Gene transfer by nonviral methodologies (i.e., lipofection) are not efficient in nondividing cell populations. Unfortunately, cells are not actively dividing in many tissues that are potential clinical targets for gene therapy. While receptor targeting, fusigenic peptides, or endosome disrupting agents help overcome some of the first barriers that limit liposome-based gene delivery, virus-free gene transfer using liposomes has limited clinical utility because of the difficulty of transporting genetic material into the nucleus of a nondividing cell. The investigators propose research to understand and potentially overcome this final rate limit to nuclear entry encountered with lipofection of nondividing cells, by developing methodologies for delivering large genetic packages into the nucleus of nondividing cells. This will be critical for the success of nonviral mediated gene therapy in vivo and for various tissue engineering applications where the low mitotic rate of target cells would greatly limit the impact of many potential therapies. This research will use cultured bovine endothelial cells, established liposome chemistries, and viral and cellular components that may facilitate nuclear penetration of plasmids. Also, epifluorescence microscopy and fluorescence spectroscopy will be used to identify cellular localization of proteins, and to quantify fluorescent reaction assay products. Liposome-mediated gene transfer is a potentially important clinical alternative to viral routes because there is less risk of immune response. From a regulatory, manufacturing, economic, and ease-of-use standpoints, liposomal routes offer many advantages over viral routes. For lipofection routes to succeed, however, a major problem to overcome is low transfection efficiency of nondividing cells. Similarly, retrovirus gene transfer may benefit from such approaches.
