KUOPION YLIOPISTON JULKAISUJA G. - A.I. VIRTANEN -INSTITUUTTI 54

KUOPION YLIOPISTON JULKAISUJA G. - A.I. VIRTANEN -INSTITUUTTI 54
KUOPIO UNIVERSITY PUBLICATIONS G.
A.I. VIRTANEN INSTITUTE FOR MOLECULAR SCIENCES 54
OUTI RAUTSI
Hurdles and Improvements in
Therapeutic Gene Transfer for Cancer
Doctoral dissertation
To be presented by permission of the Faculty of Natural and Environmental Sciences of
the University of Kuopio for public examination in Mediteknia Auditorium,
Mediteknia building, University of Kuopio,
on Saturday 6 th October 2007, at 12 noon
Department of Biotechnology and Molecular Medicine
A.I. Virtanen Institute for Molecular Sciences
University of Kuopio
JOKA
KUOPIO 2007
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Series Editors:
Research Director Olli Gröhn, Ph.D.
Department of Neurobiology
A.I. Virtanen Institute for Molecular Sciences
Research Director Michael Courtney, Ph.D.
Department of Neurobiology
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
FI-70211 KUOPIO
FINLAND
Tel. +358 17 163 790
E-mail: [email protected]
Supervisors:
Docent Jarmo Wahlfors, Ph.D.
Department of Biotechnology and Molecular Medicine
A. I. Virtanen Institute for Molecular Sciences
University of Kuopio
Riikka Pellinen, Ph.D.
Department of Biotechnology and Molecular Medicine
A. I. Virtanen Institute for Molecular Sciences
University of Kuopio
Reviewers:
Docent Mikko Savontaus, M.D., Ph.D.
Turku Centre for Biotechnology
Department of Medicine
University of Turku
Docent Mika Rämet, M.D., Ph.D.
Institute of Medical Technology
University of Tampere
Opponent:
Professor Kalle Saksela, M.D., Ph.D.
Department of Virology
Haartman Institute
University of Helsinki
ISBN 978-951-27-0613-6
ISBN 978-951-27-0435-4 (PDF)
ISSN 1458-7335
Kopijyvä
Kuopio 2007
Finland
Rautsi, Outi. Hurdles and Improvements in Therapeutic Gene Transfer for Cancer. Kuopio University
Publications G. - A.I. Virtanen Institute for Molecular Sciences 54. 2007. 79 p.
ISBN 978-951-27-0613-6
ISBN 978-951-27-0435-4 (PDF)
ISSN 1458-7335
ABS TRACT
Over the past decades, gene therapy has emerged as representing a potential treatment modality for
cancer. One promising cancer gene therapy approach is based on introducing a gene that encodes for an
enzyme called a suicide protein into tumor. This enzyme converts a normally harmless prodrug into a
toxic form that induces tumor cell death. Cell killing is observed also in the surrounding, non-transduced
cells, which is a benefit since all tumor cells do not need contain the therapeutic gene. This phenomenon
is called the bystander effect. Nevertheless, true success in clinical trials has not been achieved mainly
due to insufficient gene delivery rate of the current vectors and inadequate bystander effect in many
tumors. In the present study we evaluated various methods to overcome these problems; first by
characterizing factors that may influence efficient therapeutic gene transfer and further by modifying
viral vectors and the therapeutic gene with the aid of cell penetrating peptides.
A number of factors, including host cell immune responses, can influence the gene transfer efficiency of
viral and non-viral vectors. For that reason, we studied the contribution of the type I interferon response,
an arm of innate immune system, to the therapeutic gene transfer. The commonly used viral vectors, with
the exception of Semliki Forest virus, succeeded in avoiding the induction of the type I IFN response.
However, the delivery of plasmid DNA and particularly most forms of RNA triggered the response in a
variety of studied cell lines. In order to improve delivery of therapeutic gene into the tumor cells, we
evaluated the feasibility of using cell penetrating peptides derived from Drosophila Antennapedia
homeodomain and HIV-1 transactivator protein (TAT). These cationic peptides enhanced transduction
efficiency of adeno- and lentiviral vectors significantly in most of the tested human tumor cells.
However, the property of a commonly used commercial transduction enhancer was found to be even
better at boosting efficacy than the cell penetrating peptides. In another study included in this thesis, the
cell penetrating peptide TAT was linked to suicide-marker fusion gene (TAT-TK-GFP and TK-GFP) to
extend the cytotoxic impact of suicide gene therapy to adjacent cells and thus to compensate for the poor
gene delivery rate. Against our original hypothesis, we found that the TAT containing fusion proteins
were not trafficking between the cells. Despite the lack of intercellular movement, TAT-mediated
increased cell killing was observed in some of the tested human tumor cell lines. However, in many cell
lines the killing efficiencies of TAT-TK-GFP and TK-GFP were similar and in some cell lines the
efficiency of TK-GFP was even better.
In conclusion, these results indicate that the gene delivery can induce undesired immune responses in
target cells and thus may represent a barrier against efficient therapeutic gene transfer. Although cell
penetrating peptides improved the viral transduction rate, the utility of these peptides as general
enhancers will most likely be limited by their high manufacturing costs compared to commercially
available and clinically approved compound. Even though TAT -containing suicide fusion protein
showed some enhancement of cell killing in certain tumor cell lines, no overall difference in efficacy
between TAT-TK-GFP and TK-GFP was seen. Therefore, this concept needs to be further refined if it is
to be considered as a potential supplement for cancer suicide gene therapy.
National Library of Medicine Classification: QZ 52, QZ 266, QU 470, QU 475, QU 68
Medical Subject Headings: Neoplasms/therapy; Gene Therapy; Gene Transfer Techniques; Transduction,
Genetic; Genetic Vectors; Viruses; Cell Death; Bystander Effect; Immunity; Interferon Type I;
Drosophila Proteins; Antennapedia Homeodomain Protein; Gene Products, tat; Peptide Fragments;
Transcription Factors; Genes, Transgenic, Suicide; Thymidine Kinase; Ganciclovir
ACKNOWLEDGEMENTS
This study was carried out in the Department of Biotechnology and Molecular Medicine, at A.I.
Virtanen Institute for Molecular Sciences, University of Kuopio, during the years 2002-2007.
I express my sincere thanks to my supervisors Docent Jarmo Wahlfors, PhD, and Riikka
Pellinen, PhD. Jarmo, I thank you so much for the opportunity to join the Gene Transfer
Technology Group and to learn so much science from you. You have always been such a kind,
understanding and encouraging groupleader. Riikka, particularly during last year, I cannot thank
you enough for all the support, encouragement and as well as patience that you have given to
me. Once again, I thank you from the bottom of by heart!
I wish to thank the official reviewers Docent Mikko Savontaus, MD, PhD, and Docent Mika
Rämet, MD, PhD, for their valuable comments and constructive criticism in improving this
thesis. I also want to thank Ewen MacDonald, PhD, for the linguistic revision of this thesis.
It has been a great pleasure to work with all the past and current, super people in the Gene
Transfer Technology Group. Particularly I wish to thank my close colleagues and friends: Ann
Marie Määttä, PhD, for all the scientific, and particularly non-scientific, actitivities and
discussions that we have had, Tiina Wahlfors, PhD, and ex-room mate Tanja Hakkarainen, PhD,
for teaching me basic laboratory skills and my room mate Anna Ketola, MD for the
brainstorming sessions. Tuula Salonen, Saara Lehmusvaara, MSc and Katja Häkkinen BSc, I
indeed appreciate for your friendship and all the help you have given. I also want to thank
Marko Björn BSc, Anna Laitinen, MSc, Päivi Sutinen, MSc, Agnieszka Pacholska, BSc, AnnaKaisa Hytönen, MSc, and Anita Lampinen, BSc.
I am grateful to Professor Juhani Jänne, MD, PhD and Professor Leena Alhonen, PhD, for their
valuable support and advice. I also want to thank all the wonderful and always so helpful
persons of the JJ/LA-group, not forgetting enjoyable moments in the coffee room. Special thanks
to Maija Tusa, MSc, for teaching me numerous useful tips concerning word processing, Riitta
Sinervirta for helping with a multitude of practical problems, Arja Korhonen for sequencing,
Anne Karppinen for synthetizing the oligonucleotides and Sisko Juutinen for teaching me the
basics of histology.
I also wish to thank all the helpful people at AIVI, especially Pekka Alakuijala, Phil Lic, and
Jouko Mäkäräinen, who have helped me on countless occasions, Helena Pernu and Riitta
Laitinen for secretarial assistance and coordinator of the AIVI graduate school Docent Riitta
Keinänen, PhD, for all the good advice.
I wish to express my warmest thanks to all my friends for their friendship and all the great times!
Especially Katri, thank you for the long, enlightening, therapeutic phone calls no matter what
was the matter.
I wish truly to express my gratitude to all co-authors; my colleagues from the Gene Transfer
Technology Group and collaborators for their important contribution to this thesis. Once again,
thank you all!
I am deeply grateful my parents as well as my siblings and all the closest persons like
"Lampelan väki" and my mother-in-law for their love, support and help within my whole life as
well as during this thesis.
Finally, my deepest gratitude belongs to my loving husband Tomi and to our daughter Sanni for
the love, joy and significance that you have brought to my everyday life and also for the support
during this project. Simply, words cannot express my gratitude and appreciation for you.
This study was supported by the Graduate School of the Ministry of Education, the Graduate
School of Molecular Medicine of A.I. Virtanen Institute, the National Technology Agency of
Finland (TEKES), the Kuopio University Foundation, the North Savo Cancer Foundation and
the Finnish Cultural Foundation of Northern Savo.
Kuopio, September 2007
Outi Rautsi
ABBREVIATIONS
AAV
adeno-associated virus
Antp
Antennapedia
CAR
coxsackie- and adenovirus receptor
CpG
cytosine and guanine separated by a phosphate
CPP
cell penetrating peptide
CTL
cytotoxic T-cell
CXCR4
CXC chemokine receptor 4
DC
dendritic cell
DFMO
-difluoromethylornithine
dsRNA
double stranded RNA
GCV
ganciclovir
GFP
green fluorescent protein
HIV
human immunodeficiency virus
HSPG
heparan sulphate proteoglycan
HSV-TK
herpes simplex virus thymidine kinase
IFN
interferon
IL
interleukin
IRF
interferon regulatory factor
ISG
interferon stimulated gene
ISGF
interferon stimulated gene factor
ISRE
interferon stimulated response element
Jak
Janus kinase
LTR
long terminal repeat
MDA-5
melanoma differentiation associated gene-5
MHC
major histocompatibility complex
MOI
multiplicity of infection
mRNA
messenger RNA
MxA
myxovirus resistance protein A
NK cell
natural killer cell
OAS
2´-5´ oligoadenylate synthetase
ODD
oxygen dependent degradation
PAMP
pathogen associated molecular pattern
PCR
polymerase chain reaction
pDC
plasmacytoid dendritic cell
pDNA
plasmid DNA
PFA
paraformaldehyde
PKR
double stranded RNA dependent protein kinase R
PRR
pattern recognition receptor
PTD
protein transduction domain
qPCR
quantitative polymerase chain reaction
RIG-1
retinoid acid inducible gene-1
RNAi
RNA interference
SFV
Semliki Forest virus
shRNA
short hairpin RNA
SIN
self-inactivating lentiviral vector
siRNA
short interfering RNA
Smac
second mitochondria-derived activator of caspase
ssRNA
single stranded RNA
STAT
signal transducer and activator of transcription
TAR RNA
trans-activation responsive RNA element
TAT
trans-activator of transcription
TAT PTD
protein transduction domain of trans-activator protein
TNF
tumor necrosis factor
TLR
Toll-like receptor
TRAIL
tumor necrosis factor-related apoptosis inducing ligand
Tyk
tyrosine kinase
VP22
tegument protein from herpes simplex virus
VSV-G
vesicular stomatitis virus glycoprotein
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following publications, which are referred to by their corresponding
Roman numerals:
I
Rautsi O, Lehmusvaara S, Salonen T, Häkkinen K, Sillanpää M, Hakkarainen T, Heikkinen
S, Vähäkangas E, Ylä-Herttuala S, Hinkkanen A, Julkunen I, Wahlfors J and Pellinen R
(2007) Type I interferon response against viral and non-viral gene transfer in human tumor
and primary cell lines. J Gene Med. 9: 122-135
II
Lehmusvaara S, Rautsi O, Hakkarainen T and Wahlfors J (2006) Utility of cell-permeable
peptides for enhancement of virus-mediated gene transfer to human tumor cells.
BioTechniques. 40: 573-574, 576
III Meriläinen O, Hakkarainen T, Wahlfors T, Pellinen R and Wahlfors J (2005) HIV-1 TAT
protein transduction domain mediates enhancement of enzyme prodrug cancer gene
therapy in vitro: a study with TAT-TK-GFP triple fusion construct. Int J Oncol. 27: 203208
IV Rautsi O, Lehmusvaara S, Ketola A, Määttä A-M, Wahlfors J and Pellinen R (2007)
Characterization of HIV-1 TAT-peptide as an enhancer of HSV-TK/GCV cancer gene
therapy. Submitted.
TABLE OF CONTENTS
1
INTRODUCTION ...................................................................................................................................... 13
2
REVIEW OF THE LITERATURE............................................................................................................ 15
2.1 CANCER GENE THERAPY............................................................................................................. 15
2.1.1 OVERVIEW ............................................................................................................................ 15
2.1.2 GENE DELIVERY TOOLS ..................................................................................................... 16
2.1.3 CANCER GENE THERAPY APPROACHES .......................................................................... 19
2.1.4 HSV-TK/GCV SUICIDE GENE THERAPY ............................................................................ 21
2.1.4.1 BYSTANDER EFFECT ............................................................................................. 22
2.1.4.2 STRATEGIES TO IMPROVE HSV-TK/GCV TREATMENT.................................... 24
2.2 TYPE I INTERFERON RESPONSE AGAINST THERAPETUIC GENE TRANSFER ................ 26
2.2.1 OVERVIEW ............................................................................................................................ 26
2.2.2 TYPE I INTERFERONS .......................................................................................................... 26
2.2.3 CHALLENGES CAUSED BY THE TYPE I IFN RESPONSE AGAINST
THERAPEUTIC GENE TRANSFER ....................................................................................... 30
2.2.4 MECHANISMS TO AVOID INDUCTION OF TYPE I IFN RESPONSE................................. 33
2.3 UTILITY OF HIV-1 TAT PROTEIN TRANSDUCTION DOMAIN IN CANCER GENE
THERAPY ......................................................................................................................................... 36
2.3.1 OVERVIEW ............................................................................................................................ 36
2.3.2 HIV-1 TAT PROTEIN TRANSDUCTION DOMAIN .............................................................. 36
2.3.3 MECHANISM OF TAT PTD INTERNALIZATION ................................................................ 38
2.3.4 ANTICANCER APPROACHES UTILIZING TAT PTD .......................................................... 39
2.3.5 MOVEMENT OF TAT-FUSION PROTEINS BETWEEN CELLS ........................................... 40
2.3.6 FUTURE CONSIDERATIONS OF TAT-MEDIATED DELIVERY ......................................... 42
3
AIMS OF THE STUDY.............................................................................................................................. 44
4
MATERIALS AND METHODS ................................................................................................................ 45
5
RESULTS AND DISCUSSION .................................................................................................................. 49
5.1 TYPE I INTERFERON RESPONSE AGAINST VIRAL AND NON-VIRAL GENE
TRANSFER IN HUMAN TUMOR CELL LINES AND PRIMARY CELLS (I)............................. 49
5.1.1 COMMONLY USED VIRAL VECTORS EVADE THE TYPE I IFN RESPONSE................... 49
5.1.2 VARIOUS pDNA DELIVERY METHODS INDUCE THE TYPE I IFN RESPONSE .............. 52
5.1.3 ALL TYPES OF RNA, EXCLUDING siRNA, TURN ON THE TYPE I IFN
PRODUCTION ........................................................................................................................ 53
5.1.4 TYPE I INTERFERON RESPONSE IN HUMAN PRIMARY CELLS...................................... 56
5.2 CATIONIC CELL-PERMEABLE PEPTIDES ENHANCE TRANSDUCTION
EFFICIENCY OF VIRAL VECTORS IN HUMAN TUMOR CELL LINES (II)............................ 56
5.3 UTILITY OF TAT-TK-GFP TRIPLE FUSION PROTEIN IN HSV-TK/GCV BASED
SUICIDE GENE THERAPY (III, IV) ............................................................................................... 59
5.3.1 EXPRESSION OF TAT-TK-GFP TRIPLE FUSION PROTEIN................................................ 59
5.3.2 TAT-TK-GFP DOES NOT SUPPORT INTERCELLULAR TRAFFICKING............................ 61
5.3.3 COMPARISON OF CELL KILLING EFFICIENCIES OF TAT-TK-GFP AND TK-GFP
IN HUMAN TUMOR CELL LINES......................................................................................... 62
5.3.4 FEATURES INVOLVED IN THE TAT-MEDIATED INCREASED CELL KILLING ............. 64
6
SUMMARY AND CONCLUSIONS........................................................................................................... 67
7
REFERENCES ........................................................................................................................................... 69
APPENDIX: ORIGINAL PUBLICATIONS I-IV
1 INTRODUCTION
Cancer is a very complex disease results from of genetic alterations and failure of DNA repair
mechanisms and/or the immune system to correct the defects and eliminate the transformed
cells. It is well established fact that several environmental factors can induce genetic mutations,
but also inherited factors can increase individual´s susceptibility to cancer. During the past
decades, the biotechnological revolution has provided a broad range of new tools to identify
these malfunctioning genes responsible for malignant cell growth and to characterize their role
during carcinogenesis. As of today, approximately 300 genes have been recognized to be
associated with cancer and mutations in these genes have been detected in somatic- or germline
cells or in both cell types (Futreal et al., 2004). The two well known types of genes associated
with cancer are proto-oncogenes and tumor suppressor genes, both of which are essential
components in regulating cell cycle homeostasis under normal circumstances. However, failure
in the function of these genes can lead to uncontrolled progression of the cell cycle and the
ability to avoid programmed cell death, respectively (Hanahan and Weinberg, 2000). In addition
to the disorders in the genome, epigenetic changes play a substantial role in the progression of
carcinogenesis. These epigenetic alterations are mainly associated with gene expression
controlled by histone modifications or DNA methylation, which are crucial for the development
and differentiation processes in general (Jones and Baylin, 2007). Abnormal functions, however,
can lead to altered gene expression e.g. of oncogenes or tumor suppressor genes. Transformation
from a normal cell to a malignant cell is rarely a consequence of a single mutation, but usually
several genetic alterations are required for the development of cancer and further changes are
emerging constantly during tumorigenesis. Consequently, the cell loses the capability to respond
to growth inhibitory signals, but is able to produce the growth signal by itself to maintain its
replication. Further phenotypical changes that are developed during carcinogenesis are the
ability to sustain angiogenesis, the capability to invade tissues and the tendency to metastasize
(Hanahan and Weinberg, 2000).
