Development of a Therapeutic Adenoviral Vector for Cholangiocarcinoma Combining Infectivity-Enhancement and Tumor-Restricted Gene Expression.
Abstracts
|
Background. Cholangiocarcinoma is an aggressive malignancy that is most often unresectable upon diagnosis and unresponsive to chemotherapy and radiation. Adenoviral gene therapy has shown promise in many cancers and represents a novel approach to this devastating disease. In cholangiocarcinoma, low tumor transduction efficiency and systemic toxicity have hampered the application of gene therapy. Aim. To overcome these difficulties, we have constructed an adenoviral vector utilizing infectivity-enhancement, with RGD-motif in the fiber knob, and a tumor-specific promoter (TSP), for selective gene expression. Methods and Results. In seeking a TSP for cholangiocarcinoma, adenoviral vectors that express the reporter gene luciferase under the control of six different candidate promoters were constructed. Secretory Leukoprotease Inhibitor, Midkine, Gastrin Releasing Peptide, VEGF, Cox-2M, and Cox-2L vectors were evaluated to determine relative strength and selectivity of transgene expression. This was based on the useful "tumor on/liver off" phenotype embodied in these promoters. In cholangiocarcinoma cells lines (Oz and SkCha-1), luciferase assays demonstrated that Cox-2 promoters (M and L) showed the highest transgene expression levels relative to CMV, with Cox-2M appearing slightly stronger than Cox-2L. RT-PCR analysis indicated that these cell lines were Cox-2 positive. Next, infectivity-enhanced vectors with RGD-motif in the fiber knob were constructed with the lucerifase transgene driven by Cox-2M and Cox-2L promoters. Subsequent luciferase assays comparing the unmodified vectors to the RGD-modified versions demonstrated higher levels of luciferase activity the RGD-infected cells. This concept was then applied to the HSV-TK/GCV paradigm by constructing RGD-enhanced HSV-TK vectors driven by Cox-2 promoters. MTS assay confirmed the RGD-modified, Cox-2 (M and L) driven vectors were superior to unmodified versions in specificity and cell killing efficiency. Conclusion. The achievement of tumor targeting for cholangiocarcinoma has been hampered by the absence of defined TSPs relevant to this disease. he Cox-2 promoter demonstrates a favorable selectivity profile for this disease and RGD-modification further enhances transfection efficiency. This combination holds promise in overcoming the obstacles to clinical application of gene therapy in cholangiocarcinoma. |
