Cancer Gene Therapy (2008) 15, 140–153
r 2008 Nature Publishing GroupAll rights reserved 0929-1903/08 $30.00

www.nature.com/cgt

ORIGINAL ARTICLE

New highly potent and specific E6 and E7 siRNAs for treatmentof HPV16 positive cervical cancer
K Yamato1, T Yamada2, M Kizaki3, K Ui-Tei4, Y Natori5, M Fujino5, T Nishihara6,
Y Ikeda3, Y Nasu7, K Saigo4 and M Yoshinouchi8
1

Molecular Cellular Oncology and Microbiology, Graduate School, Tokyo Medical and Dental University,
Tokyo, Japan; 2Division of Pathology, Keio University, School of Medicine, Tokyo, Japan; 3Division of
Hematology, Keio University, School of Medicine, Tokyo, Japan; 4Department of Biophysics andBiochemistry, Graduate School of Science, University of Tokyo, Tokyo, Japan; 5RNAi Company Ltd., Tokyo,
Japan; 6Department of Oral Microbiology, Kyushu Dental College, Fukuoka, Japan; 7Department of Urology,
Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Science, Okayama, Japanand 8Department of Physiology, Okayama University Graduate School of Medicine, Dentistry, andPharmaceutical Science, Okayama, Japan
Persistent infection by high-risk types of human papillomaviruses (HPV) is a necessary cause of cervical cancer, with HPV16 themost prevalent, accounting for more than 50% of reported cases. The virus encodes the E6 and E7 oncoproteins, whose expression
is essential for maintenance of the malignant phenotype. To select efficacious siRNAs applicable to RNAi therapy for patients with
HPV16 þ cervical cancer, E6 and E7 siRNAs were designed using siDirect computer software, after which 10 compatible with all
HPV16 variants were selected, and then extensively examined for RNAi activity and specificity using HPV16 þ and HPV16cells.
Three siRNAs with the highest RNAi activities toward E6 and E7 expression, as well as specific and potent growth suppression of
HPV16 þ cancer cells as low as 1 nM were chosen. Growth suppression was accompanied by accumulation of p53 and p21WAF1/CIP1,
as well as morphological and cytochemical changes characteristic of cellular senescence. Antitumor activity of one of the selected
siRNAs was confirmed by retarded tumor growth of HPV16 þ cells in NOD/SCID mice when locally injected in a complex withatelocollagen. Our results demonstrate that these E6 and E7 siRNAs are promising therapeutic agents for treatment of virus-related
cancer.
Cancer Gene Therapy (2008) 15,140–153; doi:10.1038/sj.cgt.7701118; published online 21 December 2007
Keywords: siRNA therapy; HPV16; cervical cancer

Introduction

Cervical cancer is the second most common type of cancer
encountered in females, with an incidence rate of up to
18.7 per 100 000.1 Epidemiological and experimental
studies have shown that persistent infections with highrisk types of human papillomaviruses (hrHPVs; HPV16,
18, 31, 33, 45) are a necessary cause of cervical cancer,
with HPV16 accounting for approximately 50% ofreported cases of HPV-related cervical cancer. Theseviruses encode the E6 and E7 oncogenes, whose expression is essential for virus replication.2–4 Furthermore,deregulated E6 and E7 expression leads to malignant

Correspondence: Dr K Yamato, Molecular Cellular Oncology andMicrobiology, Graduate School, Tokyo Medical and Dental
University, 1-5-45 Yushima, Bunkyo-kyu, Tokyo 113-8549, Japan.
E-mail: yamato.mcom@tmd.ac.jp
Received 10 September 2007; revised 15 November 2007; accepted
25 November 2007; published online 21 December 2007transformation, and is pivotal for maintenance of themalignant phenotype of cervical cancer.
Cellular targets for these hrHPV oncogenes have been
investigated, with the p53 tumor suppressor protein thefirst identified E6 target and still the most important. The
E6 protein forms a tertiary complex with E6AP functioning as a ubiquitin ligase and its substrate, p53, which leads
to degradation of p53. Other E6 targets include the PDZ
family of proteins, such as PSD-95, hDlg and ZO-1, as
well as the co-activator p300/CBP. E6 also induces theexpression of human telomerase reverse transcriptase(hTERT), a catalytic subunit of human telomerase, whose
activity may be involved in immortalization.
Targets of the E7 protein include the pocket protein
family of retinoblastoma protein (Rb) and the cyclindependent kinase inhibitors p21WAF1/CIP1 and p27KIP1.2–4
Since these molecules are negative regulators of the cell
cycle, their inhibition by E7 results in upregulation ofgenes required for G1/S transition and DNA synthesis,
which is essential for viral genome replication and amplification.5 However, E7 also induces genome instability

Efficacious siRNAs targeting HPV16 oncogenes
K Yamato et al

and genetic alterations, which subsequently accumulate
and cause transformation of infected cells into malignant
cells. Recently, E7 was shown to form a complex
with promyelocytic leukemia protein (PML), which has
been implicated in controlling cellular senescence as apro-senescence factor.6
Because of the strong relationships between theexpression of HPV oncogenes and cervical cancer
carcinogenesis, several technologies have been applied totarget virus oncogenes to develop novel therapies for
cervical cancer. Antisense oligo-deoxynucleotides andribozymes targeting virus oncogenes were found to
suppress growth of virus-related cancer cells both in vitro
and in vivo.7–9 Introduction of a short double-stranded
RNA with a duplex region of 19 base pairs and two
nucleotide 30 overhangs, known as a short-interfering
RNA (siRNA), induces sequence-specific destruction ofendogenous mRNA in mammalian cells, a phenomenon
known as RNA interference (RNAi). Since the discovery
of siRNA-induced RNAi in 2001,10,11 siRNAs have beenutilized in reverse genetics and gene-targeting therapy.
siRNA technology is completely different from others, inthat siRNAs utilizes endogenous RNAi machineries thatexert normal cellular functions. Studies have shown that
introduction of those targeting E6 and E7 of hrHPVs
resulted in growth suppression of virus-positive cancer
cells.12–19 These results indicate the possibility of applying
RNAi technology to therapeutic options for HPV þ cancer.
Although siRNA-induced target gene suppression was
initially assumed to be highly sequence specific, it has
become clear that siRNAs suppress unintended genes
through RNA-induced silencing complex (RISC)mediated cleavage of target mRNA or translation
suppression, which is analogous to miRNA-mediatedRNAi,20–22 and stimulation of innate immune by type I
interferon (IFN-I) and inflammatory cytokine responses.23,24
Suppression of such unintended genes by siRNAs is
known as an off-target effect. RISC-mediated off-target
effects are caused by a partial sequence complementarity
between the guide strand and unintended genes, especiallywhen excessive RISC is present.25 Furthermore, somesiRNAs have been reported to induce innate immune
responses in a sequence-specific manner.23,24 For the
purpose of applying siRNA technology to therapy for
hrHPV-related cancers, it is mandatory to select siRNAsequences targeting E6 and E7 that have high levels ofRNAi activity and minimal off-target effects. However, todate, no known HPV16 E6 or E7 siRNA sequence has
been analyzed in detail in regard to those factors.
For the present study, we selected siRNA sequencestargeting mRNA coding HPV16 E6 and E7 using siDirect
software, based on guidelines established by Ui-Tei
et al.26,27 From those findings, we developed highly
effective siRNA sequences with maximum target specificity, and examined the siRNAs for RNAi activity andspecificity using HPV16 þ and HPV16 cancer cells. Our
results identified three E6 and E7 siRNAs compatible
with most HPV16 variants, which were potent andspecific in suppression of E6 and E7 expression, as wellas growth of HPV16 þ cancer cells.

