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Unpublished data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