Cancer Gene Therapy (2006) 13, 445–450

r 2006 Nature Publishing Group All rights reserved 0929-1903/06 $30.00

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REVIEW
Papillomaviruses as targets for cancer gene therapy
EJ Shillitoe
Department of Microbiology & Immunology, Upstate Medical University, State University of New York,
Syracuse, NY, USA

The human papillomaviruses (HPVs) are a diverse group of infectious agents, some of which are a causative agent of human
cancers. Cervical cancer and oral cancer are closely associated with specific types of HPV, and the tumors grow only if there is
continual expression of the viral E6 and E7 genes. Evidence from in vitro studies shows that when expression of these genes is
inhibited by gene therapy approaches such as antisense RNA, ribozymes, or siRNA, the transformed phenotype of the cells is lost.
Although it seems possible that clinical applications of this approach could help in the management of cervical and oral cancers
there have been no clinical trials of gene therapy for HPV-associated cancers. Since the basic information is now available, a shift to
translational research would be greatly welcomed.
Cancer Gene Therapy (2006) 13, 445–450. doi:10.1038/sj.cgt.7700926; published online 9 December 2005
Keywords: review; papillomaviruses; cervical cancer; oral cancer; gene therapy

Introduction Agency for Research on Cancer concluded that around

The last 30 years have seen the discovery of many types of
papillomavirus as well as the demonstration of their role
in human cancer and their significance as targets for
diagnosis and therapy.1 These viruses comprise a large
variety of small DNA viruses, and they can infect many
species. They were found originally in skin lesions of
rabbits, which were shown to be both infectious and
prone to malignant progression.2 The human papilloma-
viruses (HPVs) were originally found in warts3 and these
are now known as the ‘low-risk’ HPVs. Related viruses
were later found to be associated with some carcinomas
and are known as the ‘high-risk’ HPVs. Between them the
two groups of HPVs now comprise over 200 viral types
and new ones are still being identified.4
Essentially 100% of cervical cancers contain HPV of
the high-risk types, and persistent infection of the cervix
with these viruses is considered to be a necessary cause of
cervical cancer.5 Other ano-genital cancers also contain
papillomaviruses in almost 90% of cases.6 In addition to
cervical cancer, there is reason to believe that oral cancer
might have an etiological association with the high-risk
HPVs. A meta-analysis of 94 reports concluded that HPV
is indeed expressed more frequently in oral cancer than
in normal oral mucosa, and should be considered as a
risk factor.7 A multinational study by the International
Correspondence: Dr EJ Shillitoe, Department of Microbiology &
Immunology, Upstate Medical University, State University of New
York, 750 East Adams Street, Syracuse, NY 13210, USA.
E-mail: shillite@upstate.edu
Received 24 July 2005; revised 23 September 2005; accepted 4
October 2005; published online 9 December 2005
18% of oropharyngeal cancers contain high-risk HPVs.8
The prognosis for patients that are diagnosed with
HPV-associated carcinomas has not improved dramati-
cally in recent years. For cervical cancer the 5-year
survival rate has improved by only 4% over the last 25
years, and is now around 73%. For oral cancer the 5-year
survival rate has improved by 5% over the same interval,
but is still only around 59%. Screening programs for
premalignant cervical lesions appear to have reduced the
incidence of cervical cancer over the last few decades, but
in the US there are still 10 000 cases and 3700 deaths per
year.9 For oral cancer, screening programs do not have a
proven benefit10 and there has been no recent reduction in
the incidence, which remains at 30 000 cases with 7000
deaths.9 Thus there is a clear need for improvements in
prevention and management of HPV-associated cancers.
The potential for gene therapy was reviewed here in the
past,11 and the 10th anniversary of that review seems to be
an opportune time to re-examine the development of the
field.
Mechanisms of malignant transformation by HPVs
As with most oncogenic DNA viruses, the high-risk HPVs
encode proteins whose expression is continually needed
for the cell to remain malignant. These are termed E5, E6
and E7. In the animal papillomaviruses other proteins
are important but in humans it seems clear that these
three, and especially E6 and E7, play the major role. The
expression of these proteins is controlled from a region
of the viral genome known as the Upstream Regulatory
Region (URR), and the functions of this region are
regulated by factors that are found mostly in epithelial
 Papillomaviruses as targets for cancer gene therapy
EJ Shillitoe
446
cells. This explains the epithelial-specificity of HPVs.
