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Development of a DNA breast cancer vaccine
Tumor Cell Biology III
DNA vaccines for cancer is attractive and has been a goal for some time. Viral vectors are popular due to having high transfection efficiency. However, there are many disadvantages. A non-viral vector is a safer option. Unfortunately, most of them do not offer high transfection efficiency. We have studied extensively the use oxidized mannan (oxman) (targeting the mannose receptor - MR) as a carrier for protein-antigen vaccines. By using a cationic polymer conjugate with oxman we may more efficiently deliver MUC1 DNA (MUC1 a protein present on >95% of breast cancers) into MR cells by fast tracking the DNA from the endosome into the cytoplasm. The specific aims are: 1. Optimization of MUC1-DNA complexation to oxman-PLL 2. Optimization of cationic polymer linkers between MUC1 DNA and oxman 3. In vivo efficacy of oxman-linker-MUC1 DNA complexes Research plan: Aims 1 and 2: In our preliminary studies we conjugated oxman to DNA using a PLL intermediary. We achieved 50% complexation efficiency and intend to optimize the complexation by modifying ratio of PLL:DNA and salt concentration. We will also use cationic polymers, PEI and starburst dendrimers as linkers between DNA and oxman. All the conjugates will be compared for efficiency of conjugation, in vitro eGFP transgene expression, duration and amount of transgene expression by DC, macrophages, cell lines, using flow cytometry, immunofluorescense and fluorimetry. Aim 3: The in vivo immune responses of the optimized conjugates will be tested. A number of variables such as dose, route and frequency of immunization will be investigated. Immune responses will be monitored using ELISpot, proliferation, CTL, CTLp and ELISA and tumor protection / therapeutic experiments will be performed. Outcome: The outcome will be a new DNA vaccine strategy based on a non-viral gene delivery method targeting MR cells (DCs). This method will be applicable to any tumor antigen and in this proposal the outcome is a DNA vaccine for MUC1 expressing adenocarcinomas. This will form the basis of a future clinical trial. The ability of oxman to selectively induce CD4/CD8 responses depending on the dose will remove the necessity to introduce cytokines either by co-injection or bi-cistronic DNA vectors to alter immune responses. A direct utility of this technology will be for the development of gene therapies (not in this grant) which introduces genes expressing cytokines, enzymes and inhibitors for the treatment of various diseases.
The incidence of breast cancer in women is 1 in 8 and the frequency of other cancers are rising. Even with conventional approaches such as surgery, cytotoxic therapy, radiotherapy and combination therapy only a few cancers are treatable. The development of a cancer vaccine will greatly benefit humanity similar to childhood and adult vaccinations for preventing infectious disease. We have previously generated a vaccine for breast and other adenocarcinomas by linking a breast cancer associated protein, MUC1, to a sugar called mannan. This complex was capable of eradicating tumors in mice and its efficacy has been evaluated in human clinical trials (12 in total). As an extension to these studies we have now found that this sugar, mannan, when combined with DNA expressing a model antigen, is also very effective in inducing enhanced immune responses in mice. The current project will further investigate the DNA vaccine incorporating MUC1. In addition, we will optimize the various components of the vaccine to further increase its effectiveness. The optimized vaccine will then be used in animal models of breast cancer to evaluate its efficacy. If the studies are successful the DNA vaccine can be further developed for clinical trials.