KUOPION YLIOPISTON JULKAISUJA G. – A.I.

VIRTANEN –INSTITUUTTI 35
KUOPIO UNIVERSITY PUBLICATIONS G.
A.I.VIRTANEN INSTITUTE FOR MOLECULAR SCIENCES 35

TANJA HAKKARAINEN

Enhancement of Cancer Gene Therapy With

Modified Viral Vectors and Fusion Genes

Doctoral dissertation

To be presented with permission of the Faculty of Natural and Environmental Sciences

of the University of Kuopio for public examination in Auditorium, Tietoteknia building,
University of Kuopio, on Friday 13th May 2005, at 1 pm

Department of Biotechnology and Molecular Medicine

A.I. Virtanen Institute for Molecular Sciences,
University of Kuopio
Distributor: Kuopio University Library
P.O.Box 1627
FIN-70211 KUOPIO
FINLAND
Tel. +358 17 163 430
Fax +358 17 163 410
http://www.uku.fi/kirjasto/julkaisutoiminta/julkmyyn.html

Serial Editors: Professor Karl Åkerman

Department on Neurobiology
A.I.Virtanen Institute for Molecular Sciences

Research Director Jarmo Wahlfors

Department of Biotechnology and Molecular Medicine
A.I. Virtanen Institute for Molecular Sciences

Author’s address: Department of Biotechnology and Molecular Medicine

A.I. Virtanen Institute for Molecular Sciences
University of Kuopio
P.O.Box 1627
FIN-70211 KUOPIO
FINLAND
Tel. +358 17 163 790
Fax +358 17 163 030
Email: tanja.hakkarainen@uku.fi

Supervisors: Docent, Research Director Jarmo Wahlfors, PhD

Department of Biotechnology and Molecular Medicine
A.I. Virtanen Institute for Molecular Sciences
University of Kuopio

Docent Akseli Hemminki, MD, PhD

Rational Drug Design, University of Helsinki and
Department of Oncology, Helsinki University Central Hospital

Reviewers: Professor Veli-Matti Kähäri, MD, PhD

Department of Medical Biochemistry and Molecular Biology
University of Turku

Docent Anu Jalanko, PhD

Department of Molecular Medicine
National Public Health Institute

Opponent: Dr. Michael Blaese, MD

PreGentis, Inc.
Newtown, PA, USA

ISBN 951-781-394-5
ISBN 951-27-0098-0 (PDF)
ISSN 1458-7335

Kopijyvä
Kuopio 2005
Finland
Hakkarainen, Tanja. Enhancement of cancer gene therapy with modified viral vectors and fusion genes.
Kuopio University Publications G. – A.I.Virtanen Institute for Molecular Sciences 35. 2005. 63 p.
ISBN: 951-781-394-5
ISBN: 951-27-0098-0 (PDF)
ISSN: 1458-7335

ABSTRACT

Even though several approaches have been developed for improvement of gene transfer efficiency, one
major obstacle in to successful gene therapy is insufficient transduction efficiency and consequently poor
therapeutic effect. However, several approaches have been investigated to circumvent this problem. In the
following study, we evaluated the potential use of various lentiviral vectors in cancer gene therapy. In
addition, we utilized CD40 for targeting adenoviral vectors to ovarian cancer cells. Further, to compensate
for the low gene transfer efficiency and to enhance therapeutic outcome by spreading the therapeutic
element, the feasibility of VP22 protein transduction domain (PTD) and conditionally replicative
adenoviruses were studied.

Our results demonstrated that several different types of human cancer cell lines can be efficiently
transduced in vitro with lentiviral vectors. These vectors also showed the potential for efficient gene
transfer vehicle in vivo. Further, increased gene transfer rates were achieved in ovarian cancer cells by
targeting adenoviral vectors to the CD40 receptor. In addition, the use of VP22 was able to enhance tumor
cell killing under certain conditions in vitro, although no intercellular trafficking was observed. VP22
coupled therapeutic protein also significantly reduced tumor growth in vivo. However, the incorporation of
VP22 did not significantly increase the antitumor effect when compared to control proteins. Finally, the use
of conditionally replicative adenovirus resulted in effective oncolysis in ovarian cancer cells in vitro.
Moreover, this virus possessed a remarkably efficient antineoplastic activity in vivo. When combined with
suicide gene/prodrug therapy, cell killing was further augmented in vitro, but this was not observed in vivo.

In summary, these results show that several approaches can be exploited to enhance gene transfer in tumor
cells in order to achieve better therapeutic responses and simultaneously reduce the side effects of cancer
gene therapy. Further, by combining various approaches more efficient tools for gene therapy purposes can
be developed.

National Library of Medicine Classification: QZ 50, QZ 52, QZ 266, QW 160

Medical Subject Headings: neoplasms; gene therapy; gene transfer techniques; genetic vectors;
adenoviridae; lentivirus; viral fusion proteins; virus replication; viral structural proteins; thymidine kinase;
ganciclovir
ACKNOWLEDGEMENTS

This study was carried out in the Department of Biotechnology and Molecular Medicine,
A.I.Virtanen Institute for Molecular Sciences, University of Kuopio and in the Division of Human
Gene Therapy, University of Alabama at Birmingham during the years 2000-2004.

I wish to express my deepest gratitude to my principal supervisor, Docent Jarmo Wahlfors, PhD, for
giving me the opportunity to become a part of the fascinating world of science and gene therapy
research. I also thank him for his expert guidance, for teaching the philosophy of scientific thought
and for being such a kind, fair, understanding and encouraging groupleader. I am also grateful to my
second supervisor Docent Akseli Hemminki, MD, PhD, for multiple excellent ideas, his enthusiasm
for research, his determination and “we are ready to rock 'n 'roll” –attitude of conducting science.
He also receives my gratitude for providing an opportunity to start working in his group while still
finishing my thesis.

I am also thankful to David T.Curiel, MD, PhD, University of Alabama at Birmingham who offered
me the opportunity to work in his laboratory in a truly scientifically inspiring environment.

I am indebted to Docent Anu Jalanko, PhD, and Professor Veli-Matti Kähäri. MD, PhD, for
reviewing this thesis and for their constructive criticism.

My sincere and warm thanks belong to my working mates Tiina Wahlfors, MSc, Outi Rautsi (née
Meriläinen), MSc, Ann-Marie Määttä, MSc, Riikka Pellinen, PhD, Anna Ketola, MD, Saara
Lehmusvaara, MSc, Sanna Korja, MSc, Tuula Salonen and Katja Häkkinen for excellent team spirit,
good laughs, interesting discussions, help and their friendship. It has been a great pleasure to work
within this colorful, genuine and lively group. Especially I want to thank my room mate Outi, for
enlightening my day with all her funny coincidences and Google’s Search -results. In addition, I
want to thank “FACS-guru” Mikko Mättö, MSc, for collaboration and his priceless help with flow
cytometry. I am also thankful to my collegues Terhi Pirttilä, MSc, and Eija Pirinen, MSc, for their
friendship and discussions, which have enlightened my days.

I am also thankful to the whole personnel of the A.I.Virtanen Institute for creating such a pleasant
working environment. My special thanks go to Riitta Laitinen and Helena Pernu for secretarial
assistance, to Riitta Keinänen, PhD, for multiple advice and encouragement, to Kaija Pekkarinen for
help and discussions over coffee cups, and to Pekka Alakuijala, Phil. Lic., and Jouko Mäkäräinen
for technical assistance.

I thank Ewen MacDonald, PhD, for the linguistic revision of this thesis.

People at the UAB-team, Gerd Bauerschmitz, MD, Anna Kanerva, MD, PhD, Angel A Rivera, PhD,
Shannon Barker, PhD, and John Lam, MD, receive my gratitude for creating a friendly, enthusiastic
and unique atmosphere and working environment. Especially I want to thank Gerd, for his
friendship and all the help he has provided in different circumstances.

I am also thankful to the new lab crew in Akseli’s group, Anna, Tuuli, Mari, Merja, Lotta, Maria,
Tommi and Kilian for taking me to be a part of their team and for creating an energetic and fun
working environment.
I express my warm gratitude to all my friends for their support, discussions, long phone calls, laughs
and cries and enjoyable moments, which have made possible to temporarily forget the science and
the meaning of the word “work”.

I owe my deepest thanks to my whole family. To my parents for teaching me the basic things in life,
love, support, trust and just being there for me whenever I’ve needed. I want to thank my brother’s
family for love and support and especially my niece Anna for being such a great and hilarious
personality. My warmest thanks go to my beloved partner Markku for loving and believing in me
and making me complete.

This study was supported by The Saastamoinen Foundation, The Finnish Cultural Foundation of
Northern Savo and The Cancer Society of Finland.

Kuopio, April 2005

Tanja Hakkarainen
ABBREVIATIONS

AAV adeno associated virus

ADP adenovirus death protein
APC gene adenomatous polyposis coli –gene
CAR coxsackie adenovirus receptor
CMV cytomegalovirus
Cox-2 cyclo-oxygenase-2
cPPT central polypurine tract
CRAd conditionally replicative adenovirus
EGR1 early growth response gene 1
FITC fluorescein isothiocyanate
GCV ganciclovir
GFP green fluorescent protein
HIV-1 human immunodeficiency virus type 1
HSV-1 herpes simplex virus type 1
hTERT human telomerase reverse transcriptase
ITR inverted terminal repeat
LTR long terminal repeats
MDR-1 multidrug resistance gene 1
MMR mismatch repair
MRS magnetic resonance spectroscopy
PET positron-emission tomography
PFA paraformaldehyde
pfu plaque forming unit
PSA prostate specific antigen
PSMA prostate specific membrane antigen
PTD protein transduction domain
SIN self-inactivating type vector
TK herpes simplex virus thymidine kinase
t.u. transducing unit
VEGF vascular endothelial growth factor
VEGF-R vascular endothelial growth factor receptor
VSV-G vesicular stomatitis virus G protein
VP viral particle
(W)PRE (woodchuck hepatitis virus) posttranscriptional regulatory
element
LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following publications referred to their corresponding Roman numerals:

I Pellinen R., Hakkarainen T., Wahlfors T., Tulimaki K., Ketola A., Tenhunen A., Salonen
T. and Wahlfors J. (2004). Cancer cells as targets for lentivirus-mediated gene transfer and
gene therapy. International Journal of Oncology, 25:1753-1762.

II Hakkarainen T., Hemminki A., Pereboev A., Barker S., Asiedu K., Strong T., Kanerva A.,
Wahlfors J. and Curiel D.T. (2003). CD40 is expressed on ovarian cancer cells and can be
utilized for targeting adenoviruses. Clinical Cancer Research, 9: 619-624.

III Hakkarainen T., Wahlfors T., Meriläinen O., Loimas S., Hemminki A. and Wahlfors J.
(2005) VP22 does not significantly enhance enzyme prodrug cancer gene therapy as a part
of VP22-HSVTk-GFP triple fusion construct. Journal of Gene Medicine, 9:

IV Hakkarainen T., Hemminki A., Curiel D.T. and Wahlfors J. (2005) Α conditionally
replicative adenovirus that codes for a TK-GFP fusion protein (Ad5-∆24TK-GFP) for
evaluation of the potency of oncolytic virotherapy combined with molecular
chemotherapy, Submitted.
TABLE OF CONTENTS

1 INTRODUCTION........................................................................................................................... 13
2 REVIEW OF THE LITERATURE............................................................................................... 14
2.1 Cancer.......................................................................................................................................... 14
2.2 Ovarian cancer............................................................................................................................. 15
2.3 Cancer associated genes .............................................................................................................. 15
2.3.1 Overview ......................................................................................................................... 15
2.3.2 Proto-oncogenes .............................................................................................................. 16
2.3.3 Tumor suppressor genes .................................................................................................. 16
2.3.4 DNA mismatch repair (MMR) genes .............................................................................. 17
2.4 Cancer gene therapy .................................................................................................................... 17
2.4.1 Overview ......................................................................................................................... 17
2.4.2 Cancer gene therapy approaches...................................................................................... 18
2.5 Challenges of gene therapy ......................................................................................................... 21
2.6 Improvements in cancer gene therapy ......................................................................................... 23
2.6.1 Overview ......................................................................................................................... 23
2.6.2 Optimal gene transfer tools for specific purposes: various viral vectors ......................... 23
2.6.2.1 Adenoviral vectors ...................................................................................................... 23
2.6.2.2 Retro- and lentiviral vectors ....................................................................................... 26
2.6.3 Enhanced gene transfer and increased specificity: adenoviral targeting.......................... 30
2.6.4 Improved therapeutic outcome by spreading the therapeutic element:
oncolytic viruses and protein transduction domains ........................................................ 31
2.6.4.1 Conditionally replicative adenoviruses (CRAds)........................................................ 32
2.6.4.2 Protein transduction domains..................................................................................... 33
2.6.5 Combination therapies ..................................................................................................... 34
3 AIMS OF THE STUDY.................................................................................................................. 35
4 MATERIALS AND METHODS ................................................................................................... 36
4.1 Fusion gene constructs (III)......................................................................................................... 36
4.2 Viral vectors (I-IV)...................................................................................................................... 36
4.3 Cell lines and patient samples (I-IV)........................................................................................... 36
4.4 Protein expression levels ............................................................................................................. 38
4.4.1 Expression of VP22-fusion proteins ................................................................................ 38
4.4.2 CD40 expression (II) ....................................................................................................... 39
4.5 Determination of lentiviral transduction efficiency by flow cytometry (I) ................................. 39
4.6 Gene transfer with CD40-targeted adenovirus (II) ...................................................................... 39
4.7 Verification of viral replication and oncolysis (IV)..................................................................... 40
4.8 Ganciclovir sensitivity assays (I, III, IV)..................................................................................... 40
4.9 Monitoring of VP22-mediated intercellular trafficking and adenoviral
spreading (III, IV) ............................................................................................................................. 41
4.9.1 Flow cytometry................................................................................................................ 41
4.9.2 Fluorescence microscopy................................................................................................. 41
 4.10 In vivo studies (I, III and IV)....................................................................................................... 41
5 RESULTS AND DISCUSSION ..................................................................................................... 43
5.1 Cancer cells are potential targets for lentiviral gene transfer (I) ................................................. 43
5.1.1 Transduction efficiency of lentiviral vectors in human cancer cells................................ 43
5.1.1.1 Impact of accessory proteins....................................................................................... 44
5.1.1.2 Impact of WPRE and cPPT......................................................................................... 44
5.1.2 Lentivirus vector -mediated TK/ganciclovir gene therapy in vitro.................................. 45
5.1.3 Lentiviral tumor transduction in vivo............................................................................... 45
5.2 CD40 can be exploited for transductional targeting of adenovirus vectors (II)........................... 45
5.2.1 CD40 expression in ovarian cancer cells ......................................................................... 46
5.2.2 Gene transfer with CD40 -targeted adenovirus................................................................ 46
5.3 VP22 does not enhance suicide gene therapy as a part of triple fusion protein (III) ................... 47
5.3.1 Expression of fusion proteins .......................................................................................... 47
5.3.2 Monitoring of VP22 -mediated intercellular trafficking.................................................. 48
5.3.3 VP22-TK-GFP mediated cell killing in vitro................................................................... 48
5.3.4 Efficiency of VP22-TK-GFP in vivo ............................................................................... 49
5.4 Ad5-∆24-TK-GFP: a useful tool for evaluation of combined oncolytic virotherapy
and suicide gene therapy (IV) ........................................................................................................... 49
5.4.1 Verification of viral replication and oncolysis................................................................. 49
5.4.2 Monitoring of viral spreading .......................................................................................... 50
5.4.3 Effect of TK/GCV system on oncolysis in vitro.............................................................. 50
5.4.4 Efficiency of Ad5-∆24TK-GFP in vivo ........................................................................... 51
6 SUMMARY AND CONCLUSIONS ............................................................................................. 52
7 REFERENCES................................................................................................................................ 54

APPENDIX: ORIGINAL PUBLICATIONS I-IV

1 INTRODUCTION

Cancer is a world wide health problem which was responsible for the deaths of almost 7 million people in
2002. Even though our knowledge of molecular mechanisms, diagnostic methods and traditional treatments
of cancer have all improved during the last decades, most cancer types still have a poor prognosis and
evoke a high mortality, this being especially the case with metastatic types. Thus, more efficient
approaches and improved therapeutic strategies are needed for the treatment of cancer.

