| Objective: Cancer is a major public health problem around the globe, and gene therapy has shown promising results in preclinical cancer treatment in recent years. The challenge for cancer gene therapy will be to successfully delivery therapeutic gene to target tissue, and the therapeutic gene selectively, efficiency express in target tissue, which will help to achieve the goal of completely eradicating tumor cells with minimal toxicity to normal cells.Replication competent viruses, termed oncolytic viruses are essentially tumor-specific, self-replicating, and lysis-inducing cancer killer, and the replication of virus also increase the amount of therapeutic gene. For study more efficiency method of cancer gene therapy, we designed a novel chimeric oncolytic adenoviral vector armed with therapeutic gene for the treatment of aberrant wnt signaling cancer and show its higher infection efficacy, selectively replication in tumor cells, anti-tumor effect, and systemic biodistribution in vitro and in vivo.Methods: Ad5 requires the coxsackie-adenovirus receptor (CAR) on the cell membrane as a primary receptor for infection, while Ad11 requires CD46 as a primary receptor. The CAR, CD46 expression levels are different in normal tissues and tumors. Therefor, the expression levels of CAR, CD46 in different normal tissues and tumors were examined by immunohistochemistry or Real-time PCR. Infected cells with Ad5-Luc and Ad5F11b-Luc (a chimeric Ad5 with Ad11 fiber), the infection efficiency and transgene expression levels were assayed by Luciferase Assay System. Intratumoral injecting Ad5F11b-Luc or Ad5-Luc in mouse xenografts or intravenous injecting Ad5F11b-Luc or Ad5-Luc into normal nude mice, tumor local gene expression, systemic biodistribution and blood clearance properties of Ad5F11b and Ad5 vectors were analyzed by Live Cell Imaging System.To determine if aberrant wnt signaling in tumor cells can be used to selectively drive viral replication, we have analyzed?-catenin mutation status of tumor tissues as well as cell lines. And we screened for?-catenin/TCF-hyperactivated (aberrant wnt signaling) tumor cell by transfected M50 Super 8X TOPFlash plasmid which contain an luciferase expression cassette driven by 8 copies of TCF/LEF binding site (named wnt promoter). Based on these findings, a novel chimeric oncolytic adenoviral vector containing an E1A expression cassette driven by an artificial wnt promoter have been constructed to selectively replicate in aberrant wnt singaling tumor cells.For more increase the anti-tumor effect of this novel oncolytic adenovirus, an apoptosis inducing gene, interleucin-24 expression cassette driven by cytomegalovirus promoter was inserted into its E3 region. Therefore, a novel chimeric oncolytic adenoviral vector containing an E1A expression cassette driven by an artificial wnt promoter, pseudotyped fiber by replacing Ad5 with Ad11 Fiber and delivering mda-7/IL24 gene (Ad5F11.wnt-E1A-hIL24) or not (Ad5F11.wnt-E1A) was designed for treatment aberrant wnt signaling tumor. The therapeutic effect in vitro was determined by MTT, crystal violet staining, ELISA, Annexin V staining assay, and anti-tumor effect mechanism was evaluated by analysis the activation of caspase3, caspase7 and PARP using western blot assay. And the anti-tumor effect in vivo was evaluated in mouse xenograftsResults: We subjected 20 different normal human tissues to immunohistochemical analysis of CAR expression, the results showed that CAR expression high in esophagus and gastric epithelium, prostate; intermediate in intestinal epithelium, and myocytes of the heart; and low in liver, lung. Tumor tissues were subjected Real-time PCR analysis of CAR and CD46 expression, the results showed that CD46 expression levels were higher in almost all tested tumor than their adjacent normal tissues. In contrast, CAR expression levels were lower in almost all tested tumor tissues than their adjacent normal tissues (colorectal tumor:12/13; gastric tumor:10/17). The infection efficiency and transgene expression levels of Ad5F11b and Ad5 infected cells were determined in vitro using Luciferase Assay System, the results showed that the luciferase expression of Ad5F11b-Luc infected cells were significantly improved compared to non-pseudotyped Ad5-Luc control viruses. And the differential gene transduction of Ad5-Luc and Ad5F11b-Luc infected cells were further determined in vivo using Live Cell Imaging System, the results showed that the tumor local luciferase expression levels and time of intratumoral injection Ad5F11b-Luc were significantly increased compared to that of Ad5-Luc, and intravenous injection Ad5F11b-Luc exhibited a less amount and shoter-term in liver than that of Ad5. Due to the higher tumor local gene transduction and lower liver infection efficieny of Ad5F11b, it would be less side-effect than Ad5.Sequencing analysis of?-catenin mutations showed that five out of 13 (38.5%) colorectal and eight out of 17 (47.1%) gastric tumors showed point mutations in tumor tissues but not in their adjacent normal tissues. BEL7402,HCT116/4016, HCT116/379.2 also showed point mutation, but HepG2 showed deletion mutation. Cell lines carrying?-catenin mutations (HepG2, HCT116/4016 and HCT116/379.2) exhibited?-catenin/TCF- hyperactivated activity compared to cell lines with wildtype?-catenin. Ad5F11.wnt-E1A, Ad5F11.wnt-E1A-hIL24 oncolytic adenovirus can selectively replicate in aberrant wnt signaling tumor cells, and displayed a powerful efficacy in killing aberrant wnt signaling tumor cells. This selectivity cytotoxicity is cosistant to the dose of virus and its infection time. And the anti-tumor effect of Ad5F11.wnt-E1A-hIL24 was significantly improved compared to Ad5F11.wnt-E1A due to the expression of MDA-7/IL24. The therapeutic effect was associated with increased apoptosis through caspase 3 activation. Intratumoral injection Ad5F11.wnt-E1A-hIL24 resulted in profoundly inhibition of tumor growth and longterm survival.Conclusions: Aberrant wnt signaling was identified in a variety of tumors and it can be introduced to targeting cancer gene therapy. The primary receptor of Ad11 high-level expresses in a variety of tumors and Ad11 can efficiently infect tumors. Our modified chimeric oncolytic adenoviruses expressing mda-7/IL24 (Ad5F11.wnt-E1A-hIL24) could exert potential anti-tumor activity and significant apoptosis in aberrant wnt signaling tumor and offer a novel approach to cancer gene-viral therapy. |
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