RESULTS1) Oligofectamine ® allows effective transfection of siRNA scramble

2) siE6 and siE7 decrease mRNA encoding for both oncoproteins

3) siE7 and association of siE6+siE7 induce apoptosis on CaSki cells

4) Lipoplexes (DOTAP/Chol/DOPE 1/0,5/0,5 and 2/0,5/0,5 at N/P=5) can transfect CaSki cells (siRNA scramble = 100nM)

DEVELOPMENT OF ANTI-HPV LIPOPLEXES FORMULATIONS FOR THE TREATMENT OF CERVICAL CANCER

Contact : anna.lechanteur@student.ulg.ac.be

Anna Lechanteur1, Tania Furst1, Brigitte Evrard1, Philippe Delvenne2, Patrick Roncarati2, Geraldine Piel1, Pascale Hubert2

1Laboratory of Pharmaceutical Technology and Biopharmacy-CIRM, 2Laboratory of Experimental Pathology, GIGA-CANCER

University of Liege, Liège, Belgium

INTRODUCTION – PURPOSE

RESUTLSFIRST TARGET: Oncoproteins E6 and E7.siRNA E6 and siRNA E7 induce the extinction of mRNA E6 and E7 AND decrease the expression of oncoproteins.

SECOND TARGET: Anti-apoptotic protein MCL-1 siRNA anti-MCL-1 used in combination with siRNA E6 and siRNA E7.MCL-1 protein is an interesting target: this protein is overexpressed in high grade lesions compared to low grade lesions and healthy cervix.

The combination of 3 siRNA decreases proliferation and induces apoptosis on SiHa cells (HPV16+).

LIPOPLEXES can cross the cellular membrane LIPOPLEXES can release siRNA into the cytoplasm

Figure 1 – Extinction of mRNA E6 Figure 2 – Extinction of mRNA E7

Figure 13 - Extinction of mRNA E7

- Transfection agent: Oligofectamine®

- Cell line: CaSki (HPV16+)- qPCR analysis: SYBR Green detection- siRNA concentration:100nM- 2 days after transfection- n=5

- 2 days after transfection- Cell line: CaSki (HPV16+)- qPCR Analysis (SYBR Green detection)- siRNA anti-E6- n=4

Figure 7 - SiHa cells two days after the transfection of siE6 + siE7 + siMCL-1

Figure 6 - SiHa (blank) day 2 Figure 8 - Proliferation assay during four days Figure 9 - Apoptosis assay during four days

- Transfection agent: Oligofectamine® - Cell line: SiHa (HPV16+)- siRNA concentration:100nM- Proliferation compared to Blank: alamar Blue®

- Apoptosis: FITC Annexin V Apoptosis Detection kitI- n=2

Figure 3 - Normal exocervix Figure 4 - High grade Squamous Intra-epithelial Lesion = HSIL Figure 5 - Score of MCL-1 in different cervical biopsies

- Immunohistochemistry staining (cervical biopsy)-Antibody: MCL-1

- EXO N: Normal Exocervix

- LSIL: Low grade Squamous Intra-epthelial Lesion

- HSIL: High grade Squamous Intra-epthelial Lesion

- SCC: Squamous Cell Carcinoma

Interestingly, siRNA E6 turn off mRNA E6 and mRNA E7. In the same way, siRNA E7 turn off mRNA E6 and mRNA E7. Best extinctions are obtained when siRNA are used in combination.

Figure 12 - Extinction of mRNA E6

CaSKi cellsOligofectamine® siRNA 100nM

Lipoplexes siRNA 50nM

77% 98%

- Transfection time: 24 hours- Cell line: CaSki (HPV16+)- FACS Canto II- siRNA scramble fluorescent- Lipoplexes DOTAP/DOPE/Chol 1/0,5/0,5

- N/P ratio: 2,5- For Oligofectamine and Lipoplexes: - Mean Fluorescence Intensity: ±4000 - death cells: 5% (Fixable Viability Stain 450)

CONCLUSIONS / PERSPECTIVES1)Selected siRNA anti-E6 and anti-E7 can induce apoptosis and decrease proliferation of HPV16+ cells (by their action against mRNA and proteins). 2)siRNA against anti-apoptotic MCL-1 protein allow higher induction of apoptosis on SiHa cells. The effect of this siRNA must be tested on CaSki cells.3)First result obtained with lipoplexes indicate that they can cross the cellular membrane and release siRNA into the cytoplasm. Other tests will be performed to analyze the toxicity on healthy cells. 4)We would like to analyze the effect of siRNA on a 3D model of cervical lesion.

Human Papillomaviruses (HPV) 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). Gene therapy is a promising strategy to treat cancer in order to decrease side effects against healthy cells. We focused on RNA interference (siRNA) to target mRNA coding for both HPV E6 and E7 oncoproteins. Moreover, we targeted an anti-apoptotic protein (MCL-1) to increase apoptosis of HPV 16 positive cells. To protect siRNA, to allow the diffusion into the cervical mucus and to cross the anionic cellular membrane, we use nanotherapy: siRNA is encapsulated in lipidic nanovectors to form LIPOPLEXES.