Every phenotypical alteration, however, offers a target to to prevent cancer progression. The
more detailed understanding of molecular mechanisms behind carcinogenesis has consequently
offered novel strategies to treat malignant diseases. Nowadays, based on the knowledge of gene
diagnosis and abnormal gene expression patterns, several clinically approved and specific anticancer drugs have been developed (such as the monoclonal antibodies, e.g. trastuzumab for
metastatic breast cancer and different types of inhibitors, e.g. imatinib for certain types of
13
leukemia) (Collins and Workman, 2006). There have been advances not only cancer therapies,
but also in the diagnostic methods along with the discovery of novel cancer biomarkers. Though
not yet a reality, one of the future diagnostic techniques could possibly be based on expression
analysis of tumor cells, which would allow customized therapy based on the genetic disorder
present in the individual's genome (Hemminki, 2002).
Despite the remarkable advances in cancer diagnostics and therapeutics, no curative treatment
for most advanced malignant diseases has been developed yet. Even though the efficacy and
accuracy of traditional treatment forms, including chemo- and radiotherapy have improved
significantly over the past years, they have a number of side effects and the risk of developing
other cancerous diseases still remains. Therefore, also completely new therapies along with the
current treatment forms are required in the battle against cancer. In addition to other options, the
delivery of genetic material may provide novel solutions. Several gene therapy approaches for
cancer, such as the activation of the anticancer immune response, corrective gene therapy and
suicide gene strategies have been introduced. However, one common factor for the lack of
clinical success of these methods has thus far been insufficient delivery and expression of the
therapeutic gene. In the present study, we focused on this problem by characterizing whether the
type I interferon response might have influence on efficient therapeutic gene transfer.
Furthermore, we evaluated mechanisms to improve the expression of the therapeutic gene by
modifying viral vectors and therapeutic gene products with cell penetrating peptides derived
from human immunodeficiency virus type 1 (HIV-1) transactivator protein (TAT) and
Drosophila Antennapedia (Antp) homeodomain.
14
2 REVIEW OF THE LITERATURE
2.1
2.1.1
CANCER GENE THERAPY
OVERVIEW
The objective of human gene therapy is to introduce genetic material into somatic cells in order
to achieve a therapeutic effect. Originally gene therapy was designed to treat monogenic
inherited diseases by correcting the defective gene function by delivering the corrected genes
into the cells. However, at present it is well established that in addition to monogenic disorders,
several acquired diseases, including neurological-, cardiovascular-, infectious- and particularly
malignant diseases, are relevant candidates to be treated by means of gene therapy (Morgan and
Anderson, 1993). The first gene therapy clinical trial for malignant disease was conducted in the
1990s, when tumor-infiltrating lymphocytes were isolated from patients, transduced with a
marker gene- carrying retroviral vector ex vivo and returned to the patient (Rosenberg et al.,
1990). Currently, there have been or are ongoing 1260 approved clinical gene therapy trials
worldwide, the vast majority (97%) of which are phase I, I/II and II trials. Of these clinical
studies,
over
70%
are
intended
for
the
treatment
of
cancer
(January
2007,
http://www.wiley.co.uk/genetherapy/clinical/). Effective treatment for many types of cancer
exists, but one problem frequently encountered is that when cancer is diagnosed, it has already
metastasized and the treatment options are therefore limited. A number of efforts have been
directed to develop various cancer gene therapy strategies during the past years and the near
future will reveal their true potential. In 2003, the first gene medicine, Gendicine, was approved
fo sale on the market in China. A recombinant adenovirus carrying the p53-gene was approved
by State Food and Drug Administration of China for the treatment of head and neck squamous
carcinoma (Peng, 2005). However, outside China, there was a slight concern that more data
should have been collected before permitting Gendicine on the market. Thus, some researchers
claimed that the approval of Gendicine was more evidence of China´s permissive regulatory
system than a demonstration of the efficacy of the gene medicine itself (Jia, 2006).
Despite the high level of enthusiasm for cancer gene therapy, there is still much to be learned.
The delivery of genetic material has turned out to be more difficult than predicted and
particularly for that reason, many cancer gene therapy approaches have been found lacking in
clinical studies. Furthermore, a number of studies have shown efficacy, but in many cases, no
target cell specificity has been achieved (Palmer et al., 2006). To resolve these problems, the
15
current research in cancer gene therapy field is mainly focused on developing gene delivery
systems and therapeutic genes by improving the existing tools instead of creating new vectors
and cancer gene therapy strategies. On the other hand, now that a lot of data has been generated
in pre-clinical studies, one major issue is how to apply these strategies into clinical trials to
prove their real efficacy and safety (Gottesman, 2003). However, none of the cancer gene
therapy approaches may represent be the ultimate solution in curing malignant diseases as such,
but rather these concepts will be utilized in combination with conventional therapies (Vile et al.,
2000).
2.1.2
GENE DELIVERY TOOLS
Genetic material, nucleic acids DNA or RNA, are readily degraded by nucleases and
furthermore, this kind of material permeates the cell membrane very inefficiently mainly due to
its large size and highly negative net charge. Thus, nucleic acids usually need a carrier in order
to be delivered inside the target cell. A high number of cells expressing the therapeutic protein is
essential for most cancer gene therapy approaches and therefore, much research effort has been
focused on developing gene delivery methods. The crucial parameters for therapeutic gene
transfer are, in addition to number of cells expressing therapeutic protein, the level and duration
of gene expression. The therapeutic gene expression should be strictly regulated to take place
only in the target cells, leaving other cells intact without inducing toxic or unwelcome
immunological responses. Moreover, manufacturing of high titer viral preparations is required
for clinical applications (Pfeifer and Verma, 2001). Gene transfer methods can be distinguished
into two major gropus; viral and non-viral vectors. The majority of the clinical cancer gene
therapy studies are conducted using viral vectors due to their higher gene transfer efficiency and
transgene expression level compared to non-viral gene delivery systems. Furthermore, sustained
expression of therapeutic gene expression can be achieved with certain viral vectors, an
advantage over the current non-viral vectors. The benefits of non-viral gene delivery methods
compared to viral vectors are often related to safety aspects, such as the lack of risk for wildtype virus generation or insertional mutagenesis. In addition, the large-scale production of nonviral vectors and their use is considered easier than with the viral vectors (Niidome and Huang,
2002; Ohlfest et al., 2005). A summary of commonly used gene delivery methods and their
general characteristics is shown in Table 1. However, the advantages and disadvantages of
different vectors depend on the approach being taken to treat the cancer.
16
Non-viral gene delivery methods can be subdivided into two main groups; physical and chemical
strategies. Physical methods, such as direct injection of naked nucleic acid, particle
bombardment (gene gun) and electroporation, have been demonstrated to be useful, for example
in treating melanoma by means of immunotherapy (Lucas et al., 2002; Heinzerling et al., 2005;
Cassaday et al., 2007). Liposomes complexed with nucleic acids, an example of a chemical
transfection method, have shown some promise e.g. in the delivery of an immunostimulatory
protein or a cytokine for the treatment of melanoma and malignant glioma, respectively (Stopeck
et al., 2001; Yoshida et al., 2004).
Table 1. Commonly used gene transfer vectors in cancer gene therapy.
VECTOR
ADVANTAGES
DISADVANTAGES
RETROVIRUS
(ssRNA virus)
Low immunogenicity
Long term gene expression
Transgene capacity ~8 kb
Relatively low vector titers
Transduces only dividing cells
Possibility of insertional mutagenesis
ADENOVIRUS
(dsDNA virus)
Very high titers
Transduction of both dividing and
non-dividing cells
High level transgene expression
Transgene capacity ~8-10 kb
Immunogenicity
Cytotoxicity
Many people have pre-existing
neutralizing antibodies
Transient gene expression
LENTIVIRUS
(ssRNA virus)
Transduction of both dividing and
non-dividing cells
Long-term gene expression
Low immunogenity
Transgene capacity ~8 kb
Possibility of wild type virus
formation (HIV-1)
Possibility of insertional mutagenesis
ADENOASSOCIATED
VIRUS
(ssDNA virus)
Low immunogenity
Long term gene expression
Transduction of dividing and nondividing cells
Difficult to produce helper virus free
high titer AAV vector
Low transgene capacity; up to 5 kb
Requires helper virus for production
HERPES
SIMPLEX TYPE 1
-VIRUS
(dsDNA virus)
Large transgene capacity; up to 30 kb
Neurotropism
Long-term transgene expression
possible in neurons
Immunogenicity
Cytotoxiciy
Usually transient transgene
expression
pDNA alone or
complexed with
e.g. cationic
liposomes
Unlimited transgene capacity
No risk for wild type- or replication
competent virus formation
Easy to produce and use
Low gene transfer efficiency in vivo
Short term gene expression
The basic idea of constructing viral vectors for gene therapy is to remove viral genes in order to
prevent the appearance of viral pathogenic features and virus replication, but to leave intact
those sequences that are essential for viral vector production and transduction. The deleted
17
sequences can be then replaced by a therapeutic gene (Thomas et al., 2003). Today, two the most
popular viral vectors in clinical cancer gene therapy trials have been adeno- and retroviral
vectors, the latter comprising almost entirely of Moloney murine leukemia virus –based vectors
(January 2007, http://www.wiley.co.uk/genetherapy/clinical/). The characteristics of these two
viral vectors differ fundamentally from each other; adenovirus based vectors transduce both
quiescent and dividing cells and express the transgene at a high level, but the expression is
transient, whereas retroviruses are able to integrate into the host cell genome and thereby the
gene expression can be stable (Vile and Russell, 1994). Integration, however, may represent a
safety risk due to random retroviral vector integration. The first report from clinical trials that
the insertion of the retroviral genome can indeed activate oncogenes was reported in 2003
(Hacein-Bey-Abina et al., 2003). Production of the high-titer viral vector preparations is attained
easily with adenoviral vectors. However, adenovirus based vectors tend to be highly
immunogenic and to some extent also toxic, whereas one beneficial feature of retroviral vectors
is their low toxicity (Rainov and Ren, 2003; Kaplan, 2005).
Lentiviruses belong to the retroviridae family, but lentivirus derived vectors are able to
transduce also quiescent cells (Naldini et al., 1996), whereas retroviral vectors only transduce
low levels of non-dividing cells (Miller et al., 1990). The vast majority of lentiviral vectors are
derived from the human immunodeficiency virus 1 (HIV-1), although HIV-2 based and certain
other primate as well as non-primate immunodeficiency virus -based vectors have been
developed (Federico, 1999). Lentiviruses possess several properties that render them ideal tools
for gene delivery: relatively large transgene capacity, low toxicity and immunogenicity as well
as long-term transgene expression (Thomas et al., 2003). HIV-1 envelope protein is most
commonly substituted (through a procedure called pseudotyping) with vesicular stomatitis virus
glycoprotein (VSV-G), which allows transduction of a wide range of different cell types
(Naldini et al., 1996). Thus far, the utility of lentiviral vectors has not been studied as
extensively in the field of cancer gene therapy as some other viral vectors, but they have
demonstrated efficiency in numerous pre-clinical studies (De Palma et al., 2003; Kikuchi et al.,
2004; Pellinen et al., 2004; Dullaers et al., 2006). Other widely tested viral vectors for cancer
gene therapy include adeno-associated viruses (Lalani et al., 2004; Li et al., 2005a) and different
types of tumor-selective, replicative oncolytic viruses, like adeno-, vaccinia-, reo- and herpes
simplex type I viruses (Everts and van der Poel, 2005).
18
2.1.3
CANCER GENE THERAPY APPROACHES
In a view of the fact that cancer is a complex genetic disorder, there are several potential targets
to disrupt vital functions of tumors by delivering therapeutic genetic material (summarized in
Table 2.). Since it is konwn that the immune response is the most powerful and natural
mechanism against all kinds of cellular threats including malignancies, the stimulation of
immune responses has been extensively harnessed in anticancer gene therapy. The majority of
clinical cancer gene therapy studies are based on different immunotherapy strategies (January
2007, http://www.wiley.co.uk/genetherapy/ clinical/). Tumor cells can naturally express
antigens, ideally they would be recognized and eliminated by the host immune system. In cancer
immunotherapy this has been utilized for example by engineering antigen presenting cells or
lymphocytes with tumor antigens ex vivo or in vivo in order to enhance tumor cell recognition
and killing by the immune system (Conry et al., 1998; Vollmer et al., 1999; Morgan et al.,
2006). Alternatively, tumor immunogenicity has been improved by delivering genes encoding
cytokines (e.g. interleukin-12) into tumor cells (Caruso et al., 1996; Lucas et al., 2002;
Heinzerling et al., 2005). Unfortunately, many tumor cells have developed sophisticated
mechanisms to evade the immune system, for instance by down-regulating expression of MHC
(major histocompatibility complex) class I expression (Natali et al., 1989), and this may be one
of the reasons for the unimpressive therapeutic outcome in clinical trials.
Table 2. Examples of cancer gene therapy strategies.
APPROACH
MECHANISM
EXAMPLE
REFERENCE
IMMUNOTHERAPY
Activation of immune
system to recognize and
kill tumor cells
Tumor associated antigen
alpha-fetoprotein,
cytokine; interleukin-12
(Vollmer et al.,
1999; Heinzerling
et al., 2005)
REPAIR OF CELL
CYCLE DAMAGES
Suppression of oncogenes
or delivery of tumor
suppressor gene
Anti-apoptotic Bcl-2,
tumor suppressor p53
(Swisher et al.,
1999; Waters et
al., 2000)
SUICIDE GENE
THERAPY
Killing of tumor cells by
delivering gene encoding
prodrug activating enzyme
Herpes simplex virus type
I thymidine kinase
(Immonen et al.,
2004)
CHEMOPROTECTION Transfer of drug resistance
gene to protect bone
marrow from
chemotherapeutic agent
Multidrug resistance
protein 1
(Cowan et al.,
1999)
VIROTHERAPY
Killing of tumor cells with
lytic, replicative viruses
Replication-selective
adenovirus
(Nemunaitis et al.,
2001)
ANTIANGIOGENESIS
Inhibition of formation of
new blood vessels into the
tumor
Angiostatin
(Lalani et al.,
2004)
19
Although cancer is generally a disorder of multiple genes and dozens of genes are usually
expressed aberrantly, promising results have been obtained via correcting defects induced by
loss of tumor suppressor genes or excessive activation of oncogenes. Overexpression of
multifunctional tumor suppressor protein p53, whose loss of function is associated with about
50% of cancer types, has been evaluated for example in the treatment for non-small-cell lung
cancer (Swisher et al., 1999; Nemunaitis et al., 2000). On the other hand, inactivation of
oncogenes, such as anti-apoptotic bcl-2, with antisense oligonucleotides has shown some
potential ability to induce antitumoral activity against diseases like non-Hodgkin’s lymphoma
(Waters et al., 2000; Tamm et al., 2001). Furthermore, the discovery of RNA interference
(RNAi) has provided novel, promising tools with which to silence oncogenes. This approach has
thus far been tested only in pre-clinical experiments (Pai et al., 2006). However, one
considerable disadvantage of these corrective treatment forms is that extremely high gene
transfer efficiencies are required for complete recovery, since every tumor cell has to contain the
therapeutic molecule.