The first clinical gene therapy trial was carried out in the early 1990s. From that beginning, gene therapy
has become a widely studied concept for treatment of various diseases. Even though gene therapy was
initially thought to be more suitable for the treatment of inherited monogenic diseases, gene therapy has
been increasingly utilized in the treatment of acquired and complex diseases such as cancer. In fact, by the
year 2004, the majority of clinical gene therapy trials (66%) have focused on cancer diseases.

Even though several approaches have been successfully developed to improve the gene transfer efficiency,
one major obstacle in cancer gene therapy is still insufficient transduction of the gene and consequently a
poor therapeutic effect. There are several possibilities that can be utilized to circumvent this problem. First,
new alternative viral vectors can be explored to find optimal gene transfer vehicles for each purpose.
Secondly, viral vectors can be re-targeted to cancer cells, which simultaneously enhance gene transfer rates
in tumors and diminish undesired side effects in healthy tissue. In addition, it is possible to exploit viral
replication per se to destroy cancer cells. To avoid side effects and to increase the safety of these oncolytic
agents, replication can be restricted to tumor cells by partially deleting the viral genome or by using tissue
specific promoters to drive the viral genes responsible for replication. Instead of modifying the gene
transfer vector, one possibility is to modify the therapeutic gene so that the resulting therapeutic protein can
spread to surrounding cells and thus to compensate for the low gene transfer efficiency and to enhance the
therapeutic outcome.

In this study, we examined several potential approaches to enhance cancer gene therapy. First, we
explored the feasibility of using lentiviral vectors in cancer gene therapy. In addition, we targeted
adenoviral vectors to an alternative cellular receptor to increase the specificity and efficacy of gene
transfer. Furthermore, we tested the feasibility of intercellularly trafficking protein transduction domains to
achieve enhancement of the therapeutic response. Finally, we generated a novel conditionally replicative
adenovirus and evaluated its oncolytic potency for cancer gene therapy purpose.

13
2 REVIEW OF THE LITERATURE

2.1 Cancer
Cancer is a world wide health problem. In 2002, approximately 11 million people were estimated to have
been diagnosed with cancer and almost 7 million people will die from cancer (Ferlay et al., 2004). In
Finland alone, over 23 000 new cancer diagnoses were made in 2002 (Finnish Cancer Registry,
www.cancerregistry.fi). The number of cancer patients has increased partly due to advances in medical
sciences, such as the reduction of deaths from infectious and cardiovascular causes but also to improved
diagnostic methods being better able to detect cancer. Further, many factors in the Western lifestyle may
predispose to carcinogenesis. The development of a malignant tumor always involves an interaction
between the environment and heredity (Tamura et al., 2004). In most cases, the causative reason for
carcinogenesis is unknown, but the mechanism always involves mutations to cellular DNA. Somatic
mutations can be caused by environmental factors including radiation, chemicals and viruses or by
imperfection of the DNA copying machinery, which leads to abnormal cellular functions (Vogelstein and
Kinzler, 1998; Zhang et al., 1995). Genetic mutations contributing to transformation of the normal cell can
be also inherited in a Mendelian fashion (Knudson, 1991). These genes are typically recessive in
oncogenesis. Thus, according to Knudson’s two-hit hypothesis, an inherited mutation is rarely sufficient to
initiate tumorigenesis alone and an additional mutation or loss of a second allele is required (Knudson,
1971). Subsequently, Knudson classified the etiology of cancer and the role of environmental and genetic
factors on tumorigenesis (Knudson, 1985). Based on that classification, most common tumors are sporadic
(i.e. non-inherited) and only a small fraction (1-5%) of all cancer types belongs to the group of hereditary
i.e. familial, cancers (Table 1). Currently, the familial cancer syndrome is best characterized in ovarian,
breast and colorectal cancers which have the greatest heritability in their causation (Knudson, 1995).

Table 1. Knudson’s classification (modified from Tamura et al., 2004).

Environment Genetic factors Etiological viewpoint % of all causes
Basic Basic Accidental 20%
Main A little Environmental related 75%
Considerable Considerable Multifactorial 75%
A little Main Hereditary 1-5%

Even though cancer can be considered as a genetic disease, it is rarely caused by a defect in a single gene
(Tamura et al., 2004). It has been estimated that three to seven mutations are required for carcinogenesis
(Vogelstein and Kinzler, 1998). These mutations typically cause increased and irregular proliferation
activity, lack of apopotosis, deficient cell cycle control, and ability to metastasize and create

14
vascularisation into the tumor (Hanahan and Weinberg, 2000). When these defects accumulate, the
aberrant features lead to creation of cells which can proliferate unrestrictedly, form preneoplastic lesions,
invade local tissues and eventually establish distant metastasis (Rieger, 2004; Zhang et al., 1995). It has
been postulated that the number of mutations can influence the aggressiveness of the tumor. Benign tumors
seem to contain fewer mutations than malignant, progressive neoplasms. Solid tumors (i.e. tumors in solid
organs) require a greater number of mutations than diffuse tumors (i.e. leukemias), which probably
explains the longer time period before solid tumors appear after the initial mutagenic stimuli when
compared to diffuse tumors. (Vogelstein and Kinzler, 1998).

2.2 Ovarian cancer

Ovarian cancer is the most lethal gynecologic cancer. Worldwide it has been estimated that, over 200 000
new ovarian cancer patients are diagnosed and approximately 120 000 people died from ovarian cancer in
2002 (Ferlay et al., 2004). In Finland, there were 497 new cases in 2002, making ovarian cancer the fourth
most common cancer in women (Finnish Cancer Registry, www.cancerregistry.fi). Although the 5-year
survival rate in Finland is 49%, this statistic remains much lower than the overall 65% cancer survival for
women (Finnish Cancer Registry, www.cancerregistry.fi).

Due to its mild symptoms, ovarian cancer is difficult to detect in its early stages. Thus, the majority of
ovarian cancer cases are unfortunately diagnosed after the cancer has disseminated beyond the ovaries. In
advanced ovarian cancers, the standard treatment is usually surgical cytoreduction followed by
chemotherapy (Agarwal, and Kaye, 2003; DiSaia, and Bloss, 2003). Typically, a platinum/taxane
combination has been considered as the post-surgical first-line chemotherapy (Coleman, 2002). Although
this combined chemotherapy has been shown to result in good response rates (70-80%) in ovarian cancer
patients, the majority of these patients will relapse due to the development of drug resistance to these
chemotherapeutic agents (Balch et al., 2004; Stuart, 2003). Further, ovarian cancer drug resistance has been
believed to result in a treatment failure and death of more than 90% of patients with metastatic disease
(Balch et al., 2004).

2.3 Cancer associated genes

2.3.1 Overview

Three major gene types have been shown to be closely related to tumorigenesis: proto-oncogenes, tumor-
suppressor genes and DNA mismatch repair genes (Figure 1). Since cancer is a group of neoplastic
diseases involving multiple steps and genetic events, tumor development usually requires alterations in a
number of cancer-associated genes. A transformed phenotype when combined with a metastatic capability
typically involves both oncogene activation and tumor suppressor gene loss or inactivation (Bast et al.,

15
2000). Furthermore, it is noteworthy that the same genes causing genetic predisposition to cancer are most
often associated and involved in sporadic forms of carcinogenesis (Knudson, 1991).

2.3.2 Proto-oncogenes

The proto-oncogenes encode proteins that have a crucial role in cell signaling pathways. The proto-
oncogenic products (including various growth factors and their receptors, signal transducers and
transcription factors) control the growth and differentiation of normal cells (Aaronson, 1991; Bast et al.,
2000). These proto-oncogenes can be activated to carcinogenic oncogenes through mutation, gene
amplification or chromosomal rearrangement resulting in an altered proto-oncogene structure or an
increased proto-oncogene expression (Bast et al., 2000). Thus activation of oncogenes offers a growth
advantage to the cell and eventually normal cells become transformed.

M Oncogenes
Tumor Suppressor
genes

G2 G0
G1
Restriction point

Oncogenes
S
Tumor Suppressor
genes

MMR genes

Figure 1. Cancer associated genes and cell cycle. Oncogene products and mutated tumor suppressor and
DNA mismatch repair (MMR) proteins alter the cell cycle in various phases (in G1, S and G2) allowing
abnormal and unregulated cellular growth and finally tumor cell formation.

2.3.3 Tumor suppressor genes

Tumor suppressor genes, or antioncogenes can prevent cell growth in many ways e.g. by blocking the cell
cycle or by promoting cell cycle arrest and apoptosis (McCormick, 2001). In cancer cells, these genes are
inactivated and this blocks the control of normal cell cycle and thus increases the probability of tumor

16
formation (Knudson, 2001). The best characterized and most widely studied tumor suppressor genes are
RB1 (Francke and Kung, 1976; Friend et al., 1986; Knudson et al., 1976), WT1 (Call et al., 1990; Gessler
et al., 1990), TP53 (Finlay et al., 1989), colon cancer associated APC (Ichii et al., 1992; Nishisho et al.,
1991) and breast- and ovarian cancer associated BRCA1 and 2 (Miki et al., 1994; Steichen-Gersdorf et al.,
1994; Wooster et al., 1995).

2.3.4 DNA mismatch repair (MMR) genes

The DNA mismatch repair (MMR) system is one of the three cellular mechanisms involved in DNA repair
(Charames and Bapat, 2003). MMR proteins recognize and eliminate potential misincorporated nucleotides
on the newly synthesized DNA strand during DNA replication (Fedier and Fink, 2004). Hence, MMR
machinery is a crucial mechanism for maintaining genome integrity during cell proliferation. Mutations in
genes encoding MMR proteins attenuate or inactivate the DNA repair machinery and thus interfere with
genetic stability and increase the incidence of further mutations during the DNA synthesis (Fedier and
Fink, 2004). This increased mutation frequency thus augments the susceptibility to undergo cellular
transformations and tumor formation (Eshleman and Markowitz, 1996). Mutated MMR proteins have been
identified and associated with various cancer types including hereditary nonpolyposis colon cancer
(Bronner et al., 1994; Fishel et al., 1993; Risinger et al., 1993), breast (Balogh et al., 2003; Seitz et al.,
2003) and bladder cancer (Kassem et al., 2001) as well as gliomas (Wei et al., 1997). In addition, tumor
cells with a defective MMR system have been shown to display reduced sensitivity to the cytotoxic, DNA
damaging chemotherapeutic drugs such as cisplatin (Brown et al., 1997), doxorubicin (adriamycin)
(Drummond et al., 1996) and topotecan (Fedier et al., 2001).

2.4 Cancer gene therapy

2.4.1 Overview

Conventionally cancer is treated with chemo- and radiotherapy, hormonal therapies or surgery. In addition
to the growth advantage, the genetic mutations responsible for carcinogenesis may increase the resistance
of malignant cells to various conventional treatments, thus ensuring survival of cancer cells and
uninterrupted tumor growth. Cancer cells have been shown to develop resistance to chemotherapeutic
agents in a variety of ways (Fojo and Bates, 2003). Furthermore, cancer cells are often relatively resistant
to radiation. One fundamental mechanism mediating resistance to radiation involves activation of NF-
KappaB (Orlowski and Baldwin, 2002). In normal cells, a lethal radiation dose activates p53, which in turn
down-regulates NF-KappaB-controlled survival circuits, resulting in apoptosis (Chen et al., 2002; Rocha et
al., 2003). However, most advanced tumors feature a defective p53/p14ARF pathway (Sherr, 1996), which
inhibits apoptosis. In addition to failure due to therapeutic resistance, another disadvantage of conventional
treatments is their lack of selectivity. Especially chemo- and radiation therapies also damage healthy tissue,

17
leading to severe side effects and decreased therapeutic response. Surgical treatment of cancer has its own
limitations: tumors have to be solid, well confined and accessible. However, conventional therapies have
developed enormously due to technological improvements and increased knowledge of cancer biology and
genetics. In radiation therapy, the development of novel biological imaging systems (mainly positron-
emission tomography (PET) and magnetic resonance spectroscopy (MRS) have provided tools to obtain
information about tumor cell proliferation, the presence of specific tumor markers, metabolism and
vascularisation of the tumor (Ling et al., 2000). These advanced imaging techniques allow radiation doses
to be calculated more precisely and to be tumor cell specific avoiding damage to healthy tissue (Bernier et
al., 2004). In addition, knowledge of genetic mutations has been exploited to re-sensitize resistant cancer
cells to radiation in various cancer types including prostate, breast and lung cancer (Cowen et al., 2000; Mu
et al., 2004; Sakakura et al., 1997; Coleman, 1999; Shinohara et al., 2004). Further, more specific
chemotherapeutic drugs have been designed including trastuzumab (Herceptin), an antibody against HER-
2, which is known to be over expressed in breast cancer cells (Wong, 1999). Despite the development of
conventional therapies, most disseminated cancers are unfortunately relatively resistant to these treatments
resulting in relapse and mortality. Thus, more efficient approaches and tools are needed for the treatment of
cancer.