Expression from the URR is impaired by the viral E2
protein which prevents uncontrolled expression of trans-
forming proteins in all infected cells.
After a prolonged premalignant infection, part of the
HPV genome becomes integrated into the cell genome.
The chromosomal site of integration is not specific, but
the effect on the viral genome is to preserve the URR,
the E6 and E7 genes, and the 50 end of the E2 gene.4 In
the malignant cell there is thus a permanent expression of
the E6 and E7 RNAs and proteins, often with the amino-
terminal part of the E2 protein.
The major effect of the E6 protein is the loss of the
cellular protein p53. This happens through binding of E6
to p53, along with a variety of other proteins. p53 is then
degraded by the cellular ubiquitin enzyme system.12 The
effect of the E7 protein is, with other factors, to bind the
pRB protein.13 This leads to degradation of the protein14
and the release of transcription factors that are then free
to transactivate the expression of cellular genes.
In addition to the loss of functional p53 and pRB, the
expression of HPV proteins causes other changes in the
cell that are understood less well. These include the loss
of DNA-repair pathways, leading to an increased rate
of random mutations and chromosome damage.15 These
random changes result in some inconsistent effects in
the cells, which can include upregulation of Notch ligand
and Jagged 1 gene, and downregulation of a negative-
regulator, Manic Fringe.16
Those cervical and oral cancers that lack HPVs have
rather different genetic changes. They usually have muta-
tions in the p53 gene17–19 as well as deletions of whole or
large parts of chromosomes.20 The resulting changes in
the cell phenotype appear to be the same, but of course
such cells would lack the HPV targets for gene therapy.
Vaccines
The recent development of vaccines for papillomaviruses
has represented a major advance in preventive methods
for human cancer. The basis of vaccines has been the
observation that patients with cervical cancer have high
levels of antibody to the L1 gene product of papilloma-
viruses, implying that the viral particle is exposed to the
immune system at some point in the infection. Further-
more, papillomavirus infections of animals can be
prevented by vaccination, using the L1 gene product as
antigen. On this basis, two different human vaccines have
been developed and tested in controlled clinical trials.
Each has consisted of virus-like particles, which expose
the immune system to L1 antigens from more than
one HPV-type. Thus they could be expected to prevent
infection by several HPVs but could not improve a
response to tumor cells since the L1 gene is lost when
the cells become transformed. Each vaccine was effective
in reducing persistent infection with HPV and reducing
the incidence of cervical abnormalities.21,22 It is thus
expected that routine use of vaccines will soon be
approved.
Cancer Gene Therapy
The result of vaccination will obviously depend on the
degree to which it is put into use. It is likely that HPV
vaccines will be restricted to adolescent females, and thus
will not have any effect on HPV-associated cancers of
males. Even in vaccinated females, it is unlikely that oral
cancer could be reduced soon, since that cancer develops
in middle age.9 Questions have also been raised as to
whether vaccines for sexually-associated diseases could be
seen as providing excuses for promiscuity. Although this
concern might slow their acceptance in some commu-
nities, it appears to be fairly limited at the present time.23
It seems therefore that although a significant reduction in
cervical cancer can be expected from vaccines, other
methods of managing HPV-associated cancers will also be
needed for the foreseeable future.
Established tumors might be treatable by an immune
reaction to the E7 protein. One recent study has described
the use of the bacterial adenylate cyclase protein of
Bordetella pertussis to target dendritic cells with E7 in
mice and thus cause rejection of grafted E7-expressing
tumor cells.24
Gene therapy for HPV-associated cancers
With the causative nature of HPVs having become clear
for several cancers, and with the molecular pathways
having been described, it seems obvious that targets for
gene therapy now exist and that the time has arrived to
make practical use of the information. The essential
features of HPV-associated cancers, and the possible ways
that these could be used in gene therapy, are summarized
in Figure 1.
