Open access

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]