141

Materials and methods

Cell lines and plasmid transfection
SiHa (HPV16 þ cervical cancer), CaSki (HPV16 þ
cervical cancer), HeLa (HPV18 þ cervical cancer) and
SK-OV-3 (HPV ovarian cancer) cell lines were purchasedfrom American Type Culture Collection (ATCC,
Rockville, MD). SiHa, CaSki and HeLa cells weremaintained in DMEM containing 10% fetal calf serum(FCS), while SK-OV-3 cells were cultured in RPMI1640
containing 10% FCS. The incubations were performed at
37 1C in an atmosphere of 5% CO2. Plasmid transfection
was performed using Lipofectamine 2000 transfection
reagent (Invitrogen Corporation, Carlsbad, CA) according
to the manufacturer’s instructions.
siRNAs and transfection
The siRNAs used in this study were as follows: control
siRNA passenger strand, 50 -GUACCGCACGUCAUUCGUAUC-30 ; control siRNA guide strand, 50 -UACGAAUGACGUGCGGUACGU-30 ; FLuc siRNA passenger
strand, 50 -CCGUGGUGUUCGUGUCUAAGA-30 ; FLucsiRNA guide strand, 50 -UUAGACACGAACACCACG
GUA-30 ; lamin A/C siRNA passenger strand, 50 -CU
GAAAGCGCGCAAUACCAAG-30 ; lamin A/C siRNA
guide strand, 50 -UGGUAUUGCGCGCUUUCAGC
U-30 ; 222 passenger strand,12 50 -GAGGUAUAUGACUUUGCUUUU-30 ; 222 guide strand, 50 -AAGCAAAGUCAUAUACCUCAC-30 ; 660 passenger strand,12 50 -AGG
AGGAUGAAAUAGAUGGUC-30 ; 660 guide strand,
50 -CCAUCUAUUUCAUCCUCCUCC-30 ; 375 passenger
strand,14 50 -UACAACAAACCGUUGUGUGAU-30 ; 375
guide strand, 50 -CACACAACGGUUUGUUGUAU
U-30 ; 186 passenger strand,13 50 -GAAUGUGUGUACUGCAAGCAA-30 ; 186 guide strand 50 -GCUUGCAGUACACACAUUCUA-30 ; 203 passenger strand,16 50 -GCAACAGUUACUGCGACGUGA-30 ; 203 guide strand, 50 -A
CGUCGCAGUAACUGUUGCUU-30 ; 535 passenger
strand,16 50 -CACGUAGAGAAACCCAGCUGU-30 ; and535 guide strand, 50 -AGCUGGGUUUCUCUACGU
GUU-30 .
The sequences of siRNAs 233, 243, 244, 493, 497, 501,
573, 698, 707 and 752 are shown in Table 1. Twenty-onebase ribonucleotides were synthesized, annealed in buffer(100 mM potassium acetate, 30 mM HEPES-KOH at pH
7.4, 2 mM magnesium acetate) and column-purified(Proligo Co. Ltd, Boulder, CO).
One day before transfection, the cells were plated at adensity of 5  104 to 1  105 cells per well in a six-wellplate containing 2 ml of Dulbecco’s modified Eagle’s
medium (DMEM) with 10% FCS. Various amounts ofthe siRNA were added with Opti-MEM I reduced serum
medium (Invitrogen Corporation) up to 50 ml. For SiHa
cells, SK-OV-3 cells, and their derivatives, 1.6 ml of
Lipofectamine 2000 was diluted with Opti-MEM I
reduced serum medium to 50 ml. For HeLa cells and theirderivatives, 0.4 ml of Lipofectamine 2000 was diluted with
Opti-MEM I reduced serum medium to 50 ml. Diluted
siRNA and Lipofectamine 2000 were combined, incubatedat room temperature for 20 min and then added to a 2-ml

Cancer Gene Therapy

Efficacious siRNAs targeting HPV16 oncogenes
K Yamato et al

142

Table 1 siDirect-selected siRNAs targeting HPV16 E6 and E7
siRNA

Nucleotide
position

Mismatchtolerance

Sequence (upper, passenger strand;
lower, guide strands)

Incompatible HPV16class and subclass117

117–1393

50 -ACUUUCUGGGUCGCUCCUGUG-30
50 -CAGGAGCGACCCAGAAAGUUA-30

128

128–1503

50 -CAGAAAGUUACCACAGUUAUG-30
50 -UAACUGUGGUAACUUUCUGGG-30

EG(131)Af1Af2EG(131)
Af1
Af2

233

233–2553

243

243–2653

244

244–2662

324

324–3463

326

326–3483

493

493–5153

497

497–5193

501

501–523

2

573

573–595

3

583

583–6053

615

615–6373

625

625–6473

698

698–7203707

707–7293752

752–774

4

50 -CUUUGCUUUUCGGGAUUUAUG-30
50 -UAAAUCCCGAAAAGCAAAGUC-30
50 -CGGGAUUUAUGCAUAGUAUAU-30
50 -AUACUAUGCAUAAAUCCCGAA-30
50 -GGGAUUUAUGCAUAGUAUAUA-30
50 -UAUACUAUGCAUAAAUCCCGA-30
50 -GAGUAUAGACAUUAUUGUUAU-30
50 -AACAAUAAUGUCUAUACUCAC-30
50 -GUAUAGACAUUAUUGUUAUAG-30
50 -AUAACAAUAAUGUCUAUACUC-30
50 -GGUGGACCGGUCGAUGUAUGU-30
50 -AUACAUCGACCGGUCCACCGA-30
50 -GACCGGUCGAUGUAUGUCUUG-30
50 -AGACAUACAUCGACCGGUCCA-30
50 -GGUCGAUGUAUGUCUUGUUGC-30
50 -AACAAGACAUACAUCGACCGG-30
50 -CACCUACAUUGCAUGAAUAUA-30
50 -UAUUCAUGCAAUGUAGGUGUA-30
50 -GCAUGAAUAUAUGUUAGAUUU-30
50 -AUCUAACAUAUAUUCAUGCAA-30
50 -CAACUGAUCUCUACUGUUAUG-30
50 -UAACAGUAGAGAUCAGUUGUC-30
50 -CUACUGUUAUGAGCAAUUAAA-30
50 -UAAUUGCUCAUAACAGUAGAG-30
50 -CCGGACAGAGCCCAUUACAAU-30
50 -UGUAAUGGGCUCUGUCCGGUU-30
50 -GCCCAUUACAAUAUUGUAACC-30
50 -UUACAAUAUUGUAAUGGGCUC-30
50 -CUUCGGUUGUGCGUACAAAGC-30
50 -UUUGUACGCACAACCGAAGCG-30

Mismatched nucleotide

131132, 136
132
131
132, 136, 143, 145
132, 143, 145

Af1, Af2, NA1, AA

335

Af1, Af2, NA1, AA

335

As, Af2

618

siRNAs were designated by the nucleotide position number of the 30 end of the guide strand. HPV16 classes and subclasses carrying amismatch to the guide strand sequence are shown in the column of incompatible HPV16 class and subclass, with mismatchednucleotide positions also shown. The HPV16 classes and subclasses were as follows: Prototype (accession number K02718); European
German 131 (EG131; accession number AF536179); East Asian (As; accession number AF534061); African 1 (Af1; accession number
AF472508, AF536180); African 2 (Af2; accession number AF472509); Asian-American (AA; accession number AF402678); and North
American I (NA1; accession number AF486325).
Underlines indicate an immunostimulatory motif.24

culture. The amount of Lipofectamine 2000 was changed
in proportion to the culture medium volume. Transfectionof siRNAs using Oligofectamine (Invitrogen Corporation) was performed according to the manufacturer’s
instructions.

Plasmid construction
The HPV16 E6E7 region from nt 231 to 858, DNE6E7(231–858), was obtained by PCR amplification using
pSV2-E6E7 (a gift from Dr T Kanda, National Institutefor Infectious Disease, Tokyo, Japan)28 as a template, andE6 sense and E7 antisense primers containing NotI sites atthe 50 end were subcloned at the NotI site of the psiCheck-2
plasmid, with the resultant plasmid designated as

Cancer Gene Therapy

16NDE6E7/psiCheck-2. DNE6 region (231–559) and E7
region (562–858) were similarly cloned, and inserted
between the XhoI and NotI sites of psiCheck-2, with theresultant plasmids designated as 16DNE6/psiCheck-2 and16E7/psiCheck-2, respectively. An hLuc cDNA fragmentisolated from psiCheck-2 was cloned into pcDNA3 at the
HindIII and XbaI sites, and the resultant plasmid was
designated as hLuc/pcDNA3. E7 of 16E7/psiCheck-2 waschanged to an E7 variant carrying cytosine instead
thymine at nt760 using GeneTailor Site-Directed Mutagenesis System (Invitrogen Corporation), and the resultant
plasmid was designated as 16E7(760C)/psiCheck-2.
The primers used for plasmid construction were as
follows: Not-HPV16_231s, 50 -GCGGCCGCATGACTT

Efficacious siRNAs targeting HPV16 oncogenes
K Yamato et al

TGCTTTTCGGGAT-30 ; Xho-HPV16_231s, 50 -CTCGAGATGACTTTGCTTTTCGGGAT-30 ; Not-HPV16_E6as,
50 -GCGGCCGCTTACAGCTGGGTTTCTCTAC-30 ; XhoHPV16_E7s, 50 -CTCGAGATGCATGGAGATACACC
TAC-30 ; and Not-HPV16_E7as, 50 -GCGGCCGCTTAT
GGTTTCTGAGAACAGA-30 . Underlines indicate the
NotI and XhoI sites.