Increased knowledge of the molecular basis of cancer has also provided the possibility to exploit gene
therapy as a novel strategy to treat cancer. The principle of gene therapy is rather straightforward: i.e. one
introduces a therapeutic gene, whose product should stop or slow down the progression of disease, into a
target cell (Mountain, 2000). Therapeutic genes can be transferred to target cells using modified viruses as
vectors (Somia and Verma, 2000) or with non-viral methods including electroporation, gene gun and
cationic lipid or polymer coating (Li and Huang, 2000). Gene transfer technology theoretically make it
possible to treat the source of the disease whereas many conventional therapies are mainly palliative i.e.
treat the symptoms. This makes gene therapy an attractive and potentially revolutionary tool for modern
medicine. The era of gene therapy was initiated in the early 1990s with the first somatic gene therapy
clinical trial for the treatment of inherited genetic disease (Blaese et al., 1995). During the last fifteen years,
there have been dramatic advances in the modern medicine and biosciences. Since 1990 nearly 990 clinical
trials have been approved, the vast majority (~97%) of them being phase I or II trials
(http://www.wiley.co.uk/genetherapy/clinical/). More than half (66%) of the approved trials have focused
on cancer diseases (http://www.wiley.co.uk/genetherapy/clinical/).

2.4.2 Cancer gene therapy approaches

Since cancer is a group of complex neoplastic diseases caused by multiple genetic factors, it is not feasible
to correct all of the defective genes. Despite the deficiency of several genes in tumor cells, inactivation of
one particular oncogene or restoration of a single tumor suppressor gene has been shown to be adequate to
significantly inhibit tumor growth. It has been reported that blockade of oncogenic HRAS –protein

18
expression induced tumor regression in animal models (Barrington et al., 1998; Chin et al., 1999).
Furthermore, Druker et al. demonstrated that inhibition of BCR-ABL -protein exhibited antineoplastic
activity in a phase I clinical trial in patients with chronic myeloid leukemia (Druker et al., 2001).
Restoration of tumor suppressor protein p53 has been shown to reduce tumor growth both in patients with
lung and advanced head and neck cancers (Roth et al., 1996; Schuler et al., 1998; Clayman et al., 1998;
Clayman et al., 1999).

Instead of correcting mutated genes or suppressing active oncogenes, a more widely utilized approach has
been to introduce exogenous therapeutic genes into tumor cells. The inserted therapeutic genes can be
immunotherapeutic, anti-angiogenetic, chemoprotective or so called suicide genes.

In immunotherapy, tumor cells are destroyed via the immune response raised against tumor specific
antigens (Pardoll, 2000). Tumor antigens are typically tissue specific proteins (i.e. tyrosinase), unique
mutated proteins (i.e. CDK4 variant proteins), over-expressed proteins (i.e. prostate specific antigen, PSA)
or proteins from viral origin (i.e. human papilloma virus E6 and E7) (Pardoll, 2002; Vakkila and Pihkala,
1999; van der Bruggen et al., 1991). An immune response has been raised in humans against a tumor by
inserting genes encoding stimulatory proteins such as cytokines into the cancer cells (Nemunaitis et al.,
1999; Stingl et al., 1996) or by introducing tumor antigens to dendritic or other antigen presenting cells
(Brossart et al., 2000; Simons et al., 1999).

One prerequisite for tumor formation is neovascularization (i.e. angiogenesis) of the tumor. (Vile et al.,
2000). Reduced oxygen levels and certain cellular factors up-regulate the production of vascularisation
stimulating factors and their receptors (such as VEGF/VEGF-R) in cancer cells (Kong and Crystal, 1998),
which make these molecules attractive targets for gene therapy. By means of gene therapy, the expression
or function of angiogenetic proteins can be inhibited (Im et al., 2001; Kong et al., 1998) or anti-
angiogenetic proteins (such as angiostatin) can be introduced to cancer cells (Sacco et al., 2000; Tanaka et
al., 1997; Tanaka et al., 1998).

In several cases, the efficacious treatment of cancer would require the use of high-dose chemotherapy,
which is impracticable due to the cytotoxicity of these drugs to normal cells, especially to hematopoietic
cells. To enable the use of larger and more efficient drug doses, chemoprotective genes can be transferred
to hematopoietic stem cells to increase their resistance to chemotherapy (D'Hondt et al., 2001). The most
extensively studied genes have been multidrug resistance 1 (MDR-1) and multidrug resistance-associated
protein genes (Abonour et al., 2000; Cowan et al., 1999; Machiels et al., 1999).

Suicide genes encode enzymes that can convert a harmless, separately administered prodrug into a
cytotoxic molecule (McCormick, 2001). The most widely used enzyme-prodrug combination is herpes
simplex virus type 1 thymidine kinase (TK)/ganciclovir (GCV). TK phosphorylates, in concert with other
cellular kinases, GCV into its triphosphate form, which can then be incorporated into DNA during cell

19
Table 2. An overview of clinical gene therapy trials.

Cancer Type Therapeutic approach/gene Delivery method Phase Reference

Brain cancer

suicide gene therapy/ thymidine kinase adenovirus I (Eck et al., 1996; Smitt et al., 2003)

immunotherapy/IFN-β adenovirus I (Eck et al., 2001)

immunotherapy/ IL-12 semliki forest virus I/II (Ren et al., 2003)

tumor suppressor gene therapy/ p53 adenovirus I (Lang et al., 2003)

virotherapy herpes simplex virus I (Markert et al., 2000)

Colon cancer
(metastatic)
suicide gene therapy/ cytosine deaminase adenovirus I (Crystal et al., 1997)
suicide gene therapy/ thymidine kinase adenovirus I (Sung et al., 2001)

immunotherapy/HLA-B7 liposomes I (Rubin et al., 1997)

virotherapy adenovirus I (Reid et al., 2002)

Lung cancer

tumor suppressor gene therapy/ p53 adenovirus I (Roth et al., 1998; Schuler et al., 1998)

Melanoma

suicide gene therapy/ thymidine kinase adenovirus I (Morris et al., 2000a)

immunotherapy/ IL-2 adenovirus I (Stewart et al., 1999)

immunotherapy/IFN-γ retrovirus I (Nemunaitis et al., 1999)

tumor suppressor gene therapy/ p53 adenovirus I (Dummer et al., 2000)

Ovarian cancer

suicide gene therapy/ thymidine kinase adenovirus I (Alvarez et al., 2000)

immunotherapy/ BRCA-1 retrovirus I, II (Tait et al., 1997; Tait et al., 1999)

tumor suppressor gene therapy/ p53 adenovirus I/II (Buller et al., 2002)

virotherapy adenovirus I (Vasey et al., 2002)

Prostate cancer

suicide gene therapy/ thymidine kinase adenovirus I, I/II (Herman et al., 1999; Miles et al.,2001)

DNA-lipid complex, (Belldegrun et al., 2001;

immunotherapy/ IL-2 I
adenovirus Trudel et al., 2003)

immunotherapy/MUC1 and IL-2 vaccinia virus I (Pantuck et al., 2004)

virotherapy adenovirus I (Freytag et al., 2002)

20
division and blocks DNA replication, preventing cell proliferation (Moolten, 1986). In addition,
triphosphorylated GCV can diffuse into neighboring cells via gap-junctions and spread the cytotoxic effect,
creating the so called bystander effect (Mesnil et al., 1996; van Dillen et al., 2002). The efficiency of
TK/GCV system has been demonstrated successfully in patients with malignant glioma; the mean survival
of patients treated with adenovirus mediated TK/GCV therapy after surgical tumor resection was prolonged
by 81% when compared to the conventionally treated control group (Immonen et al., 2004; Sandmair et al.,
2000). Several other combinations have been also evaluated in clinical trials including cytosine
demaminase/5-Fluorocytosine (Crystal et al., 1997; Pandha et al., 1999) and nitroreductase/CB1954
(Palmer et al., 2004).

One strategy which has been extensively studied for cancer gene therapy purposes during the last ten years
is so called virotherapy. This approach does not necessary involve therapeutic gene transfer since it is
based on tumor cell killing caused by viral replication and it will be discussed later (see chapter 2.5.4.1
Conditionally replicative adenoviruses). An overview of clinical cancer gene therapy trials and the used
gene therapeutic approaches is presented in table 2.

2.5 Challenges of gene therapy

Although the theory laying the foundations for gene therapy is rather simple and good safety and promising
efficiency data have been achieved from preclinical research and clinical trials, there are still several
problems encountered with this technique. The key to successful gene therapy is efficient and specific gene
transfer, which is a sum of multiple factors. Due to inefficiency of non-viral gene transfer, the following
review will focus on viral vector mediated gene therapy.

In order to deliver genes into target tissue, gene transfer vectors first have to escape destruction by the host
immune system. It has been shown that 55% of adult humans have pre-existing neutralizing antibodies
against adenovirus serotype 5, which is one of the most frequently used gene transfer vectors (Chirmule et
al., 1999). In contrast, retroviruses, another group of viral vectors, do not elicit host immune response but
they can be rapidly degraded by the complement system (Takeuchi et al., 1994). In addition, the route of
viral vector administration plays an important role in gene delivery to target tissues. It has been shown in
several preclinical studies that irrespective of the administration route, adenoviral vectors almost invariably
evoke neutralizing antibodies directed against adenoviruses (Gahery-Segard et al., 1997; Setoguchi et al.,
1994; Smith et al., 1996; Van Ginkel et al., 1995). However, the proteins of the viral capsid have been
reported to be differentially recognized depending on the route of administration (Gahery-Segard et al.,
1997). In contrast, in humans adenoviral vectors can cause variable, administration route dependent,
humoral immune responses (Harvey et al., 1999). In cancer gene therapy, viral vectors are often
administered directly into tumor tissue (intratumorally) to achieve therapeutically relevant gene transfer
rates. In fact, the majority of reported clinical studies have been carried out using intratumoral

21
administration. However, the intratumoral route is possible only if tumor mass is local and accessible.
Some tumors are located primarily within specific body cavities which enables the intracavitary
administration (e.g. intraperitoneal) of gene transfer vectors. Intracavitary administration has been used in
several clinical trials e.g. for ovarian cancer (intraperitoneal) (Alvarez et al., 2000; Buller et al., 2002) and
for malignant mesothelioma (intrapleural) (Sterman et al., 1998). Nevertheless, if one wishes to treat
metastatic cancer, then systemic administration of the gene transfer vector is invariably required.
Intravenous and intra-arterial administrations have been used in clinical studies e.g. for metastatic
osteosarcoma (Benjamin et al., 2001) and hepatic metastases for colorectal cancer (Reid et al., 2002),
respectively. Further, for immunotherapeutic purposes, viral vectors have been delivered intramuscularly in
humans in order to achieve systemic humoral immune response against prostate cancer cells (Pantuck et
al., 2004).

The second challenge is to reach the target cells and deliver the therapeutic genes into these cells. Since
viral vector uptake requires the binding of the vector to cellular receptors, one limiting factor is the
expression levels of viral receptors on the target cell. For example, the expression level of coxsackie-
adenovirus receptor (CAR), which mediates adenoviral attachment to their target cells, has been shown to
be variable and often very low in tumor cells (Asaoka et al., 2000; Li et al., 1999a; Li et al., 1999b). Thus,
this receptor deficiency makes cancer cells rather refractory to adenoviral mediated gene transfer. Further,
viral receptors are often widely expressed in normal cells and this means that, healthy tissue can also be
susceptible to gene transfer. This naturally leads to the appearance of side effects and decreased gene
transfer into target cells.

The final critical step after viral uptake into the target cell is transgene expression. In order to achieve an
adequate therapeutic response, the transgene expression has to be at a sufficient level for a sufficient time.
When using non-integrating vectors such as adenoviral vectors, transgene is maintained
extrachromosomally in the nucleus, resulting in only transient expression (Somia and Verma, 2000). Long
term expression is typically required for a successful therapeutic outcome, especially when correcting
inactive or partially functioning genes. Even though there are various viral vectors which integrate their
transgene containing DNA into the host cell genome (including retroviral- and AAV-vectors), the use of
these vectors does not necessarily ensure long term gene expression (Kay et al., 2001). Since integration
into the host cell genome is a random process, the therapeutic gene may become integrated into an inactive
part of the genome, resulting in silenced transgene expression (Chen and Townes, 2000). The integrated
transgene may also inactivate host genes, which are crucial for normal cellular function or activate harmful
genes such as proto-oncogenes (Bushman and Miller, 1997; Shiramizu et al., 1994). In addition, the host
immune system may recognize transgene -encoded proteins as foreign and evoke an immune response,
which is likely to suppress the transgene expression (Tripathy et al., 1996).

22
2.6 Improvements in cancer gene therapy

2.6.1 Overview

Although there still are many obstacles for successful cancer gene therapy, several approaches have been
investigated to circumvent the major problems. One advance has been the characterization of different
viruses and their utilization and further modification into safe and efficient gene transfer vehicles (Kootstra
and Verma, 2003). Furthermore, development of various targeting techniques means that it has been
possible to modify the viral vectors to recognize cancer cells and thus cause minimal transduction to
healthy, non-target tissues (Nettelbeck et al., 2000; Peng and Russell, 1999). Thus, targeting can achieve
more efficient and accurate gene transfer. In addition, the natural replication capability of various viruses
including adeno, herpes simplex and alpha viruses has been exploited in cancer gene therapy (Alemany et
al., 2000; Post et al., 2004; Lundstrom, 2001). These oncolytic, replicative viruses can destroy tumor cells
via replication which can be limited into target cells by genetic modification. Instead of viral vector
modification, also transgenes can be modified so that the resulting therapeutic protein can spread to
surrounding cells and thus compensate for the initially low gene transfer efficiency. Several so called
translocatory proteins are currently known and their features have been characterized and evaluated for
cancer gene therapy purposes (Leifert and Whitton, 2003).

2.6.2 Optimal gene transfer tools for specific purposes: various viral vectors

Viruses need to transfer their genomes efficiently into host cell in order to replicate. Thus, viruses are
evolutionarily optimized gene transfer machines. In order to use viruses as safe gene transfer vehicles,
virulence genes and genes responsible for viral replication have to be eliminated. In addition to increasing
the safety of the viral vector, partial genomic deletions also enable the insertion of foreign genetic material
e.g. transgenes.

Viral vectors can be divided into different categories based on their genome (DNA vs. RNA), structure
(enveloped vs. non-enveloped) or integration (Fields et al., 1996). The most frequently used viral vectors in
human clinical gene therapy trials are based on adeno- and retroviruses and the less common vectors on
adeno associated-, herpes simplex-, pox- and alphaviruses (http://www.wiley.co.uk/genetherapy/clinical/).

