A number of imaging marker genes have been developed for the much required need of detecting and quantitating gene expression in vivo. The overall goal of this exploratory research project is to investigate tyrosinase as new bifunctional therapeutic prodrug/imaging enzymatic marker system. The main reasons for choosing tyrosinase are 1) the potential for imaging tyrosinase expression by different imaging modalities including MR imaging(e.g. tyraminyl-DOTA-Gd) and nuclear imaging (e.g. labeled tyrosine), 2) the availability of several non-cytotoxic prodrugs and 3) the lack of expression in non-melanotic cells. We hypothesized that certain tyrosinase mutants could be obtained that would have low intrinsic toxicity, similar or even higher specific enzyme activity compared to the wild-type enzyme or could be positioned on the cell surface or be secreted for more efficient interaction with the prodrug. So far we have constructed three C-terminal tyrosinase deletion mutants lacking different elements of transmembrane and sorting domains. The novel tyrosinase variants indeed had higher specific enzyme activity compared to the wild type and sensitized transfected cells to hydroxyphenyl-propanol and N-acetyl-4-S-cysteaminylphenol treatments, i.e. fwo of several available model therapeutic prodrugs. We have furthermore confirmed that tyrosinase expressing cells accumulate/convert 3H-tyrosine opening the possibility of nuclear imaging using alternative isotopes (e.g. aboutC-tyrosine). Finally, we have synthesized a novel paramagnetic tyrosinase substrate (tyrarninyl-DOTA-Gd) which significantly changes R1 relaxivity after tyrosinase interaction. The proposed studies build on these preliminary data will test the main hypothesis that engineered tyrosinase mutants are subject to differential intracellular sorting and can be utilized both as efficient prodrugconverting biocatalysts and imaging markers. The long-term goal of this research is to explore and develop novel and useful bifunctional """"""""imaging/therapeutic marker genes"""""""" for in vivo use.
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