Polyethylenimine (PEI) aerosol delivery is non-invasive direct delivery to the airway epithelial and other pulmonary surfaces, which potentially avoid problems associated with intravenous delivery such as interactions with serum proteins and difficulties penetrating endothelial barriers. The primary goal of this proposal is to test the efficacy of PEI aerosol delivery of ssDNA expression vector for the production of PKC( targeting DNA enzyme in a mice tumor metastasis model. 1. Production of PKCa targeting DNA enzyme in cells by ssDNA expression vector. B16-F10 melanoma cells will be transfected with the expression vector, pssXE(PKC)DZ or the control vector, pssXE(PKC)DZm and the expression of PKCa targeting DNA enzyme in B16-F10 melanoma cells will be assessed using primer extension assay. If successful, the effect of DNA enzyme produced in cells on the level of PKCalpha gene expression will be then assessed by both Northern and Western blotting analysis. 2. PEI aerosol delivery of PKCalpha targeting DNA enzyme expression vector. C57BL/6 mice will be injected through the lateral tail vein with 25,000 B16-F10 melanoma cells. The mice will then be treated with PEI-p53 aerosol complexes twice a week starting the day after inoculation of the cancer cells. The experiment will be terminated as soon as the control animals start dying. Tumor metastasis will be examined under a dissection microscope for tumor foci in lungs. Control groups will include untreated mice, mice treated with PEI or with PEI-control vector, pssXE(PKC)DZm complexes. The efficacy of PKCalpha targeting DNA enzyme produced by pssXE(PKC)DZ will also be evaluated against tumors already shown to be responsive to aerosol treatment using PEI-based formulations of antitumor gene p53. At the completion of this study, our expectation is that we will have established the ability of PEI aerosol delivery system for delivering single-stranded DNA expression vector for the production of PKC-( DNA enzyme in vivo that will inhibit tumor metastasis. The success of this work may lead to a new tool for gene down regulation in vivo and may eventually provide a new approach for the treatment of metastatic tumor.
