DEVELOPMENT OF ANTI-HPV LIPOPLEXES FOR THE TREATMENT OF CERVICAL CANCER
Lechanteur A1 , Tania Furst1, Brigitte Evrard1, Patrick Roncarat2, Philippe Delvenne2, Géraldine Piel1,
Pascale Hubert2
1Laboratory of Pharmaceutical Technology-CIRM, University of Liège, Liège, Belgium 2GIGA-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 essental players in order to immortalize keratnocytes 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 prophylactcs 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 associaton is essental 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-actng siRNA antE6 and/or antE7 that would be encapsulated in catonic 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 transfecton agent (Oligofectamine®). We observed that:
- The percentage of transfecton of both cell lines is high (FACS analysis) - The extncton of E6 and E7 mRNA is very efficient (qPCR analysis) - An efficient decrease proliferaton 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 rato. Lipoplexes allow a better transfecton than Oligofectamine®. Nevertheless, the extncton 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 ant-apoptotc protein with another siRNA. Moreover, we would like to analyze the effect of siRNA on a 3D model of cervical lesion. Finally, the formulaton of lipoplexes will have to be optmized to enhance the E6 and E7 mRNA extncton.
1 Jiang, M. (2002) Selectve silencing of viral gene expression in HPV-positve 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 Yang, S. (2012) Advancements in the field of Intravaginal siRNA Delivery. Journal of Controlled Release. 167, 29-39
4 Kapoor, M. (2012) Physicochemical characterizaton techniques for lipid based delivery systems for siRNA.
