DEVELOPMENT OF ANTI-HPV LIPOPLEXES FOR THE TREATMENT OF CERVICAL CANCER
Lechanteur A1, Tania Furst1, Brigitte Evrard1, Patrick Roncarati2, Philippe Delvenne2, Géraldine Piel1,
Pascale Hubert2
1
Laboratory of Pharmaceutical Technology-CIRM, University of Liège, Liège, Belgium
2
GIGA-CANCER, Laboratory of Experimental Pathology, University of Liège, Liège, Belgium
Human Papillomaviruses (HPV) are responsible for several diseases and some of them (such as HPV16
and HPV18) can induce cervical cancer. In this case the two HPV E6 and E7 oncoproteins are essential
players in order to immortalize keratinocytes by decreasing tumor suppressor genes (p53 and pRb).
Nowadays cervical cancer is known to be the third most frequent cause of death in women and only
prophylactics vaccines exist. Except classical cancer therapy (surgery and radio/chemotherapy), there
is no more treatment if the lesion is already developed.
We would like to find a therapy based on RNA interferences (siRNA). These small interfering RNA can
target mRNA coding for both HPV E6 and E7 oncoproteins and thereby induce apoptosis by increasing
tumor suppressor gene expression. siRNA would be encapsulated in cationic lipidic nanovectors to
form lipoplexes. This association is essential to protect siRNA, to allow the diffusion into the vaginal
mucus and to cross the anionic cellular membrane.
The aim of this study is to develop a local treatment of cervical cancer by long-acting siRNA antiE6
and/or antiE7 that would be encapsulated in cationic nanovectors.
The first step was to choose two siRNA sequences and to check their efficacy on SiHa and CaSki cells
by the use of a commercially transfection agent (Oligofectamine®). We observed that:
The percentage of transfection of both cell lines is high (FACS analysis)
The extinction of E6 and E7 mRNA is very efficient (qPCR analysis)
An efficient decrease proliferation of cells (Alamar Blue test)
The both sequences induce an more or less efficient apoptosis of cells (Apostain
ssDNA test)
Western blot will be performed to follow the E6/E7/p53 proteins level.
The second step was to replace Oligofectamine® by lipoplexes. Liposomes were formed with three
lipids: DOTAP, DOPE and cholesterol at different N/P ratio. Lipoplexes allow a better transfection than
Oligofectamine®. Nevertheless, the extinction of E6 and E7 mRNA seems to be lower probably
because of a worst leakage of siRNA in cytoplasm.
In conclusion, even if the results are hopeful, some other tests will have to be done to confirm
apoptosis. To enhance the rate of cell death, we will trigger an anti-apoptotic protein with another
siRNA. Moreover, we would like to analyze the effect of siRNA on a 3D model of cervical lesion.
Finally, the formulation of lipoplexes will have to be optimized to enhance the E6 and E7 mRNA
extinction.
1
Jiang, M. (2002) Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated
with siRNA, a primer of RNA interference. Oncogene. 21, 6041-6048
2
Chang, JT-C. (2010) Highly potent and specific siRNAs against E6 or E7 genes of HPV16- or HPV18-infected cervical
cancers. Cancer Gene Therapy. 17, 827-836
3
4
Yang, S. (2012) Advancements in the field of Intravaginal siRNA Delivery. Journal of Controlled Release. 167, 29-39
Kapoor, M. (2012) Physicochemical characterization techniques for lipid based delivery systems for siRNA.
International Journal of Pharmaceutics. 427, 35-57