Construction of non-viral (synthetic) DNA vectors has been a major focus in gene therapy research in recent years. Several novel formulations of synthetic vectors have been developed in the applicant's laboratory including pH-sensitive immunoliposomes, DC-chol cationic liposomes, lipopolylysine, liposome/polylysine/DNA ternary complexes (LPDI), etc. Recently, the applicant developed a novel targetable DNA vector which consists of a polycation-condensed DNA core and an anionic lipid coating (LPDII). Preliminary data show that negatively charged LPDIIs are compact (70-80 nm in diameter) non-aggregating particles which can be conjugated to a targeting ligand for highly efficient and tissue-specific transfection in cultured breast cancer cells. The high-affinity receptor for folate (Kd approximately 1 nm) has recently been identified as a marker for a variety of human tumors including breast carcinomas and is found to be absent in most normal tissues. Folate was chosen as the targeting ligand for tissue-specific delivery to breast cancer due to its small size, stability, and possible lack of in vivo immunogenicity. There are three main objectives for the proposed study. First, a folate-targeted LPDII vector will be optimized for reporter gene delivery in vitro. MDA231 cells, a breast cancer cell line overexpressing the folate receptor, and a plasmid carrying the luciferase reporter gene will be used. Factors such as polylysine-chain length, lipid composition and the incorporation of a fusogenic and/or nuclear localization signal peptide on the transfection activity and other vector properties will be examined. Secondly, the folate-targeted LPDII will be optimized for tissue-specific gene transfer in vivo. Biodistribution and tissue-specific expression of the reporter genes will be examined in normal mice and nude mice carrying xenographic implants derived from MDA231 cells following intravenous injection of the vector. The effect of lipid composition and PEG-coating on the plasma stability, specificity and the transfection efficiency of the vector will be examined. Finally, the applicant will co-administer intravenously a plasmid carrying the herpes simplex thymidine kinase (HS-TK) gene in the optimized tumor-specific LPDII vector and the antiviral drug ganciclovir into the tumor-carrying animals. Treatment protocols will be varied to achieve maximum tumor suppression. These studies should allow the applicant to critically evaluate the potential for the use of folate-targeted LPDII vectors in tumor-specific gene transfer and the gene therapy treatment of human breast cancer.
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