Background: A major obstacle to improving patients' survival with advanced prostate cancer is progression of the cancer to androgen-independence. Therefore, methods such as gene therapy capable of delaying or stopping this progression may have a significant impact on improving patients' health. However, many challenges lie ahead for gene therapy, including improving DNA transfer efficiency to cancer cells, enhancing levels of gene expression, and overcoming immune responses. To address these problems, we propose the development of a gene delivery system that can (1) shield itself from the immune system, (2) target prostate cancer cells specifically, and (3) transfect and kill prostate cancer cells efficiently.Objective/Hypothesis: The objective of this research is to design and develop a nanocarrier that is able to evade the immune system, circulate in the blood stream, find its target prostate cancer cells, and transfer therapeutic genes into prostate cancer cells efficiently. The gene carrier is composed of: (a) histone H2A peptide (H2A) to condense pDNA into nano-size particles (nanocarriers), (b) a PC-3 specific targeting motif (TM) to target prostate cancer cells, (c) an endosome disrupting motif (EDM) to disrupt endosome membrane and facilitate escape of the cargo into the cytosol, and (d) a nuclear localization signal (NLS) to actively translocate pDNA towards the nucleus of cancer cells. An elastin-like polymer (ELP) has also been engineered in the vector structure to provide a hydrophilic shield and protect the vector/pDNA complex in the blood stream from the immune system.Specific Aims: The specific aims of this project are:1) Biosynthesize the recombinant vector (i.e., EDM-H2A-NLS-ELP-TM) in E. coli, complex with pDNA, and characterize nanoparticles.2) Inject the nanocarriers in mice bearing xenograft tumor model of prostate cancer and evaluate the transfection efficiency and therapeutic efficacy.3) Examine the immune system response to the shielded nanocarriers in male immuno-competent mice.Study Design:Study design for Specific Aim 1: The vector will be biosynthesized in E. coli expression system using recombinant DNA techniques and purified. The vector will be characterized in terms of purity, expression, exact molecular weight, and amino acid composition. The purified vector will be complexed with pDNA to form nanoparticles and characterized in terms of size and charge at various pH (5-8), temperature (25- 37oC), and salt concentrations. The in vitro gene transfer efficiency of vector/pDNA (pEGFP and pCMV-luc) complexes in PC-3 human prostate cancer and RWPE-2 normal epithelial human prostate cells will be evaluated. The vector toxicity will be evaluated in normal epithelial prostate cells (RWPE-2).Study design for Specific Aim 2: To evaluate transfection efficiency in vivo, vectors will be complexed with pCMV-luc to form nanocarriers and injected systemically in nude mice bearing xenograft tumors of prostate cancer (PC-3). The luciferase expression in tumors and other tissues will be measured using an in vivo imaging system. To evaluate therapeutic efficacy, plasmid DNA encoding HSV-TK (pSR39) will be complexed with the targeted vector and injected systemically in nude mice bearing xenograft tumors of prostate cancer (PC-3) followed by GCV administration. The tumor size reduction will be measured using a pressure-sensitive caliper. Apoptosis in tumors will be measured by apoptosis assay.Study design for Specific Aim 3: To test the immunogenicity of the vector, the production of anti-vector antibodies will be evaluated in intact male C57Bl/6 mice, in which the immune system is fully functional and can develop immune response.Innovation: The innovative aspect of this project rests in the novel design of the vector and application of the ELP as a non-immunogenic shielding motif along with prostate-specific ligand. Genetically engineered elastin is a derivative of human tropoelastin, which is an extracellular matrix protein known to be non-immunogenic. This shield will not only mask the surface of the nanoparticles from the MPS but will also increase the circulation time. The proposed delivery system is designed to address both safety and efficiency problems.Impact: This technology can be used to not only treat but image prostate cancer in one platform. Furthermore, this technology could easily evolve into a multi-platform nanosystem to image and treat various sub-populations of prostate cancer cells expressing different biomarkers. In addition, in combination with cytotoxic drugs, this system can be custom designed and equipped with various targeting motifs to target and kill cancer stem cells which are hypothesized to be the source of cancer recurrence.
|Effective start/end date||7/1/09 → 7/31/13|
- Congressionally Directed Medical Research Programs (CDMRP)
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