One of the most exciting strategies is based on simply harnessing the virus itself to eradicate the
tumor, without any involvement of a therapeutic gene. This so-called virotherapy approach
utilizes oncolytic viruses that replicate specifically in tumor cells and induce cell death through
viral propagation. Some of the oncolytic viruses, like the attenuated Semliki Forest virus (SFV),
appear to replicate naturally in tumor cells, (Vaha-Koskela et al., 2006; Maatta et al., 2007).
Also, a number of other RNA viruses, such as reovirus and Newcastle disease virus that utilize a
deficient pathway in malignant cells have been tested (Russell, 2002). Alternatively, certain
oncolytic viruses have been engineered genetically to be tumor selective, so that they also
operate via inactive signaling pathways in the tumor cell. Examples of this class of virotherapy
are attenuated herpes simplex virus type 1 (Markert et al., 2000; Detta et al., 2003) and
conditionally replicating adenoviruses (Bischoff et al., 1996; Nemunaitis et al., 2001;
Hakkarainen et al., 2006).
The generation of new blood vessels is a prerequisite for tumorigenesis and therefore inhibition
of angiogenesis in order to prevent tumor growth and metastasis has received attention. The
utility of different angiogenesis regulatory factors have been evaluated; molecules which inhibit
angiogenesis in tumor endothelial cells (like angiostatin) (Lalani et al., 2004) and molecules that
inhibit angiogenic inducers (e.g. vascular endothelial growth factor inhibitor) (Yu et al., 2001).
20
In this type of therapy, long-term gene expression is needed; this can be obtained by using e.g.
lentiviral or AAV vectors.
One well-studied cancer gene therapy approach is cytotoxic therapy or suicide gene therapy. The
method is based on introducing an enzyme into tumor cells, which catalyzes the conversion of a
harmless prodrug into a toxic form that leads to death of the cell expressing the suicide enzyme.
An essential feature of this therapy is that cell death is induced also in surrounding, nontransduced tumor cells, since the toxic form of the prodrug can diffuse into the neighboring cells.
This indirect cell killing is called the bystander effect and it means that a high gene delivery rate
is not mandatory for successful therapy. Several prodrug activation systems have been
developed, all mediating their action through DNA replication. Therefore, this therapy form has
impact on only proliferating cells and operates most efficiently in rapidly dividing cells,
especially tumor cells (Aghi et al., 2000).
2.1.4
HSV-TK/GCV SUICIDE GENE THERAPY
Today, the best and also the first characterized suicide/prodrug system is based on herpes
simplex virus type 1 thymidine kinase gene (HSV-TK) combined to ganciclovir (GCV) (Fig. 1)
(Moolten, 1986). Although thymidine kinase activity is also present in eukaryotic cells as well,
the viral TK differs since it possesses the ability to efficiently phosphorylate also nucleoside
analogues such as ganciclovir, a drug that has been used against infections caused by herpes
simplex viruses (Field et al., 1983).
TRANSFER OF HSVTK GENE INTO
TUMOR CELLS
ADMINISTRATION OF
PRODRUG (GCV)
TK
TK
TK CONVERTS GCV
INTO A
TOXIC FORM g
CELL DEATH
DIFFUSION OF
PHOSPHORYLATED
FORM OF GCV INTO
ADJACENT CELLS
TK
TK
ENHANCED CELL DEATH
(=BYSTANDER EFFECT)
Figure 1. Principle of HSV-TK/GCV-mediated cell killing.
HSV-TK, herpes simplex thymidine kinase; GCV, ganciclovir
21
HSV-TK gene is most commonly delivered using viral vectors directly to tumors or (particularly
in the treatment of brain tumors) to the surrounding tissue. After administration of GCV, the first
step is phosphorylation of GCV to GCV monophosphate by viral TK (Field et al., 1983).
Thereafter different host cell kinases are able to phosphorylate GCV into the diphosphate and
then to the toxic triphosphate form, which will act as a substrate for DNA polymerase. The
triphosphorylated form of GCV can inhibit DNA polymerase by being incorporated into DNA.
Since GCV has complete lack of sugar ring, it means that incorporation of GCV triphosphate
into newly-synthesized DNA leads to termination of chain synthesis immediately or after
incorporating one additional nucleotide beyond GCV triphosphate (Ilsley et al., 1995).
This approach has been studied for 20 years, and there are several theories of the mechanism of
cell killing after DNA damage. Not only induction of apoptotic pathways but also non-apoptotic
mechanisms have been shown to be involved in HSV-TK/GCV-mediated cell killing. Some
studies indicate that the cell cycle arrest at G2 phase (Kaneko and Tsukamoto, 1995) or at G2-M
transition (Halloran and Fenton, 1998) eventually triggers cell death. The role of apoptosis was
ruled out in these studies, since no DNA laddering was detected. The frequency of necrotic cell
death has been shown to remain low, approximately 10% of cells being necrotic in vitro (at GCV
concentration of 1 µM) (Thust et al., 2000; Tomicic et al., 2002). Nevertheless, the main
mechanism to explain the cell death during HSV-TK/GCV treatment occurs via apoptosis
(Freeman et al., 1993; Bai et al., 1999; Beltinger et al., 1999; Wei et al., 1999; Tomicic et al.,
2002). The HSV-TK/GCV induced apoptosis has shown to be involved e.g. there is evidence of
increased level of p53 and death receptor aggregation or induction of caspase-9 mediated
cleavage of Bcl-2 protein (Beltinger et al., 1999; Wei et al., 1999; Tomicic et al., 2002).
However, these pathways behind GCV induced cell death vary in different cell types.
2.1.4.1
BYSTANDER EFFECT
It was found in early studies that the expression of HSV-TK was not required for cell
destruction. Complete cell death was detected in cultured cells with only about every tenth cell
expressing the HSV-TK gene (Moolten, 1986). This phenomenon was named the bystander
effect. Similar results were obtained in animal models, in which complete tumor eradication was
achieved when 10-50% of cells were carrying HSV-TK gene (Culver et al., 1992; Freeman et al.,
1993). Interestingly, the effect was not shown be restricted to similar tumor cells, but the effect
was seen to operate between different cell types in vitro and even between different cells types
originating from diverse tissues in vivo (Ishii-Morita et al., 1997; Vrionis et al., 1997; Arafat et
22
al., 2000). The presence of a bystander effect is needed for successful cancer gene suicide
therapy, since gene transfer capability of current vectors is not high enough to deliver HSV-TK
gene into all cells in the tumor. Without the bystander effect, the therapeutic impact of HSVTK/GCV therapy would remain at the level of the HSV-TK gene delivery rate.
Some theories to explain the mode of the bystander action have been proposed. Close cell-to-cell
contact has shown to play important role in several studies. It has been demonstrated that cell
viability of HSV-TK negative cells co-cultured with HSV-TK positive cells in the presence of
GCV, is dependent on cell density. Namely, cells sharing physical contact will die, whereas cells
lacking contact are able to survive (Moolten, 1986; Freeman et al., 1993). Freeman et al showed
that death in nearby adjacent cells was mediated by phagocytosis of apoptotic vesicles that
contain cytotoxic products, released into the medium from the HSV-TK carrying cells (Freeman
et al., 1993). Another popular theory postulates that instead of cytotoxic vesicles, the bystander
effect is transmitted by diffusion of phosphorylated forms of GCV through gap junctions, which
are channels that are composed of proteins called connexins and permit the traffic of ions and
certain small molecules between cells. The extend of the cytotoxic effect in adjacent cells has
been shown to be dependent on the connexin expression and gap junctional intercellular
communication between the cells (Fick et al., 1995; Vrionis et al., 1997) and can be decreased
with chemicals that interfere with gap junction function (Touraine et al., 1998a). However, it has
also been shown that HSV-TK negative cells lacking physical contact with HSV-TK positive
cells are killed if they are grown in the same medium or in the medium collected from the HSVTK -expressing cells. The authors suggested that bystander effect was mediated by
internalization of soluble toxic agents from the medium released by HSV-TK expressing cells
(Princen et al., 1999).
Particularly in animal studies, immune responses have been shown to play a significant role in
mediating bystander effect induced cytotoxicity. Freeman et al have reported that certain
cytokines are produced during GCV-treatment, and the agents can evoke necrosis in tumor cells
(Freeman et al., 1995). Furthemor, a reduction of cytotoxicity has been demonstrated in
immunodeficient animal models (both in immunocompetent compared to athymic mice and in
athymic mice compared to completely immunodeficient mice) (Vile et al., 1994; Bi et al., 1997).
Massive infiltration of macrophages and T-lymphocytes has been observed in rat liver
metastases after direct injection of cells expressing HSV-TK and treatment with GCV, pointing
to a role of local inflammation in HSV-TK/GCV-mediated cytotoxicity (Caruso et al., 1993).
23
Interestingly, Bi et al showed that in addition to local response, naive tumor cells distant from
the tumor treated with the HSV-TK expressing cells (two tumors at the opposite flanks of a
mouse) can be destroyed after GCV administration. Furthermore, concomitant treatment with an
immunosuppressive agent impaired the antitumor effect in non-treated tumor. The study of Bi
and co-workers was performed in athymic mice, evidence also for a role for macrophages and/or
natural killer (NK) cells (Bi et al., 1997).
2.1.4.2
STRATEGIES TO IMPROVE HSV-TK/GCV TREATMENT
Despite impressive results in pre-clinical studies, the therapeutic effect has been a
disappointment in many human trials and therefore further progress is required to make realize
the full potential of HSV-TK/GCV treatment in gene therapy. Although inadequate gene transfer
efficiency is the major reason for the lack of efficiency in clinical trials, several improvements
have been claimed to raise the cytotoxicity of HSV-TK/GCV treatment. The suicidal gene itself
has been engineered to enhance the catalytic activity for the prodrug. The HSV-TK variants with
mutated sequences at or near the catalytic sites have shown improved sensitivity for GCV in
vitro and in vivo (Black et al., 1996; Kokoris et al., 1999). In addition to enhanced tumor cell
killing, higher activity of HSV-TK would allow the use of lower GCV concentrations. GCV has
several toxic side effects, such as hematological-, liver-, kidney- and neurotoxic effects.
Alternatively, other nucleoside analogues, such as acyclovir, have been evaluated for substrates
to HSV-TK instead of GCV. Acyclovir is less toxic than GCV in vivo, but on the other hand,
acyclovir is also a less efficient substrate for HSV-TK compared to GCV (Field et al., 1983).
Since the bystander effect plays such an important role in HSV-TK/GCV therapy, several
improvements have been directed towards attempting to increase cytotoxicity to adjacent cells.
Enhancement of the bystander effect has been obtained by increasing the gap junctional
communication by co-transfecting genes encoding for the gap junction protein (e.g. connexin
43) or exposing cells to pharmacological agents that increase gap junctional communication
(Mesnil et al., 1996; Touraine et al., 1998b). Alternatively, the anti-tumoral impact of immune
response has been utilized by introducing a gene encoding for an immunostimulatory protein,
such as interleukin-2, into the tumor cells in combination with HSV-TK gene. This strategy
demonstrated systemic antitumoral immunity to secondary tumors implanted subcutaneously at
sites distant to the primary tumor at the end of GCV administration (Chen et al., 1995). The
feasibility of using replication competent viruses has also been evaluated in combination with
HSV-TK/GCV treatment; boosting the efficacy of HSV-TK/GCV treatment on therapeutic
24
effect has been observed in some studies (Wildner et al., 1999), whereas some studies have not
detected any increased cytotoxicity in vivo using combination therapy with oncolytic viruses
(Morris and Wildner, 2000; Hakkarainen et al., 2006). An alternative method to enhance the
cytotoxic impact of HSV-TK/GCV therapy is to modify the HSV-TK gene so that it contains a
polypeptide domain that promotes HSV-TK protein movement from expressing cells to the
surrounding, HSV-TK negative cells. The cell penetrating properties of herpes simplex virus
tegument protein VP22 (Dilber et al., 1999; Liu et al., 2001) and HIV-1 transactivator protein
(TAT) have been shown to extend GCV cytotoxity when they are fused with HSV-TK (Tasciotti
et al., 2003; Tasciotti and Giacca, 2005). More detailed description about the utility of HIV-1
TAT cell penetrating peptide in cancer gene therapy is provided in chapter 2.3.
In the future, the HSV-TK/GCV therapy most likely will not provide a cure as such, but rather
be used as a combination therapy with conventional treatment forms or together with other gene
therapy methods. Promising results have been obtained in the treatment of malignant glioma. A
significant prolongation in life expectancy was obtained using HSV-TK/GCV therapy as an
adjuvant after surgical removal of the solid tumor (Sandmair et al., 2000; Immonen et al., 2004).
Malignant glioma represents an important target to be treated with HSV-TK/GCV-mediated
suicide gene therapy. This cancer form does not metastasize beyond the central nervous system
and vector administration directly to the desired location is possible, without inducing systemic
toxic responses (Pulkkanen and Yla-Herttuala, 2005). Moreover, the utility of HSV-TK/GCV
has been evaluated for the treatment of patients suffering from many other malignant diseases
such as metastatic melanoma and ovarian cancer (Klatzmann et al., 1998; Alvarez et al., 2000).
The clinical studies performed so far have been mainly phase I or II trials, which assess safety
and also preliminary efficacy. In these trials, the safety of adeno- and retroviral vectors was
confirmed and some evidence of HSV-TK/GCV treatment efficacy was demonstrated (Ram et
al., 1997; Klatzmann et al., 1998; Alvarez et al., 2000). Unfortunately, one of the first phase III
trials conducted using HSV-TK/GCV therapy as an adjuvant to surgical resection and
radiotherapy in a treatment for malignant glioma, did not show any clinical benefit (Rainov,
2000). There may be many reasons for the poor therapeutic outcome, nevertheless, the most
important limiting factor for successful results seems to be the low percentage of tumor cells
expressing HSV-TK and furthermore an inadequate bystander effect.
25
2.2
2.2.1
TYPE I INTERFERON RESPONSE AGAINST THERAPEUTIC GENE TRANSFER
OVERVIEW
Combatting against extracellular pathogens, such as bacteria and viruses, is one of the key
functions of the human immune system. Current gene therapy vectors are, however, mainly
based on viruses or employ plasmid DNA (pDNA), which is of bacterial origin. Although vector
development has been designed to avoid immunogenicity and toxicity, the viral genomic
components, viral proteins, transgene products or unmethylated bacterial CpG -rich pDNA
might trigger undesired host cell defense mechanisms (Zhou et al., 2004). While these responses
may have certain beneficial properties in cancer immunotherapy; in the context of other gene
therapy approaches, immune responses may be considered as harmful side-effects, since they
can lead to decreased gene delivery rate, shortened transgene expression time and
ineffectiveness of vector re-administration. Furthemore, a strong immune response can even lead
to severe side-effects in clinical trials, as was tragically seen in the case of Jesse Gelsinger, who
died during a phase I clinical trial after receiving adenoviral vector (Raper et al., 2003). Several
components of adaptive and innate immune system are involved in mediating these immune
responses; neutralizing antibodies, cytotoxic T-cells and various cytokines (Bessis et al., 2004),
including interferons.
2.2.2
TYPE I INTERFERONS
The human immune system consists of two components; the innate and adaptive immune
systems. The latter system represents more specific immunity mediated by two types of
lymphocytes; T-cells and B-cells. The innate immune system provides less specific, but
immediate defense mechanism and consists of the induction of inflammation, plus activation of
complement system and the cells of the innate immune system such as NK cells, macrophages
and dendritic cells (DCs) (Lydyard and Grossi, 1998; Male and Roitt, 1998). Proteins and
peptides called cytokines mediate the signaling in both adaptive and innate immune systems by
binding to their specific receptors. Interferons (IFNs) belong to a multigene family of inducible
innate cytokines, which have a central role in modulating the immune system, having a
particularly important role in the early defense against viral infections (Vilcek and Sen, 1996).
Traditionally, IFNs have been distinguished into two main groups, type I and type II IFNs, based
on their recognition of specific receptors and producing cell type. Recently, it was reported that
there is a third subgroup called IFN s or alternatively interleukin 28 or 29 (IL28 and IL29)
(Kotenko et al., 2003). Type II IFN, which comprises only IFN , is induced by mitogenic or
26
antigenic stimuli and is synthesized only by particular cells of the immune system; NK cells and
certain T-lymphocytes, and this interferon possesses only modest antiviral activity (Farrar and
Schreiber, 1993). The type I IFNs, which consist mainly of IFN and IFN , are primarily
responsible for the host cell defense mechanism against viruses.
The type I IFNs are produced by almost every cell type during viral infection, although
compared to other blood cell types, certain specialized immune cells, plasmacytoid dendritic
cells (pDC), are capable of producing a huge amount of type I IFNs in response to viral infection
(100-1000 times more) (Liu, 2005). It has been known some time that the key factor triggering
the production of type I IFNs is double stranded RNA (dsRNA) which, in addition to occurring
in the genomes of dsRNA viruses, is produced at some point during the replication of many
viruses. This foreign intracellular dsRNA has been shown to be recognized by receptors called
dsRNA dependent protein kinase R (PKR) or 2´, 5´-oligoadenylate synthetase (OAS). The PKR
is able to combat directly against viruses by inhibiting translation, while activation of OAS leads
eventually to cleavage of viral RNA (Williams, 1999; Samuel, 2001). In addition to the fact that
both PKR and OAS are activated by an interaction with dsRNA independently of induction of
type I IFNs, they also belong to the IFN inducible genes. Of these proteins, only PKR acts as a
signal transducer in a pathway that initiates the production of type I IFNs.
