Rational Design of Degradable Polymers for Gene Delivery

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INTELLECTUAL MERIT: This project explores a strategy for the delivery of genetic material to target cells for gene therapy. Viral vectors provide one system capable of delivering DNA to the nucleus of a cell. Alternative delivery systems, free of the possible adverse effects of viral infection, are of considerable interest. An alternative strategy involves complexing and condensing the negatively charged DNA with a positively charged biocompatible polymer. The resulting "polyplex" has been shown to be capable of entering a cell, penetrating the nucleus, and releasing some portion of the complexed DNA, thus making it available for transfection. The efficiency of this process remains low, presumably because an ionically stabilized polyplex does not readily release its DNA component. In this work the PI plans to synthesize a series of cationic polymers, similar in backbone structure but differing in their susceptibility to hydrolysis of beta-thioester, beta-aminoester, beta-amino, and thioamide linkages built into the polymer backbone. Dissociation of the polyplex, once delivered to the cell, will be facilitated by degradation of the polymer through cleavage of the susceptible linkages. Rates of degradation can in principle be tuned by adjusting the proportions of hydrolysable linkages and the substitutent patterns near such linkages. The degradation kinetics and ability to complex, condense, and protect DNA will be investigated in vitro for a library of polycations. The challenge will be to tune the hydrolytic susceptibility of the polycations so that the stability of the polyplex is retained in the cytoplasm but not in the nucleus. Finally the dissociation, intracellular trafficking, and in vitro transfection efficiency of the polyplexes will be investigated in selected cell lines. The polymers will be labeled with appropriate fluorescence tags to facilitate study of trafficking within the cells using fluorescence resonance energy transfer spectroscopy.BROADER IMPACTS: The work has the potential for substantial impact in the gene therapy domain. The program includes a program of educational outreach, provision of undergraduate research opportunities, and curriculum development. The PI will work with the University of Wyoming Office of Multicultural Affairs to recruit high school students from throughout the rural Rocky Mountain area (plus Nebraska and the Dakotas) to participate in a paid , seven-week Summer Apprentice Program. Special emphasis will be placed on recruiting from the substantial population of Native Americans in the region. The PI will provide opportunities for two undergraduates to work on the project in his laboratory. He is also establishing the first course in Polymer Synthesis, Engineering, and Applications on the Wyoming campus. This will include a laboratory component that incorporates experiments in various synthetic approaches, determination of polymer molecular weights and molecular weight distributions, preparation of responsive polymers, preparation and characterization of amphiphilic block copolymers, and cellular uptake of nanoparticles prepared by the students.