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Infectivity Enhanced Adenovirus as a Strategy for Improving the Efficiency of RNA Interference in an Ovarian Cancer Model. T Michael Numnum, MD International Gynecologic Cancer Society Santa Monica, CA October 15, 2006. Background –Ovarian Cancer. - PowerPoint PPT Presentation
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Infectivity Enhanced Adenovirus as a Strategy for Improving the
Efficiency of RNA Interference in an Ovarian Cancer Model
T Michael Numnum, MD
International Gynecologic Cancer SocietySanta Monica, CAOctober 15, 2006
Background –Ovarian Cancer
• 70% complete response with radical surgery + platinum based combination chemotherapy.
• Majority will recur and die of disease.
• Gene knockdown strategies may a therapeutic model
Gene Knockdown Strategies
• Antisense Oligonucleotides
• Ribozymes• Drugs• RNA
interference
RNA Interference (RNAi)
• Short interfering RNA (siRNA)
• Post transcriptional gene silencing
• Inhibits gene expression via degradation of corresponding mRNA.
• Cancer, infectious disease research
Why RNAi?
• Sequence specific– Parallels specificity of
antigen-antibody
• Stable• Delivery to the target
still the rate limiting step.– Lipid based infection– Electroporation– Adenoviral delivery
The Infectivity Enhanced Adenovirus
• Genetically engineered to overcome poor infectivity of Adenovirus in cancer cells
• May enhance delivery of siRNA to tumor cells
CAR
Normal cell Tumor cell
Hypothesis
• The infectivity enhanced adenovirus as a delivery mechanism for RNAi is an attractive vector for gene knockdown strategies in an in vitro ovarian cancer model.
Hec1 (Highly Expressed in Cancer)
• Essential in chromosome segregation
• Modulates G2/M phase of the cell cycle
• Disruption of Hec1 by genetic deletion leads to cell death
• Potential target in actively replicating cells
Martin et al., 2002
Ad-siRNA Hec1 inhibits tumor growth
• High Infectivity (LacZ)• 50-68% translational
knockdown at 48, 72 hours
• 40% in vivo knockdown
Gurzov et al., 2005
Expression of Hec1 in vitro
Unpublished data
Quantitative RNA Expression of Hec1 in Ovarian Carcinoma Cell Lines
0
20
40
60
80
100
120
140
HFBC OV4 Hey SKOV3ip1 OV3 PA-1
Hec
1 co
pie
s/ n
g R
NA
Materials
• siRNA oligo sequences designed– Hec1: Gen Bank Accession # NM_006101 – GAPDH: Gen Bank Accession # NM_002046
• Negative control
• Sequences cloned into– Adenovirus 5
• Wild type
– Adenovirus F5/3• Chimeric virus designed to enhance infectivity
(Kanerva et al., 2003)
Reagents Ad-siRNA-Hec1 ΔE1 ΔE3
CMV >>siRNA>>polyA
Ad-siRNA-Hec1 F5/3 ΔE1 ΔE3 F5/3
Ad-siRNA-GAPDH F5/3 ΔE1 ΔE3 F5/3
CMV >>siRNA>>polyA
CMV >>siRNA>>polyA
METHODS
• Infectivity enhancement– Cell lines infected Ads– DNA purified after 3 hours– Quantitative PCR for
Adenoviral E4 gene
• RNA knockdown– Cell lines infected with Ads
(500 vp/cell)– RNA purified after 48 hours– Quantitative PCR for Hec1
• Translational Inhibition– Cell lines infected with Ads
(500 vp/cell)– Protein isolated after 72
hours– Western Blot for Hec1/ β-
actin
Methods
• Apoptosis Assay– Cell lines infected with Ads– Cells collected after 96