Recently, it was discovered that there are two types of, mainly intracellular, detector systems in
mammalian cells that initiate the cascade leading to the expression of type I IFNs. Proteins
called pattern recognition receptors (PRRs) have been shown to recognize specific motifs in
genome components or glycoproteins that are called pathogen associated molecular patterns
(PAMPs). Depending on the entry route of the pDNA, RNA or viruses and their surface
glycoprotein composition, it seems that the recognition of PAMP can be associated with a
specific PRR. Of the two subsets of PRRs, on important class is the Toll-like receptors (TLRs),
which are found primarily in the cells of innate immune system, including macrophages and
dendritic cells (Kawai and Akira, 2006; Saito and Gale, 2007). However, also other cell types,
including tumor cells, appear to express different TLRs at variable levels (Nishimura and Naito,
2005; Perry et al., 2005; Hou et al., 2006). The TLR4 has been shown to detect viral proteins on
the cell surface, for example vesicular stomatitis virus glycoprotein (Georgel et al., 2007). The
TLR3, TLR7/8 and TLR 9 recognize dsRNA, single stranded RNA (ssRNA) or double stranded
CpG -rich DNA, respectively, on endosomal membranes and genome components of viruses that
enter the cell via endocytosis (Hemmi et al., 2000; Alexopoulou et al., 2001; Diebold et al.,
27
2004; Heil et al., 2004). In addition to recognition of viral genomic dsRNA in endosomes, there
are other subsets of PRR sensors, including members of the RNA helicase family; retinoid acid
inducible gene-1 (RIG-1) and melanoma differentiation associated gene-5 (MDA-5) that detect
cytosolic dsRNA (Yoneyama et al., 2004; Yoneyama et al., 2005). Once PAMP is recognized by
its specific PRR, this interaction activates an intracellular signaling cascade via several adaptor
and signaling molecules, leading to the activation of certain transcription factors. The
accumulation of transcription factors into the nucleus finally initiates expression of type I IFNs
(Fig. 2) (Takaoka and Yanai, 2006; Uematsu and Akira, 2007).
The secreted type I IFNs contribute in an autocrine and paracrine fashion by binding to their
specific receptor, which activates the Janus -family tyrosine kinases, Jak1 and Tyk2, the signal
transducers and activators of transcription STAT1 and STAT2 and the interferon regulatory
factor 9 (IRF9), finally leading to the formation of a transcription factor known as interferonstimulated gene factor 3 (ISGF3). In the nucleus, binding of ISGF3 complex to ISRE element of
DNA initiates expression of hundreds of cellular genes known as interferon-stimulated genes
(ISGs) (Fig. 2) (Samuel, 2001; Smith et al., 2005; Takaoka and Yanai, 2006). These genes
encode many proteins such as PKR, OAS, adenosine deaminase, myxovirus-resistance proteins
(Mx), interferon regulatory factors 5 and 7 etc. They mediate antiviral actions either directly or
indirectly; for example by inhibiting translation, degrading and editing of viral RNA or by
interfering with viral nucleocapsids (Stark et al., 1998; Samuel, 2001).
28
RECOGNITION OF
VIRAL PROTEINS ON
CELL SURFACE
RECOGNITION OF VIRAL
GENOME AND
REPLICATION PRODUCTS
IN CYTOPLASM
RECOGNITION OF VIRAL
NUCLEIC ACIDS AS WELL
AS ssRNA, dsRNA AND CpGrich DNA IN ENDOSOMES
TLR4
TLR7
TLR3
RIG-1
TLR8
MDA-5
PKR
IFN
TRANSCRIPTION OF
TYPE I IFNs
TLR9
IFN
IFN
IFN
IFN
IFN
Jak-STAT pathway
OAS
IRF7
ISGF3
TRANSCRIPTION OF ISGs
SUCH AS:
ISRE
MxA
IRF5
PKR
Figure 2. Schematic representation of the activation of type I IFN response that different proteins
and genomic components of gene delivery vehicles may induce in a variety of cell types.
Abbreviations: dsRNA, double stranded RNA; ssRNA, single stranded RNA; CpG-rich pDNA,
(unmethylated) cytosine- and guanosine rich DNA; TLR, Toll-like receptor; MDA-5, melanoma
differentiation associated gene-5; RIG-1, retinoid acid inducible gene-1; PKR, double stranded RNA
dependent protein kinase R; Jak, Janus kinase; STAT, signal transducer and activator of transcription;
ISGF3, interferon stimulated gene factor 3; ISRE, interferon stimulated response element; ISG, interferon
stimulated gene; OAS, 2´,5´-oligoadenylate synthetase; IRF, interferon regulatory factor; MxA,
myxovirus resistance protein A. References: (Stark et al., 1998; Samuel, 2001; Sen, 2001; 2004; Perry et
al., 2005; Kawai and Akira, 2006; Takaoka and Yanai, 2006; Saito and Gale, 2007)
29
2.2.3
CHALLENGES CAUSED BY THE TYPE I IFN RESPONSE AGAINST THERAPEUTIC
GENE TRANSFER
In addition to antiviral functions, type I IFNs have various biological functions. They have a role
in activating lymphocyte differentiation, enhancing NK-cell cytotoxicity as well as promoting
the maturation of antigen presenting cells. Type I IFNs also have potent anti-proliferative, antiangiogenic and pro-apoptotic activities (Pfeffer et al., 1998; Theofilopoulos et al., 2005).
Therefore, they have been widely harnessed in the treatment of different types of cancer such as
certain hematological malignancies, melanomas, renal cell carcinoma and Kaposi's sarcoma
(Pfeffer et al., 1998). In addition, type I IFNs, particularly IFN , have been utilized also in gene
therapy approaches for cancer (Ferrantini and Belardelli, 2000).
Several cancer gene therapy applications lack efficiency, for the most part due to the poor
transgene delivery rate and consequently inadequate expression of the therapeutic protein. It is
well known that in many cases this is a result adaptive and innate immune responses induced by
therapeutic gene transfer, but only a small number of studies have characterized the role of type I
IFNs in this process. Bearing in mind the function of type I IFN response in resistance to viruses,
it is not surprising that also commonly used viral vectors may well be able to activate the
expression of type I IFNs. However, secretion of type I IFNs is not a challenge in the context of
cancer therapy. Nonetheless it is crucial to determine, whether these responses can influence the
expression of the therapeutic protein.
Although lentiviral vectors are considered to be less immunogenic than many other viral vectors,
systemically injected VSV-G pseudotyped lentiviral vectors have been shown to rapidly trigger
the production of IFN stimulated genes in mouse liver and spleen. Studies with isolated cells
from spleen suggested that pDCs were primarily responsible for the production of type I IFNs in
response to lentiviral vectors. The observed IFN response was not dependent on the envelope
glycoprotein which was used. Furthermore, it was shown that improved transduction efficiency
and more stable transgene expression were obtained in the absence of the type I IFN response
(studied in type I IFN receptor knock-out mice). Although, lentiviral vector gene expression is
considered to be stable due to its integration into the host cell genome, the type I IFN response
seems to play a role also in decreasing the long-term expression of the therapeutic protein by
inducing vector clearance (Brown et al., 2006). On the other hand, another recent study showed
that the induction of a type I IFN response by lentiviral vectors is dependent on the envelope
protein. VSV-G pseudotyped vectors that were generated using lipofection based transient
30
transfection, induced the production of type I IFNs by pDCs. The authors suggested that
lentiviral preparations had been contaminated with the tubulo-vesicular structures that were
generated in transfected producer cells with the aid of VSV-G protein. These structures had then
been co-purified along with lentiviral vectors during concentration and were able to carry
plasmid DNA (pDNA) into the transduced pDC cells, thus activating the type I IFN response.
Interestingly, the production of type I IFNs was detected even when using a very low
multiplicity of infection (MOI 0.2) (Pichlmair et al., 2007).
Adenoviral vectors are known to be highly immunogenic and able to activate both adaptive and
innate immune responses, which has represented a barrier to their clinical use (Bessis et al.,
2004). The importance of type I IFNs has been less well understood, but recent studies have
demonstrated that the first generation E1/E3 -deleted adenoviral vectors could induce the
production of type I IFNs (Huarte et al., 2006; Nociari et al., 2007; Zhu et al., 2007). The
adenovirus-mediated gene delivery was shown to trigger a type I IFN response in cultured DCs
as well as in splenic DCs in vivo after intravenous injection of the vector. In spite of the release
of type I IFNs, re-administration of adenoviral vector was succeeded. Furthermore, exposing
cultured HeLa cells to IFN
did not decrease the transgene expression from the adenoviral
vector. Both IFN and IFN proteins were detected also from the sera of patients treated with
intratumoral injection of HSV-TK carrying adenoviral vector into the liver (Huarte et al., 2006).
Similar results have been obtained by Zhu and co-workers who demonstrated a high level
expression of type I IFNs particularly by pDCs, but also by conventional DCs and macrophages
in vitro induced by adenoviral gene transfer. Moreover, the production of IFN was detected in
mice sera after intravenous adenoviral vector administration. However, in contrast to the
observations by Huarte et al, they reported that the response was critical for adenoviral function
in vivo. An increased copy number of adenoviral DNA, more stable transgene expression and
reduced inflammation was observed in liver of IFN
receptor knock-out mice compared to
their wild type counterparts. Blocking the type I IFNs with neutralizing antibodies was followed
by improved transgene expression, decreased virus-specific T-cell response and a reduced
inflammation response compared to non-treated mice (Zhu et al., 2007).
A dose-dependent reduction in the transgene expression caused by type I IFNs has been reported
when murine muscle cells or tissue were treated with type I IFNs prior to exposure with the
adenoviral vector (Acsadi et al., 1998). Type I IFNs have also been shown to suppress transgene
expression after retrovirus-mediated gene delivery. Expression of
31
-galactosidase was
significantly down-regulated in keratinocytes when the cells were treated with IFN
(Ghazizadeh et al., 1997). However, in these studies it was not tested whether retro- or
adenoviral gene delivery induced the production of type I IFNs (Ghazizadeh et al., 1997; Acsadi
et al., 1998).
Non-viral vectors are considered to be less immunogenic and safer than viral vectors in general,
however, delivery of pDNA can induce the release of various cytokines (Rudginsky et al., 2001;
Zhou et al., 2007). The rapid induction of inflammatory cytokines (e.g. tumor necrosis factor
alpha, TNF ) for their part is associated with toxicity of cationic-liposome-DNA complexes
(Niidome and Huang, 2002). These immune responses are induced by unmethylated CpG motifs
present in the bacterial pDNA in contrast to mammalian DNA, which has a lower frequency of
these unmethylated CpGs. Although the immunostimulatory effect of CpG-rich DNA has been
known for some time, it was discovered quite recently that cellular responses induced by CpGrich DNA are mediated by TLR9 (Hemmi et al., 2000). Transfection of cationic liposome-DNA
complexes has been shown to induce the expression of type I IFNs in vitro by macrophages and
fibroblasts. Further, high concentrations of IFN and IFN have been detected in mouse serum
after intravenous injection of cationic liposome-DNA-complexes. The transfection efficiency of
cationic liposome-DNA complexes was shown to be dose-dependently decreased in vitro when
the cells were treated with IFN or IFN shortly after transfection. Moreover, increased reporter
gene expression was observed in several tissues of IFN
receptor deficient mice compared to
the wild type after the transfection with cationic liposome-DNA complexes, suggesting that type
I IFNs can have a harmful effect on non-viral gene delivery (Sellins et al., 2005). In addition to
lipofection, introduction of pDNA by some other non-viral gene delivery means may activate the
host cell defense system. Expression of IFN regulated proteins (e.g. IRF7) was detected in
human tumor cell lines after pDNA delivery with a commercial transfection reagent,
electroporation and calcium-phosphate precipitation. Since the medium from pDNA-transfected
cells was also able to induce expression of IRF7 in non-treated cells, the authors suggested that
these responses were mediated by soluble factors in the transfected cells, such as IFN (Li et al.,
2005b).
Delivery of mRNA instead of DNA has been considered as an alternative method to introduce
therapeutic material into cells. However, inside the target cell, mRNA may be recognized by
receptors, and their activation can lead to induction of responses that may impede production of
the therapeutic protein. It has been shown that mRNA can be recognized by TLR7 (Diebold et
32
al., 2004) or alternatively, it can form double stranded secondary structures, which thereby
renders it susceptible for recognition by dsRNA receptors e.g. PKR (Ceppi et al., 2005). Ceppi et
al demonstrated that myeloid DCs responded to lipofection-mediated delivery of mRNA coding
for GFP by producing type I IFNs dose-dependently. However, electroporation-mediated
transfection induced considerably type I IFN production less when compared to lipofection. One
explanation why electroporation induced less type I IFN production may be due to the different
intracellular localization of mRNA achieved by these transfection methods; nucleic acids that
are delivered by lipofection are mainly taken up by endocytosis, whereas electroporated material
enters directly into the cytoplasm in transient pores on the cell membrane. Nevertheless, when
the cells were treated with a PKR inhibitor (2-aminopurine), the production of type I IFNs was
blocked, pointing to a role of PKR in recognizing mRNA (Ceppi et al., 2005).
RNAi is a fascinating tool which can be used to determine gene function and to manipulate gene
expression by silencing genes with short (19-29 bp) dsRNA sequences. Although these RNA
sequences were originally thought to be too short to induce an antiviral response, recent studies
have shown that short interfering RNAs (siRNA) and short hairpin RNAs (shRNA) are able to
activate the type I IFN responses (Bridge et al., 2003; Sledz et al., 2003; Kariko et al., 2004;
Kim et al., 2004; Judge et al., 2005; Sioud, 2005; Marques et al., 2006). Silencing of genes by
RNAi is considered to be both highly efficient and specific to the targeted gene. However,
induction of a type I IFN response may lead to sequence-independent silencing of other host cell
genes and induce toxic effects (Kariko et al., 2004; Kim et al., 2004), thus activation of IFN
inducible proteins e.g. OAS or PKR, can lead to non-specific degradation cellular RNA and
inhibit host cell protein synthesis (Williams, 1999; Samuel, 2001).
2.2.4
MECHANISMS TO AVOID INDUCTION OF THE TYPE I IFN RESPONSE
Circumventing the immune responses is a major challenge for efficient gene therapy. This
relates not only to viral vectors as was usually thought, but also to non-viral gene delivery. In
order to enhance therapeutic gene transfer, different strategies have been developed to avoid
induction of these undesired immune responses. For example, as many viral proteins as possible
have been removed from the vectors with the intention of minimising the host immune response.
This might have only a modest impact on preventing the type I IFN response, since the viral
genomic components are the major inducers of type I IFN response. However, the development
of more efficient vectors is important in order to allow the use of lower MOIs. The use of
transiently immunosuppressive agents or specific inhibitors of IFN signaling pathways prior to
33
gene transfer could also create a more beneficial environment for efficient viral transduction.
Due to the transient nature of the type I IFN response, Brown et al suggested that treatment with
neutralizing antibodies targeted to IFN
proteins or the use of antagonists for IFN receptor or
PRRs could enhance lentiviral transduction and prevent short-term expression of the therapeutic
gene (Brown et al., 2006). This strategy was successfully tested by Zhu et al who described
reduced adaptive and innate immune responses that lead to prolonged transgene expression and
reduced inflammation in vivo after adenoviral gene transfer. During the course of evolution
viruses have evolved strategies to counteract the antiviral actions of IFN by interfering with a
number of components that are involved in different steps in the IFN pathway; block in IFN
signaling, synthesis and disruption of the function of IFN inducible proteins (Levy and GarciaSastre, 2001). The use of these viral elements interfering with the type I IFN system in the
context of viral or non-viral vector constructs could provide tools to inhibit the induction of type
I IFNs.
Some approaches have been introduced to reduce the immunogenicity of non-viral pDNA
delivery. Some strategies attempt to as modify CpG sequences in pDNA. Methylation of
cytosine in CpG dinucleotides by methylase has been shown to prolong the expression of certain
viral proteins from pDNA in vivo (Reyes-Sandoval and Ertl, 2004). Furthermore, employment of
a PCR (polymerase chain reaction) amplified gene instead of pDNA and reduction in the number
of CpG motifs in pDNA have led to diminished production cytokines and side effects (e.g.
inflammation and liver toxicity) in vivo (Hofman et al., 2001; Yew et al., 2002). Although these
strategies have been shown to decrease expression of cytokines other than type I IFNs, it is
likely that the above methods could be beneficial also when suppressing the type I IFN response,
since unmethylated CpG DNA is a potent stimulator of this system. Furthermore, choosing an
alternative gene transfer method for pDNA delivery instead of lipofection could provide a way
to avoid induction of type I IFN responses. Li et al showed that delivery of pDNA by
electroporation results in a reduced type I IFN induction compared to lipofection based gene
transfer (Li et al., 2005b). In another study it was proposed that since with electroporation
pDNA is delivered directly to cytoplasm, it is able to bypass recognition of CpG DNA by TLR9,
whereas liposome mediated transfection carries its cargo via the endocytotic pathway and
therefore passess through TLR9-signaling (Zhou et al., 2007).