SiHa, HeLa and SK-OV-3 clones with constitutive
expression of firefly luciferase
SiHa, HeLa and SK-OV-3 cells were transfected with afirefly luciferase (Fluc) expression plasmid (hLuc/
pcDNA3) using Lipofectamine 2000 according to the
manufacturer’s instructions, and selected by cultivating in
the presence of G418. The resultant stable clones expressing FLuc were designated as FL-SiHa-2, FL-SKOV-5 and
FL-HeLa-1, respectively.
Reverse transcription–PCR
Isolation of DNA-free total RNA, synthesis of singlestranded cDNAs and PCR amplification were performed
as previously described.13 cDNA from mock-transfected
cells was serially diluted and used as the standard. PCR
assays were performed under the following conditions:denaturation at 94 1C for 5 min, followed by 22–26 PCR
cycles (denaturation at 94 1C for 30 s, primer-annealing at
58 1C for 30 s, primer-extension at 72 1C for 30 s) andincubation at 72 1C for 5 min. Amplified products wereseparated on 2.5% agarose gels, stained with SYBRGOLD (Molecular probes, Eugene, OR) and analyzed
using an FLA-2000 fluoro-image analyzer (Fuji Film,
Tokyo, Japan). Band intensities were quantified using
Image Gauge software (Fuji Film), and normalized tothose of the b-actin band or those of 18S ribosomal RNA.
PCR primers used for detection of E6 and E7 cDNAs areshown in Table 2. Those for detection of b-actin cDNA
were as follows: b-actin forward primer, 50 -CTCAC
CATGGATGATGATAT-30 and b-actin reverse primer,
50 -TGGGTCATCTTCTCGCGGTT-30 . A pair of PCRprimers for 18S ribosomal RNA was purchased from
Applied Biosystems (Foster City, CA).
Immunoblotting
Cells at 2–5  105 were washed with Dulbecco’s phosphate-buffered saline (PBS) once, directly dissolved in
100 ml of sodium dodecyl sulfate (SDS)-gel loading buffer
and incubated for 5 min at 95 1C. Ten microliters of eachsample was then separated on a polyacrylamide gel
containing 0.1% SDS. Immunoblot analysis was perTable 2 PCR primers for HPV16 E6 and E7
PCR
primerDirection Nucleotide
position

E6s104 Forward
E6as375 Reverse
E6as558 Reverse
E7s562 Forward
E7as855 Reverse

Sequence

104–123 50 -ATGTTTCAGGACCCACAGGA-30
375–356 50 -TGCTGTTCTAATGTTGTTCC-30
558–539 50 -TACAGCTGGGTTTCTCTACG-30
562–581 50 -ATGCATGGAGATACACCTAC-30
855–836 50 -TTATGGTTTCTGAGAACAGA-30

formed as described previously.13 Bands were detectedusing an FLA-2000 fluoro-image analyzer (Fuji Film).
Band intensities were quantified using Image Gauge
software (Fuji Film) and normalized to those of thelamin A/C band. Anti-p21WAF1/CIP1 (clone 70) and anti-lamin
A/C monoclonal antibodies (clone 14) were purchasedfrom BD Biosciences (San Jose, CA), while anti-p53
monoclonal antibody (DO-1) came from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA).

Analysis of in vitro cell growth and senescenceassociated b-galactosidase staining
Cell viability was examined with a colorimetric assay
using WST-8 (Cell Counting Kit 8, Dojin Laboratories,
Kumamoto, Japan) according to the manufacturer’s
instructions. Senescence-associated b-galactosidase staining
was performed using a Senescent Cell Staining Kit(Sigma-Aldrich Corporation, St Louis, MO).
In vivo tumor growth
To study the effects of the siRNAs on tumor growth invivo, SiHa cells (2  107 cells) were subcutaneously
inoculated into 6-week-old NOD/SCID mice, after whichtumors slowly formed at the injected site. Six weeks after
the inoculation, siRNAs complexed with AteloGene(Koken Co. Ltd, Tokyo, Japan) according to the
manufacturer’s instructions were directly injected into
the tumors at a dose of 500 pmole per tumor every 7 days(n ¼ 6 for each group). Thirty-five days after the firstsiRNA injection, the mice were killed according to the
UKCCCR guidelines, and each tumor was excised andweighed. Tumor weight differences were analyzed forsignificance using a Student’s t test, with Po0.05
considered to indicate statistical significance.

Results

Selection of HPV16 E6 and E7 siRNA sequences
A total of 17 siRNA sequences targeting the E6 and E7
coding regions of HPV16 (Table 1, Figure 1a) wereselected using siDirect software,27 which is based on
guidelines for designing siRNA sequences for mammalianRNAi.26 All of the sequences had structural characteristics
of functional siRNA26,30,31 and contained at least threemismatches in the guide strand with the nonredundantsequence set of human genes (mismatch tolerance 3).27
Five siRNAs (117, 128, 324, 326, 615) containing
nucleotides that differed among HPV16 classes andsubclasses were excluded (Table 1, Supplementary Table1), and a total of 10 siRNAs targeting different regions ofE6 and E7 were chosen from the remaining 12 for thisstudy, as follows: 233, 243 and 244, targeting E6-E7
mRNA, but not E6*I-E7 or E6*II-E7 mRNA; 493, 497
and 501, targeting E6-E7 and E6*I-E7 mRNA, but not
E6*II-E7 mRNA; 573, 698, 707 and 752, targeting all
three splicing variants. After revision of the nonredundantsequence database, two siRNAs (244, 501) were reclassified
as mismatch tolerance 2.

Cancer Gene Therapy

143

Efficacious siRNAs targeting HPV16 oncogenes
K Yamato et al

144
244

752

501

243

siRNAs

707
698

497
493

233

A

573

AA

AA

AA

97
E6-E7 mRNA

A

97

226

97

226

AA

AA

AA

409

E6*I-E7 mRNA
A

A
AA

A
AA

526

E6*II-E7 mRNA
104

556
562

E6

855
E7

16E6s104

16E6as375

16E6as558

PCR primers
16E7s562
556
125

562 855

16E7
16∆NE6E7

HSV-TK
promoter
FLuc
Syntheticpoly(A)

RLuc

psiCheck-2

SV40 Latepoly(A)

intronrelative RLuc activity (%)

231
16∆NE6

125

75
50
25

0.01
0.1
1
10
siRNA concentration (nM)

100

0.01
0.1
1
10
siRNA concentration (nM)

100

125
relative RLuc activity (%)

relative RLuc activity (%)

100

0
0.001

SV40 earlyenhancer/promoter

100
75
50
25
0
0.001

16E7as855

0.01
0.1
1
10
siRNA concentration (nM)

100

100
75
50
25
0
0.001

Figure 1 siDirect-selected siRNAs targeting HPV16 E6 and E7, and their effects on luciferase-E6 and -E7 fusion gene expression. (a) E6 andE7 siRNAs, mRNA splicing variants, genomic organization and PCR primers. siRNAs targeting E6 and E7 are depicted as short lines (top). Thickbars represent naturally occurring E6E7 mRNA species with introns within E6 (middle). The closed boxes show the locations of the E6 and E7
coding regions of the HPV16 prototype (accession number K02718) (bottom). Positions of the PCR primers used in this study are indicated asarrows. The numbers refer to the locations of the transcription initiating site and splicing junctions.29 (b) Illustration of Renilla luciferase (Rluc)E6E7 fusion gene expression plasmids. SiHa cells were sequentially transfected with an RLuc-DNE6E7 (c), RLuc-DNE6 (d) or RLuc-DNE6E7
expression plasmid (e) and various concentrations of E6 and E7 siRNAs. Forty-eight hours after transfection, the cells were assayed for firefly
luciferase (Fluc) and RLuc activities. RLuc activity was normalized to FLuc activity, with RLuc activity of mock-transfected cells defined as 100%.
Closed triangle represents siRNA 233; closed square, siRNA 243; closed circle, siRNA 244; open diagonal, siRNA 493; closed diagonal, siRNA573; open triangle, siRNA 698; open square, siRNA 707 and open circle, siRNA 752. Experiments were performed in triplicate. Error bars
represent the s.d. from the mean.

