Genetic Engineering for Production of Curcumin in Human Cells

Exposure to space radiation in long-term missions is estimated to increase the chance of lethal cancer 5-21%. Efforts to reduce radiation exposure to permissible limits include engineering countermeasures via shielding materials and time management. Currently, no biological countermeasures to mitigate or prevent the detrimental effects of radiation exposure in space have been approved. Curcumin, a polyphenol extracted from the rhizome of several curcuma plant species, has been studied extensively in recent years for its potent antioxidant and radioprotective properties. However, the ability for curcumin to act as an effective therapeutic agent is mainly limited by its very poor bioavailability. The overall goal of this proposal is to develop a non-viral gene therapy approach for the local production of curcumin in mammalian cells. By producing curcumin within the cellular environment, bioavailability may be increased and larger therapeutic effects may be realized. The specific aims of this project are to: (1) Develop polyplexes (polymer-DNA complexes) that enable efficient transfection of mammalian cell lines and facilitate local production of curcumin as determined in vitro. A series of cationic polymers will be tested for their ability to condense plasmid DNA into nanoparticles with size and charge that permit endocytosis. Plasmid DNAs that encode for the five proteins necessary in the production of curcumin will be complexed with the polymers, administered to model cell lines, and evaluated for subsequent protein expression. (2) Optimize the ratio of plasmid delivery and evaluate the cytotoxicity of produced curcumin in vitro. The five plasmids will be delivered in varying ratios to tune curcumin production levels. Cell viability levels will be evaluated across the resulting range of production levels to ensure no cytotoxic responses are observed. (3) Evaluate the antioxidant activity and effects on cancer cell migration and proliferation of locally produced curcumin in vitro. Serum and lysates from cells will be assayed to evaluate the antioxidant potential of the produced curcumin across the concentration standards developed in Aim 2. MDA-MB-231 cells will be transfected with the appropriate ratios of plasmids to determine the effects of local curcumin production on cancerous cell migration.