2.6.2.1 Adenoviral vectors

Adenoviruses are a family of viruses (over 50 serotypes) that most commonly cause benign respiratory
illnesses in humans (Volpers and Kochanek, 2004). Adenoviruses are nonenveloped, double stranded DNA
viruses, whose genome is surrounded by an icosahedral protein capsid comprising of three major proteins,
hexon, penton base and knobbed fiber (Russell, 2000). The linear virus genome is about 36 bp in size and
consists of immediate early (E1A), early (E1-E4), intermediate and late genes (L1-L5) (Figure 2).

23
Transcription of these genes can be divided into early and late phases, i.e. those occurring before and after
DNA replication (Kootstra and Verma, 2003).

L5
L4
L3
MLP L2
E1B L1
E3
E1A

LITR Ψ Adenoviral genome RITR

E2B
IX E2A E4
IVa2

Figure 2. Adenoviral genome. Adenoviral genome contains early (E1-4), intermediate (IX and IVa2) and
late (L1-5) genes flanked by left and right inverted terminal repeats (LITR and RITR, respectively). MLP:
major late promoter, Ψ packaging signal.

In order to penetrate inside the host cell, adenoviruses first attach to their primary cellular receptor, the
coxsackie- adenovirus receptor (CAR) (Bergelson et al., 1997) followed by interaction with cellular
integrins resulting in internalization of the virus via receptor-mediated endocytosis (Wickham et al., 1993)
(Figure 3). In the endosomes, the viral genome is released from the viral capsid and thereafter transported
into the nucleus. The adenoviral replication cycle is initiated by transcription of the E1A gene followed by
transcription of other early genes (Volpers and Kochanek, 2004). Early gene products interfere with
antiviral host cell defense mechanism, alter the cell cycle and modulate cellular metabolism in favor of
viral replication (Russell, 2000). The linear DNA is flanked by ITRs, which contain the sequences required
for the DNA replication (Hay et al., 1995). After the onset of DNA replication mediated by E2 and E4 gene
products, intermediate genes are expressed at high levels followed by the expression of late genes driven
by the major late promoter (Kay et al., 2001; Russell, 2000). Late genes encode for structural viral proteins
that assemble together with viral genomes in the nucleus followed by cell lysis and release of newly
synthesized virions (Volpers and Kochanek, 2004).

24
 1. Binding 2. Internalization

Penton base

Fiber knob αυβ integrins

CAR
3. Endosytosis
8. Cell lysis and release of
Cytoplasm new adenoviruses

4. Release of 7. Viral assembly

viral genome 5. DNA replication

Adenoviral genome

Nucleus 6. Viral protein

production

Figure 3. Adenoviral replication cycle. Viruses first attach to the coxsackie- adenovirus receptor (CAR)
followed by an interaction with cellular integrins resulting in internalization of the virus via receptor-
mediated endocytosis. In the endosomes, the viral genome is released from the viral capsid and thereafter
transported into the nucleus for DNA replication. Structural viral proteins assemble together with viral
genomes in the nucleus followed by cell lysis and release of newly synthesized virions.

Since their first description in the 1950s, adenoviruses have been studied extensively and they have
become one of the most widely used gene transfer tools in human gene therapy. From the gene therapy
standpoint, adenoviruses offer numerous advantages: 1) good characterization and reasonable
understanding of their biology, 2) low pathogenicity in humans, 3) capability to infect both dividing and
quiescent cells, 4) capacity to accommodate relatively large transgenes, 5) low risk for insertional
mutagenesis due to an inability to integrate into the host cell genome and 6) relatively easy manipulation
and high-titer production (Danthinne and Imperiale, 2000).

The most frequently used adenoviral vectors are based on serotype 5 virus. Adenoviruses can be modified
to viral gene transfer vehicles by partial deletion of the viral genome. The first generation adenoviral
vectors were made by deleting the E3 and E1 region, which is responsible for initiation of viral replication.
These deletions enabled insertion of ~8 kbp of foreign DNA into the first generation vectors (Danthinne
and Imperiale, 2000). To increase the safety and transgene capacity of the adenoviral vectors, additional
deletions have yielded second generation adenoviral vectors (E1-4 regions deleted) (Armentano et al.,

25
1995; Gorziglia et al., 1996) and so called gutless vectors (all viral genes deleted) (Kochanek et al., 1996).
Although deletion of various viral proteins have decreased the immunogenicity and toxicity of adenoviral
vectors and prolonged the persistence of transgene expression (Engelhardt et al., 1994; Kochanek et al.,
1996; Wang et al., 1997), the existing viral structural proteins still elicit an immune response, which
prevents repeated administration of these vectors (Somia and Verma, 2000). One possibility to circumvent
the immune system would be to use vectors based on various human adenovirus serotypes (Barouch et al.,
2004; Mack et al., 1997) or animal adenoviruses (Moffatt et al., 2000; Rasmussen et al., 1999) for
readministration. Another disadvantageous feature of adenoviral vectors is their propensity to accumulate
into liver and cause hepatotoxicity. This problem can be partially circumvented by targeting the viral
vectors to cancer cells (see chapter 2.5.3 Enhanced gene transfer and increased specificity: adenoviral
targeting).

Due to the nonintegrating nature of adenoviruses, transgene expression from adenoviral vectors is only
transient. Therefore, use of these vectors has mainly focused on treatment of cancer types, where high
level, short-term gene expression is sufficient to evoke a therapeutic response. In humans, adenoviral
vectors have been widely used in various cancer types including lung-, ovarian-, prostate- and breast cancer
and malignant glioma (Tursz et al., 1996; Alvarez et al., 2000; Herman et al., 1999; Stewart et al., 1999;
Immonen et al., 2004).

2.6.2.2 Retro- and lentiviral vectors

Retroviruses are lipid-enveloped, single stranded RNA viruses, which can be divided into oncoretro-, lenti-
and spumaviruses (Fields et al., 1996). The enveloped viral particle contains the viral genome consisting of
two copies of 8-12 kbp -sized RNA strands surrounded by the nucleocapsid (Kootstra and Verma, 2003).
The retroviral genome flanked by long terminal repeats (LTR) contains three essential genes: gag encoding
viral structural protein, pol encoding reverse transcriptase and integrase and env encoding viral envelope
glycoprotein, which mediates virus entry (Figure 4). In the lentiviral genome, there are additional accessory
genes: for example HIV-1 has vif, vpr, vpu, tat, rev and nef genes that encode for the proteins necessary for
efficient viral replication and persistence of infection in the natural target cells of this virus (Kootstra and
Verma, 2003).

26
 ψ

Oncoretrovirus LTR GAG POL ENV LTR

ψ
NEF
Lentivirus LTR GAG POL VIF ENV LTR
VPR
VPU
TAT
REV

Figure 4. Genome structure of oncoretrovirus and HIV-1 lentivirus. Both genomes contain gag, pol
and env genes flanked by long terminal repeats, LTRs. The HIV-1 genome contains six additional genes
encoding vif, vpr, vpu, tat, rev and nef. Ψ Packaging signal.

In the early onset of retroviral replication cycle (Figure 5), the retrovirus binds to its receptor followed by
membrane fusion and release of RNA genome from the viral capsid (Fields et al., 1996). In the cytosol, the
viral genome is copied to double-stranded DNA by the viral reverse transcriptase (Jolly, 1994). The viral
DNA is then translocated to the nucleus (retroviruses with passive migration and lentiviruses with active
transport), where it becomes integrated into the host-cell genome by its own integrase enzyme to yield a
provirus. The cellular machinery is then utilized to make viral RNA, using the provirus as a template. The
viral RNA also serves as mRNA, which is translated into viral proteins (Kootstra and Verma, 2003). For
viral particle formation, translated viral proteins or their precursors assemble together with two viral RNA
strands followed by budding from the plasma membrane. During the budding process, the virus acquires its
lipid-coated envelope by incorporating env -glycoproteins from the host cell membrane (Jolly, 1994).

27
 1. Binding
2. Fusion of plasmamembranes

Retroviral
3. Endocytosis Cytoplasm
receptor
9. Viral assembly

4. Release of viral
RNA genome
Retroviral genomes
Retroviral
genome
(RNA) 5. Reverse
transcription 7. Production of viral RNA

6. Integration 8. Viral protein

Retroviral genome
Nucleus production
(dsDNA)

Figure 5. Retroviral replication cycle. Retrovirus binds to its receptor followed by membrane fusion and
release of the RNA genome from the viral capsid. In the cytosol, the viral genome is copied into double-
stranded DNA by viral reverse transcriptase. The viral DNA is then translocated to the nucleus where it
integrates into the host cell genome. Cellular machinery is then utilized to make new viral genomes and
proteins. Translated viral proteins assemble together with two viral RNA strands followed by budding from
the plasma membrane. During the budding process virus acquires a lipid-coated envelope (with
incorporated env–glycoproteins) from the host cell membrane.

Retroviruses were the first viral vectors used in clinical studies (Blaese et al., 1995). At present,
retroviruses have become the most widely used gene transfer vectors in gene therapy. Several features have
lead to the wide use of retroviral vectors. First, their genome is rather simple, making the genetic
modification required for vector production relatively straightforward. Second, retroviral vectors are able
to integrate into the host cell genome, enabling long-term expression of the transgene in target cells and
also in their progeny. However, integration does not necessarily ensure stable transgene expression, in fact
it may increase the risk for insertional mutagenesis (Bushman and Miller, 1997; Kay et al., 2001;
Shiramizu et al., 1994). Third, retroviral vectors do not elicit an immune response, which minimizes their
cytotoxicity and allows the readministration of these vectors. On the other hand, retroviral vectors are
susceptible to rapid degradation by the complement system (Takeuchi et al., 1994). The main factor
limiting the use of most retroviral vectors (including the most widely used Moloney murine leukemia virus
based vectors) is their inability to transduce into non-dividing cells (Barquinero et al., 2004). In contrast,
vectors based on lentiviruses are capable of transducing both into dividing and quiescent cells (Delenda,
2004). Another limiting factor for retroviral vectors is their inefficient production at high titers (Romano et

28
al., 1999). However, by replacing the region responsible for initiation of transcription (U3 –region) from
the 5’ LTR with the CMV promoter, higher vector titers have been obtained (Finer et al., 1994). In
addition, modification of viral glycoproteins has generated more stable viral particles allowing them to be
concentrated to higher titers (Burns et al., 1993). Nevertheless, retroviral titers still lag far behind, for
example adenoviral titers; adenoviruses can be routinely produced in titers 2-3 orders of magnitude higher
than the best retroviral-/lentiviral titers.

Due to the relatively small size of the retroviral genome, their transgene capacity is a mere 8 kbp
(McCormick, 2001). If one wishes to utilize retroviruses as gene transfer vectors, then the viral genes are
completely replaced with the desired transgene and in many cases also with an internal promoter. The viral
proteins required for functionality of the vector are produced from separate packaging constructs, which
minimize the probability for generation of replication competent viruses and thus increase the safety of
these vectors (Romano et al., 1999). Instead of using the packaging cell lines, transient transfection can be
used to deliver the required constructs to the producer cells to obtain efficient virus production (Pear et al.,
1993; Soneoka et al., 1995). Self-inactivating type vectors (SIN) have been developed to further increase
the safety of retroviral vectors (Yu et al., 1986). These vectors contain a deletion on the 3’ LTR, which
inactivates the functionality of both enhancer and promoter. When the vector genome is reversely
transcribed, this deletion is transferred to the 5‘LTR, abolishing transcriptional activity of the integrated
provirus. In the context of HIV-1 based lentiviral vectors, safety aspects have to be more carefully
addressed. Thus, all the HIV-1 accessory genes have been deleted and the virus production components
have been divided into 3-4 separate parts (Zufferey et al., 1997). Additionally, self-inactivating deletions
have been introduced into the vector backbones (Zufferey et al., 1998).

Various approaches have been developed to enhance the transduction rates of retroviral vectors. Retroviral
particles have been pseudotyped to broaden the host cell tropism, e.g. the env-glycoprotein has been
replaced by other viral proteins such as glycoprotein from vesicular stomatitis virus (VSV-G), a technique
which has also been shown to stabilize the vector particles (Yee et al., 1994). Furthermore, oncoretroviral
vectors have been retargeted by fusing polypeptides into the envelope glycoproteins (Peng et al., 1999;
Peng et al., 2001). Transduction efficiency and transgene expression of lentivirus vectors has also been
enhanced by incorporating central polypurine tract (cPPT) and posttranscriptional regulatory elements
(PRE) into the vector constructs. The cPPT has been reported to act by increasing nuclear transport of the
viral preintegration complex (Follenzi et al., 2000; Zennou et al., 2000) but it may also facilitate nuclear
import of viral RNA species and in their way improve the lentiviral transduction efficiency (Van Maele et
al., 2003). Further, PREs from human or woodchuck hepatitis viral origin have been shown to stabilize
viral vector RNA improving transgene expression (Patzel and Sczakiel, 1997; Zufferey et al., 1999).
Although the use of retroviral vectors has mainly focused on inherited genetic disease where stable, long-
term transgene expression is required, also several clinical studies have also been reported for cancer
diseases. Due to the inability of retroviral vectors to transduce non-proliferating cells (e.g. neurons),

29
retrovirally mediated suicide gene therapy has been proposed for the treatment of malignant brain tumors
(Culver et al., 1994). Retroviral gene delivery has also been exploited in studies, where extended gene
expression is required for therapeutic outcome including introducing wild-type tumor suppressor gene into
lung tumors (Roth et al., 1996). In preclinical studies, lentiviral vectors have been evaluated against several
cancer types including ovarian (Indraccolo et al., 2002), prostate (Bastide et al., 2003; Zheng et al., 2003)
and bladder cancer (Kikuchi et al., 2004). In addition, a cancer gene therapy approach based on lentiviral
vector targeting to tumor endothelium has been introduced (De Palma et al., 2003).

2.6.3 Enhanced gene transfer and increased specificity: adenoviral targeting

Adverse side effects caused by unspecific gene transfer to non-target organs can be avoided by targeting
the viral vectors and/or the transgenes into cancer cells. Additionally, such maneuvers allow enhancement
of gene transfer rates in tumor tissue resulting in an enhanced therapeutic outcome. Especially in
adenoviral gene transfer, the biggest obstacle is the variable and often low expression level of CAR in the
tumor cells, making these cells rather refractory to adenoviral gene transfer. Further, it would be also
important to minimize the adverse side effects by targeting the vectors to avoid the liver. Targeting
strategies can be based on transductional or transcriptional approaches.

