119. Intraperitoneal Transduction of Adeno-Associated Virus 2 Expressing Angiostatin and Endostatin Synergistically Augments Paclitaxel Therapy and Tumor-Free Survival in a Mouse Model of Epithelial Ovarian Cancer

Abstract

We have recently demonstrated that stable expression of angiostatin and endostatin by intramuscular (i.m.) injection of recombinant adeno-associated virus 2 (rAAV) provides significant protection against the growth of human ovarian cancer cell line SKOV3.ip1 as subcutaneous implants in athymic mice. However, when a similar approach was used to control the intraperitoneal (i.p.) growth of SKOV3.ip1 cells, the effect was only moderate. Unlike organ defined solid tumors, the i.p. growth of epithelial ovarian cancer is highly disseminative, accompanied by excessive ascites and exfoliation of tumor cells in the peritoneal cavity, which limits the efficacy of drugs and other therapeutic molecules from reaching tumor cells. To overcome this limitation, in the present study, we sought to determine the effects of rAAV angiostatin and endostatin therapy by i.p. transduction. Further, the effect of i.p. rAAV therapy as an adjuvant therapy to chemotherapy was determined in athymic nude mice. Cohorts of female athymic nude mice received either no virus or 1011 particles of rAAV encoding GFP or angiostatin plus endostatin by i.p. injection. Three weeks later, the mice were challenged with 107 human epithelial ovarian cancer cell line SKOV3.ip1 i.p. Chemotherapy was administered with paclitaxel (20 mg/kg body weight). As a measure of effectiveness of the therapy, tumor weight, abdominal distension, ascites volume and vascular endothelial growth factor (VEGF) level were determined. Immunohistochemistry was performed to determine tumor cell proliferation, apoptosis, and microvessel density. Real-time PCR analysis was performed to determine the biodistribution of vector DNA in peritoneal organs. Tumor-free survival was recorded as endpoint. Results indicated that i.p. rAAV anti-angiogenic therapy alone provided anti-tumor effects, comparable to that of chemotherapy. Ascites volume in rAAV endostatin and angiostatin treated mice was significantly lower than na|[iuml]|ve mice and contained less hemorrhage and tumor conglomerates. The level of VEGF in the ascites of anti-angiogenic vector treated mice was also significantly less compared to untreated or rAAV-GFP treated mice. Immunohistochemical analyses indicated increased tumor cell apoptosis and decreased blood vasculature following rAAV endostatin and angiostatin treatment. Analysis of liver toxicity following rAAV-E+A therapy did not show significant difference from control mice without tumor injection. Interestingly, combination of rAAV-E+A and paclitaxel dramatically increased long-term tumor-free survival, which was significantly greater than either of them alone. These results indicate that anti-angiogenic gene therapy by i.p. administration of rAAV expressing angiostatin and endostatin in combination with paclitaxel therapy can be used against intraperitoneal ovarian cancer growth and dissemination.