1. Development and optimization of lipoplex vectors for an antisens therapeutic approach in the context of HPV induced lesions Contact : anna.lechanteur@student.ulg.ac.be Anna Lechanteur 1,Brigitte Evrard 1,Philippe Delvenne 2, Patrick Roncarati 2,Pascale Hubert 2, Geraldine Piel 1 1 Laboratory of Pharmaceutical Technology-CIRM, 2 Laboratory of Experimental Pathology, GIGA-CANCER University of Liege, Liège, Belgium HPV 16 and 18 are responsible for cervical cancers, in over 70% of cases. These viruses integrate into keratinocyte cells and induce the expression of oncogenes E6 and E7. These prevent the expression of tumor suppressor genes (p53 and pRb) and lead keratinocytes transformation into tumor cells. The purpose of this study is to target locally mRNA encoding for E6-E7 oncoproteins with siRNA. In order to protect and to optimize their penetration through the vaginal mucus and into the cytoplasm, siRNA will be incorporated into cationic liposomes. HPV 16 and 18 are responsible for cervical cancers, in over 70% of cases. These viruses integrate into keratinocyte cells and induce the expression of oncogenes E6 and E7. These prevent the expression of tumor suppressor genes (p53 and pRb) and lead keratinocytes transformation into tumor cells. The purpose of this study is to target locally mRNA encoding for E6-E7 oncoproteins with siRNA. In order to protect and to optimize their penetration through the vaginal mucus and into the cytoplasm, siRNA will be incorporated into cationic liposomes. Cervical cancer  Human Papillomavirus responsible for cervical cancer Histological development of cervical cancerDevelopment of cervical cancer Frequently two (DOTAP and DOTMA) Biodegradable (DOTAP = ester) Essential CATIONIC charged (but toxic) Liposomes  Cationic lipids used: general structure Lipid bilayer Internal aqueous compartment  Characteristics required High transfection Selective for E6 and E7 proteins Induce APOPTOSIS of cancer cells  Design rules Double strand (17-25 nucleotides) + 2dTdT at the 3’end of the antisens sequence GC content less than 50% … siRNA 2 sequences selected : - siRNA targeting E6 (against HPV16) : - siRNA targeting E7 (against HPV16) : 1) Cells transfection using Transfectine ® HPV16+ : SiHa, CaSki HPV16- : C33A HPV18+ : C4 II 2) Tests % transfection (FACS) % decrease of oncoproteins E6 and E7 (QRT-PCR) % apoptosis: Annexin V-PI (FACS) 5’-------CUAGGCAAACAACUAUACAUGAUAdTdT-----3’ 3’-----dTdTGAUCCGUUUGUUGAUAUGUACUAU---------5’ 5’-------AGGAGGAUGAAAUAGAUGGdTdT-----3’ 3’-----dTdTUCCUCCUACUUUAUCUACC----------5’  Physico-chemical characterization: Morphology, size (<200nm), zeta potential (0-40mV), physical stability over the time, encapsulation rate, capacity to release their content,…  Tests on cells: HPV16+ : SiHa, CaSki HPV16- : C33A HPV18+ : C4-II  Tests on organotypic cultures to evaluate penetration efficiency  Validation of the concept in vivo (mice)  Tools to improve the efficiency of lipoplexes Add neutral lipids (DOPE, cholesterol), PEG, change proportions of compounds,… Lipoplexes Evaluation of the transfection efficiency and cytotoxicity