development and optimization of lipoplex vectors for an antisens therapeutic approach in the context...
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Development and optimization of lipoplex vectors for an Development and optimization of lipoplex vectors for an antisens therapeutic approach in the context of HPV induced antisens therapeutic approach in the context of HPV induced
lesionslesions
Contact : [email protected]
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.
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 Development of cervical cancer
Frequently two (DOTAP and DOTMA)
Biodegradable (DOTAP = ester)
Essential CATIONIC charged (but toxic)
LiposomesCationic 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 efficiencyValidation 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