Cancer Gene Therapy

Efficacious siRNAs targeting HPV16 oncogenes
K Yamato et alRNAi activities of E6 and E7 siRNAs toward Renillaluciferase-E6E7 fusion mRNA
The RNAi activities of the E6 and E7 siRNAs selectedusing siDirect software were examined with a reporter
gene assay. SiHa HPV16 þ cancer cells were transfectedwith a Renilla luciferase (RLuc)-DNE6E7 fusion gene
expression plasmid (Figure 1b), along with various
concentrations of the different siRNAs (233, 243, 244,
493, 573, 698, 707, 752). After 48 h of incubation, RLucand FLuc activities were determined (Figure 1c). The
RNAi activities of siRNA 493 and 573 toward RLucDNE6E7 were low (data not shown), and thus reevaluatedusing RLuc-DNE6 and RLuc-E7, respectively (Figures 1dand e). All the siRNAs examined showed high RNAi
activities with IC50 values between 0.012 and 0.380 nM,
which suggested that the selected siRNAs efficiently
formed an RISC and degraded the artificial RLuconcogene fusion mRNAs.
Effects of HPV16 E6 and E7 siRNAs on endogenous
mRNAs coding E6 and E7
To examine the effects of the siDirect-selected siRNAs on
endogenous E6 and E7 expression, SiHa cells weretransfected with the siRNAs (50 nM) for 48 h, then E6
and E7 expression was analyzed by RT–PCR. As shownin Figure 2a, PCR amplification of cDNA from control
siRNA-transfected cells with a pair of E6-specific primers(E6_104s and E6_558as in Figure 1a, Table 2) gave rise to
three bands representing E6, E6*I and E6*II (top panel).
Transfection with siRNA 233, 243 and 244 decreased thelevels of the upper band representing mRNA coding E6,
but not those of the PCR bands of E6*I and E6*II.
Furthermore, transfection with 493, 497 and 501
decreased the bands of E6 and E6*I, while that with573, 698, 707 and 752 decreased all three bands. RT–PCR
analysis using a pair of E7-specific primers (E7s562 andE7as855 in Figure 1a, Table 2) showed a decrease in E7mRNA level in cells transfected with 493, 497, 501, 573,
698, 707 and 752 (middle panel). The levels of the E7 band
were proportional to the sum of the E6, E6*I and E6*II
levels. Each sample was also examined for b-actin mRNAlevel and the results were used as an internal control(bottom panel). Transfection with siRNA 497, 573, 698,
707 and 752 decreased the expression of either E6 or E7 toa level below 25%, thus they were chosen for further
analyses.
Effects of HPV16 E6 and E7 siRNAs on the growth of
HPV16 þ and HPV16 cancer cells
HPV16 þ (SiHa) and HPV16 (HeLa, SK-OV-3) cancer
cells were stably transfected with a FLuc expression
plasmid (hLuc/pcDNA3), and designated as FL-SiHa-2,
FL-HeLa-1 and FL-SKOV-5 cells, respectively. Using an
siRNA targeting FLuc mRNA (FLuc siRNA) and
Lipofectamine 2000, siRNA transfection conditions forthese cells were optimized to achieve comparable transfection efficiencies. Transfection with 5 nM of FLucsiRNA suppressed the FLuc activities of FL-SiH-2, FLSKOV-5 and FL-HeLa-1 cells by 96.3±0.5, 93.8±0.3
and 93.3±0.5%, respectively (data not shown), which

demonstrated that the cells were transfected by the siRNAat similar levels of efficiency. These results enabled us toanalyze the specificity of E6 and E7 siRNA-induced
growth suppression.
Under the optimized transfection condition, FL-SiH-2,
FL-SKOV-5 and FL-HeLa-1 cells were transfected withfive different siDirect-selected siRNAs (497, 573, 698, 707,752) as well as four others previously reported (186, 203,
222, 535)12,14,16 and analyzed for cell viability. As shownin Figure 2b, all tested siRNAs showed moderate-tostrong growth suppression toward FL-SiHa-2 cells, while
they also inhibited the growth of HPV cells (FL-HeLa1, FL-SKOV-5) to various degrees. Among them, 698,
186 and 535 strongly suppressed the growth of HPV16
cells, especially FL-HeLa-1 cells by more than 70%,
suggesting that these siRNAs had a strong nonspecificgrowth inhibition characteristic.
To determine if decreased siRNA concentrations
alleviated nonspecific growth suppression without compromising the specific growth inhibition, the dose effectsof siRNA 497, 573, 707, 752, 203 and 222 were examined.
At concentrations from 1 to 5 nM, all siRNAs suppressed
the growth of FL-SiHa-2 cells in a dose-dependent
manner (Figure 3c), except for 573, which exhibited
similar levels of growth suppression at these concentrations.
At a concentration of 1 nM, 497, 573 and 752 inhibited
FL-SiHa-2 cell growth by more than 90%, while thegrowth suppression of HPV16 cells decreased to lessthan 35% (Figures 3a and b). These results suggest that497, 573 and 752 were able to induce a potent and specificgrowth inhibition toward HPV16 þ cells at 1 nM. In
contrast, 707, 203 and 222 showed less potent growth
inhibition of FL-SiHa-2 cells at 1 nM.

Dose-dependent effects of E6 and E7 siRNAs on
endogenous E6–E7 mRNA
To confirm that the observed siRNA-induced growth
inhibition was associated with E6 and E7 downregulation,
FL-SiHa-2 cells were transfected with mock, control, E6
and E7 siRNAs (752, 203, 535) for 72 h, after which E6
and E7 expression was examined by RT–PCR using
primer pairs for each (E6; 16E6s104 and 16E6as375,
shown in Figure 1a, Table 2 and E7; 16E7s562 and
16E7as855). As shown in Figure 3d, siRNA 752
suppressed the level of E7 mRNA to 11, 19 and 43% at
25, 5, and 1 nM, respectively, as compared to the control
siRNA-transfected cells. Furthermore, siRNA 752
decreased the E6 mRNA level to 29, 48 and 52%,
respectively. In addition, two previously reported siRNAs(203, 535) showed comparable RNAi activities toward E6
and E7 mRNA. Also, 186 decreased the expression levelsof both E6 and E7 mRNA, whereas 222 decreased onlythat of E6 (Figure 3e), which has been reported
previously.12,13 siRNA 37514 and 66012 were less potent
and suppressed E6 and E7 expression by only 50% (datanot shown).
Induction of p53 and p21WAF1/CIP1 by E6 and E7 siRNAs
Next, we examined the downregulation of E6 protein in
the siRNA-transfected cells by analyzing the expression ofCancer Gene Therapy

145

Efficacious siRNAs targeting HPV16 oncogenes
K Yamato et al

146

Figure 2 Effects of E6 and E7 siRNAs on E6 and E7 expression and cell growth. (a) Suppression of endogenous E6 and E7 expression in SiHa
cells by E6 and E7 siRNAs. SiHa cells were transfected with mock, control, E6 or E7 siRNA (50 nM) for 48 h. cDNA was synthesized from total
RNA and subjected to PCR. The intensity of the bands for E6 and E7 from the siRNA-treated cells were normalized to the b-actin band, and therelative E6 and E7 mRNA levels were calculated by comparing with those of serially diluted samples from mock-transfected cells. The mRNAlevel in control siRNA-treated cells was defined as 100%. (b) Effects of transfection with HPV16 E6 and E7 siRNA on growth of HPV16 þ andHPV16 cells. Effects of siDirect-designed (497, 573, 698, 707, 752) and reported E6 and E7 (186, 203, 222, 535) siRNAs on the growth of
HPV16 (FL-SKOV-5, FL-HeLa-1) and HPV16 þ (FL-SiHa-2) cells were examined. The cells were transfected with mock, control, E6 or E7
siRNA at 5 nM, with the media changed and siRNA transfection repeated every 3 or 4 days. In 8–12 days after the first transfection, cell viability
was examined using a WST-8 assay. Cell viabilities relative to mock-transfected cells are shown. Closed bar represents FL-SKOV-5; gray bar,
FL-HeLa-1; open bar, FL-SiHa-2. All experiments were performed in triplicate. Error bars represent the s.d. from the mean.