Decoys
The expression of the genes of HPV is controlled by
factors that bind the viral URR, and therefore it is clear
Figure 1 Gene therapy approaches to papillomavirus-transformed
cancer cells. In tumor cells from HPV-associated cancers the virus
sequences (green) are integrated into the cell chromosome (orange)
in such a way as to preserve the URR, the E6 and E7 genes, and the
50 end of the gene for E2. The E2 truncated protein, together with
cellular transcription factors, act on the URR to promote expression
of E6, E7 and E2. The E6 and E7 genes prevent correct functioning
of the p53 and pRB genes, respectively. Approaches to gene therapy
(red) include: (1) Decoy proteins to inhibit the promoter/enhancer
effects of the URR, (2) subversion of the URR to control expression
of antitumor genes or replication of antitumor viruses, (3) blocking of
the expression of E6 and E7 by antisense, ribozymes or siRNA, and
(4) replacement of the missing p53 and pRB proteins.

that if decoy factors could bind to the URR in place of the
specific factors, then expression of the viral oncogenes
would stop. A major protein that binds this region is the
E2 protein, and two research groups have shown that
the E2 protein from the bovine papillomavirus can bind
the URR but repress expression in the human cervical
cancer cell line, HeLa. This arrests the growth of these
cells in vitro.25,26 This seems to be a very promising, yet
under-explored area for future research.
Subversion of the URR
Since the URR of papillomaviruses is specifically active in
particular cell types, especially squamous carcinoma cells,
it might be expected that the URR could be used to direct
carcinoma-specific expression of genes that are thera-
peutic. The strength of the URR of HPV-16 and -18 is
rather less than that of promoters from the human cyto-
megalovirus or the SV40 virus, but it is stronger than
the promoters of the mouse mammary tumor virus or
the human mdr-1 gene, all of which have been proposed
for similar uses.27 The URR does have the important
advantage of showing specificity for squamous cells which
the more potent promoters do not. Indeed, in one study,
the URR was used to drive expression of the herpes
simplex thymidine kinase gene in an adenovirus vector,
and an effective suicide phenotype was conferred on the
cancer cells.27
An alternate use for the URR might be to control
the replication of an oncolytic virus within a squamous
cell carcinoma. One virus that could be used is the
herpes simplex virus, which is a cytolytic virus whose
natural host tissue is epithelium. When the promoter of an
essential gene of herpes simplex virus type-1 was replaced
with the URR of HPV-16 the resulting herpes/papillo-
mavirus chimera replicated in oral cancer cells and killed
them, but had significantly less toxicity for cells of other
tissues.28 This virus may have anticancer potential.
Blocking of viral gene expression
Since the main tumorigenic effects of the high-risk HPVs
have been attributed to expression of the E6 and E7 genes,
much of the effort to develop gene therapy for these
infections has been directed at blocking their expression.
Antisense RNA was the original method for specific
blocking of the translation of RNA to protein. Small
single-stranded antisense RNA molecules can bind RNA
of the opposite strand, leading to a duplex, which is soon
degraded. Thus there is a reduction in gene expression.
The original HPV antisense constructs were expressed
from plasmids that contained the E6 and E7 genes in
the opposite orientation to the virus, relative to their
promoter, and were tested in the C4-1 cervical cancer cell
line. This indeed showed a loss of many features of
transformed cells.29 The observations were confirmed in
other cervical cancer cells and in oral cancer cells that
contained HPV sequences.30 These studies established the
feasibility of blocking expression and thus eliminating the
malignant phenotype.
