Ovarian cancer gene therapy using HPV-16 pseudovirion carrying the HSV-tk gene

Abstract

Ovarian cancer is the leading cause of death from all gynecological cancers and conventional therapies such as surgery, chemotherapy, and radiotherapy usually fail to control advanced stages of the disease. Thus, there is an urgent need for alternative and innovative therapeutic options. We reason that cancer gene therapy using a vector capable of specifically delivering an enzyme-encoding gene to ovarian cancer cells will allow the cancer cell to metabolize a harmless prodrug into a potent cytotoxin, which will lead to therapeutic effects. In the current study, we explore the use of a human papillomavirus (HPV) pseudovirion to deliver a herpes simplex virus thymidine kinase (HSV-tk) gene to ovarian tumor cells. We found that the HPV-16 pseudovirion was able to preferentially infect murine and human ovarian tumor cells when administered intraperitoneally. Furthermore, intraperitoneal injection of HPV-16 pseudovirions carrying the HSV-tk gene followed by treatment with ganciclovir led to significant therapeutic anti-tumor effects in murine ovarian cancer-bearing mice. Our data suggest that HPV pseudovirion may serve as a potential delivery vehicle for ovarian cancer gene therapy.

Figure 1. Comparison of HPV pseudovirion infectivity in naïve and murine ovarian tumor-bearing mice.

5–8 weeks old C57BL/6 mice were challenged with MOSEC tumor cells (1×10⁶ cells/mouse). One week later, tumor-bearing mice were intraperitoneally injected with or without HPV-16/luc pseudovirions (20 µg HPV-16 L1 protein/mouse, equivalent to 120 ng of DNA/mouse). Naïve mice infected with or without HPV-16/luc pseudovirions served as a control. Mice were imaged by non-invasive luminescence imaging 1 day after infection. Data shown are representative of 2 experiments performed.

Figure 2. Characterization of the infectivity of HPV pseudovirion in naïve nude mice and human ovarian tumor-bearing nude mice.

5–8 weeks old nude mice were injected intraperitoneally with ES2 human ovarian tumor cells (1×10⁶ cells/mouse). One week later, tumor-bearing mice were intraperitoneally injected with wild-type (wt) HPV-16/Luc psV or mutant L2 (mtL2) HPV-16L1mtL2-Luc pseudovirions (20 µg HPV-16 L1 protein/mouse, equivalent to 120 ng of DNA/mouse). Naïve mice infected with wt or mt HPV-16/luc pseudovirions served as controls. Mice were imaged by non-invasive luminescence imaging 1 day after infection. Data shown are representative of 2 experiments performed.

Figure 3. HPV-16/luc psV preferentially infects ovarian tumors in MISIIR-TAG transgenic mouse.

C57BL/6 mice and MISIIR-TAG transgenic mice were injected with 20 µg of HPV-16/luc psV by intraperitoneal injection 10 weeks after birth. Luminescence images were taken 1 day after HPV-16/luv psV injection. Top, Representative luminescence images of psV-infected C57BL/6 mice or MISIIR-TAG transgenic mice (left) and their harvested organs (right). Note: White arrow indicates only ovarian cancer from MISRII transgenic mice can be infected by HPV-16/luc psV. Bottom, Representative bar graphs of luminescence imaging in MISIIR-TAG transgenic mice or C57BL/6 mice. Data representative of 2 experiments performed.

Figure 4. In vitro cytotoxicity mediated by HPV16-tk pseudovirions and ganciclovir.

MOSEC-Luc cells were infected HPV16-GFP psV or HPV16/HSV-tk psV at a concentration of 1 µg L1 protein/ml for 48 hours. The infected cells were seeded in 96 well plates and then treated with 0 µg/ml, 0.1 µg/ml, 1 µg/ml, or 10 µg/ml of Ganciclovir for 72 hours. Luciferase expression was examined by the IVIS 200 system (Xenogen Corp., Alameda, CA, USA). Data shown are representative of 2 experiments performed.

Figure 5. In vivo cytotoxicity mediated by HPV16-tk pseudovirions and ganciclovir.

C57BL/6 mice (5 per group) were intraperitoneally injected with 1×10⁶ MOSEC-Luc cells per mouse on day 1. Luciferase activity was examined on day 2 as indication of number of tumors in the mice. Mice were injected HPV16-GFP psV (L1 protein 20 µg) or HPV16/HSV-tk psV at a dose of L1 protein (20 µg/mouse) on day 3. Mice were treated daily with ganciclovir at a dose of 50 mg/kg or PBS from day 5 to day 18. Mice were imaged by non-invasive luminescence imaging techniques on day 20. Data shown are representative of 2 experiments performed.