. We have cloned and expressed in mammalian cells a prototype artifcial CEST protein (Lysine Rich Protein- LRP), which has been shown to give signifcant contrast enhancement on CEST MR images. As compared to control cells, LRP-transfected cells have shown no difrence in proliferation and metabolic rate. Other CEST reporter genes, e.g. argenine-rich-protein (ARP) are being developed. Due to the specifc radio frequencies, the CEST reporter system allows double or -triple labeling and detection strategies using difrent artifcial proteins. The CEST reporters can be applied to label and track the biodistribution and migration of mammalian cells, including hematopoietic cells, stem cells, and tumor cells. It also has the potential to be applied in developmental studies of transgenic mice, and as a reporter for activation of a specifc promoter under external regulation (ie hypoxia). The overall concept of CEST reporter genes is similar to existing endogenous reporter genes such as EGFP. However, the CEST reporter genes will permit non-invasive and repetitive monitoring with 3D whole body imaging without dependence on light penetration. Unlike conventional reporter genes in PET and bioluminescent imaging, the CEST gene is an endogenous reporter gene, and no administration of substrate is required, obviating the need of substrate-tissue penetration including crossing of the blood-brain barrier. As compared to conventional MR labeling techniques using contrast agents the CEST reporters wil allow unlimited monitoring of rapidly dividing cells, and no foreseen confounding artifacts when cells die. We foresee a fundamental use of these genes in basic research as well as its potential clinical use as reporter genes for evaluating the effciency of gene therapy and monitoring of cell therapy. Description (Set) Proposed Use (Set) The new genes can be integrated into an expression vector (plasmids, viruses) and can be used as reporter for gene delivery into cells (transftction or infection). Cells transftcted with the CEST reporter genes can also be used to track the migration and homing of transplanted or infused stem cells and hematopoietic cells. Furthermore, these genes can be used for tagging other proteins by creating a fusion protein or under IRES (internal ribosome entry site) regulation, and than can be used for screening of trans gene-expressing cells using MRI. In addition, these reporter genes can be delivered together with therapeutic genes of interest in gene therapy and then be used for assessing the effciency of gene transftr and treatment, and for imaging the transfected tissue areas. Finally, transftcted bacteria, viruses, and parasites could be studied for their biodistribution and sites of natural homing following local or systemic infection.
Van Zijl, Peter
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