Development and optimization of lipoplex vectors for an antisens therapeutic approac h in the context of HPV induced lesions Anna Lechanteur1,Brigitte Evrard1,Philippe Delvenne2, Patrick Roncarati2 ,Pascale Hubert2, Geraldine Piel1 1Laboratory of Pharmaceutical Technology-CIRM, 2Laboratory 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. Cervical cancer Human Papillomavirus responsible for cervical cancer Histological development of cervical cancer Liposomes Development of cervical cancer Cationic lipids used: general structure Lipid bilayer Frequently two (DOTAP and DOTMA) Biodegradable (DOTAP = ester) Internal aqueous compartment Essential CATIONIC charged (but toxic) siRNA 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% •… Lipoplexes 2 sequences selected: 1) Cells transfection using Transfectine ® - siRNA targeting E6 (against HPV16) : • HPV16+ : SiHa, CaSki • HPV16- : C33A • HPV18+ : C4 II 5’-------CUAGGCAAACAACUAUACAUGAUAdTdT-----3’ 3’-----dTdTGAUCCGUUUGUUGAUAUGUACUAU---------5’ 2) Tests - siRNA targeting E7 (against HPV16) : 5’-------AGGAGGAUGAAAUAGAUGGdTdT-----3’ 3’-----dTdTUCCUCCUACUUUAUCUACC----------5’ • % transfection (FACS) • % decrease of oncoproteins E6 and E7 (QRT-PCR) • % apoptosis: Annexin V-PI (FACS) 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 Evaluation of the transfection efficiency and cytotoxicity 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,… Contact : [email protected]