Transductional targeting is based on altered viral tropism by modifying the viral proteins mediating
receptor binding. In adenoviral vectors, the re-targeting moieties allowing CAR- independent delivery can
be linked physically to fiber knob or introduced genetically by incorporating the necessary changes into the
viral genome (Figure 6). Simultaneously, the binding to the primary viral receptor is blocked. Alternative
receptors are typically expressed at high levels in cancer cells but to a lesser extent in normal cells, and this
is a one way to improve the tumor cell specificity of the gene transfer. There are several reports of
successful adenoviral targeting in vitro and in animal models which have evaluated many alternative
cellular receptors including αv integrins (Wickham et al., 1996), CD3 (Wickham et al., 1997), CD40
(Tillman et al., 1999), adenovirus serotype 3 receptor (Kanerva et al., 2002) and prostate specific
membrane antigen, PSMA (Kraaij et al., 2005). Further, several studies have shown that adenoviral vector
mediated liver toxicity can be reduced by targeting the vectors to cancer cells (Einfeld et al., 2001; Printz et
al., 2000).

30
 Integrins

Alternative Alternative
CAR cellular receptor cellular receptor

Untargeted adenovirus Genetically targeted adenovirus Physically targeted adenovirus

Figure 6. Transductional targeting of adenoviruses. Transductional targeting can be used to re-route the
adenoviral to alternative cellular receptors instead of its primary coxsackie-adenovirus receptor (CAR).
Targeting can be based on genetically modified knobs or bi-specific ligands, which bind both to viral knob
and alternative receptor.

In addition to re-routing the viral vectors to alternative receptors, expression of therapeutic genes can be
limited into tumor tissue. The expression of transcriptionally targeted genes is driven by tissue specific
promoters, which are activated in target cells by tissue specific transcription factors. Several tissue specific
promoters have been recently characterized and studied for cancer gene therapy purposes including α-
fetoprotein for hepatomas (Kanai et al., 1997), cyclo-oxygenase-2 (Cox-2) for ovarian and gastric cancer
(Casado et al., 2001; Yamamoto et al., 2001) and osteocalcin for metastatic prostate cancer (Koeneman et
al., 2000). Additionally, radiation or drug inducible promoters have been exploited for transcriptional
targeting such as early growth response gene 1 (EGR-1) promoter (Manome et al., 1998).

2.6.4 Improved therapeutic outcome by spreading the therapeutic element: oncolytic viruses and
protein transduction domains

One possibility to circumvent the initially low gene transfer efficiency is to spread the therapeutic element
inside the tumor tissue. One approach is based on oncolytic adenoviruses, which replicate in tumor cells,
killing the host cell and spreading to the neighboring cells, eventually throughout the tumor. Another
possibility is to exploit protein transduction domains (PTD), which can spread the fused therapeutic protein
from one cell to another throughout the tumor.

31
2.6.4.1 Conditionally replicative adenoviruses (CRAds)

The use of conditionally replicative adenoviruses (CRAds) is based on their ability to spread throughout
the tumor theoretically as long as tumor cells persist by virtue of viral replication and concomitant cell lysis
(Alemany et al., 2000; Post et al., 2003).

To minimize adverse side effects and to increase the safety of these anti-cancer agents, replication can be
limited to tumor tissue by genetically modifying the CRAd genome. This can be achieved either by partial
deletions in the E1 region or by using tissue specific promoters to drive the genes responsible for viral
replication (Alemany et al., 2000). Partial viral genome deletions allow virus to replicate selectively in
cells with defective p53/p14ARF (Bischoff et al., 1996) or Rb-p16 pathway (Fueyo et al., 2000) that are
hallmarks of many cancer cells. Replication has also been limited by using tissue specific promoters such
as human telomerase reverse transcriptase (hTERT) (Irving et al., 2004), hepatocellular carcinoma specific
α-fetoprotein promoter (Hallenbeck et al., 1999) and melanoma specific tyrosinase promoter (Nettelbeck et
al., 2002) to drive the E1A region expression.

To increase the specificity and antitumoral activity of these oncolytic agents, several targeting approaches
have been developed. CRAds have been successfully targeted to alternative cellular receptors, e.g. the
adenovirus serotype 3 receptor and integrins (Kanerva et al., 2003; Suzuki et al., 2001). In addition,
antitumoral activity of CRAds has been improved by exploiting existing viral genes. It has been reported
that retaining the adenovirus E3 region (Suzuki et al., 2002) and overexpression of its adenovirus death
protein (ADP) (Yun et al., 2004) can increase the oncolytic activity of CRAds. Also, deletion of
unnecessary viral genes such as the gene encoding the apoptose inhibitor E1B-19 kDa protein, can enhance
oncolysis (Liu et al., 2004).

The most widely studied and first clinically tested CRAd was ONYX-015 (dl1520), from which the p53-
inhibitory protein encoding E1B-55 kDa gene had been deleted (Bischoff et al., 1996). It was hypothesized
that due to this deletion, ONYX-015 would only be able to replicate in those tumor cells which have lost
p53 function, a common occurrence in many cancer cells. In initial preclinical studies, ONYX-015 was
reported to replicate selectively in p53-deficient tumor cells (Bischoff et al., 1996) and in addition, it was
shown not to replicate in normal epithelial and endothelial cells (Heise et al., 1997). However, subsequent
studies showed that there was no correlation between the viral replication and p53-status of the tumor cells
and the mechanism of selectivity was far more complex than initially proposed (Goodrum, and Ornelles,
1998; Rothmann et al., 1998). ONYX-015 has also been tested extensively in humans; over 10 clinical
trials (phase I-III) have enrolled approximately 300 patients with head and neck cancer (Ganly et al., 2000;
Nemunaitis et al., 2000; Nemunaitis et al., 2001a), metastatic colorectal cancer (Reid et al., 2002),
pancreatic cancer (Mulvihill et al., 2001) and ovarian cancer (Vasey et al., 2002). ONYX-015 has also been
studied in patients with lung metastasis (Nemunaitis et al., 2001b). The results from these studies showed
that although ONYX-015 does seem to be a safe, well tolerated vector that can be administered by various

32
routes (intratumoral, intraperitoneal, intra-arterial and intravenous), the majority of treated tumors did not
respond to the therapy and no significant antitumoral effects were detected. However, it should be noted
that many of these patients were not responding to any standard therapies, which could also potentially
influence to these results.

Also a PSA -selective CRAd (CV706) has been studied in humans as a single anti-cancer agent for prostate
cancer (DeWeese et al., 2001). The results were parallel with those obtained from the ONYX-trials: the
virus was well tolerated and evidence of its replication was obtained but the achieved antitumoral effect
was modest.

Taken together, these results suggest that early generation CRAds are safe anticancer agents, but they have
a limited ability to completely destroy tumors as a single agent. However, improved antitumoral activity
has been obtained by combining CRAds with conventional therapy and suicide gene therapy (for details
see chapter 2.6.5 Combined therapies).

2.6.4.2 Protein transduction domains

There are several so called translocatory proteins which are reported to be secreted from cells via a non-
classical Golgi-independent route and to move intercellularly in a receptor- and transport –independent
manner (Schwarze and Dowdy, 2000). The best known translocatory proteins are herpes simplex virus type
1 (HSV-1) tegument protein VP22 (Elliott and O'Hare, 1997), HIV-1 tat (Frankel and Pabo, 1988; Green
and Loewenstein, 1988) and Drosophila antennapediae (Joliot et al., 1991). These intercellularly
trafficking proteins share several features: all of them appear to migrate to the nucleus and each of them
has a highly basic region (Leifert and Whitton, 2003). Although the exact mechanism of intercellular
spreading is still unknown, domains responsible for protein transduction have been identified. These small
PTDs contain several basic amino acid residues, which have been suggested to mediate their cellular
binding and penetration (Gratton et al., 2003; Lundberg et al., 2003).

The most widely studied PTD is VP22, a 38 kDa sized major protein of HSV-1 tegument encoded by UL49
gene (Elliott and Meredith, 1992). Several reports indicate that VP22 is able to retain its trafficking
capacity even when fused to other proteins. VP22 has been shown to spread intercellularly in vitro when
fused to GFP (Aints et al., 1999; Brewis et al., 2000; Elliott and O'Hare, 1997; Elliott and O'Hare, 1999;
Wybranietz et al., 1999) and enhance the antitumoral effect in vivo when fused to HSV-1 thymidine kinase
and tumor suppressors p53 and p27 (Dilber et al., 1999; Wills et al., 2001; Zavaglia et al., 2003). On the
other hand, however, there are also a few reports suggesting that VP22 mediated intercellular trafficking
might be an artifact, related to the fixation process (Fang et al, 1998; Lundberg et al., 2003), thus making
the exploitation of protein transduction approach for gene therapy purposes rather dubious. According to
recent findings by Lundberg and co-workers, positively charged PTDs e.g. VP22 only mediate cell surface

33
adherence via electrostatic interactions and the observed translocation across the cell membrane is an
artifact introduced during the fixation procedure (Lundberg et al., 2003).

2.6.5 Combination therapies

Although several gene therapy approaches have been demonstrated to destroy tumor cells, clinical trials
suggest that the early generation viral vectors have a rather limited ability to completely eradicate advanced
tumor masses. In order to achieve an increased antitumoral effect, combining novel approaches and
conventional therapies might be useful.

Conventional therapies have been efficiently exploited in cancer gene therapy. In patients with malignant
gliomas, the remaining malignant cells have been shown to be destroyed by suicide gene therapy following
surgical tumor resection (Immonen et al., 2004). In addition, introduction of suicide, tumor suppressor or
immunotherapeutic genes into the patients receiving chemotherapy has been studied in ovarian and lung
cancer and multiple myeloma (Hasenburg et al., 2001; Schuler et al., 2001; Trudel et al., 2001). However,
in these studies only a minor additional benefit was obtained with combination therapy. The transfer of
MDR-1 gene into hematopoietic stem cells to reduce toxic effects of cancer chemotherapy has also been
evaluated (Hesdorffer et al., 1998). Combining radiotherapy with therapeutic genes encoding e.g.
thymidine kinase and p53 has been studied in prostate and lung cancer (Teh et al., 2004; Swisher et al.,
2003). Furthermore, utilizing chemotherapy and radiotherapy in conjunction with CRAds has been
suggested to augment antitumor activity in pre-clinical studies with colon cancer (Rogulski et al., 2000a)
and malignant glioma (Lamfers et al., 2002) and in clinical studies with head and neck cancer (Khuri et al.,
2000) and metastatic gastrointestinal carcinoma (Reid et al., 2002).

In pre-clinical studies, immunotherapy combined with suicide genes has been demonstrated to induce
antitumoral immunity and to enhance tumor regression in lung, colon and metastatic breast cancer (Park et
al., 2003; Jones et al., 2000; Majumdar et al., 2000).

Although the ability of PTDs to translocate fused therapeutic proteins is still debatable, PTDs have been
successfully employed to enhance both lenti- and adenoviral transduction in vitro and in vivo (Gratton et
al., 2003; Kretz et al., 2003). In addition, PTDs have been demonstrated to improve the viral uptake and
replication of tumor-specific oncolytic adenoviruses in vitro (Kuhnel et al., 2004). Another approach to
enhance the antineoplastic activity of oncolytic adenoviruses is to utilize their ability to multiply and
amplify inserted therapeutic genes during their replication process. Several pre-clinical and clinical studies
have indicated that combining therapeutic genes, such as p53, cytosine deaminase and thymidine kinase
with replicative viruses might improve their oncolytic potency and increase the anti-tumoral effect (van
Beusechem et al., 2002; Fuerer and Iggo, 2004; Wildner et al., 1999; Freytag et al., 2002; Freytag et al.,
2003).

34
3 AIMS OF THE STUDY

The main purpose of this study was to evaluate different approaches to enhance gene delivery systems and
therapeutic genes for cancer gene therapy.

Specific aims of this thesis were:

1. To study the utility of lentiviral vectors as gene transfer vehicles for human cancer cells in vitro
and in vivo (I)
2. To enhance adenoviral transduction and target cell specificity by utilizing the CD40 receptor for
adenoviral re-targeting (II)
3. To study the VP22-mediated intercellular trafficking and its feasibility for cancer gene therapy
application (III)
4. To evaluate the oncolytic potency and monitor viral spreading of a novel conditionally replicative
adenovirus (IV)

35
4 MATERIALS AND METHODS

4.1 Fusion gene constructs (III)

VP22-GFP and VP22-TK fusion genes were kind gifts from P. O’Hare, Marie Curie Research Institute,
UK and TK-GFP fusion gene was constructed earlier in our laboratory (Loimas et al., 1998). The triple
fusion gene VP22-TK-GFP was constructed in two parts as described in the original publication. Briefly, a
1.7-kb fragment containing VP22 and half of the TK and the 1.15-kb fragment containing the other half of
the TK and GFP were ligated simultaneously into pUC19 to create a VP22-TK-GFP.

4.2 Viral vectors (I-IV)

Lenti - and adenoviral vectors and oncolytic adenoviruses used in studies I-IV are listed in Tables 1 and 2.
All viral vectors were constructed in our lab except AdLuc1 (Kanerva et al., 2002), ∆24 (Fueyo et al.,
2000) and the SIN type lentiviral vectors (kind gifts from D. Trono, University of Geneva, Switzerland).
Lentiviral vectors were propagated in 293T cells and adenoviral vectors in 293 or A549 cells using
standard techniques as described in the original publications. Functional lentiviral titers (transducing units,
t.u.) were determined with flow cytometry and the number of viral particles (VP) in each preparation was
determined with the aid of ELISA kit (Alliance HIV-1 p24 ELISA kit, Perkin Elmer Life Sciences)
according to manufacturer’s instructions. The adenoviral titers were determined with a spectrophotometer
(VP-titer) and with the plaque assay (plaque forming units, pfu-titer).

4.3 Cell lines and patient samples (I-IV)

Cell lines representing various cancer types and the viral vector producer cells used in studies I-IV are
listed in Table 3. Cell lines were obtained mainly from ATCC (Manassas, VA) or were kindly provided by
collaborators and were cultured as recommended at 37°C under 5% CO2 (for further details see the original
publications).

Ovarian tumor samples (II) were collected from patients undergoing surgical evaluation at the University
of Alabama at Birmingham for suspected epithelial ovarian carcinoma or primary peritoneal carcinoma.
Specimens were collected in a sterile fashion at the time of surgery and immediately frozen at -70°C.

