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 (siRNA E6 and siRNA E7). Moreover, we targeted an anti-apoptotic protein (MCL-1) to increase apoptosis of HPV 16 positive cells. MCL-1 protein is an interesting target: this protein is overexpressed in high grade lesions compared to low grade lesions and healthy cervix.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. DEVELOPMENT OF ANTI-HPV LIPOPLEXES FORMULATIONS FOR THE TREATMENT OF CERVICAL CANCER Anna Lechanteur 1,2, Tania Furst 1, Brigitte Evrard 1, Philippe Delvenne 2, Géraldine Piel 1, Pascale Hubert 2 1 Laboratory of Pharmaceutical Technology and Biopharmacy - CIRM, University of Liege, Liege, Belgium 2 Laboratory of Experimental Pathology, GIGA-Cancer, University of Liege, Liège, Belgium 1. INTRODUCTION 2. RESULTS AND DISCUSSION 3. CONCLUSION / PERSPECTIVES 000 Figure 1 – siRNA E6 and siRNA E7 alone or in combination induce the extinction of mRNA E6 and mRNA E7. Interestingly, siRNA E6 turn off mRNA E6 and mRNA E7. In the same way, siRNA E7 turn off mRNA E7 and mRNA E6. Best extinctions are significantly obtained by the combination of both siRNA. Results presented here wereperformed on CaSki cells but had been also performed on SiHa cells. Cells were transfected with Oligofectamine ® (as transfection agent) and siRNA at 100nM. Two days after transfection, they were harvested and analyzed by qRT-PCR assay (SYBR Green detection). Data were compared to Mock samples (siRNA scramble). Figure 2 – siRNA E6 and siRNA E7 induce the reactivation of p53 protein traduction. CaSki and SiHa cells were transfected with Oligofectamine ® (as transfection agent) and siRNA at 100nM. Two days after transfection, they were harvested and analyzed by Western Blot assay. Β-Actine antibody was used as controlled protein.  Combination of siRNA E6, siRNA E7 and a siRNA MCL-1 (an anti-apoptotic protein) induces cells apoptosis and decreases cell proliferation. Figure 3 – siRNA E6, siRNA E7 and siRNA MCL-1 induce cells apoptosis and decrease cells proliferation. The number of cells dramatically decreases and also, they were in severely suffering. Results presented here were performed on SiHa cells after two days but, have been also performed on CaSki cells and until four days. Blank SiHa cells are represented on the left and on the right, cells were treated with the combination of siRNA E6, E7 and MCL-1 at 100nM. Control SiHa cellssiRNA E6 + siRNA E7 + siRNA MCL-1 Figure 4 – The combination of siRNA E6, siRNA E7 and siRNA MCL-1 is significantly more effective on proliferation and apoptosis compared to binary combination (siRNA E6 + siRNA E7). alamar Blue ® is used for proliferation assay and FITC Annexin V Apoptosis Detection kitI (Flow cytometry assay) is used to measure apoptosis. After four days, 16% (±3,6) of SiHa cells remain in proliferation with the three siRNA compared to 41% (±6,3) with the two siRNA. Moreover, on the few remaining cells, 65% (±11,1) are in apoptosis with the three siRNA compared to 56% (±16) with the two siRNA. On CaSki cells, at day four, 31% (±8,6) and 50% (±10) of cells proliferate for siRNA E6 + siRNA E7 + siRNA MCL-1 and siRNA E6 + siRNA E7 respectively. About apoptosis, there is no difference between binary or ternary combinations. Proliferation assay Apoptosis assay B) Lipoplexes formulations cross cellular membrane and release siRNA into the cytoplasm. (For more information on lipoplexes characterizations see Poster n° Tania FURST) Figure 5 – Lipoplexes DOTAP/DOPE/Chol 1/0,5/0,5 (N/P 2,5) cross easily the cellular membrane of CaSki cells. These results are obtained by flow cytometry (FACS Canto II) with a fluorescent siRNA, one day after transfection. At the concentration of 50nM, transfection efficiency is similar to Oligofectamine ® at 100nM. Moreover, the Mean Fluorescence Intensity is higher with lipoplexes 100nM compared to Oligofectamine ® 100nM but similar with lipoplexes 50nM. Figure 6 – Lipoplexes can release siRNA into the cytoplasm. Indeed, siRNA E6 was transfected compared to Mock sample (siRNA scramble) and CaSki cells were analyzed two days after transfection. As previously seen in Figure 1, siRNA E6 induce the extinction of mRNA E6 and mRNA E7 (qRT-PCR assay). With Oligofectamine ® 100nM, 28% (±6,1) and 40% (±10,2) of mRNA E6 and mRNA E7 respectively, remains active. With lipoplexes 50nM, 44% (±9,7) and 46% (±9,7) of mRNA E6 and mRNA E7 respectively, remains active.  siRNA E6 and siRNA E7 decrease expression of oncoproteins E6 and E7 and activate the re-expression of p53 protein on CaSki and SiHa cells.  The combination of siRNA E6, siRNA E7 and siRNA MCL-1 induce apoptosis and reduce dramatically proliferation of CaSki and SiHa cells.  Lipoplexes formulation (50nM) transfect a high percentage of CaSki cells.  Lipoplexes formulation (50nM) release siRNA into the cytoplasm and induce the extinction of mRNA.  Action of lipoplexes formulation on p53 protein, apoptosis and proliferation will be performed (with one or several siRNA)  Moreover, lipoplexes will be test on a 3D model of cervical lesion.  PEG polymers will be added on lipoplexes (for vaginal administration) A) Validation of siRNA efficiency with Oligofectamine ® on two HPV16 positive cell lines.  siRNA E6 and E7 induce mRNA E6 and mRNA E7 extinction and reactivation of p53 traduction on CaSki and SiHa cells. Blank Mock siE6 siE7 siE6+siE7 p53 β Actine CaSki SiHa