p53, since there is no high affinity anti-E6 antibody
capable of detecting low E6 expression presently available.
p53 is an E6 target protein that degrades p53 through theubiquitin-proteasome pathway,2,3 while E6 knockdown

Cancer Gene Therapy

restores its expression. The dose-dependent effects ofsiRNA 497, 573 and 752 on p53 levels were examinedin FL-SiHa-2 cells using immunoblot analysis (Figures 4a–c).
siRNA 497 and 573 caused a substantial accumulation ofEfficacious siRNAs targeting HPV16 oncogenes
K Yamato et alp53 and its downstream target gene product p21WAF1/CIP1
at concentrations from 1 to 25 nM. Transfection of siRNA752 also caused accumulations of p53 and p21WAF1/CIP1 atconcentrations from 1 to 25 nM, with a slightly lower
induction of p53 at 1 nM.
The effects of the E6 and E7 siRNAs (573, 752, 186,
222, 660, 203, 535) on the expressions of p53 andp21WAF1/CIP1 were also examined in SiHa and CaSki
cells, both of which are HPV16 þ cervical cancer celllines, using immunoblotting (Figures 4d–f). As shown in
Figures 4d and e, accumulations of p53 and p21WAF1/CIP1
were seen in SiHa cells transfected with 752, 573, 186, 222,
203 and 535, but not in those transfected with 375 and660, which was consistent with the low RNAi activities of

FL-HeLa-1120%cell viability

100
80
60
40
20
0
control

497

573

707

752

203

222

497

573

707

752

203

222

497

573

707

752

203

222

FL-SKOV-5120%cell viability

100
80
60
40
20
0
control
FL-SiHa-2
120%cell viability

100
80
60
40
20
0
control

Figure 3 Dose-dependent effects of HPV16 E6 and E7 siRNAs on growth of HPV16 and HPV16 þ cancer cells, and endogenous E6 and E7
expression. Dose-dependent effects of E6 and E7 siRNAs on growth of FL-HeLa-1 (a), FL-SKOV-6 HPV16 (b) and FL-SiHa-2 HPV16 þ cells(c). Cells were plated in 96-well plates, then transfected with mock, control, E6 or E7 siRNAs (497, 573, 707, 752, 203, 222) at 5 nM (closed bar),
2 nM (gray bar) and 1 nM (open bar). Transfection was repeated every 3–4 days and a WST-8 assay was performed 7–11 days after the firsttransfection. Cell viabilities relative to mock-transfected cells are shown. All experiments were performed in triplicate. Error bars represent the s.d.
from the mean. Dose-dependent effects of E6 and E7 siRNAs on E6 and E7 mRNA expression were analyzed in FL-SiHa-2 cells. FL-SiHa-2 cells
were transfected with mock, control, E6, E7 or firefly luciferase (Fluc) siRNA at the indicated concentrations for 72 h, then analyzed for E6 and E7
expression (RT–PCR) and FLuc activity. The E6 and E7 siRNAs used in this experiment were 752, 203 and 535 (d), and 186 and 222 (e). 18S
ribosomal RNA was used for normalizing the mRNA quantity. mRNA levels in siRNA-treated cells are shown relative to the level in control siRNAtreated cells. FLuc siRNA decreased FLuc activity by 96, 97 and 95% at 25, 5 and 1 nM, respectively.

Cancer Gene Therapy

147

Efficacious siRNAs targeting HPV16 oncogenes
K Yamato et al148
0standard (arbitrary unit)
12.5 25
50 100 200

25

cont
5

1

25

752
5

1

25

203
5

1

25

535
5

1

siRNA
nM

E6

E7

18S

E6
E7
18S
E6(%)
E7(%)

120 92
153 101
80
91
100 100
100 100

Standard (arbitrary unit)
0

12.5

25

50

100 200

99
87
83
100
100

37
18
86
29
11

control
25

5

40
17
84
48
19

70
41
90
52
43

77
42
84
61
26

186
25

5

67
37
72
93
46

94
44
82
96
51

222
1

25

5

47
26
80
39
17

29
21
85
34
22

54
44
85
54
50

siRNA
1

nM

E6

E7

18S
E6

174 188

70

64

63

95

E7

162 178

69

65

75

176 209 206

18S

83

103 124 127 118 121 102

E6(%)

100 100

32

25

24

38

42

50

E7(%)

100 100

34

27

30

76

88

103

88

106 108

Figure 3 Continued

those siRNAs toward E6 expression. The level of p53
level induced by 535 was disproportionate to its effect on
E6 mRNA, which might have been caused by an offtarget effect. CaSki cells were transfected with Oligofectamine, since Lipofectamine 2000 is cytotoxic to CaSki
cells. Transfection of lamin A/C siRNA (50 nM) decreased

Cancer Gene Therapy

the level of lamin A/C to 35%, suggesting that at least
two-thirds of the cells were transfected with Oligofectamine (Figure 4f). The accumulation of p53 by siRNA 573
and 752 was obscure in CaSki cells as compared to SiHa
cells, which was likely due to dilution of accumulated p53
by nontransfected cells containing low levels of p53.

1

(nM)

25

5

1

(nM)

control

5

573

mock

25

control497

mock

controlmock

Efficacious siRNAs targeting HPV16 oncogenes
K Yamato et al

149
752
25

5

1

(nM)

p53

p21WAF/CIP1

p53

1.0

4.2 4.3

2.6

control752

mocklaminA/C

control

573

752

4.4 4.3 3.7

535

1.0

203

p53

mock

573

660

752

375

3.1 3.3 2.8

222

1.0

186

p53

controlmocklamin A/C

p53

p21WAF/CIP1

lamin A/C

p53

1.0 3.3 2.4 3.0

2.4 0.7 1.6

p53

1.0 5.1 4.2 13.4

p53
1.0 1.0
lamin A/C 0.3 1.0
p53 fold- 1.0
increase

1.5
1.2

1.3
1.1

1.3

1.2

Figure 4 Effects of E6 and E7 siRNAs on p53 and p21WAF1/CIP1 expression in HPV16 þ cancer cells. FL-SiHa-2 cells were transected withmock, control, 497 (a), 573 (b) or 752 siRNA (c) at the indicated concentrations. SiHa cells were transfected with mock, control, 752, 573, 186,
222, 375 or 660 siRNA (d), or 203 or 535 siRNA (e) at 25 nM. CaSki cells were transfected with mock, control, 573 or 752 siRNA at 50 nM (f). The
cells were incubated for 72 h, then analyzed for the expression of p53, p21WAF1/CIP1 and lamin A/C by immunoblotting. p53 levels werenormalized to those of lamin A/C. Lamin A/C siRNA was used to assess siRNA transfection efficiency in CaSki cells.

However, both siRNAs induced p21WAF1/CIP1 expression
in those cells, suggesting that their suppression of E6
expression caused p53 accumulation in cells effectivelytransfected with the siRNAs.

Induction of senescence in cervical cancer cells treatedwith siRNA 573 and 752
To clarify the mechanism by which E6 and E7 siRNAs
induced growth suppression in cervical cancer cells,
FL-SiHa-2 cells were transfected separately with siRNA
573 and 752 at 5 nM every 3 days, with cell morphologicalchanges closely observed under an inverted microscope.
Seven days after the first transfection, both 573- and 752siRNA-transfected cells become larger and flatter,
whereas mock and control siRNA-transfected cells did
not change (data not shown). In 12–14 days after the firsttransfection, most E7 siRNA-treated cells were found tohave long cytoplasmic projections and became positive
for senescence associated b-galactosidase (SA b-gal)
staining (Figure 5a). Furthermore, SA b-gal activity was
detected in the perinuclear areas of the E7 siRNA-treatedcells. These results suggested that the E7 siRNAs caused

growth inhibition in HPV16 þ cells by inducing cellularsenescence.