In addition to plasmid expression vectors that cause
the antisense molecules to be synthesized in the cell, it is
Papillomaviruses as targets for cancer gene therapy
EJ Shillitoe
447
possible to use short antisense oligonucleotides, which are
taken up by cells spontaneously. Such oligonucleotides
will also inhibit the transformed phenotype of HPV-
containing cervical and oral cancer cells.31
In addition to established cell lines, HPV antisense
RNA has been shown to inhibit the growth of cervical
cancer primary cells in culture.32 In a mouse model it has
been reported that intratumoral introduction of antisense
RNA to E6 and E7 by an expression plasmid produced
inhibition of tumor growth by downregulation of expres-
sion of E6 and E7.33
Virus vectors have been used to transfer antisense
molecules to cancer cells. For example, antisense
sequences to HPV-16 E6/E7 were transferred to cervical
cancer cells in one study using an adenovirus vector, and
the transfected cells showed less tumorigenicity when
transplanted to nude mice.34 Similarly a retrovirus vector
has been used to transfer antisense to E7 into the cervical
cancer cell line CaSki, and this inhibited the expression of
the E7 protein with upregulation of RB, downregulation
of E2F-1, and inhibition of the tumorigenicity of the cells
in nude mice.35
Ribozymes were an advance on the use of antisense
RNA, in that they comprise an antisense molecule with
a secondary structure that provides the enzymatic ability
to cleave the target molecule. Anti-HPV ribozymes
are capable of digesting the E6/E7 RNA from HeLa cells
although different target sites on the transcript show
different susceptibilities.36 The most effective ribozyme,
targeted to a site at nucleotide 309 in the HPV-18
transcript, can inhibit the transformed phenotype of
HeLa cells when expressed in the cells from a plasmid
expression vector.37 Others have shown that a hairpin
ribozyme against the E6 region of the E6/E7 transcript
prevents immortalization of cells by HPV-16.38 However,
even noncatalytic versions of the ribozyme have some
effect, implying that the antisense component of their
effect is critical, and the digestion of the target is less
important.
In addition to HPV-16 and -18, a self-processing
ribozyme that targets HPV-11 as well as the hepatitis B
virus has been shown to reduce intracellular concentra-
tions of the E6/E7 transcript of HPV-11.39 The use
of anti-HPV ribozymes seems to have been restricted to
in vitro experiments, and practical applications are still
awaited.
Another advance on the principle of antisense RNA
and ribozymes is the development of small-interfering
RNAs (siRNAs). These also lead to digestion of their
target molecule, although by a different mechanism
from ribozymes. Silencing of E6 and E7 mRNA
transcripts by siRNAs has been shown to be feasible.40
Silencing of E6 by siRNA caused apoptosis of HeLa cells
in one study.41 Another group did not find apoptosis
from silencing E6 alone, but the cells did become
more sensitive to the effects of chemotherapy.42 In a
different line of cervical cancer cells, silencing of E6
caused them to lose anchorage-independence and the
cells made significantly smaller tumors in immuno-
suppressed mice.43
Cancer Gene Therapy
 Papillomaviruses as targets for cancer gene therapy
EJ Shillitoe
448
Other approaches to the prevention of gene expression
are possible, but have received less attention. The E6
protein itself might be blocked from functioning by an
RNA molecule that binds it specifically, known as an
aptamer. One group showed that this could cause
apoptosis in HPV-16-positive cancer cells but not HPV-
negative cells.44 The aptamer was encoded by an
expression-plasmid that was transfected into the cells.
Thus, although this provides an alternative method of
blocking the effects of E6 the method of delivery would be
the same as for antisense, ribozyme or siRNA molecules.
In another approach, the HPV E6 gene can be targeted
directly by peptide nucleic acids. One group has shown
that this approach may have potential45 although the
introduction of the peptide nucleic acid to the cell and to
its molecular target does require some additional special-
ized methods.
There appear to have been no direct comparisons
between the effects of antisense RNA, ribozymes, siRNA
and the other approaches. Each seems to have its
individual benefits and drawbacks. None have been
reduced to clinical practice, or have yet been shown to
have an effect on human carcinomas that are growing as
animal xenografts.
Replacement therapy
Since the effects of E6 and E7 are the loss of function of
p53 and pRB respectively, it seems likely that HPV-
associated tumors could be controlled by replacement of
wild-type p53 or pRB proteins in a functional form. This
indeed can be done, but always results in uncontrolled
overexpression of the proteins and death of the target
cells. In cervical cancer cells the overexpression of p53 is
toxic to the cells, independently of the presence of HPV
genes.46 Similarly in oral cancer cells the expression of p53
from an adenovirus vector has the effect of killing tumor
cells regardless of the presence or absence of mutations in
the p53 gene.47,48 The potential use of p53 or pRB as gene
therapy has been examined for many types of cancers,
quite apart from those that are associated with HPV, and
has progressed to the stage of multiple human trials.49
Other approaches
The increase in the mutation frequency of cells that are
infected by HPV leads to expression changes in many
genes that might contribute to the malignant phenotype.
Thus these genes provide novel targets for gene therapy,
but might be restricted to sub-sets of tumors.