34
Although the role of innate immune response in RNAi technology was recognized recently,
much of research has been done to identify the factors that make RNAi molecules
immunostimulatory and furthermore, to characterize host cell recognition systems for RNAi
sequences. For example induction of type I IFN response by siRNAs has been shown to be
dependent on the sequence (Judge et al., 2005; Sioud, 2005). Judge et al demonstrated that
guanosine (G)- and uridine (U) rich sequences (5´ UGUGU 3´) in synthetic siRNA constructs
are immunostimulatory (Judge et al., 2005). Furthermore, siRNA molecules delivered by
electroporation are shown to avoid induction of type I IFN response, while the same siRNA
sequences transfected by lipofection are likely to induce this response (Sioud, 2005). Exogenous
RNA interfering molecules are produced by chemical synthesis or by in vitro transcription using
bacteriophage RNA polymerase. Kim et al showed that siRNAs as well short ssRNAs, which are
synthesized using bacteriophage polymerases, induce an intense type I IFN response compared
to chemically synthesized RNA molecules. The induction was most likely due to the presence of
initiating 5´ triphosphate in the RNA strands, since the response was abolished by removal of the
5´ triphosphate (Kim et al., 2004). However, it is noteworthy that in some other studies also
chemically synthesized siRNAs have been shown to induce the type I IFN response (Sioud,
2005). Finally, avoidance of known immunostimulatory structures and choosing an alternative
delivery route when designing siRNA constructs could provide a mechanism of circumscribing
the induction of the type I IFN response. However, in anticancer therapy approaches, the gene
silencing is in most cases targeted to disrupted pathways to directly induce tumor cell death (Pai
et al., 2006). Therefore, innate immune responses induced by siRNA may not be as significant a
problem in cancer gene therapy as in other applications.
In summary, it is notable that much of the research characterizing the role of type I IFN response
against therapeutic gene transfer has been conducted in pDCs or in other types of immune
system cells, which produce substantially more type I IFNs than other cell types. This holds true
particularly with commonly used tumor cells that may have a defective IFN response. However,
depending on the administration route of gene delivery vehicle, the cells of the innate immune
system may be confronted. Therefore, these challenges need to be considered on a case by case
basis, since the type I IFN response may decrease the gene transfer efficiency and the duration of
therapeutic gene expression with either viral or non-viral methods.
35
2.3
UTILITY OF HIV-1 TAT PROTEIN TRANSDUCTION DOMAIN IN CANCER GENE
THERAPY
2.3.1
OVERVIEW
The eukaryotic plasma membrane is largely impermeable to proteins and peptides. However,
over the past decades, several studies have demonstrated the existence of a novel group of
proteins/peptides that do have the ability to penetrate through plasma membrane and even to
move from cell to cell. Consequently, these proteins/peptides have been named as protein
transduction domains (PTDs), cell penetrating peptides (CPPs), translocatory proteins or
alternatively as messenger proteins. This fascinating property has prompted several researchers
to investigate whether these proteins could be utilized to deliver therapeutic agents into the
target cell. When different macromolecules have been chemically linked or fused with PTDs,
these molecules have been shown to be taken up by cells both in vitro and in vivo (Schwarze and
Dowdy, 2000; Ford et al., 2001). The mechanism of membrane translocation is not fully
understood, but a common feature for these peptides is their cationic charge. The three most
widely studied CPPs are domains from herpes simplex virus structural protein VP22 (Elliott and
O'Hare, 1997) Drosophila homeotic transcription factor Antennapedia (Antp) (Joliot et al.,
1991) and HIV-1 trans-activator protein (TAT) (Frankel and Pabo, 1988; Green and
Loewenstein, 1988).
2.3.2
HIV-1 TAT PROTEIN TRANSDUCTION DOMAIN
The trans-activator of transcription (TAT) is a pleiotropic protein of human immunodeficiency
virus 1 (HIV-1) and it has a central role in controlling viral gene expression and replication. The
transcription from the HIV-1 provirus promoter is inefficient; however, TAT does possess the
ability to increase greatly the transcription of provirus DNA. This transactivation has been
shown to be mediated by binding of the TAT protein into so-called transactivation responsive
(TAR) RNA element in the 5´ end of nascent mRNA, which is proposed to promote elongation
of newly initiated transcriptants. TAT has also been claimed to increase the amount of stable
RNA polymerase II initiation complexes (Luciw, 1996). The full-length TAT protein is encoded
by two exons that encode a 101 amino acid -long protein comprising an acidic domain, a
cysteine-rich domain, a core region and a basic region (Fig 3.) (Fittipaldi and Giacca, 2005).
Despite the lack of the secretory signal and nuclear export signal, the full-length TAT protein is
secreted from HIV-1 infected cells without cell death and exerts multiple functions in the
extracellular milieu after its release (Ensoli et al., 1990; Rubartelli et al., 1998).
36
EXON I
N
ACIDIC
CYS-RICH
CORE
EXON II
BASIC
C
PROTEIN TRANSDUCTION DOMAIN
(YGRKKRRQRRR)
Figure 3. Schematic representation of HIV-1 transactivator protein (TAT) and the 11 amino acid
protein transduction domain located in the basic region.
One of the extracellular features of TAT protein is its ability to cross the plasma membrane and
to translocate into the nucleus in order to transactivate the long terminal repeat (LTR) of the
HIV-1 provirus. This was discovered simultaneously by two independent research groups
(Frankel and Pabo, 1988; Green and Loewenstein, 1988). When this feature was studied in more
detail, it was found that also the truncated form, containing mainly amino acids from the basic
region, could pass through the plasma membrane. It supports both the cytoplasmic and nuclear
localization (Mann and Frankel, 1991) and has the ability to promote the delivery of
heterologous (e.g. -galactosidase) proteins in vitro and in vivo when they are chemically linked
with TAT (Fawell et al., 1994). Further, it has been shown that the 11 amino acid basic region of
TAT47-57 alone is able to confer cellular delivery of in-frame fusion proteins into mammalian
cells (Nagahara et al., 1998). The TAT47-57 sequence is usually what is ment by the term TAT
protein transduction domain (TAT PTD), although even shorter sequences (8 amino acids) have
been shown to possess cell penetrating activity (Cascante et al., 2005). Today, the feasibility of
using TAT PTD to transport therapeutic agents into cultured cells and tissues has been
demonstrated in a number of diverse applications, some of which are illustrated in Fig. 4. The
experiments have used liposomes, adenoviruses, pDNA, bacteriophage and a number of proteins
and peptides (Fig. 4) as cargo.
37
ISCHEMIA
Protection against
ischemic brain injury
using anti-apoptotic protein
(Cao et al., 2002)
MANIPULATION OF STEM CELLS
Delivery of transcription factor and induction
of insulin production in human embryonic
stem cells (Kwon et al., 2005)
PHAGE DISPLAY
Introduction of phage and
expression of marker genes
in vitro and in vivo
(Eguchi et al., 2001)
VIRAL GENE DELIVERY
Enhanced transduction efficiency
(Gratton et al., 2003)
MONOGENIC DISORDER
Correction of purine nucleoside
phosphorylase deficiency in vivo
(Toro and Grunebaum, 2006)
NON-VIRAL GENE TRANSFER
Improved efficiency and reduced
cytotoxicity of liposome-mediated
transfection (Torchilin et al., 2003)
Figure 4. Examples of applications utilizing HIV-1 TAT protein transduction domain.
2.3.3
MECHANISM OF TAT PTD INTERNALIZATION
The underlying mechanism to explain how TAT PTD containing molecules enter into cells is not
yet fully understood. There are a number of conflicting theories, but there is major agreement
that the initial step for membrane translocation is the occurence of an ionic interaction between
the positively charged TAT PTD and the negatively charged molecules on cell membrane. This
event takes place particularly with heparan sulphate proteoglycans (HSPG) (Console et al.,
2003; Richard et al., 2005), which also initiates membrane translocation of the full-length TAT
protein (Tyagi et al., 2001). However, all studies do not confirm the proposal that expression of
HSPG is essential for TAT-mediated entry (Silhol et al., 2002; Violini et al., 2002). The
pioneering translocation studies proposed different mechanisms for TAT-mediated entry, such as
direct penetration, since internalization appeared to occur via in an endosome independent
manner (Vives et al., 1997). However, until now, the strongest evidence of TAT internalization
indicates that TAT PTD is taken up by different forms of endocytosis and after internalization it
is released into cytoplasm to at least to some extent. In addition to the most common type of
endocytosis in mammalian cells, the clathrin-mediated endocytosis (Richard et al., 2005), also
caveolae-mediated endocytosis (Ferrari et al., 2003) as well as macropinocytosis (Wadia et al.,
2004) have been demonstrated to participate in TAT PTD-mediated entry. It seems most likely
that the TAT PTD can enter cells using a variety of pathways, which are apparently dependent
on the type of cargo, as well as on the target cell type (Mai et al., 2002; Brooks et al., 2005;
38
Tunnemann et al., 2006). In addition, the degree of intracellular delivery has been also shown to
be dependent on the type of cargo and target cell as well as concentration of the TAT PTD
containing molecules (Mai et al., 2002; Silhol et al., 2002).
2.3.4
ANTICANCER APPROACHES UTILIZING TAT PTD
The majority of studies that have demonstrated the utility of TAT PTD have been performed by
adding exogenously synthesized (produced mainly in bacterial cells) fusion proteins in cell
culture or in vivo. These studies have mostly been translocation studies, attempting to
characterize the mechanism of protein transduction. A wide range of different cargos have been
successfully delivered mostly to cultured cells but also to animal tissues. However, the ultimate
goal of many of these studies has been to discover new tools to treat various diseases.
Neurobiological disorders have been a focus of particular interest, presumably due to the
observation that TAT PTD is able to pass through the blood brain barrier (Schwarze et al.,
1999). In the context of cancer gene therapy, one attractive feature of TAT PTD is its versatile
mode of action. Several anticancer peptides or proteins, either enhancing apoptosis or correcting
defective pathways, have been introduced successfully into tumor cells, with promising
therapeutic outcomes in pre-clinical animal studies (Fulda et al., 2002; Harada et al., 2002;
Snyder et al., 2004; Snyder et al., 2005). In addition to direct killing of tumor cells, nucleic acid
cancer vaccines utilizing TAT-mediated antigen delivery to DC cells have been shown to evoke
antitumoral effect (Shibagaki and Udey, 2002; Shibagaki and Udey, 2003). Not only does TAT
PTD represent a novel method to deliver therapeutic material into the malignant cells, but this
property has also been harnessed to improve the efficacy of existing gene delivery vehicles and
cancer gene therapy strategies, including suicide gene therapy (Tasciotti et al., 2003; Kuhnel et
al., 2004; Cascante et al., 2005; Tasciotti and Giacca, 2005). Selected examples of applications
of TAT PTD in cancer protein/gene therapy are shown in Table 3.
39
Table 3. Examples of applications of TAT PTD in anticancer therapy
APPLICATION
CARGO
EFFECT
REFERENCE
Targeted TATmediated anti-cancer
therapy
CXCR4-ligand and
anticancer peptide
linked with TAT
Enhanced killing of tumor
cells expressing CXCR4
receptor in vitro
(Snyder et al., 2005)
DC based cancer
vaccine
Model tumorassociated antigen
Induction of antitumoral
CTLs and partial tumor
regression in mice
(Shibagaki and
Udey, 2002)
Delivery of antitumoral peptide
Modified p53 peptide
Tumor growth regression
and extension of survival in
mice
(Snyder et al., 2004)
Delivery of hypoxia
stabilizing domain
and anti-tumoral
protein
ODD- caspase3 fusion
protein
Cell death in hypoxic tumor
regions
(Harada et al., 2002)
Delivery of antitumoral peptide
Smac,
co-administered with
TRAIL
Synergistic antitumoral
effect and extension of
survival in mice
(Fulda et al., 2002)
Viral gene delivery
Replication deficient
and -competent
adenoviral vector
Enhancement of
transduction efficiency and
oncolysis
(Kuhnel et al., 2004)
HSV-TK suicide gene
Increased cell killing in
vitro and in vivo
(Tasciotti et al.,
2003; Cascante et
al., 2005; Tasciotti
and Giacca, 2005)
Suicide gene therapy
Abbreviations: CXCR4, CXC chemokine receptor 4; DC, dendritic cell; CTL, cytotoxic T-cell; ODD,
oxygen dependent degradation; Smac, second mitochondria-derived activator of caspase; TRAIL, tumor
necrosis factor-related apoptosis inducing ligand; HSV-TK, herpes simplex virus thymidine kinase
2.3.5
MOVEMENT OF TAT-FUSION PROTEINS BETWEEN CELLS
In most cancer gene therapy approaches, it is essential that one achieves a high percentage of
tumor cells containing the therapeutic protein if one wishes to achieve successful treatment.
Initially it was thought that TAT PTD would be able to move intercellularly and that particular
capability would be highly beneficial in cancer gene therapy. Though the full-length TAT
protein can exit infected cells via some yet uncharacterized secretory pathway (Ensoli et al.,
1990), this property does not seem to be shared by TAT PTD, which appears to remain in the
nucleus due to its cationic charge and strong nuclear localization signal. Since TAT PTD does
not possess either a nuclear export signal or a secretion signal, it apparently does not have any
transcellular capacity itself (Chauhan et al., 2007). Cashman and co-workers did not detect any
intercellular trafficking to adjacent cell from cells expressing the fusion protein, even though
40
membrane translocation was observed when fusion proteins were added exogenously (Cashman
et al., 2003). This is in agreement with a study indicating that after plasmid transfection, de novo
synthesized TAT-fusion proteins are not trafficked between the cells and do not enhance the
therapeutic effect (Leifert et al., 2002). Furthermore, it has been noted that under certain
conditions, the membrane translocation property of TAT-fusion proteins may represent an
artifactual phenomenon associated with the cell fixation procedure (Leifert et al., 2002;
Lundberg et al., 2003; Richard et al., 2003). It has been demonstrated that due to strong ionic
interactions, TAT-peptide is bound onto the cell surface, and cannot be internalized. During
fixation, the cell membrane can become porous, allowing a redistribution of TAT-peptide into
cytoplasm and further into the nucleus, where it binds to the negatively charged DNA.
Therefore, in the case of fluorescent fusion proteins or labels, if cells are not treated properly
with protease digestion prior to fluorescence microscopy or flow cytometric analysis, false
positive results may arise, since TAT-peptide binds tightly to cell membrane (Richard et al.,
2003). These pieces of evidence doubting the veracity of protein transduction should be
considered critically, particularly when evaluating validity of the TAT PTD-mediated protein
transduction in combination with fluorescent fusion proteins or conjugates.
Nevertheless, there are a few studies demonstrating the intercellular spread of TAT containing
fusion proteins. TAT PTD fused with -glucuronidase expressed by adenoviral vector or adenoassociated vector has been shown to improve the biodistribution of therapeutic protein (Xia et
al., 2001; Elliger et al., 2002). However, a small amount of -glucuronidase protein is secreted
also under normal conditions and furthermore, in the latter study the export of the enzyme was
enhanced by the insertion of a secretion signal peptide into the fusion construct (Elliger et al.,
2002). Fusion of TAT PTD to HSV-TK has enhanced cell killing of cultured tumor cells and
tumor eradication. In these studies TAT-TK fusion genes were delivered into tumor cells using
adeno-associated viral vector or electroporation mediated plasmid DNA transfer (Cascante et al.,
2005; Tasciotti and Giacca, 2005). Once again, intercellular spreading most likely does not
occur, instead the fusion protein is presumably released from the GCV treated, degraded cells
and thereafter taken up by adjacent cells. Finally, if TAT-mediated anti-tumoral effect is targeted
directly to kill tumor cells, the lack of an intercellular capacity will not impede its therapeutic
properties. However, if an enhanced therapeutic effect is sought by affecting the adjacent cells,
the TAT-fusion protein needs to contain a secretory signal or a boosting effect should appear
after cell destruction, such as that occuring in the context of suicide gene therapy.
41
2.3.6
FUTURE CONSIDERATIONS OF TAT-MEDIATED DELIVERY
If, or when TAT PTD is internalized by endocytosis, it is subsequently localized in endosomal
vesicles before fusion with lysosomes. Therefore, the critical question concerning different types
of cargos except for those that are being targeted to the lysosomes, is how can TAT PTD escape
from endosomes to cytoplasm before it is degraded in the lysosome? It is obvious that a small
fraction of internalized TAT-peptide containing fusion proteins does escape from endosomes,
since therapeutic effect has been demonstrated in several reports without using agents that
disrupt endosomes. However, some studies have described that the efficacy of TAT PTDmediated protein delivery can be enhanced by using lysosomotropic agents, such as chloroquine
(Caron et al., 2004; Wadia et al., 2004). Alternatively, the escape of TAT fusion proteins from
macropinosomes has been enhanced by co-delivering fusogenic domain from influenza virus in
trans (Wadia et al., 2004). Therefore, the development of constructs that allow escape from
endosomes could be considered as one way to enhance TAT PTD-mediated delivery.
