This exploratory grant application is intended to support an innovative approach to the design and development of novel biomaterials for systemic cancer gene therapy using recombinant techniques. The genetic engineering, characterization and in vitro evaluation of targetable polymeric gene carriers are proposed. The carriers contain, at precise locations on the polymeric backbone, lysine (K) residues to condense plasmid DMA, a targeting ligand (fibroblast growth factor, FGF2) for biorecognition by cancer cells, histidine (H) residues to allow escape from the endosomes, and cysteine (C) residues to allow intracellular bioreduction and DMA release. Compared to chemical synthetic strategies used to-date, genetic engineering of polymers will allow a more precise correlation of structure with function.
The Specific Aims of this project are: (1) To synthesize and characterize polymeric analogs of lysine-histidine-cysteine-FGF2 by recombinant techniques. A series of copolymers with the general structure of (KHKHKHKHKKC)n-(FGF2) will be synthesized where n will be varied to evaluate the influence of molecular weight on transfection efficiency. Control polymers without the cysteine residues or targeting moieties will also be synthesized. The copolymers will be characterized for amino acid content, sequence and molecular weight. (2) To complex the copolymers with plasmid DMA and characterize the physicochemical properties of the complexes. The copolymers will be complexed with model plasmid DMA and the complexes will be evaluated for their size, charge, protection against nuclease degradation, stability and disulfide bond reduction. (3) To evaluate the transfection efficiency and cytotoxicity of the complexes in vitro. The copolymer/DNA complexes with slightly positive, neutral, and slightly negative charges, and compact structure will be evaluated for their cytotoxicity and transfection efficiency in cancer cell lines that overexpress FGF2 receptors. Results from this study will allow the correlation of the influence of targeting moiety, cysteine residues, polymer length and complex charge on transfection efficiency. In the long-term these studies will lead to the development of clinically acceptable targetable gene carriers with appropriate transfection efficiency profiles. ? ? ?
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| Ghandehari, Hamidreza (2008) Recombinant biomaterials for pharmaceutical and biomedical applications. Pharm Res 25:672-3 |