Inhibition of tumor growth initiated by SiHa cells in
NOD/SCID mice by siRNA 752
SiHa cells at 2  107 were subcutaneously inoculated into
NOD/SCID mice. Six weeks after inoculation, palpable
tumors had formed in all of the mice, and control or 752
siRNA complexed with atelocollagen was directly injected
into each tumor every 7 days. Thirty days after the firstsiRNA injection, the tumors were excised and weighed.
As shown in Figure 5b, tumors treated with siRNA 752
had significantly lower weights than those treated with
control siRNA (Po0.001, mean weight: 2.73±0.66 gin the control siRNA-treated group; n ¼ 6 versus
1.03±0.29 g in the 752-treated group; n ¼ 6), whichshowed that treatment with siRNA 752 decreased in vivo
tumor growth caused by SiHa cells.
Effects of E7 siRNAs on E7 variant expression
Three siRNAs (497, 573, 752) were chosen from 10
siDirect-selected siRNAs based on their high levels ofRNAi activity and specificity. We then performed a

Cancer Gene Therapy

Efficacious siRNAs targeting HPV16 oncogenes
K Yamato et al

150

Figure 5 Induction of senescence and in vivo tumor growth of HPV16 þ cancer cells by HPV16 E7 siRNA. (a) FL-SiHa-2 cells were transfectedwith mock, control, 573 or 752 siRNA at 5 nM using Lipofectamine 2000 every 3 days for 14 days, and then stained for senescence-associated bgalactosidase activity. Representative microscopy photographs are shown. (b) Suppression of in vivo tumor growth of SiHa cells by E7 siRNA.
SiHa cells were subcutaneously inoculated into NOD/SCID mice (n ¼ 6). Six weeks after transplantation palpable tumors had developed, andthen the control or 752 siRNA (500 pmole) complexed with atelocollagen was directly injected into the tumors every 7 days. Thirty-five days after
the first siRNA injection, all tumors were excised and weighed. Bars represent the mean results (n ¼ 6).

literature search for E6 and E7 variant sequences that hada mismatch with these siRNAs.32–37 A minor E7 variant,E7(760c), which contained cytosine instead of thymine at
nucleotide 760,35 was found to have a mismatch withsiRNA 752. We tested the RNAi activity of 752 toward
the expression of E7(760c) using a reporter gene assay.
SiHa cells were transfected with the RLuc-E7(760c) fusionmRNA expression plasmid along with siRNA 752 or
752c, which was perfectly matched to E7(760C) (Supplementary Figure 1). Although both 752 and 752c
suppressed the expression of RLuc-E7(760c), the 1-basemismatch decreased the IC50 value by fivefold (0.021 nM
of 752c siRNA versus 0.107 nM of 752 siRNA). This result
suggested that perfect sequence complementarity between
the guide strand and target mRNA, especially in the
middle portion of the guide strand, is important for RISC
enzyme efficiency.

Discussion

With the aim of identifying new drug candidates for
HPV16 infection and virus-related cancer, we designed
siRNAs targeting HPV16 E6 and E7 oncogenes using
siDirect computer software,27 and analyzed their RNAi
activities and specificity. The siDirect software is based on
guidelines for the selection of effective siRNA sequencesdeveloped by Ui-Tei et al.26 and enables selection ofsiRNAs with structural advantages for efficient loadingof guide strands into RISC,26,30,31 as well as a minimal

Cancer Gene Therapy

number of off-target candidates from a database of
nonredundant sequence sets of human genes. In thepresent study, reporter gene assays using luciferase-target
fusion gene expression plasmids and our designed siRNAs
revealed that all siDirect-designed siRNAs possessed highRNAi activity, as expected. However, the levels of
suppression of endogenous E6-E7 mRNA in HPV16 þcancer cells varied among them. Five of the 10 siRNAs
suppressed the expression of target mRNA by more than
75%, while the others showed suppression of 35–65%.
These results suggest that RISC loading is not a soledeterminant of RNAi activity and other factors, such asaccessibility of RISC to the target sequence,38–40 target
cleavage and release of cleaved RNA, might be involvedin the RNAi activities of designed siRNAs. Target
accessibility is assumed to be due to the RNA secondary
structure and interaction of target mRNA with othermolecules. Thus, structural prediction of target mRNA
would be beneficial for designing siRNAs.
Despite initial reports showing a high specificity for
siRNA-directed RNAi,10,11 recent studies have revealed
that siRNAs induce off-target effects by several mechanisms.20,25,41 An siRNA can tolerate some mismatches with
its target mRNA when undergoing directed target
cleavage, though enzyme efficiency decreases with increases in the number of mismatches.25 In fact, weobserved that a single nucleotide mismatch in the central
portion of an siRNA targeting mRNA caused a fivefold
decrease in the value of IC50. siRNA sequences selectedby siDirect software have at least three base mismatcheswith nonredundant sequence sets of human genes.27

Efficacious siRNAs targeting HPV16 oncogenes
K Yamato et al

Therefore, some off-target effects exerted through RISC
cleavage may be attenuated by lowering the siRNAconcentration.
mRNA that has a 30 untranslated region containing asequence complementary to nucleotide 2–7 of the 50 endof the guide strand, analogous to the seed region of
miRNA, can be a collateral target for siRNA-mediatedtranslation inhibition.20–22 Those studies also suggested
that all siRNAs potentially possess miRNA-like activities.
To minimize such off-target effects, complementarity
between the guide strand and human genes should be
carefully analyzed. Chemical modifications of siRNAs,
such as a 20 O-methyl ribose modification, have beenreported to reduce the miRNA-like off-target effectwithout compromising RNAi activity.42
siRNAs can also exert unintended effects through IFN-I
and inflammatory cytokine production. Receptors ofsiRNAs for immunostimulation include double-stranded
RNA-dependent protein kinase (PKR) and toll-likereceptors (TLRs), such as TLR3, TLR7 and TLR8.
siRNAs with sequences of 50 -GUCCUUCAA-30 , 50 UGUGU-30 and 50 -UGUCU-30 have been shown tostimulate mouse TLR7 and, most likely, human
TLR8.23,24 Among the E6 and E7 siRNAs selected inour study, siRNA 497 contained the 50 -UGUCU-30
sequence. Activation of TRLs may be bypassed bydelivery of an siRNA in a cholesterol-conjugated43 or
atelocollagen-complexed form,44 as well as following
chemical modification of the RNA backbone.45
The mechanism by which the present siRNAs exhibited
their off-target effects on HPV16 cells was not revealed.
However, nonspecific growth suppression of three of thesiRNAs (497, 573, 752) was significantly improved
without compromising their strong growth suppressionof HPV16 þ cells by reducing the siRNA concentration
to as low as 1 nM. To further improve the off-target effect,
we are now working on backbone modification of thesesiRNAs, which has been reported to attenuate miRNA-like
activity and cytokine response.42,45,46
miRNA has been implicated in diverse regulation
pathways, including control of cell differentiation, apoptosis,
cell proliferation and organ development. Long-term useof a high dose of siRNA might disturb these normal
functions of miRNA by saturating the limited source ofRNAi machinery. Therefore, it is important to administera minimal dose of synthetic siRNA with high RNAi
activity. A molecular abundance of human miRNA in
HeLa cells has been reported.47 The calculated intracellular concentrations of different miRNAs, including
miR-22, miR-16, let-7 and miR-21, in HeLa cells ranges
from about 0.1 to 2 nM, when assuming that the cellvolume is about 10 pl. Therefore, administration of E6
and E7 siRNAs that do not exceed an intracellularconcentration of 2 nM should safely exert an antitumor
effect without disturbing endogenous RNAi.
In the present experiments, we identified three newsiRNAs (497, 573, 752) that possessed high RNAi
activities toward E6 and E7 expression, and whose
growth inhibition effects were potent and specific to
HPV16 þ cancer cells. Furthermore, the sequences of

