Development of anti-HPV lipoplexes for the treatment of cervical cancer

Contact : anna.lechanteur@student.ulg.ac.be

Anna Lechanteur1,Tania Furst1 ,Brigitte Evrard1,Philippe Delvenne2, Patrick Roncarati2 ,Geraldine Piel1 ,Pascale Hubert2

1Laboratory of Pharmaceutical Technology-CIRM, 2Laboratory of Experimental Pathology, GIGA-CANCER University of Liege, Liège, Belgium

INTRODUCTION – PURPOSE HPV 16 and 18 are responsible for cervical cancers, in over 70% of cases. The tumourigenic potential of HPV is based on the expression of the E6 and E7 oncoproteins. Their transforming properties are based on their ability to interact with cellular proteins, such as p53 and Rb which are inactivated, respectively, by E6 and E7 viral proteins.Even if, at the moment, vaccines have been developed, they protect against the two genotypes most frequently encountered and they have no therapeutic effect. So morbidity and mortality caused by HPV infection still remain a major health problem. The aim of this study is to develop a treatment based on local administration of siRNA anti-E6 and/or E7 by the use of cationic liposomes (LIPOPLEXES). These nanovesicles allow to trigger specifically cancer cells, protect and transport siRNA.

MATERIALS and METHODS1) Transfection efficacy: . Cell lines: SiHa and CaSki (HPV16+) . Transfection medium and reagent: OptiMEM® and Oligofectamine®

. siRNA scramble (100nM) coupled to fluorescein . FACS analysis 24H after transfection

2) siRNA efficacy . siRNA(100nM) targeting E6(siE6)

. siRNA(100nM) targeting E7(siE7)

. RNA extraction: Macherey-Nagel protocol

. Reverse transcription: Invitrogen protocol . qRT-PCR: SYBR Green detection .Transfection medium and reagent: OptiMEM® and

Oligofectamine® .Time of transcription: 1, 2, 3, 4, 5, 6 days3) Apoptosis: . AnnexinV-PI analyzed by FACS4) Lipoplexes .Lipids: DOTAP/Cholesterol /DOPE 1/0,5/0,5 DOTAP/Cholesterol /DOPE 2/0,5/0,5 .N/P: 5; 7,5;10 .siRNA scramble (100nM) coupled to fluorescein

5’-------CUAGGCAAACAACUAUACAUGAUAdTdT-----3’ 3’-----dTdTGAUCCGUUUGUUGAUAUGUACUAU---------5’

5’-------AGGAGGAUGAAAUAGAUGGdTdT-----3’ 3’-----dTdTUCCUCCUACUUUAUCUACC----------5’

Depletion of mRNA E6 by siRNA at three different concentrations 48H after transfection on SiHa

Depletion of mRNAE7 by siRNA at three different concentrations 48H after transfection on SiHa

Apoptosis on CaSki cells until six days after siE6 and siE7 transfection (AnnexinV-PI by FACS)

• J4: siE7 12% apoptosis siE6+siE7 14% apoptosis• J5: siE7 17,5% apoptosis siE6+siE7 22,5% apoptosis• J6: siE7 16% apoptosis siE6+siE7 46% apoptosis

CONCLUSIONS / PERSPECTIVES-siE6 and siE7 decrease E6 and E7 mRNA with Oligofectamine® on HPV16 cell lines. We will now test the activity of both siRNA on HPV18 cell line and the efficacy of the lipoplexes. -siE7 and siE6+siE7 lead CaSki apoptosis, we will now test the same kinetic on SiHa cell and on organotypic culture. To enhance apoptosis level, we will trigger MCL-1 protein (apoptosis antagonist protein) with another siRNA.

Depletion of mRNA E6 by siRNA (100nM) until six days after trasnfection on CaSki

Depletion of mRNA E7 by siRNA (100nM) until six days after trasnfection on CaSki

CaSki: 83% of transfected cells (MFI 3000 )

J1 J2 J3 J4 J5 J6

SiHa: 92% of transfected cells(MFI 3800)

CaSki: blank CaSki: 72% transfected with Oligofectamine ® (MFI 1950)

CaSki: 92% transfected with lipoplexes1/0,5/0,5 (MFI 3800)

CaSki: 88% transfected with lipoplexes 2/0,5/0,5 (MFI 9000)

RESULTS1) Oligofectamine ® allows effective transfection of siRNA scramble

2) siE6 and siE7 decrease mRNA encoding for both oncoproteins

3) siE7 and association of siE6+siE7 induce apoptosis on CaSki cells

4) Lipoplexes (DOTAP/Chol/DOPE 1/0,5/0,5 and 2/0,5/0,5 at N/P=5) can transfect CaSki cells (siRNA scramble = 100nM)

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