Gene silencing with siRNA targeting E6/E7 as a therapeutic intervention in a mouse model of cervical cancer

Objective. Selective silencing of HPV oncogenes using short interfering RNA (siRNA) blocks E6/E7 expression and restores normal p53 and Rb function. Our objective was to determine if siRNA targeting E6/E7 would inhibit the growth of established tumors in a mouse model of cervical cancer. Methods. In vitro studies were performed using unique siRNA sequences to confirm their ability to target and reduce E6/E7 mRNA and restore functioning p53. Next, siRNA targeting lamin was injected daily for three days into tumors established from HPV 16 positive CaSki human cervical cancer cells. Immunohistochemistry and branched DNA gene quantification were used to determine distribution and duration of activity of these siPNA. For our therapeutic studies tumors were directly injected with siRNA targeting E6/E7, non-targeting control siRNA, or saline. In preliminary experiments injections were daily or every three days for a total of three doses. A second therapeutic experiment utilized every three day dosing for 35 days. Tumor volume, growth curves and E7 mRNA levels were assessed. Results. The two most active siRNA sequences resulted in a 67% and 71% reduction in E6/E7 mRNA. Fluorescent lamin siRNA was visualized up to 120 h after the initial tumor injection and was evenly distributed throughout the tumors. IHC showed lamin expression to be inhibited by 68% and 75% when compared to controls at 54 and 120 h respectively. In our preliminary therapeutic intervention experiments there was no significant difference in tumor growth between the treatment groups when mice were treated with three daily injections (p = 0.41). However, when treated every third day for three injections final tumor volume was less in animals injected with siRNA sequences A (78% reduction; p<0.0001) and G (60% reduction; p = 0.005) compared to saline injection. Tumors showed a corresponding decrease in E6/E7 mRNA. Extended treatment with siRNA completely or nearly eradicated tumors in 70% of the animals. Conclusion. Therapeutic siRNA targeting E6/E7 significantly inhibits tumor growth in this mouse model of cervical cancer. Further investigation is needed to determine optimal dosing and route of delivery. (C) 2008 Elsevier Inc. All rights reserved.