hours– Annexin V/PI FITC-FACS
• Cell Viability– Cell lines infected with Ads
(500 vp/cell)– MTS assay performed at
days 2,4,6, and 8
• Crystal Violet Staining– Cell lines infected with Ads– Multiplicity of infection:
1000,100,10,1,0.1,0 vp/cell– Crystal violet staining after
10 days
RESULTSINFECTIVITY ENHANCEMENT
Infectivity of Ad-siRNA-Hec1
1.00E+001.00E+011.00E+021.00E+031.00E+041.00E+051.00E+061.00E+071.00E+081.00E+09
Hec1
Hec1
F5/3
Hec1
Hec1
F5/3
Hec1
Hec1
F5/3
SKOV3.ip1 HEY OV4
E4 c
opie
s/ n
g D
NA
p<0.001
*Log scale
RESULTSmRNA KNOCKDOWN
mRNA Knockdown
RNA Knockdown of Hec1 in OV4 Cells
0.00E+001.00E+012.00E+013.00E+014.00E+015.00E+016.00E+017.00E+01
500 vp/cell (48 hours)
Co
pie
s/ n
g R
NA
mRNA Knockdown
RNA Knockdown of Hec1 in HEY Cells
0.00E+00
5.00E+00
1.00E+01
1.50E+01
2.00E+01
Moc
kHec1
Hec1 F
5/3
GAPDH F5/
3
500 vp/cell (48 hours)
Co
pie
s/ n
g R
NA
mRNA Knockdown
RNA Knockdown of Hec1 in SKOV3.ip1 Cells
0.00E+001.00E+012.00E+013.00E+014.00E+015.00E+016.00E+017.00E+018.00E+019.00E+011.00E+02
Moc
kHec
1
Hec1
F5/3
GAPDH F
5/3
500 vp/cell (48 Hours)
Co
pie
s H
ec1/
ng
RN
A
Translational Inhibition
Western Blot
SKOV3.ip1 HEY OV4
Hec 1
βactin
Mo
ck
Hec
1
Hec
1F5/
3
GA
PD
H F
5/3
Mo
ck
Hec
1
Hec
1F5/
3
GA
PD
H F
5/3
Mo
ck
Hec
1
Hec
1F5/
3
GA
PD
H F
5/3
*96 hours
RESULTSAPOPTOSIS ASSAY
Annexin V-PI AnalysisOV4 (Day 4)
No Infection
Ad siRNA Hec1 (500vp/cell) Ad siRNA Hec1 F5/3 (500vp/cell)
Ad siRNA GAPDH F5/3 (500vp/cell)
PI
+
-Annexin V - +
Annexin V-PI AnalysisHEY (Day 4)
No infection
Ad siRNA Hec1 F5/3 (500vp/cell)
Ad siRNA GAPDH F5/3 (500vp/cell)
Ad siRNA Hec1 (500vp/cell)
Annexin V - +
PI
+
-
Annexin V-PI Analysis SKOV3.ip1 (Day 4)
Ad siRNA Hec1 F5/3 (500vp/cell)
No Infection Ad siRNA GAPDH F5/3 (500vp/cell)
Ad siRNA Hec1 (500vp/cell)
Annexin V - +
PI
+
-
RESULTSCELL VIABILITY ASSAY
Cell Viability in OV4 Cells
0.00%
20.00%
40.00%
60.00%
80.00%
100.00%
Day2 Day4 Day6 Day8
Per
cen
t V
iab
ilit
y
Ad-Hec1
Ad-Hec1-F5/3
Ad-Gapdh-F5/3
Cell Viability in HEY Cells
0.00%
20.00%
40.00%
60.00%
80.00%
100.00%
Day2 Day4 Day6 Day8
Per
cent
Via
bilit
y
Ad-Hec1
Ad-Hec1-F5/3
Ad-Gapdh-F5/3
Cell Viability in SKOV3.ip1 Cells
0.00%
20.00%
40.00%
60.00%
80.00%
100.00%
Day2 Day4 Day6 Day8
Per
cent
Via
bilit
y
Ad-Hec1
Ad-Hec1-F5/3
Ad-Gapdh-F5/3
RESULTSCRYSTAL VIOLET STAINING
Null Hec1F5/3 Hec1 Gapdh
OV4 SKOV3.ip1Hey
Crystal Violet Staining (Day 10)
Null Hec1F5/3 Hec1 Gapdh Null Hec1F5/3 Hec1 Gapdh
103
102
101
100
10-1
0
Vp/cell
Conclusions
• In an in vitro ovarian cancer model, RNA interference of Hec1 results in mRNA knockdown and apoptosis leading to cell death.
• The infectivity enhanced adenovirus is a reasonable strategy for delivery of RNAi in an ovarian cancer model.
Acknowledgements
• UAB Division of Gynecologic Oncology– Sharmila Makhija, MD– Ronald Alvarez, MD
• Division of Human Gene Therapy– David Curiel, MD, PhD– Zeng Bian Zhu, MD– Baogen Lu, MD– Minghui Wang, MD– Angel Rivera
Infectivity Enhanced Adenovirus as a Strategy for Improving the
Efficiency of RNA Interference in an Ovarian Cancer Model
T Michael Numnum, MD
International Gynecologic Cancer SocietyLos Angeles, CAOctober 15, 2006