those siRNAs showed full complementarity with all
HPV16 classes and subclasses. We also performed aliterature search to determine if the siRNAs werecompatible with E6 and E7 variants found in patient
samples from various countries (total 991 cases).32–37
Three variants were found in the same series of patientsfrom Hong Kong (255 patients),35 with E6(517G), in
which thymine at nt 517, located at the 50 end of the 497
guide strand, was changed to guanine, found in threepatients, E7(757T), which carried thymine instead ofcytosine at nt 757, found in one patient and E7(760c),
which contained thymine instead of cytosine at nt 760,
found in seven patients (3.1%). These variants were notseen in patients from other areas. Thus, siRNA 497, 573
and 752 are considered to be compatible with most
HPV16 variants.
Deregulation of E6 and E7 expression is a necessary
cause of malignant transformation, and additional genetic
alterations accumulate before progression to carcinoma
development. Studies using antisense oligo-DNA, ribozymes, transcriptional suppression by forced E2 expression, and siRNAs have revealed that inhibition of E6
and E7 expression is sufficient to cause growth suppression of HPV-related cancer cells.7–9,12–18,48 In the present
study, E6 and E7 siRNA-induced growth suppression
was associated with morphological and cytochemical
characteristics of cellular senescence. Our results are
consistent with other studies, which also demonstrated
that downregulation of E6 and E7 causes in vitro andin vivo growth suppression, as well as cellular senescence.15,16,48 Induction of senescence might be caused byreactivation of PML, which has been shown to be a
mediator of senescence and a target of E7.6 Recently,
human fibroblasts immortalized by a temperature sensitive SV40 large T antigen were reported to undergo
irreversible senescence by a reactivated p16INK4a/Rb
pathway after incubation at a nonpermissive temperature
for a specific period of time.49 The functional similarity
between SV40 large T antigen and hrHPV E6E7
suggests that siRNA-mediated senescence might beirreversible.
Infection with hrHPV causes intraepithelial precancerous
lesions in the cervix, termed cervix intraepithelial neoplasia (CIS).50 High-grade lesions (CIS grade 3) express
deregulated hrHPV oncogenes and eventually develop
into invasive cervical cancer after a long latency without
spontaneous regression. Downregulation of the virus
oncogenes using an siRNA could reverse the neoplastic
phenotype and also prevent E7-induced genetic alterations. In hrHPV-infected lesions, replication of HPVs
relies on E7 expression.5 Thus, an siRNA targeting E7
may be able to inhibit virus replication as well as
propagation. CIS lesions exist within epithelium and areeasily accessible.51 Furthermore, local siRNA administration could help to avoid its hazardous systemic offtarget effects. Therefore, we consider such lesions to beideal targets of RNAi therapy.
The US Food and Drug Administration (FDA)
approved a preventive HPV vaccine for immunizationof women between 9 and 26 years of age, with a second

Cancer Gene Therapy

151

Efficacious siRNAs targeting HPV16 oncogenes
K Yamato et al

152

vaccine now being tested. These HPV vaccines will
eventually reduce the incidence rates of viral infection
and cervical cancer. However, there are currently noeffective therapies for individuals infected with HPV anda small fraction of those patients will develop cancer in a
decade or two.50 Notably, immunosuppressed patientssuch as organ transplant recipients and HIV-infectedpatients, whose response to an HPV vaccine is unknown,
have greater propensity for cervical cancer and analcancer.52,53 It is also unclear how long the protection
provided by the HPV vaccines will last. Therefore, RNAitherapy using potent and specific siRNAs will benefit
patients infected with HPV. Also, in parallel with HPV
vaccination, RNAi therapy may speed up the reduction inrates of incidence of HPV infection and cervical cancer.

Acknowledgements

We thank Dr Hideki Matsui and Dr Kazuhito Tomizawa(Okayama University) for their helpful discussion andsuggestions. This work was supported in part by Grantsin-aid for Scientific Research (C) from Japan Society for
the Promotion of Science [15591749 (MY), 19592169(KY)].

References
1 Bosch FX, de Sanjose S. Chapter 1: human papillomavirus
and cervical cancer—burden and assessment of causality.
J Natl Cancer Inst Monogr 2003; 31: 3–13.
2 zur Hausen H. Papillomaviruses causing cancer: evasion
from host-cell control in early events in carcinogenesis.
J Natl Cancer Inst 2000; 92: 690–698.
3 Longworth MS, Laimins LA. Pathogenesis of humanpapillomaviruses in differentiating epithelia. Microbiol Mol
Biol Rev 2004; 68: 362–372.
4 Munger K, Baldwin A, Edwards KM, Hayakawa H, Nguyen
CL, Owens M et al. Mechanisms of human papillomavirusinduced oncogenesis. J Virol 2004; 78: 11451–11460.
5 Flores ER, Allen-Hoffmann BL, Lee D, Lambert PF. The
human papillomavirus type 16 E7 oncogene is required forthe productive stage of the viral life cycle. J Virol 2000; 74:
6622–6631.
6 Bischof O, Nacerddine K, Dejean A. Human papillomavirus
oncoprotein E7 targets the promyelocytic leukemia protein
and circumvents cellular senescence via the Rb andp53 tumor suppressor pathways. Mol Cell Biol 2005; 25:1013–1024.
7 Venturini F, Braspenning J, Homann M, Gissmann L,
Sczakiel G. Kinetic selection of HPV 16 E6/E7-directed
antisense nucleic acids: anti-proliferative effects on HPV 16transformed cells. Nucleic Acids Res 1999; 27: 1585–1592.
8 Alvarez-Salas LM, Cullinan AE, Siwkowski A, Hampel A,
DiPaolo JA. Inhibition of HPV-16 E6/E7 immortalization of
normal keratinocytes by hairpin ribozymes. Proc Natl Acad
Sci USA 1998; 95: 1189–1194.
9 Chen Z, Kamath P, Zhang S, St John L, Adler-Storthz K,
Shillitoe EJ. Effects on tumor cells of ribozymes that cleave
the RNA transcripts of human papillomavirus type 18.
Cancer Gene Ther 1996; 3: 18–23.

Cancer Gene Therapy

10 Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K,
Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNAinterference in cultured mammalian cells. Nature 2001; 411:
494–498.
11 Caplen NJ, Parrish S, Imani F, Fire A, Morgan RA. Specificinhibition of gene expression by small double-stranded
RNAs in invertebrate and vertebrate systems. Proc Natl
Acad Sci USA 2001; 98: 9742–9747.
12 Jiang M, Milner J. Selective silencing of viral gene expressionin HPV-positive human cervical carcinoma cells treated withsiRNA, a primer of RNA interference. Oncogene 2002; 21:6041–6048.
13 Yoshinouchi M, Yamada T, Kizaki M, Fen J, Koseki T,
Ikeda Y et al. In vitro and in vivo growth suppression ofhuman papillomavirus 16-positive cervical cancer cells by E6
siRNA. Mol Ther 2003; 8: 762–768.
14 Butz K, Ristriani T, Hengstermann A, Denk C, Scheffner M,
Hoppe-Seyler F. siRNA targeting of the viral E6 oncogene
efficiently kills human papillomavirus-positive cancer cells.
Oncogene 2003; 22: 5938–5945.
15 Hall AH, Alexander KA. RNA interference of humanpapillomavirus type 18 E6 and E7 induces senescence in
HeLa cells. J Virol 2003; 77: 6066–6069.
16 Putral LN, Bywater MJ, Gu W, Saunders NA, Gabrielli BG,
Leggatt GR et al. RNA interference against humanpapillomavirus oncogenes in cervical cancer cells results inincreased sensitivity to cisplatin. Mol Pharmacol 2005; 68:
1311–1319.
17 Niu XY, Peng ZL, Duan WQ, Wang H, Wang P. Inhibitionof HPV 16 E6 oncogene expression by RNA interference invitro and in vivo. Int J Gynecol Cancer 2006; 16: 743–751.
18 Gu W, Putral L, Hengst K, Minto K, Saunders NA, Leggatt
G et al. Inhibition of cervical cancer cell growth in vitro andin vivo with lentiviral-vector delivered short hairpin RNA
targeting human papillomavirus E6 and E7 oncogenes.
Cancer Gene Ther 2006; 13: 1023–1032.
19 Kuner R, Vogt M, Sultmann H, Buness A, Dymalla S,
Bulkescher J et al. Identification of cellular targets for thehuman papillomavirus E6 and E7 oncogenes by RNA
interference and transcriptome analyses. J Mol Med 2007;
85: 1253–1262.
20 Jackson AL, Bartz SR, Schelter J, Kobayashi SV,
Burchard J, Mao M et al. Expression profiling reveals offtarget gene regulation by RNAi. Nat Biotechnol 2003; 21:635–637.
21 Lin X, Ruan X, Anderson MG, McDowell JA, Kroeger PE,
Fesik SW et al. siRNA-mediated off-target gene silencing
triggered by a 7 nt complementation. Nucleic Acids Res 2005;
33: 4527–4535.
22 Jackson AL, Burchard J, Schelter J, Chau BN, Cleary M,
Lim L et al. Widespread siRNA ‘off-target’ transcriptsilencing mediated by seed region sequence complementarity.
RNA 2006; 12: 1179–1187.
23 Hornung V, Guenthner-Biller M, Bourquin C, Ablasser A,
Schlee M, Uematsu S et al. Sequence-specific potent
induction of IFN-alpha by short interfering RNA in
plasmacytoid dendritic cells through TLR7. Nat Med 2005;
11: 263–270.
24 Judge AD, Sood V, Shaw JR, Fang D, McClintock K,
MacLachlan I. Sequence-dependent stimulation of the
mammalian innate immune response by synthetic siRNA.
Nat Biotechnol 2005; 23: 457–462.
25 Haley B, Zamore PD. Kinetic analysis of the RNAi enzyme
complex. Nat Struct Mol Biol 2004; 11: 599–606.
26 Ui-Tei K, Naito Y, Takahashi F, Haraguchi T, OhkiHamazaki H, Juni A et al. Guidelines for the selection ofEfficacious siRNAs targeting HPV16 oncogenes
K Yamato et al

