Project Summary Breast cancer is the most common cancer in women in the U.S. The overall cure rate for breast cancer has shown only slight improvement over the past decade. Chemotherapy is the primary therapy for treating breast cancer patients along with surgery and/or radiation therapy but is limited by tumor resistance to the therapy and the lack of specificity in drug delivery causing systemic toxicity. Both cancer immune and gene therapies have shown promises in circumventing the drug resistance acquired by many breast cancers. However, immune cancer therapy is hampered as tumors gradually become less amenable to immune recognition and endogenous tumor-specific T cells become less active in tumor destruction over time. The gene therapy is hindered by the challenges in developing delivery vehicles that must simultaneously meet a series of stringent requirements, including, in particular, non-toxicity, colloidal stability, small hydrodynamic size, long serum half- life, and an ability to overcome both extracellular and intracellular barriers and to selectively transfect tumor tissues. In this project, we propose to develop a non-viral multifunction nanovector with imaging, dual targeting, and dual therapy capabilities to address the limitations of current breast cancer therapies. The dual targeting is configured to deliver therapeutic genes specifically to breast cancer cells by use of a tumor targeting ligand and to induce gene expression only in breast cancer cells via selectivity of a tumor-specific promoter (TSP). By integrating both spleen tyrosine kinase (SYK) and interferon gamma (IFNγ) genes in our design, the nanovector will simultaneously inhibit tumor growth and activate T cells for tumor eradication. The nanovector is formulated to overcome both extra- and intra-cellular barriers and to minimize potential toxicity. The superparamagnetism of the iron oxide core of the nanovector will allow for treatment monitoring by MRI. We have three specific aims: 1) Develop a colloidally stable and biologically safe non-viral nanovector composed of an iron oxide core and a biodegradable polymer shell to complex with plasmid DNAs and study its ability to overcome intracellular barriers for gene transfection. (2) Isolate TSPs from breast cancer cells and clone them into RFP encoding plasmids, and evaluate gene transfection efficiency, dual-targeting capability and cytotoxicity of the nanovector in breast cancer cells in vitro. (3) Construct therapeutic nanovectors using genes SYK, IFNγ, and their combo to replace the RFP gene in the nanovector developed in Aims 1 and 2 and investigate their therapeutic functions in both xenograft and transgenic mice in vivo. If successful, this synergetic strategy could substantially improve the treatment outcome and minimize deleterious side effects, and can be potentially generalized to develop gene vectors with different targeting ligands and specific promoters for combating various malignancies.