Although the ability of TAT PTD to deliver its cargo to all types of cells is beneficial when
compensating for the poor gene delivery rate of current vectors in vivo, unlimited entry of
therapeutic protein has obvious disadvantages in cancer gene therapy. Due to the loss of
distributed TAT PTD, doses of peptide have to be increased in order to achieve an adequate
therapeutic effect in the target tissue (Vives, 2005). Furthermore, increased concentrations TAT
PTD can cause toxicity and similar to the situtation with other anticancer therapies, non-tumor
cell specific protein transduction is undesirable. Distribution of pro-apoptotic agents or cytotoxic
compounds in all tissues would clearly be expected to induce severe side effects. Viral vectors
can be targeted by modifying the viral surface composition to restrict the host cell range or by
localizing therapeutic protein expression using tissue specific promoters, but these methods
cannot be applied to target TAT PTD. Particularly in the situation when events on the cell
membrane are not completely understood and non-specific binding, such electrostatic
interactions can occur, it is difficult to achieve targeted delivery. When fusion proteins are
administered intratumorally, this may not be a major concern, but it can become a problem when
fusion proteins are administered systemically. One approach to increase target cell specificity is
to construct fusion a protein that contains, in addition to TAT PTD and therapeutic the agent, a
ligand for receptor that is expressed on the surface of tumor cells. Snyder et al observed
increased tumor cell killing using ligand in a TAT PTD-p53 fusion construct that binds to
protein overexpressed in several tumor types (Snyder et al., 2005).
42
Clearly, there are many questions associated with TAT-mediated delivery that need to be solved
before the true potential of the TAT-peptide will be clarified. Furthermore, many technical
hurdles must be overcome in the future before we understand the exact mechanisms of action in
TAT PTD-mediated entry in a given target cell type. Although characterization of the utility of
TAT PTD in cancer gene therapy is only in its infancy, different anti-cancer strategies have been
successfully introduced in the pre-clinical studies (Table 3). The crucial feature that needs to be
resolved since it has a impact on the utility of TAT-peptide in cancer gene therapy is whether it
possesses a true ability to move intercellularly. Should it transpire that TAT PTD can move from
cell to cell, it clearly has the potential to improve the efficacy of therapeutic gene delivery in
cancer.
43
3 AIMS OF THE STUDY
The main purpose of this study was to evaluate hurdles involved with therapeutic gene transfer
and to find solutions to improve gene delivery into tumor cells.
The specific aims were:
1. To evaluate the contribution of type I interferon response to therapeutic gene transfer against
cancer (I)
2. To improve the transduction efficiency of viral vectors with the aid of cationic cell penetrating
peptides (II)
3. To enhance the HSV-TK/GCV cancer gene therapy by increasing the movement of
therapeutic protein between the cells using the HIV-1 TAT cell-permeable peptide (III, IV)
44
4 MATERIALS AND METHODS
The following tables contain a summary of the methods, cell lines, viral constructs, RNA
constructs, plasmid DNA constructs, peptide sequences and antibodies used in studies I-IV. The
detailed description of different methods is provided in original publications I-IV.
Table 4. Methods used in studies I-IV
METHOD
REF.
DNA TECHNIQUES
Construction of TAT-TK-GFP, TK-GFP and
VP22-GFP fusion genes
III
RNA TECHNIQUES
In vitro transcription of mRNA
I
RNA isolation
I
PRODUCTION OF VIRAL
VECTORS
Lentiviral vectors
I, III, IV
Adenoviral vectors
I
VIRAL GENE DELIVERY
See table 6
NON VIRAL GENE
DELIVERY
Commercial transfection reagents
I, III
Electroporation
I
ANALYSIS OF GENE
TRANSFER EFFICIENCY
Analysis GFP positive cells by flow cytometry
I, II, III, IV
Fluorescence microscopy
III
Western blotting
III, IV
ANALYSIS OF INDUCTION
OF TYPE I INTERFERON
RESPONSE
Western blotting
I, IV
Biological interferon assay
I
Quantitative RT-PCR
I
HSV-TK/GCV
CYTOTOXICITY
ANALYSIS
GCV treatment
III, IV
Cell viability determination by MTT-assay
III, IV
STATISTICAL ANALYSES
Mean and SD
II, III, IV
Analysis of variance
II, III, IV
Analysis of area under curve
IV
45
Table 5. Cell lines used in studies I-IV
CELL LINE
DEFINITION
REF.
293T
Human embryonic kidney (a gift from Garry Nolan)
I, III, IV
HUVEC
Human umbilical vein endothelial cells (CC-2517)
I
NHDF
Normal human dermal fibroblast (CC-2511)
I
HEp2F
Human hepatoma (a gift from Ilkka Julkunen)
I
U-251 MG
Human astrocytoma (JCRB IFO50288)
I
A172
Human glioma (ATCC CRL-1620)
III
HeLa
Human cervix carcinoma (ATCC CCL-2)
I, IV
SiHa
Human cervix carcinoma (ATCC HTB-35)
IV
A549
Human lung carcinoma (ATCC CCL-185)
I, IV
SW900
Human lung carcinoma (ATCC HTB-59)
IV
SKOV3.ip1
Human ovarian carcinoma (a gift from David Curiel)
I, II, III
OV-4
Human ovarian carcinoma (a gift from David Curiel)
III
Hey
Human ovarian carcinoma (a gift from Judy Wolf)
II, IV
PC-3
Human prostate carcinoma (ATCC CRL-1435)
I, II, III
MG-63
Human osteosarcoma (ATCC CRL-1427)
II
U-2 OS
Human osteosarcoma (ATCC HTB-96)
IV
SW1353
Human chondrosarcoma (ATCC HTB-94)
IV
TE671
Human rhabdomyosarcoma (ATCC HTB-139)
IV
A2058
Human melanoma (ATCC CRL-11147)
IV
SK-MEL-5
Human melanoma (ATCC HTB-70)
IV
IV
CHO
Chinese hamster ovary (a gift from Marika Ruponen)
pgsB-618
Mutant chinese hamster ovary (a gift from Marika Ruponen)
IV
pgsD-617
Mutant chinese hamster ovary (a gift from Marika Ruponen)
IV
BHK
Baby hamster kidney (ATCC CRL-1632)
I
BT4C
Rat glioma (a gift from Rolf Bjergvik)
III
COS-7
Monkey kidney fibroblast (a gift from Marika Ruponen)
II
46
Table 6. Viral vectors used in studies I-IV
VECTOR
CHARACTERISTIC
REF.
nd
WOX TK-GFP
2 generation VSV-G pseudotyped lentiviral vector, expressing
TK-GFP under EF1 promoter
II, III, IV
WOX
TAT-TK-GFP
2nd generation VSV-G pseudotyped lentiviral vector, expressing
TAT-TK-GFP under EF1 promoter
III, IV
HPT GFP
2nd generation VSV-G pseudotyped lentiviral vector,
expressing eGFP under EF1 promoter
I
Ad TK-GFP
1st generation E1/E3 deleted serotype 5 adenoviral vector,
expressing TK-GFP under CMV promoter
I, II
Ad5 TK-GFP
Conditionally replicative E3 deleted serotype 5 adenoviral
vector, expressing TK-GFP under CMV promoter
I
VSV
Vesicular stomatitis virus, Indiana strain
I
SFV (A7[74])
Attenuated strain of Semliki Forest virus
I
AAV-eGFP
Recombinant adeno-associated virus, expressing eGFP under
CMV promoter
I
Table 7. RNA constructs used in studies I-IV
RNAs
CHARACTERISTIC
REF.
SIN VSV-G TK-GFP (SIN RNA)
In vitro transcribed self replicating mRNA
I
SFV LacZ
In vitro transcribed self replicating mRNA
I
TRI-Xef
In vitro transcribed mRNA
I
GreenLantern
In vitro transcribed mRNA
I
Total RNA
Total RNA from mouse liver
I
PolyA+ RNA
mRNA from mouse skeletal muscle
I
GFP22 siRNA
siRNA targeted to GFP
I
Table 8. Plasmid DNA constructs used in studies I-IV.
pDNAs
CHARACTERISTIC
REF.
pGreenLantern
Expression plasmid, GFP gene
I
pUC19 TK-GFP
Non-expression plasmid, TK-GFP gene
I, III
pWOX TK-GFP
Expression plasmid, TK-GFP gene
III
PWOX TAT-TK-GFP
Expression plasmid, TAT-TK-GFP gene
III
PWOX VP22-GFP
Expression plasmid, VP22-GFP gene
III
47
Table 9. Peptides used in studies I-IV.
PEPTIDES
SEQUENCE
REF.
Antennapedia (Antp)
RQIKIWFQNRRMKWKK
II
HIV-1 TAT PTD (TAT1)
YGRKKRRQRRR
II, III, IV
HIV-1 TAT PTD (TAT2)
GRKKRRQRRRPPQ
II
Table 10. Antibodies used in studies I-IV
ANTIBODY
DILUTION
MANUFACTURER
REF.
Rabbit anti-MxA
1:2000
A kind gift from Prof. Ilkka Julkunen
I, IV
Rabbit anti-GFP
1:200
Santa-Cruz
III, IV
Rabbit anti-actin
1:10 000
Sigma-Aldrich
IV
48
5 RESULTS AND DISCUSSION
5.1
TYPE I INTERFERON RESPONSE AGAINST VIRAL AND NON-VIRAL GENE
TRANSFER IN HUMAN TUMOR CELL LINES AND PRIMARY CELLS (I)
Mammalian cells have evolved various strategies to combat pathogens in order to ensure host
survival. Some of the main factors mediating these defense mechanisms are the type I
interferons (IFNs), which not only possess antiviral activity but also play a role in modulating
innate and adaptive immune system. It is a fact that the majority of the current gene delivery
vectors is based on viruses or alternatively may contain bacterial pDNA or various forms of
RNA that have immunostimulatory features. Since the activation of the target cell innate
immune system may have a negative impact on the expression of the therapeutic gene, it was
decided to evaluate the contribution of the type I IFN response to gene transfer. Several
commonly used viral vectors as well as non-viral DNA or RNA delivery methods were tested for
their ability to induce the type I IFN response in human tumor and primary cells. The induction
of the type I IFN response was evaluated by analyzing the accumulation of MxA-protein
(myxovirus resistance protein A), which is expressed in cells exposed to IFN or IFN (Haller
and Kochs, 2002). Moreover, the expression of type I IFNs was determined by analyzing
secreted IFN
proteins by a biological IFN assay or alternatively by measuring IFN mRNA
levels by quantitative PCR (qPCR). Gene transfer efficiencies were determined by analyzing
expression of the marker gene, the green fluorescent protein (GFP) using flow cytometry.
5.1.1
COMMONLY USED VIRAL VECTORS EVADE THE TYPE I IFN RESPONSE
The type I IFN response induced by viral vectors and viruses was first characterized in human
lung cancer cell line A549, since it is known to produce high amounts of IFNs during viral
infection (Ronni et al., 1997). The A549 cells were transduced with replication competent
attenuated SFV (A7 [74]), adenoviral vector, conditionally replicative adenoviral vector, AAV
vector or lentiviral vector. When the cells were analyzed at different time points posttransduction, accumulation of the MxA protein was observed only in those cells transduced with
SFV, whereas all of the studied viral vectors failed to induce the MxA response (I, Fig. 1 and
Table 1). Since the MxA is an indirect marker for induction of the type I IFN response, the
amount of released IFN
proteins was measured from culture media. However, no detectable
levels (detection limit 13 IU/ml) of IFN
were observed as a result of transduction with
adeno-, AAV- or lentiviral vector (I, Table 1). Competent transduction efficiencies (as judged by
49
analyzing transduction efficiencies by flow cytometry) of these viral vectors ruled out the
possibility that the absence of the type I IFN response was due to a low level gene transfer
efficiency (I, Table 1).
Further, the ability of adenoviral vector or SFV to trigger production of the type I IFNs was
studied in four additional human tumor cell types (HeLa, U-251 MG, SKOV3.ip1 and PC-3).
Similar to the situation in the A549 cells, no expression of either MxA protein or IFN mRNA
was detected after adenovirus-mediated gene transfer (I, Fig. 8 B, qPCR data not shown). On the
other hand, SFV induced accumulation of the MxA in PC-3 and U-251 cells, but not in HeLa or
SKOV3.ip1 cells, indicating that these responses are cell line specific (I, Fig. 8 B). The IFN
mRNA analysis revealed that SFV induced transcription of IFN to the greatest extend in PC-3
cells with lower levels of IFN mRNA being produced in HeLa, SKOV3.ip1 and U-251 MG
cells (I, data not shown). Again, the lack of MxA accumulation after adenoviral gene transfer
was not due to inefficient transduction efficiency in these cell lines (I, Fig. 8 B). Interestingly,
despite the induction of MxA protein, SFV was still able to replicate and express GFP at a high
level in A549, PC-3 and U-251 MG cells. Similar data has been obtained in animal studies,
where intratumorally injected SFV was able to replicate in mouse subcutaneous A549 tumors
despite the induction of the MxA response (Maatta et al., 2007). However, our finding is in
contrast to an earlier study indicating that the expression of MxA could impair replication of
SFV. In that study, the target cells were transfected with plasmid expressing the MxA protein
prior to SFV infection (Landis et al., 1998). The virus conferred antiviral MxA protein
immediately in cytoplasm when entering cell. In our study, on the other hand, the SFV infection
first induced the production of type I IFNs that led to an accumulation of the MxA protein. Since
SFV is a positive strand RNA virus, the genome can serve as mRNA for protein synthesis.
Therefore, it is possible that replication of SFV was already so massive by the time that the MxA
protein was produced that antiviral properties were insufficient to terminate or even hamper viral
replication. Furthermore, it should be kept in mind that these responses differ depending on the
viral strain. In addition, SFV is a rapidly replicating RNA virus, capable of undergoing
beneficial mutations in a rather short time in order to sustain viral replication.
Although adeno- or lentiviral vectors did not induce the expression of antiviral MxA protein, it
has been shown that both adenovirus and lentivirus (HIV-1) infection can induce the production
of the MxA protein (Chieux et al., 1998; Gurney et al., 2004). Expression of the MxA protein in
these studies was analyzed from thymocytes or whole blood cells that contain a number of
50
immune system cells. Those cells most likely produce higher amounts of type I IFNs than tumor
cells, which can explain why the response was not seen in our studies. A recent study showed
that VSV-G pseudotyped lentiviral vectors can induce the production of type I IFNs in pDCs due
to contamination with tubulo-vesicular structures that contain pDNA (Pichlmair et al., 2007).
Although the lentiviral vector used in our study also contained the VSV-G envelope protein, it
did not trigger the type I IFN response in A549 cells (I, Table 1). This could be explained by
target cell differences, but also due to the differences in lentiviral vector production and
purification protocols. However, in our experiments it is, possible that adeno- and lentiviral
vectors did trigger the production of the type I IFNs, but the concentration was less than the
detection limit of the biological IFN assay (13 IU/ml). If the IFNs were produced, it cannot be
ruled out that other IFN inducible proteins were activated in preference to MxA, accumulation of
which was not observed. Brown et al noted that systemically administered VSV-G pseudotyped
lentiviral vectors could turn on the expression of IFN inducible protein 2´-5´ oligoadenylate
synthetase (OAS) in mouse spleen and liver. The response was shown to primarily mediated by
type I IFN production by pDCs. The response was not dependent on the VSV-G glycoprotein,
since baculovirus glycoprotein (GP64) -pseudotyped virus induced production of OAS and
TNF as well. These results further demonstrated that the activation of type I IFN response
decreased the level and duration of transgene expression (Brown et al., 2006).
Recent studies indicate that also E1/E3 deleted adenoviral vectors can elicit the type I IFN
responses. A high level production of the type I IFNs has been detected after adenovirusmediated gene transfer to the cells of the innate immune system, such as pDCs and macrophages
(Huarte et al., 2006; Nociari et al., 2007; Zhu et al., 2007). However, it is uncertain whether
these responses had an impact on the transgene expression; Zhu et al claimed that the induction
of type I IFN response was crucial for adenoviral function in vivo, whereas Huarte et al did not
detect any interference by type I IFNs on the transgene expression (Huarte et al., 2006; Zhu et
al., 2007). Acsadi et al have previously shown that exogenously added IFN and IFN impair
adenovirus-mediated transgene expression in the muscle cells. However, it was not studied
whether the adenoviral vectors could trigger the production of the IFN
(Acsadi et al., 1998).
Comparison of these results to our data is complicated, since the induction of type I IFN
response has been characterized mainly in immune system cells, whereas we have studied the
response against adenoviral vectors in human tumor cells, which respond to a viral challenge in
a very different manner. Moreover, adenoviral transductions by Huarte et al and Zhu et al were
performed using higher amount of vectors (MOIs 250-1000) (Huarte et al., 2006; Zhu et al.,
51
2007) compared to the MOI values used in our study (MOI 10).