36
Table 1. Lentiviral vectors used in studies I and III

The first generation, VSV-G pseudotyped lentiviral vector

HR’-GFP/LentiGFP I, III
expressing GFP under CMV promoter
The first generation, VSV-G pseudotyped lentiviral vector
HR’-TK-GFP/LentiTK-GFP I, III
expressing TK-GFP fusion gene under CMV promoter
HR’VP22-TK-GFP/LentiVP22- The first generation, VSV-G pseudotyped lentiviral vector
III
TK-GFP expressing VP22-TK-GFP fusion gene under CMV promoter
WOX-VP22-GFP/LentiVP22- The first generation, VSV-G pseudotyped lentiviral vector
III
eGFP expressing VP22-eGFP fusion gene under EF1α promoter
The second generation, VSV-G pseudotyped, SIN-type
HOX-GFP I
lentiviral vector expressing eGFP under EF1α promoter
The first/second generation, VSV-G pseudotyped, SIN-type
lentiviral vector expressing eGFP under EF1α promoter and
WOX-GFP I
containing woodchuck hepatitis virus posttranscriptional
regulatory element (WPRE)
The second generation, VSV-G pseudotyped, SIN-type
HPT-GFP lentiviral vector expressing eGFP under EF1α promoter and I
containing central polypurine tract (cPPT)
The second generation, VSV-G pseudotyped, SIN-type
WPT-GFP lentiviral vector expressing eGFP under EF1α promoter and I
containing both cPPT and WPRE

Table 2. Adenoviral vectors used in studies I-IV

The first generation, E1/E3 deleted serotype 5 adenoviral vector

AdGFP I
expressing GFP under EF1α promoter
I, III
The first generation, E1/E3 deleted serotype 5 adenoviral vector
AdTK-GFP and
expressing TK-GFP under CMV promoter
IV
The first generation, E1/E3 deleted serotype 5 adenoviral vector
AdVP22-TK-GFP III
expressing VP22-TK-GFP under CMV promoter
The first generation, E1/E3 deleted serotype 5 adenoviral vector
Ad5luc1 II
expressing luciferase under CMV promoter
The conditionally replicative, E3 deleted serotype 5 adenoviral
∆24 IV
vector encompassing 24 bp deletion in E1A
The conditionally replicative, E3 deleted serotype 5 adenoviral
Ad5-∆24TK-GFP vector encompassing 24 bp deletion in E1A and expressing IV
TK-GFP under CMV promoter

37
Table 3. Used cell lines in studies I-IV.

Bladder cancer J82, TCCSUP and UM-UC-3 I

A172, Hs 683, SH-SY5Y, TE 671, U-118 MG,
Brain cancer I
U-251 MG, IMR-32 and U-87 MG
Bone cancer
MG-63, Saos-2, and U-2 OS I
(Osteosarcoma)

Breast cancer MCF7, SK-BR-3, ZR-75-1 and BT-20 I, II

Cervix cancer HeLa and SiHa I,II

Colon cancer HCT 116, HT 29 and CaCo-2 I

NCI-H23, NCI-H146, NCI-H661, NCI-H520, NCI-H82,

Lung cancer I, IV
NCI-H460, NCI-H1650, SW 900, A549 and Calu-3

Melanoma A2058, SK-MEL-5, WM-115 and WM-266-4 I

Ovarian cancer Hey, SKOV3.ip1,OV-4 and OV-3 II-IV

Pancreatic
Mia PaCa-2, BxPC-3, AsPC-1 I
cancer

Prostate cancer DU 145, PC-3 and 22Rv1 I, III

Renal cancer Caki-2 III

Rat glioma 9L and BT4C III

Producer cell I, III

293T, 293 and 911
lines and IV

* If not indicated otherwise, the cell lines are of human origin.

4.4 Protein expression levels

4.4.1 Expression of VP22-fusion proteins

OV-4 cells were transduced with HR’GFP, HR’VP22-eGFP, HR’TK-GFP and HR’VP22-Tk-GFP. Four
days later, cells were trypsinized and washed once with PBS. Cells were lysed with total cell lysis buffer
(20 mM Tris/Hcl pH 8, 2 mM EDTA, 3% NP-40, 100 mM NaCl, aprotin (5 µg/ml), leupeptin (5 µg/ml)
and 50 mM NaF) and restored supernatants were used for western blot analysis. Equal amounts of proteins
were run into 7.5% SDS-PAGE gel and proteins were transferred onto InvitrolonTM PVDF membranes
(Invitrogen, Paisley, UK). The membrane was blocked overnight at +4 °C with blocking buffer (5% fat free
dry milk and 0.1% Tween in PBS), incubated for 1 hour with a 1:200 diluted rabbit anti-GFP primary

38
antibody (GFP (FL), Santa Cruz Biotechnology, Inc., Heidelberg, Germany) followed by a 1 hour
incubation with 1:200 000 diluted donkey anti-rabbit secondary antibody (ECL anti-rabbit IgG, peroxidase
(HRP) linked whole antibody, Amersham Bioscience, Buckinghamshire, UK). Chemiluminescence
detection was performed using the ECL plus Western blotting detection kit (Amersham Bioscience,
Buckinghamshire, UK) according to the manufacturer’s instructions.

4.4.2 CD40 expression (II)

To detect CD40 mRNA, isolated and DNase treated total RNA from cultured ovarian cancer cells and
primary tumor samples was used as a template for RT-PCR. Amplification was carried out with OneStep
RT-PCR Kit (QIAGEN, Valencia, CA) using CD40 specific primers (Upstream 5’ AGA AGG CTG GCA
CTG TAC GA 3’, Downstream 5’CAG TGT TGG AGC CAG GAA GA 3’). GAPDH primers were used
as a control (Upstream 5’ TCC CAT CAC CAT CTT CCA 3’, Downstream 5’ CAT CAC GCC ACA GTT
TCC 3’).

To determine CD40 expression levels by flow cytometry, trypsinized ovarian and breast cancer cells were
resuspended in PBS containing 2% FBS (FACS-buffer) and stained with fluorescein isothiocyanate (FITC)
-conjugated mouse anti-human CD40 monoclonal antibody. Cells were incubated at +4 °C for 20 min and
washed with FACS-buffer prior to flow cytometry (FACScan, Beckton Dickinson, San Jose, CA). Cells
stained with FITC- conjugated IgG1 isotype control antibody (BD Biosciences, San Diego, CA) were used
as control.

4.5 Determination of lentiviral transduction efficiency by flow cytometry (I)

Cells were plated on the 12 or 24 -well plates and transfected 24 hours later with lentiviral vectors using 3
t.u./cell. Appropriate growth media supplemented with 10% FBS and polybrene (8 µg/ml) were used for
transductions. Cells were incubated with viruses for 24 hours, followed by addition of fresh growth media
to each well. Cells were collected for determination of GFP positive cells by FACS analysis 3, 4, 8, or 14
days after viral transductions.

4.6 Gene transfer with CD40-targeted adenovirus (II)

To determine gene transfer efficiency, replication deficient luciferase expressing adenovirus Ad5luc1 was
incubated with CD40 retargeting CAR/G28 fusion protein (Pereboev et al., 2002) at a ratio of 100 ng
fusion protein/100 pfu for 45 min at RT. In the dose response assay, virus was preincubated with different
amounts of CAR/G28 fusion protein (0, 3, 10, 30, 100 or 150 ng/100 pfu). Ovarian and breast cancer cells
were infected with either non-targeted or CD40-targeted virus for 1 hour. Luciferase expression was
determined with Luciferase Assay System (Promega, Madison, WI) 24 hours after infections. For the

39
blocking assay, OV-4 and BT-20 cells were incubated either with growth media, supernatant from G28-5
hybridoma cells containing monoclonal anti-human CD40 antibody (blocking) or supernatant from
1D11.16.8 hybridoma cells containing monoclonal anti-TGF-β2 antibody (irrelevant antibody) followed by
infections with non-targeted or CD40-targeted virus for 1 hour. Luciferase expression was determined as
above.

4.7 Verification of viral replication and oncolysis (IV)

Ovarian cancer cells were transduced with AdTK-GFP, Ad5-∆24TK-GFP and ∆24 for overnight using 1,
10 and 100 pfu/cell. Growth media was replaced every other day. OV-3, OV-4, SKOV3.ip1 and Hey cells
were analyzed with MTT-assay according to the manufacturer’s instructions (Cell proliferation Kit II,
Roche Diagnostics, Indianapolis, IN) and stained with crystal violet 8, 12, 14 and 18 days post-infection,
respectively.

4.8 Ganciclovir sensitivity assays (I, III, IV)

Osteosarcoma cells were transduced either with AdTK-GFP or HR’TK-GFP (I) and BT4C, 9L, 293T, PC-
3, Caki-2 and SKOV3.ip1 cells either with HR’TK-GFP or HR’VP22-TK-GFP (III). The proportion of
GFP-positive cells was analyzed three (adeno) and four (lenti) days post-transduction by flow cytometry
(FACSCalibur, Becton Dickinson). GFP-positive cells were mixed with naïve, non-transduced cells to
obtain cell populations with varying proportions (0-20%) of fusion gene expressing cells. Cells were
cultured for five days in the presence of GCV (0 – 1000 µg/ml) followed by cell viability measurements
with the MTT assay as above.

To determine the influence of cell growth rate on VP22-mediated cell killing, a ganciclovir sensitivity
assay was performed as above with BT4C cells, except that the cells were cultured in medium with a
reduced amount of FBS (5% or 2.5%) in order to slow down the cell proliferation rate (III).

For replication virus studies, ovarian cancer cells were infected either with non-replicative AdTK-GFP or
replicative Ad5-∆24TK-GFP (10 pfu/cell) (IV). GCV containing media was added 24 hours post-infection
and cells were grown in the presence of ganciclovir (10 µg/ml) for five days. Cell viability was analyzed
with the MTT-assay as above.

40
4.9 Monitoring of VP22-mediated intercellular trafficking and adenoviral spreading (III,
IV)

4.9.1 Flow cytometry

To monitor VP22-mediated spreading, BT4C or SKOV3.ip1 cells were transduced either with lentiviruses
coding VP22-eGFP, TK-GFP or VP22-TK-GFP (1 t.u./cell). Four days later the amount of GFP-positive
cells was determined by flow cytometry (FACSCalibur, BecktonDickinson). GFP-positive cells were
mixed with naïve BT4C and SKOV3.ipI cells in order to achieve cell populations with 20-50% VP22-GFP,
TK-GFP or VP22-TK-GFP -positive cells. After 24, 48 and 72 hours, cells were harvested and either left
untreated or fixed with 4% paraformaldehyde (PFA) or 100% methanol. Cells were analyzed with flow
cytometry to monitor the percentage of GFP positive cells at different time points (III).

To monitor viral spreading, ovarian cancer cells were infected either with AdTK-GFP or Ad5-∆24TK-GFP
(5 pfu/cell). At each time point (2-10 days post-infection), cells were trypsinized and fixed with 4%
paraformaldehyde (PFA) followed by flow cytometry analysis in order to determine the proportion of GFP-
expressing cells (IV).

4.9.2 Fluorescence microscopy

To visualize VP22 mediated intercellular trafficking, BT4C and SKOV3.ip1 cells were transfected with
plasmids containing VP22-eGFP, TK-GFP or VP22-TK-GFP. Cells were washed with PBS and either left
untreated or fixed with 4% PFA or 100% methanol at 3 different time points (24, 48 and 72 hours post-
transfection). Prior to fluorescence microscopy (Axiovert 135M, Zeiss, Göttingen, Germany), cells were
washed once with PBS (III).

To visualize viral spreading, OV-4 cells were infected either with AdTK-GFP or Ad5-∆24TK-GFP and left
overnight. Cells were overlayed with 1:2 mixture of 2 x Dulbecco’s Modified Eagle’s medium (DMEM)
supplemented with 5% fetal bovine serum (FBS), 4mM glutamine and gentamicin (100 mg/l) (Qibco BRL,
Life Technologies, UK) and 1.8% Seaplaque agarose (BioWhittaker Molecular Applications, Rockland,
ME). Cells were examined under fluorescence microscope 3, 5, 7 and 9 days after infection (IV).

4.10 In vivo studies (I, III and IV)

Female NRMI nu/nu mice (age 10-12 weeks) were purchased from Harlan Netherlands. Mice were
anesthetized with phenyl-fluanisone-midazolam and three million A549 (I) or SKOV3.ip1 (III, IV) cells
per site were inoculated subcutaneously to two sites on the back of each animal. When the appropriate
tumor size for gene transfer was achieved, the following viruses were injected intratumorally: HPT-GFP
(4x106 t.u/tumor) and AdGFP (4x106pfu/tumor and 5x109 pfu/tumor) (I), AdTK-GFP and AdVP22-TK-

41
GFP (6.5x108 pfu/tumor) (III) and AdTK-GFP, Ad5-∆24TK-GFP and ∆24 (3 x 108 pfu/tumor) (IV). In
each study, tumors with no viral treatments were used as a control.

To determine the gene transfer rates in tumors, mice were sacrificed two (III, IV) or four (I) days after
viral injections. Tumors were harvested and minced with a scalpel and digested for two hours in digestive
enzyme solution [0.002% w/v Dnase I (type IV, Sigma) 0.1% w/v collagenase (type IV, Sigma) and 0.01%
w/v hyaluronase (type V, Sigma) in OptiMEM]. Single cell suspensions were created by washing the
digested cells twice with PBS and filtering with Falcon cell-strainer cap tubes (35 µm mesh,
BecktonDickinsonLabware). The cell suspensions were analyzed with flow cytometry to determine the
proportion of GFP-positive cells. In studies III and IV, only half of the tumor was used for flow cytometry
and the other half was fixed with formalin. Cryo slices were performed from formalin fixed and paraffin
embedded tumor samples and analyzed by fluorescence microscopy.

To study the therapeutic efficiency of the viruses, mice received either GCV (50 mg/kg/day) or saline
intraperitoneally for 10 (III) or 12 (IV) days. After the treatment period, mice were euthanized and tumors
were weighed. One-way analysis of variance with Bonferroni’s post hoc test for multiple comparisons was
used for statistical analysis (GraphPad Prism 3.0, GraphPad Software, Inc., San Diego, CA).

To monitor the spreading of the GFP-containing viruses in vivo (IV), mice injected either with AdTK-GFP
or Ad5-∆24TK-GFP were euthanized two animals at a time 2, 5, 9 and 12 days after viral injection.
Tumors were harvested and minced with a scalpel and digested as above followed by flow cytometry
analysis (FACSCalibur, BecktonDickinson).

All animal procedures were reviewed and approved by the animal care and use committee of the University
of Kuopio.

42
5 RESULTS AND DISCUSSION

5.1 Cancer cells are potential targets for lentiviral gene transfer (I)
Lentivirus vectors were introduced eight years ago, but their utility has already been demonstrated in a
variety of in vitro and in vivo applications. However, most of the studies have focused on the treatment of
inherited monogenic diseases. To study whether lentiviral vectors could be useful in cancer gene therapy,
we investigated in a systematic manner the efficacy of various lentiviral vectors against different cancer
types.

