Transcript Poster GIGA DAY Lechanteur

DEVELOPMENT OF ANTI-HPV LIPOPLEXES FORMULATIONS
FOR THE TREATMENT OF CERVICAL CANCER
Anna Lechanteur1, Tania Furst1, Brigitte Evrard1, Philippe Delvenne2, Patrick Roncarati2, Geraldine Piel1, Pascale Hubert2
1Laboratory of Pharmaceutical Technology and Biopharmacy-CIRM,
2Laboratory of Experimental Pathology, GIGA-CANCER
University of Liege, Liège, Belgium
INTRODUCTION – PURPOSE
Human Papillomaviruses (HPV) such as HPV16 and HPV18 can induce cervical cancer. In this case,
the two HPV E6 and E7 oncoproteins are essential players in order to immortalize keratinocytes by
decreasing tumor suppressor genes (p53 and pRb). Gene therapy is a promising strategy to treat cancer in
order to decrease side effects against healthy cells. We focused on RNA interference (siRNA) to target mRNA
coding for both HPV E6 and E7 oncoproteins. Moreover, we targeted an anti-apoptotic protein (MCL-1) to
increase apoptosis of HPV 16 positive cells. To protect siRNA, to allow the diffusion into the cervical mucus
and to cross the anionic cellular membrane, we use nanotherapy: siRNA is encapsulated in lipidic
nanovectors to form LIPOPLEXES.
RESUTLS
RESULTS
FIRST TARGET:
Oncoproteins E6 and E7.
®
1) Oligofectamine allows effective transfection of siRNA scramble
 siRNA E6 and siRNA E7 induce the extinction of mRNA E6 and E7 AND decrease the expression of oncoproteins.
Interestingly, siRNA E6 turn
off mRNA E6 and mRNA
E7. In the same way, siRNA
E7 turn off mRNA E6 and
mRNA E7. Best extinctions
are obtained when siRNA are
used in combination.
- Transfection agent:
Oligofectamine®
- Cell line: CaSki
(HPV16+)
- qPCR analysis: SYBR
Green detection
- siRNA
concentration:100nM
- 2 days after transfection
- n=5
2) siE6 and siE7 decrease mRNA encoding for both oncoproteins
Figure 1 – Extinction of mRNA E6
Figure 2 – Extinction of mRNA E7
SECOND TARGET: Anti-apoptotic protein MCL-1  siRNA anti-MCL-1 used in combination with siRNA E6 and siRNA E7.
 MCL-1 protein is an interesting target: this protein is overexpressed in high grade lesions compared to low grade lesions and healthy cervix.
- Immunohistochemistry
staining (cervical biopsy)
-Antibody: MCL-1
- EXO N: Normal Exocervix
- LSIL: Low grade Squamous Intraepthelial Lesion
- HSIL: High grade Squamous Intraepthelial Lesion
- SCC: Squamous Cell Carcinoma
Figure 3 - Normal exocervix

Figure 4 - High grade Squamous Intra-epithelial Lesion = HSIL
Figure 5 - Score of MCL-1 in different cervical biopsies
The combination of 3 siRNA decreases proliferation and induces apoptosis on SiHa cells (HPV16+).
- Transfection agent:
Oligofectamine®
- Cell line: SiHa (HPV16+)
- siRNA concentration:100nM
- Proliferation compared to Blank:
alamar Blue®
- Apoptosis: FITC Annexin V
Apoptosis Detection kitI
- n=2
3) siE7 and association of siE6+siE7 induce apoptosis on CaSki cells
Figure 6 - SiHa (blank) day 2
Figure 7 - SiHa cells two days after the
transfection of siE6 + siE7 + siMCL-1
Figure 8 - Proliferation assay during four days
LIPOPLEXES can cross the cellular membrane
Figure 9 - Apoptosis assay during four days
LIPOPLEXES can release siRNA into the cytoplasm
Oligofectamine®
Lipoplexes
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)
CaSKi cells
siRNA 100nM
siRNA 50nM
77%
- 2 days after transfection
- Cell line: CaSki (HPV16+)
- qPCR Analysis (SYBR Green
detection)
- siRNA anti-E6
- n=4
98%
Figure 12 - Extinction of mRNA E6
- Transfection time: 24 hours
- Cell line: CaSki (HPV16+)
- FACS Canto II
- siRNA scramble fluorescent
- Lipoplexes DOTAP/DOPE/Chol 1/0,5/0,5
- N/P ratio: 2,5
- For Oligofectamine and Lipoplexes:
- Mean Fluorescence Intensity: ±4000
- death cells: 5% (Fixable Viability Stain 450)
Figure 13 - Extinction of mRNA E7
CONCLUSIONS / PERSPECTIVES
1) Selected siRNA anti-E6 and anti-E7 can induce apoptosis and decrease proliferation of HPV16+ cells (by their action against mRNA and proteins).
2) siRNA against anti-apoptotic MCL-1 protein allow higher induction of apoptosis on SiHa cells. The effect of this siRNA must be tested on CaSki
cells.
3) First result obtained with lipoplexes indicate that they can cross the cellular membrane and release siRNA into the cytoplasm. Other tests will be
performed to analyze the toxicity on healthy cells.
4) We would like to analyze the effect of siRNA on a 3D model of cervical lesion.
Contact : [email protected]