5.1.2
VARIOUS pDNA DELIVERY METHODS INDUCE THE TYPE I IFN RESPONSE
Plasmid DNA (pDNA) transfection is generally considered to be a safe gene transfer method.
However, the main hurdle with this gene delivery form is its inefficiency compared to viral
vectors. To study whether the non-viral gene transfer could induce the type I IFN response,
several commonly used pDNA delivery methods, including commercial transfection reagents
and electroporation were tested in A549 cells. Induction of the type I IFN response was
determined using an expression plasmid carrying the GFP gene (4 µg per ~3 x 105 cells). All
studied transfection reagent-pDNA complexes (ExGen ™ , DreamFect™ , LipofectAMINE
PLUS™ and SuperFect®) induced accumulation of MxA protein variably at different time
points (I, Fig. 4). All transfection reagent-pDNA complexes induced also detectable levels of
released type I IFNs (above 13 IU/ml), with the exception of SuperFect® -mediated transfection
(I, Fig. 4). The transfection with ExGen™ reagent appeared to be a potent inducer, leading also
to detectable expression of IFN mRNA (I, Fig. 5).
To exclude the possibility that the induction of the IFN response was raised against expressed
transgene, we transfected A549 cells with the promoterless plasmid (pUC19 TK-GFP) or
plasmid expressing GFP (pGreenLantern) with the FuGENE transfection reagent. In addition,
we evaluated whether the induction was dependent on the amount of delivered complex by
transfecting cells with either 2 µg or 4 µg of pDNA. Induction of the MxA response appeared to
be dose-dependent and increased as a function of time (I, Fig. 3). Despite the fact that the
accumulation of MxA was observed both using expression plasmid and silent plasmid, no
detectable levels of secreted IFN
were observed (I, Fig. 3). Furthermore, the qPCR analysis
did not reveal any synthesis of IFN mRNA after transfecting A549 cells with the silent plasmid
(I, Fig. 5). The reason for the lack of expression of IFN
despite the presence of MxA protein
was most likely due to the fact that MxA Western blotting is a more sensitive detection method
for induction of type I IFN response than the biological IFN assay. Secreted type I IFNs are
rapidly taken up by adjacent cells and are known to induce the expression of MxA protein
already at low concentrations (the doses of IFN <10 IU/ml in A549 cells) (Ronni et al., 1993).
52
Even though electroporation of 4 µg of pDNA resulted in a moderate transfection efficiency in
A549 cells (I, Fig. 4), in contrast to pDNA delivery by transfection reagents, it did not induce
synthesis of IFN
mRNA (I, Fig. 5), nor did it evoke the production of type I IFNs or
accumulation of MxA (I, Fig. 4). The "naked DNA" appeared to be taken up by A549 cells to a
very low degree (I, Fig. 4), which may explain why it was not able to induce a type I IFN
response (I, Fig. 4 and 5).
The type I IFN response induced by lipofection reagent/DNA complexes was most likely raised
against unmethylated CpG -rich sequences in the plasmid DNA (Hemmi et al., 2000).
Advantage has been taken of this immunostimulatory feature of CpG pDNA in combination with
cationic liposome transfection in cancer immunotherapy to induce antitumoral immune
responses (Rudginsky et al., 2001). However, on the other hand, some researchers have reported
suppressed gene expression levels as a consequence of the type I IFN response (Sellins et al.,
2005). However, we did not detect any clear correlation between transfection efficiency and
induction of MxA response. Thus, based on this experimental setting, it cannot be concluded
unequivocally whether the induction of type I IFN response contributes to DNA transfection
efficiency.
Interestingly, we found that the pDNA delivery by electroporation did not result in induction of
the type I IFN response (I, Fig. 4 and 5). The explanation for that observation could be a
different entry route of pDNA into the target cell. During electroporation, the cell membrane
becomes porous, allowing the transit of DNA molecules into the cytoplasm independently of
endocytosis (Somiari et al., 2000). Cationic liposome/DNA complexes, for their part, are taken
up mainly by endocytosis and are localized in endosomes (Dass, 2004), where they are
susceptible to recognition by TLR9 (Hemmi et al., 2000). In accordance with our results, Zhou
and co-workers recently observed a notable reduction in the production of cytokines (e.g. IL-12,
TNF
and IFN ) induced by pDNA electroporation compared to lipofection-mediated
transfection in vitro and in vivo (Zhou et al., 2007).
5.1.3
ALL TYPES OF RNA, EXCLUDING siRNA, TURN ON THE TYPE I IFN PRODUCTION
The genome of many viruses consists of RNA and furthermore, RNA appears as an intermediate
during replication of several viruses. Therefore, it is not unexpected that mammalian cells have
developed various mechanisms to detect foreign RNA (Meylan and Tschopp, 2006). However,
the use of RNA molecules instead of DNA, such as mRNA for protein expression or RNA
53
interference for silencing of expression, is one potential cancer gene therapy approach. We
evaluated the induction of type I IFN response in A549 cells against various forms of RNA,
including siRNA targeted to the GFP gene, total RNA from mouse liver, mRNA from mouse
muscle cells, in vitro transcribed mRNA and replicative mRNA based on Sindbis- (SIN VSV-G
TK-GFP) or Semliki Forest virus replicons (SFV LacZ), all delivered using the
TransMessenger™ transfection reagent.
Transfection of 2 µg of total RNA, mRNA from mouse muscle cells, in vitro transcribed mRNA
and replicative RNA strongly induced MxA accumulation in A549 cells and the response
increased as a function of time (I, Fig. 6 A and B). The critical amount of RNA (µg) to trigger
the MxA response induced by replicative RNA (SIN VSV-G TK-GFP, hence SIN RNA), total
RNA and in vitro transcribed mRNA was variable (I, Fig. 7 A-C). An amount as low as 0.02 µg
of replicative RNA was sufficient to turn on the expression of MxA, whereas 20 µg of total
RNA was required to induce the response (I, Fig. 7 A and B). Transfection of these RNA species
(SIN RNA, in vitro transcribed RNA and total RNA) induced also the production of IFN
mRNA (I, Fig 7 E). Furthermore, transfection of SIN RNA produced a notably higher release of
biologically active IFN
in A549 cells compared to the other pDNA or RNA species (I, data
not shown). Transfer of SIN RNA into different human tumor cell lines (HeLa, SKOV3.ip1, PC3 and U-251 MG) indicated that the induction of the IFN response was cell type specific; the
accumulation of MxA protein was seen in all four tested cell lines (I, Fig. 8 A), but the response
was weakest in HeLa cells. In agreement with the MxA analyses, detectable IFN mRNA
expression levels were observed in all of the cell lines, with the exception of the HeLa cells (I,
data not shown).
Synthetic siRNA targeted against the GFP gene suppressed its expression in a GFP expressing
cell line, but failed to induce the accumulation of MxA (I, Fig. 7 D). At the time when siRNA
technology was introduced, it was thought that these dsRNA molecules would be too short to
induce type I IFN response. Nevertheless, some recent studies have indicated that also siRNA
molecules are able mediate the induction of this response (Bridge et al., 2003; Sledz et al., 2003;
Kariko et al., 2004; Kim et al., 2004; Judge et al., 2005; Sioud, 2005; Marques et al., 2006). For
example this immunostimulatory impact has been recognized to be dependent on the siRNA
sequence (Judge et al., 2005; Sioud, 2005), as well as on the production method of the siRNA
molecules (Kim et al., 2004). In our study, the reason that the GFP -targeted siRNA succeeded
in avoiding inducing a of type I IFN response could be that this siRNA did not contain the 5´-
54
UGUGU-3´ sequence which has been claimed to be responsible for induction of this type of
response (Judge et al., 2005). Furthermore, the used siRNA molecules were produced by
chemical synthesis, which is known to be less immunostimulatory, compared to in vitro
transcribed siRNAs using a phage promoter system (Kim et al., 2004).
As discussed earlier, one way that mammalian cells can distinguish non-self DNA molecules
from self-DNA, particularly DNA of bacterial origin, is through differencies in the nucleoside
modification, such as DNA methylation. Similar mechanisms may also apply in discrimination
of non-self RNA. Kariko et al have suggested that mammalian total RNA is less
immunostimulatory compared to bacterial RNA, due to the higher degree of nucleoside
modifications. They also noted that all mammalian RNA species were not equally
immunostimulatory in monocyte derived DCs. The mammalian mitochondrial RNA induced the
highest production of TNF , whereas tRNA and polyA+ mRNA were the least
immunostimulatory. Moreover, in vitro transcribed RNA appeared to induce a high level of
TNF production (Kariko et al., 2005). This observation could partially explain our finding that
notably higher amounts (µg) of total RNA were required to induce MxA accumulation compared
to in vitro transcribed mRNA and SIN RNA (I, Fig 7 A-C). Finally, the observation that delivery
of mRNA could induce a type I IFN response raises doubt of the utility of mRNA as a gene
therapy tool under certain circumstances. In addition to the fact that mRNA might be recognized
by host cell TLRs (Diebold et al., 2004), these nucleic acids may also form double-stranded
secondary structures, rendering them recognizable to other intracellular host cell dsRNA
receptors, including PKR (Ceppi et al., 2005).
In contrast to pDNA transfection, induction of the type I IFN response had a significant impact
on the gene transfer rate of in vitro transcribed SIN RNA (as measured by analyzing the GFP
expression by flow cytometry). The transfection efficiency of SIN RNA was relatively low in
several human tumor cell lines after electroporation or lipofection -mediated gene transfer. This
was most probably due to the induction of an intense IFN
response against the delivered
RNA (unpublished data). However, in BHK cells, which may produce little or no interferon
(Schlesinger and Dubensky, 1999), the expression of the transgene was high and this selfreplicating SIN RNA was able to spread throughout the BHK cell population within a few days
leading to cell death (unpublished data). To verify the role of the type I IFN response, we
exposed BHK cells to human recombinant IFN
prior to, simultaneously with or shortly after
transfection with self-replicating SIN RNA. Regardless of the time of initiation of treatment, the
55
presence of IFN
inhibited expression and replication of SIN RNA in a dose-dependent
manner (unpublished data). Delivery of SIN RNA in vivo also appeared to be highly inefficient.
Transfection of SIN RNA by electroporation into the subcutaneous BHK tumors in nude mice or
delivery by gene gun to mouse skin resulted in only a few transgene expressing cells. One
explanation for the poor gene delivery rate in vivo could be the induction of the type I IFN
response. It is possible that gene transfer to skin, a tissue where a number of dendritic cells are
known to be present, triggered antiviral innate responses and inhibited expression and replication
of SIN RNA. The same response could have been triggered in BHK tumors due to activation and
production of IFNs by other cell types present in the tumor tissue.
5.1.4
TYPE I INTERFERON RESPONSE IN HUMAN PRIMARY CELLS
Fibroblasts and endothelial cells are frequently the first cell types that confront pathogens and
therefore these cells play a role in the host cell defense system. To evaluate whether gene
transfer to these cell types could trigger an IFN response, the effects of mRNA and pDNA
transfection as well as SFV infection were studied in HUVEC (endothelial cells) and NHDF
(fibroblasts) primary cells. SFV infection and delivery of silent pDNA with both FuGENE and
ExGen transfection reagents induced accumulation of MxA in studied fibroblasts, but not in
endothelial cells (I, Fig. 9 A and B). However, the transfection of replicative SIN RNA or in
vitro transcribed mRNA initiated MxA accumulation in both primary cell lines (I, Fig. 9 A and
B). These results with human primary cells further confirmed that the induction of type I IFN
response is clearly cell type specific.
5.2
CATIONIC CELL-PERMEABLE PEPTIDES ENHANCE TRANSDUCTION
EFFICIENCY OF VIRAL VECTORS IN HUMAN TUMOR CELL LINES (II)
Cell penetrating peptides (CPPs) have been shown to possess an attractive ability to pass through
the plasma membrane alone or to deliver different molecules when these are attached to CPP.
Recently Gratton and co-workers demonstrated that peptides derived from Drosophila
Antennapedia homeodomain (Antp) and HIV-1 transactivator protein (TAT) could significantly
enhance adeno- and retroviral transduction efficiency in cultured cells (COS-7, bovine aortic
endothelial cells and HUVEC) and in vivo into mouse arteries, muscle and skin (Gratton et al.,
2003). However, they did not test the ability of these peptides to boost gene delivery to tumor
cells. Since several cancer gene therapy approaches suffer from insufficient gene transfer, we
evaluated whether these cationic CPPs could be useful tools for enhancing the gene transfer rate
56
of adeno- and lentiviral vectors to human tumor cells. The efficacy of TAT-peptide (two
different sequences, named TAT1 and TAT2) and Antp-peptide was tested in human tumor cell
lines that are generally rather resistant to adeno- and lentiviral transduction. Two different
sequences derived from HIV-1 TAT were chosen; TAT1 (YGRKKRRQRRR) which is the most
commonly used sequence, whereas TAT2 (GRKKRRQRRRPPQ) was the peptide that Gratton
et al presumably tested. Prior to transduction (MOI 1), the viral vectors were complexed with
TAT1, TAT2 and Antp-peptide or alternatively, with two commercially available and commonly
used polycationic transduction enhancers, polybrene and protamine.
First, we tested the impact of cationic peptides on viral transduction efficiency in COS-7 cells to
verify the previously demonstrated results (Gratton et al., 2003). All three tested cell-permeable
peptides (Antp, TAT1 and TAT2) enhanced significantly the transduction of both adeno- and
lentiviral vector to COS-7 cells (P<0.001), with the exception of the TAT2-peptide complexed
with adenoviral vector (II, Fig. 1 A and B). However, we found that these effects were not as
distinctive as described earlier (Gratton et al., 2003). It is possible that there were some
differences in the experimental procedures between our study and that performed by Gratton et
al. Furthermore, the quality of adenoviral vectors and/or peptides could have been different, thus
decreasing the efficacy of the CPPs in our studies. Cell lines can accumulate phenotypical
changes during extended culturing and thereby the properties of the COS-7 cell line studied in
our experiments may not have been identical to those cells used in the study of Gratton et al.
However, the presence of cationic CPPs enhanced both adenoviral and lentiviral transduction
efficiency significantly in all studied human tumor cell lines except for the MG-63 cells. These
cells were highly resistant to lentivirus transduction overall (II, Fig. 2 A). The MG-63 cells are
known to possess an ability to produce high amounts of interferon (Billiau et al., 1977).
Therefore, it is possible that the lentiviral transduction was sufficient to cause sufficient stress in
MG-63 cells, to trigger the production of type I IFNs. However, further studies are needed to test
this hypothesis. With respect to the cell penetrating peptides, Antp was the most powerful
enhancer of transduction in all of the studied cell lines (P<0.001) again with the exception of
MG-63 cells. TAT1 increased the transduction efficiency almost as efficiently as Antp, but
TAT2 had clearly the weakest impact on transduction efficiency. The influence of peptides was
similar in all of the studied cell lines, suggesting that the enhancement was based on electrostatic
interactions rather than being dependent on the target cell surface composition. Two commonly
used polycations, polybrene and protamine, were also potent transduction enhancers. Indeed, it
57
transpired that polybrene was the most effective booster compared to cationic peptides in
virtually all cell lines. Only in SKOV3.ip1 cells, did Antp increase the transduction more than
polybrene. The impact of protamine was similar than those of Antp and TAT1 (II, Fig, 2 A and
B). The reason why Gratton et al used a different TAT PTD –derived peptide (namely TAT2)
than the type commonly used, is not clear. However, the amino acid sequence had decisive role,
since impact of TAT2 peptide to transduction efficiency was significantly worse than TAT1, yet
both peptides had similar presumed net charge at pH 7.
Lentiviral vectors, particularly VSV-G pseudotyped, can transduce efficiently human tumor cells
(Pellinen et al., 2004). However, it is challenging to produce of high concentrations of lentiviral
vectors which is one limiting factor regarding the use of this vector type in clinical trials. Since
polybrene is not clinically approved, CPPs could reprensent alternative mechanism to
compensate for low viral vector titers by increasing transduction efficiency. Nevertheless,
protamine, which can be used also in a clinical setting, enhanced the gene delivery of both viral
vectors equally well as the cationic peptides.
The primary receptor for adenoviral serotype 5 –based vectors (the most widely used serotype in
adenoviral gene therapy) is the coxsackie- and adenovirus receptor (CAR) (Bergelson et al.,
1997). CAR is expressed at variable levels in different cell types and low-level expression of
CAR is considered to be one of the key factors hindering the adenoviral gene delivery. Kühnel et
al observed that the cell penetrating peptides VP22, TAT and Antp increased gene delivery rate
of replication deficient adenoviral vector and enhanced oncolysis of conditionally replicating
adenoviral vectors in vitro. The CPPs were fused with the extracellular domain of CAR receptor,
thus these fusion proteins were designed to act as adapter molecules between the target cell
membrane and adenoviral fiber knob protein. The CAR-CPP fusion proteins facilitated
adenoviral transduction to non-permissive cells (e.g. osteosarcoma SAOS-2 cells), indicating
that these fusion proteins could be used to broaden the host cell range of the adenoviral vector.