One such target is the Notch 1 protein, which
complements the function of E6/E7 in the transformation
of cells. Transformation is associated with the upregula-
tion of the Notch ligand together with a protein known
as Jagged 1, with simultaneous downregulation of a
negative-regulator, Manic Fringe. Antisense to Notch 1
reduces the growth of the cervical cancer cell line CaSki
when introduced in an adenovirus vector. siRNA to
Jagged 1 also inhibits the tumorigenicity as does over-
expression of Manic Fringe or expression of a dominant
negative Jagged 1.16 Thus this component of HPV-
Cancer Gene Therapy
associated tumorigenicity might represent a target for
gene therapy.
In another interesting approach, efforts have been
made to produce adenoviruses that replicate preferentially
in HPV-transformed cells. This could be possible if
replication-defective viral mutants could be comple-
mented by HPV oncogenes. There are DNA-sequence
similarities between the E7 protein of high-risk papillo-
maviruses and the pRB-binding regions, denoted CR1
and CR2, in the adenovirus E1A proteins. Thus, by
deleting the CR1 and CR2 domains of an adenovirus, one
mutant has been obtained that is reported to grow
preferentially in raft cultures of human keratinocytes that
were previously transfected with the E7 gene of HPV-18.50
If this method can be developed further then it might
produce a therapeutic virus that replicates in HPV-
associated tumors, and thus be used in their treatment.
Delivery of anti-HPV gene therapy
As with many of forms of gene therapy, a significant
problem, and possibly the most important problem, is the
delivery of the therapeutic molecules into the tumor of
a patient.
Since HPV-associated tumors always develop on a
body surface, the most obvious way to transfer DNA to
a primary tumor would be by physical application. One
method of achieving this is to coat the DNA onto gold
beads and propel them into tissue with compressed helium
using a ‘gene gun’. Since papillomavirus-associated
cancers develop in basal epithelial cells, just a few cells
below the surface, this is an attractive approach for the
transduction of the target. However, early-generation
gene guns have not shown sufficient efficiency, and until
this is improved, it does not seem that physical transfer
will be effective for treating human cancers.51
Antisense oligonucleotides are simple constructs to
deliver, and this can be accomplished by intravenous
infusion. This has been shown successfully in cancer
therapy for the blocking of expression of genes such as,
for example, Bcl-2. Antisense oligonucleotides that target
that gene have now been tested in several human trials.52
It should be possible, therefore, to deliver antisense E6 or
E7 RNA by intravenous infusion. Larger molecules,
including ribozymes, cannot be delivered by intravenous
infusion, and the gene that encodes them must be
delivered directly to the tumor. This has not yet been
found to be possible in the case of anti-HPV ribozymes.
Indeed even expression of antisense RNA, which is
effective when encoded by a plasmid vector, has not been
found to be effective in cells that were infected with an
adenovirus vector that carried the same expression
cassette. The reasons for this are not known. Further-
more, solid tumors, such as oral cancers, are not well
transfected by adenovirus vectors, and only cells around
the needle track are well transduced.47
Adeno-associated virus vectors can be used to deliver
anti-E6/E7 antisense or ribozyme sequences to HeLa or
SiHa cells, but injection into established SiHa tumors had

no therapeutic effect.53 The same vector was able to
express the monocyte chemoattractant protein-1 and thus
inhibit tumors, and therefore the vector itself was
effective. The reason that the anti-E6/E7 sequences were
not effective remains to be found, and this question
represents a critical feature of the development of gene
therapy for solid tumors. Adeno-associated virus does
have the added advantage of having some intrinsic anti-
HPV effects, even without any need for inserted anti-HPV
sequences.54 Although the mechanism for this is un-
known, it could certainly increase the effectiveness of any
gene therapy vector.
Despite recent progress and the various possibilities
that have emerged, it still seems that there is no effective
delivery mechanism for anti-HPV gene therapy, and this
presents a problem at least as important as the specific
targets and mechanisms.
Summary
Only a few human cancers are clearly associated with
viruses, but the ones that are present very attractive
targets for gene therapy. Cervical and other cancers that
express HPV gene transcripts are dependent on these
transcripts for their persistence. Exact molecular targets
have been shown to exist and several approaches to their
inhibition have been demonstrated. A stronger effort
in translational research could produce effective cancer
therapies in a short time.
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