5.1.1 Transduction efficiency of lentiviral vectors in human cancer cells

To evaluate the transduction efficiency of lentivirus vectors, 42 different cancer cell lines representing
cancer originating from 10 different tissue types were transduced with GFP expressing lentiviral vector
(HR’GFP) and analyzed by flow cytometry at four days post-transduction. The lentivirus vector yielded
high gene transfer rates throughout the human tumor cell line panel, varying from 50 to 95% in the
different tumor types (I, Table 2). In contrast, a few cell lines were more resistant to lentivirus mediated
gene delivery e.g. the colon cancer cell line CaCo-2, where only ~9% of cells expressed the GFP transgene
after transduction. When the same cell lines were transduced with an equivalent amount of GFP expressing
adenoviral vector (AdGFP), similar transduction rates were obtained (I, Table 3). Our results indicate that
all of these tumor types were equally good targets for lentivirus-mediated gene delivery suggesting that the
transduction efficiency in each case is dependent on the characteristics of that particular transformed cell
line, not the tissue of origin of the tumor. Although the obtained transduction rates were comparable to
adenoviral gene transfer, a direct comparison between adenoviral- and lentiviral vectors should be avoided
for several reasons. First, titering methods for these two vector types are different. When compared to
original titers, both adenoviral- and lentiviral titers varied by almost one order of magnitude depending on
the cell line (I, Table 1). Thus it is very likely that the actual concentrations of biologically active viruses
may not be equal. Second, common additives (including polycation polybrene) used for lentiviral
transduction in vitro have also an effect on the gene transfer efficiency. It has been shown that different
polycations can improve also the adenoviral gene transfer in vitro (Arcasoy et al., 1997). Since adenovirus
transductions in vitro are routinely performed without polybrene, this factor would have to be taken into
account to obtain an appropriate comparison of the gene transfer rates. Nevertheless, these results
demonstrate that different tumor cell types are highly permissive for lentiviral vectors, indicating that
lentiviral vectors could potentially possess cancer gene therapy applications. Our findings are in parallel
with recent studies showing a high lentiviral gene transfer efficiency into various human cancer cells
including hepatoma, myeloma and bladder cancer cells (Gerolami et al., 2004; Uch et al., 2003; Kikuchi et
al., 2004).

43
5.1.1.1 Impact of accessory proteins

Colon, prostate, cervix and brain cancer cell lines were transduced with both first (carries all the accessory
proteins) and second (lacking nef, vif, vpr and vpu accessory proteins) generation lentiviral vector particles
expressing GFP (WOX-GFP) and analyzed with flow cytometry to determine the transduction efficiency.
The second generation viral vector yielded slightly higher gene transfer rates and transgene expression
levels when compared to first generation vector (I, Fig. 2A and B). Our results indicate that the HIV-1
accessory proteins nef, vif, vpr and vpu are neither necessary for successful lentiviral- mediated gene
delivery nor offer any additional benefit for transduction in vitro in human cancer cells. However, the
function of the accessory proteins needs to be studied and evaluated more thoroughly in vivo.

5.1.1.2 Impact of WPRE and cPPT

Lentiviral vectors with improved transduction efficiency based on additional genetic elements that modify
either the nuclear localization of the viral genome (cPPT) or the stabilization of the viral mRNA species
(PRE) have been described recently (Barry et al., 2001; Maurice et al., 2002; Mautino and Morgan, 2000;
Salmon et al., 2000). The presence of these elements has been reported to increase transduction efficiency
(Van Maele et al., 2003) and to enhance transgene expression (Zufferey et al., 1999). To study the impact
of these elements, colon, prostate, cervix and brain cancer cells were transduced with four different self
inactivating –type lentivirus vectors (HOX-GFP with no additional elements, HPT-GFP with cPPT, WOX-
GFP with WPRE and WPT-GFP with both cPPT and WPRE). Transduction efficiency (I, Fig. 3) and
transgene expression (I, Fig. 4) were determined by flow cytometry. An approximately 90% transduction
efficiency was detected with all the cancer cell lines except for the colon cancer line CaCo-2 which yielded
only 30-50% efficiency. These results indicate that neither cPPT nor WPRE had any major effect on
lentiviral transduction efficiency. Nevertheless, the expression level increased over time with vectors
carrying at least one genetic enhancing element, suggesting that cPPT and WPRE do possess some ability
to enhance transgene expression. The presence of cPPT clearly increased the expression level in colon and
cervical cancer cells, but the same phenomenon was not seen in prostate and brain cancer cells.
Furthermore, cPPT did not increase GFP expression level over time as was the case for WPRE and there
was no cooperative effect between cPPT and WPRE. Therefore, based on our observations, WPRE alone
appears to be sufficient to increase the transgene expression levels at least in most cancer cell lines.
Although our results suggest that a lentivirus vector with a SIN-backbone and WPRE would seem to be
optimal for cancer cell transduction purposes, the results may vary due to differences in the elements used
and their relative positions and/or used promoters and transgenes. It has been shown that lentivirus vector
carrying both cPPT and PRE from human hepatitis virus yielded 2-3 times more efficient gene transfer
rates when compared to vectors with only one of these elements (Barry et al., 2001). Furthermore, in
comparison to vectors carrying only one of the enhancing elements, transgene expression levels were
approximately 6 times higher when both elements were present.

44
5.1.2 Lentivirus vector -mediated TK/ganciclovir gene therapy in vitro

To test the capability of the first generation lentivirus vector expressing TK-GFP to elicit a therapeutic
response, both HR’TK-GFP and AdTK-GFP transduced U-2-OS osteosarcoma cells were exposed to GCV
for 5 days (I, Fig. 5). The results demonstrated that the cell killing curves were almost identical and both
vector types induced similar TK/GCV cytotoxicity. Thus, these data suggest that even the first generation
lentivirus vector without any additional genetic elements can provide sufficient transgene expression level
for successful suicide gene therapy in vitro. Similar results were also obtained in our previous study with
other human osteosarcoma and chondrosarcoma cell lines (Ketola et al., 2004), suggesting that this
phenomenon is not limited to one particular cell line.

5.1.3 Lentiviral tumor transduction in vivo

To test the feasibility of the lentiviral mediated gene transfer in vivo, relatively equal amounts of HPT-GFP
(4 x 106 t.u) and AdGFP (4 x 106 pfu) were injected intratumorally to subcutaneous A549 –tumors bearing
mice. Four days after viral injections, tumor cells were analyzed by flow cytometry to determine the
transduction rates in tumors. Lentiviral vector yielded 18.8 ± 15.9% (percentage of GFP positive tumor
cells ± S.D.) gene transfer efficiency, whereas the efficiency with the respective amount of adenovirus was
only 2.1 ± 1.2%. To ensure that the adenoviral vector was functional and provided adequate gene transfer
rates, approximately a thousand times higher adenoviral dose (5 x 109 pfu) was administered, resulting in
13.7 ± 8.8% gene transfer efficiency. These results from in vitro experiments confirm our belief that
lentiviruses are potential vectors for cancer gene therapy. This data clearly demonstrates that despite being
administered at titers several orders of magnitude lower than those routinely required for adenovirus
vectors, lentiviruses can be successfully used to transduce tumor tissue in vivo. In conjunction with the
results obtained from in vitro experiments, these data show that lentiviruses are potential vectors for cancer
gene therapy applications. Although polybrene had some influence on the gene transfer efficiency in vitro,
our results in vivo (no polybrene added to the lentivirus preparation) demonstrate that this polycation was
not responsible for the striking gene transfer efficiency of lentivirus vectors.

5.2 CD40 can be exploited for transductional targeting of adenovirus vectors (II)
Even though the biological role of CD40 in carcinoma cells is mostly unknown, activation of CD40 has
been shown to prevent neoplastic cell growth and can sensitize cancer cells to undergo apoptosis
(Eliopoulos et al., 2000; Ghamande et al., 2001; Tong et al., 2001). Additionally, some studies have
suggested that CD40 could be a potential marker for malignant tumors (Moghaddami et al., 2001).
Furthermore, there is increasing evidence suggesting that ovarian (Kim et al., 2002; You et al., 2001) and

45
other types of cancer cells (Hemminki and Alvarez, 2002) do not express the primary adenoviral receptor
CAR making these cells rather refractory to adenoviral mediated gene delivery. Thus, we wanted to study
CD40 expression in ovarian cancer cells and its utilization for adenoviral targeting.

5.2.1 CD40 expression in ovarian cancer cells

The CD40 expression was determined by RT-PCR and flow cytometry. RT-PCR was performed with total
RNA isolated both from a variety of ovarian carcinoma cell lines (II, Fig. 1A) and primary ovarian tumor
samples (II, Fig 1B), because it has been suggested that cells negative for CD40 expression in vivo could
revert to a positive CD40 phenotype when cultured in vitro (Young et al., 1998). All the examined ovarian
cancer cell lines and patient samples produced a 425 bp-sized amplification product specific to CD40
mRNA, indicative of CD40 expression. Furthermore, these results provide the first evidence of CD40
expression in clinical ovarian tumor specimens. To study the CD40 expression on the cell surface, ovarian
cancer cells stained with anti-CD40 antibody were analyzed by flow cytometry (II, Fig. 1C-H). The
examined ovarian carcinoma cell lines displayed variable degrees of CD40 expression. CD40 expression
levels were considered as high in Hey (II, Fig 1C) and OV-4 (II, Fig. 1E) cells (~60% of cells CD40
positive) and moderate or low in SKOV3.ip1 (II, Fig. 1D) and OV-3 (II, Fig. 1F) cells, respectively.

These results clearly demonstrate CD40 expression in ovarian cancer cell lines and tumor specimens. In
addition, the presence of CD40 on ovarian cancer cells means that ovarian cancer is a potential target for
CD40-ligand based immunotherapy, a technique which has been shown to induce apoptosis and suppress
tumor growth in preclinical studies (Tong et al., 2001; Kikuchi et al., 2000; Eliopoulos et al., 2000;
Ghamande et al., 2001).

5.2.2 Gene transfer with CD40 -targeted adenovirus

After confirming the CD40 expression on ovarian cancer cells, we investigated the potential of this
phenomenon for transductional adenoviral targeting. First, cells were infected with three doses of non-
targeted or CD40-targeted luciferase expressing adenovirus (II, Fig. 2). With the CD40 negative HeLa
cells, CD40 -targeting conferred no advantage in gene expression (II, Fig. 2F). In contrast, CD40-targeting
enhanced the gene transfer rates in all of the CD40 expressing cell lines (II, Fig. 2A-E). In comparison to
the non-targeted virus, higher gene transfer rates (from 4 to 42 times) were obtained when 100 pfu/cell of
CD40-targeted virus was used. Second, to ensure that the increased gene expression was due to the
retargeting moiety, the virus/targeting fusion protein -ratio was varied (II, Fig. 3). In CD40 negative cells,
increasing the amount of targeting protein did not have any effect on gene transfer (II, Fig. 3F) but in
CD40 expressing cells, increasing the amount of the targeting moiety resulted in a dose dependent increase
in gene transfer rates (II, Fig. 3A-E). With some cell lines, the highest amounts of fusion protein (100 and
150 ng) may have saturated the adenoviral knobs available for binding CAR, resulting in a plateauing of

46
the transgene expression. However, with cells expressing high levels of CD40, such as OV-4, the full
targeting potential was not reached. Third, to confirm that CD40 actually was mediating the binding of
CD40 –targeted virus and eliciting increased transgene expression, cellular CD40 receptors were blocked
with an anti-CD40 antibody prior to transduction (II, Fig. 4). This blocked binding of the CD40 -targeted
virus and returned luciferase expression back towards the levels achieved without targeting.

These results suggest that utilization of CD40 to increase transduction efficiency promoted enhanced
transgene expression. Since ovarian cancer cells (including the studied cell lines) typically express low
levels of CAR (You et al., 2001; Kanerva et al., 2002), a higher frequency of CD40 receptors allows
increased binding, entry and transgene expression. Further, utilization of tumor associated receptors e.g.
CD40, for transductional targeting could help to increase transduction of tumor tissues, resulting in
augmentation of the therapeutic efficacy of adenoviral cancer gene therapy (Krasnykh et al., 2000).
Moreover, this could lower the total dose required to achieve a therapeutic outcome and reduce
transduction in non-target tissues such as liver thus minimizing adverse side effects (Einfeld et al., 2001;
Printz et al., 2000). CD40-targeted adenoviruses have also been used in immunotherapy to convert
dendritic cells so that they are susceptible to low viral doses (Pereboev et al., 2002).

5.3 VP22 does not enhance suicide gene therapy as a part of triple fusion protein (III)
The utility of PTDs for cancer gene therapy has been extensively examined during the last few years.
Several studies have suggested that the putative intercellular trafficking property of VP22 can be used to
deliver coupled therapeutic proteins from one cell to another for an increased therapeutic effect (Cashman
et al., 2002; Dilber et al., 1999; Liu et al., 2001; Wills et al., 2001; Zavaglia et al., 2003; Zender et al.,
2002). On the other hand, there are some findings suggesting that VP22-mediated spreading is a merely
fixation related artifact thus making the status of PTDs in gene therapy rather controversial (Falnes et al.,
2001; Fang et al., 1998; Lundberg and Johansson, 2002; Lundberg et al., 2003). To address the question of
whether VP22 could be employed for protein transduction -assisted gene therapy, we created a novel triple
fusion protein VP22-TK-GFP and tested its functionality both in vitro and in vivo.

5.3.1 Expression of fusion proteins

Expression of the lentivirally delivered fusion proteins in ovarian cancer cells was detected by Western
blotting using anti-GFP antibody (III, Fig. 1B). All the proteins were expressed at detectable levels and
expression of the full length, ~120 kDa triple fusion protein VP22-TK-GFP was observed. Compared to
cells expressing GFP (~37 kDa), VP22-eGFP (~75 kDa) and TK-GFP (~80 kDa), the level of VP22-TK-
GFP protein was lower, which might be due to the large size of the triple fusion protein or could be related
to the detection technique. Nevertheless, when the cells were analyzed using flow cytometry, there were no

47
remarkable differences in the intensity of mean fluorescence between TK-GFP and VP22-TK-GFP,
indicating similar expression levels of these two fusion proteins.