highly effective siRNA sequences for mammalian and chick
RNA interference. Nucleic Acids Res 2004; 32: 936–948.
27 Naito Y, Yamada T, Ui-Tei K, Morishita S, Saigo K.
siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference. Nucleic Acids Res
2004; 32: W124–W129.
28 Kanda T, Furuno A, Yoshiike K. Human papillomavirus
type 16 open reading frame E7 encodes a transforming genefor rat 3Y1 cells. J Virol 1988; 62: 610–613.
29 Smotkin D, Prokoph H, Wettstein FO. Oncogenic andnononcogenic human genital papillomaviruses generate
the E7 mRNA by different mechanisms. J Virol 1989; 63:
1441–1447.
30 Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore
PD. Asymmetry in the assembly of the RNAi enzyme
complex. Cell 2003; 115: 199–208.
31 Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs
and miRNAs exhibit strand bias. Cell 2003; 115: 209–216.
32 Matsumoto K, Yoshikawa H, Nakagawa S, Tang X, Yasugi
T, Kawana K et al. Enhanced oncogenicity of humanpapillomavirus type 16 (HPV16) variants in Japanese
population. Cancer Lett 2000; 156: 159–165.
33 Hu X, Pang T, Guo Z, Ponten J, Nister M, Bernard Afink G.
Oncogene lineages of human papillomavirus type 16 E6, E7
and E5 in preinvasive and invasive cervical squamous cell
carcinoma. J Pathol 2001; 195: 307–311.
34 Zehbe I, Tachezy R, Mytilineos J, Voglino G, Mikyskova I,
Delius H et al. Human papillomavirus 16 E6 polymorphisms
in cervical lesions from different European populations andtheir correlation with human leukocyte antigen class II
haplotypes. Int J Cancer 2001; 94: 711–716.
35 Chan PK, Lam CW, Cheung TH, Li WW, Lo KW, Chan
MY et al. Human papillomavirus type 16 intratypic variant
infection and risk for cervical neoplasia in southern China.
J Infect Dis 2002; 186: 696–700.
36 Kammer C, Tommasino M, Syrjanen S, Delius H, Hebling
U, Warthorst U et al. Variants of the long control region andthe E6 oncogene in European human papillomavirus type 16
isolates: implications for cervical disease. Br J Cancer 2002;
86: 269–273.
37 Tornesello ML, Duraturo ML, Salatiello I, Buonaguro L,
Losito S, Botti G et al. Analysis of human papillomavirus
type-16 variants in Italian women with cervical intraepithelial neoplasia and cervical cancer. J Med Virol 2004; 74:
117–126.
38 Brown KM, Chu CY, Rana TM. Target accessibility dictates
the potency of human RISC. Nat Struct Mol Biol 2005; 12:469–470.
39 Schubert S, Grunweller A, Erdmann VA, Kurreck J. Local
RNA target structure influences siRNA efficacy: systematic
analysis of intentionally designed binding regions. J Mol Biol
2005; 348: 883–893.

40 Overhoff M, Alken M, Far RK, Lemaitre M, Lebleu B,
Sczakiel G et al. Local RNA target structure influences
siRNA efficacy: a systematic global analysis. J Mol Biol
2005; 348: 871–881.
41 Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams
BR. Activation of the interferon system by short-interfering
RNAs. Nat Cell Biol 2003; 5: 834–839.
42 Jackson AL, Burchard J, Leake D, Reynolds A, Schelter J,
Guo J et al. Position-specific chemical modification ofsiRNAs reduces ‘off-target’ transcript silencing. RNA 2006;
12: 1197–1205.
43 Soutschek J, Akinc A, Bramlage B, Charisse K, Constien R,
Donoghue M et al. Therapeutic silencing of an endogenous
gene by systemic administration of modified siRNAs. Nature
2004; 432: 173–178.
44 Takeshita F, Minakuchi Y, Nagahara S, Honma K, Sasaki
H, Hirai K et al. Efficient delivery of small interfering RNA
to bone-metastatic tumors by using atelocollagen in vivo.
Proc Natl Acad Sci USA 2005; 102: 12177–12182.
45 Kariko K, Buckstein M, Ni H, Weissman D. Suppression of
RNA recognition by Toll-like receptors: the impact of
nucleoside modification and the evolutionary origin ofRNA. Immunity 2005; 23: 165–175.
46 Morrissey DV, Lockridge JA, Shaw L, Blanchard K, Jensen
K, Breen W et al. Potent and persistent in vivo anti-HBV
activity of chemically modified siRNAs. Nat Biotechnol 2005;
23: 1002–1007.
47 Lim LP, Lau NC, Weinstein EG, Abdelhakim A, Yekta S,
Rhoades MW et al. The microRNAs of Caenorhabditis
elegans. Genes Dev 2003; 17: 991–1008.
48 DeFilippis RA, Goodwin EC, Wu L, DiMaio D. Endogenous human papillomavirus E6 and E7 proteins differentiallyregulate proliferation, senescence, and apoptosis in HeLa
cervical carcinoma cells. J Virol 2003; 77: 1551–1563.
49 Takahashi A, Ohtani N, Yamakoshi K, Iida S, Tahara H,
Nakayama K et al. Mitogenic signalling and the p16IINK4a-Rb
pathway cooperate to enforce irreversible cellular senescence.
Nat Cell Biol 2006; 8: 1291–1297.
50 Snijders PJ, Steenbergen RD, Heideman DA, Meijer CJ.
HPV-mediated cervical carcinogenesis: concepts and clinicalimplications. J Pathol 2006; 208: 152–164.
51 Palliser D, Chowdhury D, Wang QY, Lee SJ, Bronson RT,
Knipe DM et al. An siRNA-based microbicide protects micefrom lethal herpes simplex virus 2 infection. Nature 2006;
439: 89–94.
52 Koshiol JE, Schroeder JC, Jamieson DJ, Marshall SW,
Duerr A, Heilig CM et al. Time to clearance of humanpapillomavirus infection by type and human immunodeficiency virus serostatus. Int J Cancer 2006; 119: 1623–1629.
53 Palefsky J. Human papillomavirus-associated malignancies
in HIV-positive men and women. Curr Opin Oncol 1995; 7:
437–441.

Supplementary Information accompanies the paper on Cancer Gene Therapy website (htttp://www.nature.com/cgt)

Cancer Gene Therapy

153