5.3.2 Monitoring of VP22 -mediated intercellular trafficking

To demonstrate, whether VP22-TK-GFP could be traffic intercellularly, TK-GFP, VP22-eGFP and VP22-
TK-GFP expressing cells were analyzed both with fluorescence microscopy and flow cytometry. To study
the effect of various fixation methods, cells were either left untreated or fixed with PFA or methanol prior
to analysis. Even though various subcellular localizations of VP22-TK-GFP were detected with
fluorescence microscopy, no typical dual pattern of distribution or intercellular transport was observed
under any conditions (III, Fig. 2). Some spreading of the fluorescence was detected after methanol fixation
around bright, clearly fragmented VP22-eGPP expressing cells (III, Fig. 2A, lower right panel).
Nevertheless, the degree of spreading was considerably lower than that reported in earlier studies (Aints et
al., 1999; Wybranietz et al., 1999). The cells were also indirectly stained with anti-GFP antibody, but no
intercellular trafficking was detected, although there are several studies demonstrating that fixation and
antibody staining can facilitate the visualization of intercellular trafficking VP22 fusion proteins (Aints et
al., 1999; Brewis et al., 2000; Elliott and O'Hare, 1999; Wybranietz et al., 1999). In addition, when cells
were analyzed at different time points with flow cytometry, the amount of VP22-TK-GFP expressing cells
decreased gradually or remained constant, showing no signs of intercellular spreading (III, Fig. 3). This is
not likely to be due to the gene delivery method, since intercellular trafficking of VP22-linked proteins
expressed from lentiviral vectors have been demonstrated (Lai et al., 2000). These results are in agreement
with the earlier findings by Fang et al., who detected no intercellular transport even when the cells were
fixed and stained with antibody (Fang et al., 1998). Additionally, our data support the theory proposed by
Lundberg et al. According to their recent findings, VP22-mediated intercellular trafficking appears to be
simply a fixation-induced artifact (Lundberg et al., 2001; Lundberg et al., 2002; Lundberg et al., 2003).

5.3.3 VP22-TK-GFP mediated cell killing in vitro

Notwithstanding the lack of direct evidence for VP22-mediated spreading of the fusion proteins, the effect
of VP22 to TK/GCV -based cell killing was evaluated (III, Fig. 4). When BT4C rat glioma cell
populations containing 10% transduced cells were cultured in the presence of GCV (1 µg/ml), TK-GFP
killed only the predicted 10% of the cells, whereas VP22-TK-GFP destroyed approximately 55% of the
cell population. Moreover, when the proportion of transduced cells was increased to 20%, VP22-TK-GFP
caused almost complete cell killing (95%) while TK-GFP was able to destroy only 75% of the cells.
However, VP22-mediated increased sensitivity to GCV was not detected in other examined cell lines,
although their bystander effect, which could significantly enhance the cell killing and overrun the VP22 –
mediated enhancement, and response to TK/GCV therapy varied from poor to good. Interestingly, when

48
the cell proliferation rate of BT4C cells was slowed down, the VP22 –mediated enhancement of
ganciclovir sensitivity was abolished (III, Fig. 5). However, when other vigorously dividing cell lines were
tested, this phenomenon was not observed, suggesting that BT4C cells harbor some yet unidentified
feature(s) that enable this cell line to support VP22 –mediated enhancement.

5.3.4 Efficiency of VP22-TK-GFP in vivo

Finally, to investigate the function of VP22-TK-GFP in vivo in three dimensional tumor structures,
SKOV3.ip1 –tumors bearing mice received intratumorally AdTK-GFP or AdVP22-TK-GFP followed by
GCV treatment. Although both TK-GFP- and VP22-TK-GFP – vectors yielded comparable and relatively
high fusion protein expression levels, no VP22-mediated spreading was observed when cryo sections were
analyzed in the fluorescence microscope (III, Fig. 6A). In addition, similar gene transfer rates (~16% in
average) (III, Table 1) which were sufficient to elicit therapeutic responses (III, Fig. 6B) were achieved
with both vectors. In comparison to the control group, mice receiving AdTK-GFP/GCV or AdVP22-TK-
GFP/GCV had significantly (p< 0.01) smaller tumors. However, when compared to TK-GFP, VP22-TK-
GFP did not significantly reduce the tumor size (p>0.05). Our findings are similar to those obtained by
Falnes et al, who concluded that no additional cytotoxic effects could be observed with the VP22-fused
diphtheria toxin A-fragment (Falnes et al., 2001). Therefore, the exact mechanism and potential utility of
VP22-mediated protein transport still remains to be resolved.

5.4 Ad5-∆24-TK-GFP: a useful tool for evaluation of combined oncolytic virotherapy

and suicide gene therapy (IV)

Although CRAds have been demonstrated to destroy tumor cells via replication, clinical trials suggest that
the ability of early generation agents have at best a limited ability to completely eradicate advanced tumor
masses. One might be able to achieve an increased antitumoral effect by combining enhancing elements to
oncolytic agents. One promising approach is to enhance the oncolytic potency of CRAds by incorporating
therapeutic genes into their genome (Chen et al., 2004; Fuerer and Iggo, 2004; Wildner et al., 1999). A
∆24-based CRAd expressing the TK-GFP fusion protein was created (Ad5-∆24TK-GFP) to examine
whether TK/GCV system could enhance the oncolytic potency of a CRAd and if a fluorescent transgene
product could be utilized to visualize viral spreading in tumor cells,

5.4.1 Verification of viral replication and oncolysis

Ovarian cancer cells transduced with Ad5-∆24TK-GFP, AdTK-GFP or ∆24 were analyzed both with
crystal violet staining and with the MTT-assay at given time points (IV, Fig.2A and B). Ad5-∆24TK-GFP

49
killed eventually all of the examined tumor cells effectively, although oncolysis was slightly delayed when
compared to ∆24. This is presumably due to the activity of the CMV promoter and subsequent TK-GFP
production, which both might divert resources away from virus replication and oncolysis (Haviv et al.,
2002; Hemminki et al., 2003; Rivera et al., 2004). The rate of replication varied between cell lines, with
the slowest replication observed in Hey cells (effective killing evident 18 days post-infection) and the most
rapid replication in OV-3 cells (apparent cytotoxicity at 8 days post-infection). This difference might be
partially attributable to the variable expression levels of CAR in ovarian cancer cells (Kanerva et al., 2002).

5.4.2 Monitoring of viral spreading

To determine the proportion of GFP-expressing cells, AdTK-GFP and Ad5-∆24TK-GFP transduced

ovarian tumor cells were analyzed both with flow cytometry and fluorescence microscopy (IV, Fig. 3A and
B). When replicative virus was used, the amount of GFP-positive cells increased from 8% to 85%, from
3% to 38% and from 15% to 28% in OV-4, OV-3 and SKOV3.ip1 cells, respectively, whereas in Hey cells
the percentage of transduced cells remained nearly constant. When compared to AdTK-GFP, the increase
in the proportion of GFP positive cells was significant (p< 0.001 or p< 0.01) after 7 days in OV-3, OV-4
and SKOV3.ip1 cells, whereas in Hey cells there was no significant difference between those two viruses.

When transduced OV-4 cells were analyzed with fluorescence microscopy, the majority of Ad5-∆24TK-
GFP treated cells exhibited strong fluorescence after 9 days indicating spread of the replicative virus
throughout the cell layer. Thus, the efficacy of viral spreading closely mirrored the oncolytic capacity of
the virus i.e. the most efficient spreading occurred in those cell lines supporting rapid replication (OV-3
and OV-4). However, although oncolysis was effective in OV-3 cells, the proportion of GFP-expressing
cells increased only up to 40%. This may be due to the narrow window of GFP expression in CRAd
infected cells – cells will exhibit fluorescence only after completion of TK-GFP production but before lysis
of the cell.

5.4.3 Effect of TK/GCV system on oncolysis in vitro

To evaluate the effect of TK/GCV therapy on oncolytic potency of Ad5-∆24TK-GFP in vitro, transduced
cells were analyzed with a CGV-sensitivity assay (IV, Fig. 4). The most dramatic impact of the TK/GCV
system was observed in the SKOV3.ip1 cell line, where cell death was over 15 times more efficient when
compared to virus only treated cells. Even though the additive effect on oncolysis was lower in OV-4 and
Hey cells, enhanced cell killing by the TK/GCV system was evident. In contrast to other cell lines, GCV
inhibited cell killing in OV-3 cells. While replicative virus alone killed almost 50% of the cell population,
only 10% of GCV-treated OV-3 cells were destroyed by oncolysis. In addition, GCV treatment combined
with the replication deficient virus caused only modest cell death despite the adequate gene transfer rate,

50
suggesting that these cells exhibit a weak bystander effect. These findings support the theory that GCV
might inhibit viral replication especially in cells, which support rapid viral replication but lack efficient
gap-junctional communication (Post et al., 2003). Thus in those cells, the antiviral action of GCV
predominates over its anticancer action resulting in inhibited viral replication which can prevent viral
spreading in the tumor tissue (Post et al., 2003; Rogulski et al., 2000b). Further, the utility of oncolysis
combined with TK/GCV might be more pronounced in primary human tumors, where cells replicate much
slower than in murine xenografts, and therefore might be more resistant to TK/GCV.

5.4.4 Efficiency of Ad5-∆24TK-GFP in vivo

In order to evaluate the antineoplastic activity of a CRAd combined with TK/GCV therapy and
intratumoral penetration in vivo, viruses were injected intratumorally to subcutaneous SKOV3.ip1 tumors
followed by GCV treatment (IV, Fig. 5A). Both replicative viruses (Ad5-∆24TK-GFP and ∆24)
significantly reduced tumor growth in vivo when compared to the replication deficient AdTK-GFP (p<
0.001), indicating efficient replication and viral penetration into the tumor. Additionally, both viral
replication and efficient transgene expression from the CRAd was confirmed by analyzing AdTK-GFP and
Ad5-∆24TK-GFP treated tumor cells with flow cytometry showing that the proportion of GFP expressing
cells remained relatively constant (~35%) in Ad5-∆24TK-GFP treated animals (IV, Fig. 5B). Although
high transgene expression levels were achieved in vivo, GCV did not reduce the tumor size significantly in
the Ad5-∆24TK-GFP treated animals (p>0.05). These results are in parallel with earlier studies, where
GCV did not increase the antitumor efficiency of TK-expressing oncolytic viruses (Freytag et al., 1998;
Lambright et al., 2001; Morris and Wildner, 2000; Rogulski et al., 2000b; Wildner et al., 1999). On the
other hand, there are several papers showing that oncolytic potency and an antitumor effect can be
enhanced by incorporation of the TK/GCV system (Freytag et al., 2003; Nanda et al., 2001; Wildner et al.,
1999). There are several features that can modify the efficacy of TK/GCV system when it is incorporated
into CRAds. It has been suggested that some cancer cells may feature abundant gap junctions (Chipman et
al., 2003), which might enhance the cell killing by allowing GCV-triphosphate to penetrate into
neighboring cells that are resistant to virus replication. Also, since TK/GCV mediated cell killing is most
effective in cycling cells, cells allowing effective viral replication are locked into the S-phase by the viral
proteins, making these cells concomitantly resistant to the TK/GCV system. Therefore, the potential utility
of the combination of CRAd and TK/GCV system may be determined by the balance between the viral
replication and anticancer activity of GCV: in cells permissive for replication, TK/GCV might not be
needed and in cells sensitive to TK/GCV killing, virus replication might not provide any additional
benefits.

51
6 SUMMARY AND CONCLUSIONS

The principal aim of this study was to evaluate various approaches for enhancement of cancer gene
therapy. The following results were obtained.

I Since the majority of the lentiviral vector applications have focused on monogenic diseases and
lentiviruses have only occasionally been used in cancer gene therapy, we decided to study the feasibility of
using various lentiviral vectors against different cancer types. All 42 examined cancer cell lines were
moderate or good targets for lentiviral mediated gene delivery. Further, the presence of accessory proteins
had conferred no advantages on gene transfer rates, suggesting that safer second generation vectors should
be used for cancer gene therapy studies. The used enhancing elements (cPPT and WPRE) had an additive
influence on the level and duration of transgene expression. Lentiviral vectors were also able to achieve
adequate gene transfer rates to elicit a therapeutic response in vitro. In addition, therapeutically relevant
gene transfer rates were obtained in vivo. Thus, our data demonstrate that lentivirus vectors are truly
potential gene transfer vehicles for different cancer types, especially when more efficient production
systems are developed to obtain higher viral titers.

II To enhance and increase the specificity of the adenoviral gene transfer, expression of CD40 was
studied in the context of ovarian cancer and utilized for transcriptional adenoviral targeting. CD40 was
shown to be expressed in ovarian cancer cell lines and for the first time also in clinical primary tumor
samples. This phenomenon was successfully utilized for adenoviral targeting. In comparison to untargeted
adenovirus, increased transduction rates were observed when CD40 -targeted adenovirus was used.
Moreover, we showed that the CD40 -targeting moiety resulted in a dose-dependent increase in gene
transfer rates and the augmentation of viral entry was due to the binding to the CD40 receptor. Therefore,
targeting to tumor-associated receptors such as CD40 could help to improve the efficacy of adenoviral-
mediated gene delivery and thus circumvent the problem caused by low CAR expression levels in many
tumor cells. Simultaneously, targeting to tissue specific receptors may be one way to reduce the adverse
side effects related to unspecific gene transfer. Moreover, CD40-targeted adenoviruses could also been
exploited in immunotherapy e.g. to make dendritic cells more sensitive to lower viral doses.

III To study the utility of VP22-mediated intercellular trafficking to enhance suicide gene therapy, a
novel triple fusion protein VP22-TK-GFP was constructed and characterized. Based on our data, the full-
length triple fusion protein was expressed both in vitro and in vivo. Although several detection methods
were used, no VP22-mediated intercellular trafficking could be observed. Thus, our results support the
earlier findings proposing that VP22-mediated intercellular trafficking may be only a fixation-induced
artifact In addition, VP22-mediated enhancement in TK/GCV cytotoxicity was shown not to be a
ubiquitous phenomenon; VP22-TK-GFP caused increased cell death only in one cell line when compared

52
to TK-GFP. This phenomenon was shown not to be related to a bystander effect or to the cell proliferation
rate. Although VP22-TK-GFP retained its thymidine kinase activity in vivo and significantly reduced
tumor growth, we did not observe any VP22-mediated spreading or any significantly increased anti-
tumoral efficiency when compared to TK-GFP. Therefore, the potential utility of VP22-mediated protein
transport for gene therapy purposes still remains rather dubious and it is questionable if PTDs offer any real
benefit in cancer gene therapy.

IV To evaluate the viral spreading and the potency of oncolytic virotherapy combined with suicide
gene/prodrug therapy, a TK-GFP encoding CRAd was constructed (Ad5-∆24TK-GFP). Efficient
replication and spreading of the Ad5-∆24TK-GFP in ovarian tumor cells was demonstrated both in vitro
and in vivo. Further, TK/GCV system was shown to enhance the oncolysis in vitro. However, under some
conditions GCV seemed to exhibit antiviral activity, inhibiting viral replication and oncolysis. Although an
efficacious therapeutic outcome was achieved for viral replication in vivo, no enhancing effects attributable
to the suicide gene/prodrug system were observed. Thus, to obtain maximum benefit with CRAds
combined to TK/GCV system, the balance between antiviral and antitumoral effect of GCV must be
determined. However, this can be viewed in a positive way- GCV mediated inhibition of oncolysis could
be utilized as a fail-safe mechanism to achieve virus inactivation and abolishment of virus-harboring cells
in case of replication associated toxicity.

53
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