CAR-Antp fusion protein appeared to have the poorest transduction enhancing property
compared to VP22 and TAT. Moreover, the CAR-VP22 and CAR-TAT fusion proteins
improved significantly adenoviral gene delivery to permissive tumor cells, like cervix carcinoma
HeLa cells and osteosarcoma U-2 OS cells. However, in that study, TAT alone (without CAR)
was not able improve the extent of adenoviral transduction (Kuhnel et al., 2004).
58
5.3
UTILITY OF TAT-TK-GFP TRIPLE FUSION PROTEIN IN HSV-TK/GCV BASED
SUICIDE GENE THERAPY (III, IV)
Suicide gene therapy using herpes simplex virus type I thymidine kinase (HSV-TK) in
combination with ganciclovir (GCV) has proved to be a promising treatment for cancer.
However, the clinical efficacy has appeared to be limited, most often due to the low number of
tumor cells expressing the therapeutic protein and a weak bystander effect to surrounding tissue.
While vector development to improve gene transfer efficiency is extremely important,
enhancement of the bystander effect could also compensate for the poor gene delivery.
Furthermore, extension of bystander effect is primarily required under circumstances where the
therapeutic gene is transferred to healthy tissue instead of tumor (e.g. in treatment of malignant
glioma). In attempt to improve the efficacy of HSV-TK/GCV therapy, we fused the sequence of
HIV-1 TAT protein transduction domain corresponding to amino acids (47-57) with the
previously constructed TK-GFP gene (Loimas et al., 1998). The intention was to enhance
suicidal protein traffic from the transduced cells to the adjacent, non-transduced cells and thus to
increase the cell death. In order to study the utility of the triple fusion protein containing TATpeptide, HSV-TK and green fluorescent protein (TAT-TK-GFP) in HSV-TK/GCV suicide gene
therapy, the TAT-TK-GFP was inserted into a second generation VSV-G pseudotyped lentiviral
vector (III, Fig. 1 A)
5.3.1
EXPRESSION OF TAT-TK-GFP TRIPLE FUSION PROTEIN
First, the expression of TAT-TK-GFP fusion protein was verified in OV-4 cells transduced with
lentiviruses carrying TAT-TK-GFP or TK-GFP fusion genes (by Western blotting using an antiGFP antibody). Both fusion proteins were expressed at a similar level, resulting in bands with
protein sizes of ~80 kDa (III, Fig. 1 B). However, when several human tumor cell lines were
analyzed by flow cytometry, the mean expression level of TAT-TK-GFP fusion proteins was
substantially lower compared to TK-GFP in most of the cell lines (IV, data not shown). We
further examined the expression of TAT-TK-GFP in A549 and A2058 cells by Western blotting
after transduction with lentiTAT-TK-GFP or lentiTK-GFP viruses. Interestingly, the expression
of TAT-TK-GFP protein was notably down-regulated in both cell lines compared to cells
expressing TK-GFP (IV, Fig. 2). The observation that in OV-4 cells both fusion proteins were
expressed at a similar level could be explained by the fact that fusion proteins had been
overloaded onto the gel and the intensity of protein band was saturated, thus making it difficult
to distinguish the differences. However, in A2058 and A549 cells, the proportions of fusion
59
proteins loaded on the gel were lower and down-regulation of TAT-TK-GFP was observed.
Interestingly, also Cashman and co-workers have reported the down-regulation of GFP protein
fused with either full-length TAT protein or TAT PTD in human conjunctiva epithelial, Chang C
cells. Down regulation of GFP was observed both after adenoviral transduction and pDNA
transfection, thus indicating that down-regulation was not due to interference of TAT protein or
TAT PTD with some unknown adenoviral components. Furthermore, TAT-induced cytotoxicity
was ruled out, since the adenoviral vector was carrying also red fluorescent protein, the
expression of which was not down-regulated. One explanation was that the fusion of TAT or
TAT PTD to GFP may have targeted fusion proteins to proteasomal degradation at a higher rate
than the native GFP protein (Cashman et al., 2003). This hypothesis might explain also our
observations. On the other hand, in certain cell, lines the viability of the TAT-TK-GFP
expressing cells was slightly lower compared to TK-GFP containing cells (IV, Fig. 1). This may
indicate TAT-peptide induced cytotoxicity at some degree. However, we did not detect any
notable cytotoxicity in various tumor cell lines in the presence of exogenously added TAT PTD
at a concentration 0.5 mM (as judged by microscopical examination) (II, data not shown).
Nevertheless, Jones et al have shown that concentrations above 100 µM of exogenously added
TAT PTD tend to be toxic to different cell lines, including A549, HeLa and CHO cells (Jones et
al., 2005).
It was also notable that the TU (transducing unit) titers of TAT-TK-GFP viruses were 2-10 fold
lower than TK-GFP viruses that were produced at the same time and under identical conditions,
whereas the particle titers of these two viruses were comparable (IV, data not shown). During
the preparation of study IV manuscript, we learned that it has been demonstrated that
exogenously added TAT PTD can compete with full-length TAT protein in binding to TAR
RNA, thus inhibiting the production of second generation lentiviral vectors (Mi et al., 2005).
Earlier, Dull et al have shown that the TAT protein is obligatory in producer cells to activate
transcription from HIV-1 LTR and to produce lentiviral vectors with high transducing activities.
However, the presence of TAT was not indispensable, since HIV-1 LTR could be substituted
with chimeric promoter, e.g. CMV promoter (Dull et al., 1998). Therefore, production of viral
particles without TAT-TK-GFP vector genome (empty particles) was most likely due to the fact
that TAT-TK-GFP was able to bind to TAR RNA and to inhibit efficient transcription of the
vector genome.
60
However, the impact of possible TAT-peptide/TAR RNA interaction on the expression of TATTK-GFP after transduction remains unclear. The lentiviral vectors used in our study were socalled self-inactivating (SIN) vectors. This means that viral RNA contains a deletion at the 3´
LTR. During reverse transcription, this deletion is transferred into 5´ LTR, leading to
inactivation of HIV-1 proviral LTR promoter (Zufferey et al., 1998). Therefore, transgene
mRNA is transcribed only if driven by an internal EF1
promoter, since full-length vector
genome mRNA is not produced in transduced cells due to the deletion in 5´ LTR. During vector
generation, TAT protein is supplied from the helper plasmid, but it is not produced in transduced
cells. In addition, the full-length TAT protein is not packaged into the generated HIV-1 virions
(Luciw, 1996). The use of SIN lentiviral vectors and the fact that TAT protein is not packaged
into wild type HIV-1 virions, suggests that the low protein expression level could not explained
by the ability of TAT PTD to inhibit transactivation by competing with full-length TAT in the
transduced cells.
5.3.2
TAT-TK-GFP DOES NOT SUPPORT INTERCELLULAR TRAFFICKING
To study the property of TAT-peptide to move intercellularly, BT4C rat glioma cells were
transfected with lentivirus vector plasmids carrying TAT-TK-GFP or TK-GFP genes. The
movement of TAT-fusion protein from the transfected cells to the adjacent cells was monitored
by fluorescence microscopy after fixation with either paraformaldehyde (PFA) or methanol.
However, we did not observe any TAT-mediated intercellular spreading with either of these
fixation methods (III, Fig. 2). Moreover, immunostaining of transfected cells with anti-GFP
antibody or flow cytometric analysis at different time points also did not reveal intercellular
spreading of TAT-fusion proteins (III, data not shown). The two fixation methods were used,
since it has been shown that spreading of TAT-fusion proteins could be an artifact caused by
methanol fixation (Leifert et al., 2002; Richard et al., 2003).
By the time when we started to evaluate the utility of TAT-peptide as a booster for HSVTK/GCV therapy, the hypothesis was that TAT-TK-GFP would exit from transduced cells with
the aid of TAT-peptide and after release, would deliver the fusion protein into neighboring nontransduced cells. However, at the present, we know that this phenomenon is unlikely to occur.
Though there is some evidence that TAT-peptide has a property to move from cell to cell, the
recent studies have tended to provide alternative explanations (Leifert et al., 2002; Cashman et
al., 2003; Lundberg et al., 2003). The TAT-peptide contains a strong nuclear localization signal
(Ruben et al., 1989), but it does not contain any signal for exiting the nucleus or for secretion.
61
Therefore it may not possess the ability to move between cells as such, but requires fusion with a
suitable domain to ferry it out of the cell (Chauhan et al., 2007). Since neither TK nor GFP
contains a secretory signal, it would explain why no intercellular spreading of TAT-TK-GFP
was observed. Interestingly, some studies have shown that TAT-containing fusion proteins can
indeed move intercellularly, as was the case with TAT- -glucuronidase (Xia et al., 2001; Elliger
et al., 2002). The explanation for the success with TAT- -glucuronidase may be due to the fact
that
-glucuronidase can be secreted from producer cells to some degree also normal
circumstances. However, Tasciotti et al detected release of TAT-TK fusion protein into cell
culture media from the expressing cells (Tasciotti and Giacca, 2005), even though TK is not a
secreted protein.
Furthermore, it has been suggested that partial denaturation of TAT containing proteins is
essential for efficient membrane translocation (Nagahara et al., 1998; Schwarze et al., 1999;
Becker-Hapak et al., 2001). However, Tasciotti et al proposed that if GFP is unfolded during
membrane translocation, it will lose its fluorescent property (Tasciotti and Giacca, 2005).
Therefore, even though TAT-TK-GFP fusion proteins were not seen to move from cell to cell,
one explanation may be that the fusion protein cannot be monitored simply by trying to follow
the GFP expression.
5.3.3
COMPARISON OF CELL KILLING EFFICIENCIES OF TAT-TK-GFP AND TK-GFP IN
HUMAN TUMOR CELL LINES
Several human tumor cell lines and BT4C rat glioma cell line were tested to study the utility of
TAT-peptide as a booster for HSV-TK/GCV suicide gene therapy. In BT4C, SKOV3.ip1 and
PC-3 cell populations with 20% proportion expressing TAT-TK-GFP or TK-GFP it appeared
that the TAT-peptide increased cell death significantly after a five-day exposure to GCV (1
µg/ml) (III, Fig. 3). When we further tested the utility of TAT-TK-GFP in several human tumor
cell lines, only three of the studied 12 tumor cell lines were sensitized to GCV better with TATTK-GFP in favor of TK-GFP. Enhancement of cell killing induced by TAT-peptide was
statistically significant only in A2058 cells (P<0.001). Since in most of the studied cell lines, the
cell killing efficiencies of TAT-TK-GFP and TK-GFP were rather similar and in some cell lines
TK-GFP turned out to be more efficientc it cannot be concluded that the TAT peptide had a
significant ability to increase overall cell death (IV, Fig. 1). All of the studied cell lines
displayed a notable bystander effect. Although a mere 20% of the cells were expressing fusion
protein, the cell-killing rate was notably higher, ranging from 30% to 70% (at GCV
62
concentration of 1 µg/ml) (IV, Fig. 1).
The reasons for the weaker cell killing efficiency of TAT-TK-GFP compared to TK-GFP in
some of the studied cell lines can be explained in several ways. Presumably, the possible downregulation of the entire TAT-TK-GFP fusion protein expression in most of the cell lines had an
impact on cell killing efficacy. Since at least expression of GFP was down-regulated, most likely
also the concentrations of active TK were lower in TAT-TK-GFP containing cells compared to
cells expressing TK-GFP. However, one interesting observation was that although the mean
expression level of TAT-TK-GFP was significantly lower in many cell lines compared to TKGFP (IV, data not shown), the sensitivity to GCV of the cell lines expressing the fusion genes
was somewhat similar.
In some studies that have utilized TAT-containing fusion proteins, the glycine residues have
been inserted between the end of TAT-peptide and the amino-terminus of fusion protein
(Nagahara et al., 1998; Becker-Hapak et al., 2001; Cashman et al., 2003; Toro and Grunebaum,
2006). This amino acid is "flexible" and allows free peptide bond rotation. However, the TATTK-GFP fusion protein did not contain any glycine residues between the TAT PTD and TK.
Therefore, it is possible that although TAT-TK-GFP was expressed correctly, the TAT-peptide
may suffer from steric hindrance by TK-GFP and thus impair or even totally prevent the TATpeptides´s cell penetrating feature.
Taking into account the fact that the TAT-peptide may not have the ability to exit the cell, we
have postulated an alternative mechanism for TAT-mediated enhancement of HSV-TK/GCV
cytotoxicity: soon after GCV challenge, cells expressing TAT-TK-GFP will die, which will
release the fusion protein into extracellular milieu. Thereafter, the fusion protein is taken up by
surrounding cells, inducing cell death also in non-transduced cells. In accordance with our
hypothesis, another research group has come to the conclusion that TAT-TK cannot spread
intercellularly and therefore GCV induced cell death is required to release fusion protein
(Cascante et al., 2005). The main mechanism to explain cells die during HSV-TK/GCV
treatment appears to be apoptosis, with the necrotic cell death seemingly playing only a minor
role (Thust et al., 2000; Tomicic et al., 2002). However, it is known that during apoptosis, some
intracellular proteins are degraded. Could it be possible that also the HSV-TK protein is
damaged at some stage in this process and loses its enzymatic activity? Therefore, the active TK
could be released solely from cells dying via necrosis and the observed modest differences in
63
cell killing by TAT-TK-GFP compared to TK-GFP could be explained by cell-specific
differences in the death pathways evoked during HSV-TK/GCV treatment. Nevertheless, studies
by Cascante et al and Tasciotti et al have shown a significant TAT-mediated increase on HSVTK/GCV treatment in vitro and in vivo (Cascante et al., 2005; Tasciotti and Giacca, 2005),
suggesting that possible degradation of TK would not be the major reason for the insufficient
action of TAT-TK-GFP.
5.3.4
FEATURES INVOLVED IN THE TAT-MEDIATED INCREASED CELL KILLING
Impact of extended GCV exposure. To verify that the effect of TAT-peptide was not attributable
to an inadequately short incubation time with the prodrug, the impact of prolonged GCV
exposure was tested in A2058 and A549 cells. A2058 was chosen to represent a cell line in
which TAT increased sensitivity to GCV whereas A549 was a cell line in which no TATmediated enhancement was observed. Extended GCV incubation increased cytotoxicity of both
fusion proteins in A5058 cells, but not remarkably in A549 cells. However, when comparing
TAT-TK-GFP to TK-GFP, the difference was not statistically significant (IV, Fig. 3).
Role of cell surface heparan sulfate proteoglycans. To study whether the cell surface heparan
sulphate proteoglycans (HSPG) have a role on TAT-mediated increased cell death, we compared
cell killing efficiencies of TAT-TK-GFP and TK-GFP in CHO-cells as well as in mutant cell
lines, which do not produce HSPG (the pgsD-677 cells) (Lidholt et al., 1992) or only produce
15% of all proteoglycans compared to wild type cells (pgsB-618) (Esko et al., 1987). In the wild
type CHO-cells in which 20% expressed the therapeutic protein, TAT-TK-GFP increased cell
death slightly more than TK-GFP (at GCV concentration of 1 µg/ml). This enhancement was not
observed in mutant cell lines pgsD-677 and pgsB-618, suggesting that the impact on increased
cell killing could be at least partially mediated via the uptake of TAT-TK-GFP by HSPG (IV,
Fig. 4). This observation is in agreement with previous findings suggesting that ionic
interactions and subsequent internalization of the TAT-peptide occurs with cell surface HSPG
receptors (Console et al., 2003; Richard et al., 2005). Mani et al recently showed that the
presence of TAT PTD in culture media could inhibit polyamine uptake in human carcinoma T24
cell line. These cells were made dependent on extracellular polyamines by treatment with difluoromethylornithine (DFMO). These workers further demonstrated that treatment with
DFMO increased the uptake of TAT-peptide in vitro and combined treatment with DFMO and
TAT PTD attenuated tumor growth in mice more than DFMO alone (Mani et al., 2007). DFMO
depletes intracellular polyamines by inhibiting ornithine decarboxylase that is one of the key
64
enzymes of the polyamine biosynthesis pathway (Metcalf et al., 1978). Consequently, depletion
of intracellular polyamines by DFMO could increase the uptake of polyamines from outside of
the cell. It has also been proposed that polyamines are taken up via HSPG receptors (Belting et
al., 1999). Therefore, TAT peptide most likely was able to compete with polyamines in binding
to HS proteoglycans (Mani et al., 2007). The mechanism behind enhanced TAT PTD
internalization as a result of treatment with DFMO was thereby most likely due to the fact that
DFMO induced polyamine depletion increased the amount of cell surface HSPG (Belting et al.,
1999).
Role of cell growth rate. To study whether a high cell division rate has an impact on the TATmeditated increased cell killing, the HSV-TK/GCV sensitivity assay was carried out in BT4C rat
glioma cells using reduced serum concentrations in cell culture media to slow down the cell
growth. In BT4C cells, of which 20% were expressing the transgene, TAT-TK-GFP rendered
BT4C cells 50% more sensitive to GCV compared to TK-GFP. The serum deprivation abolished
the effect when the cells were grown in culture media supplemented with 5% or 2.5% serum
instead of normal 10% (III, Fig. 4). However, serum deprivation may induce also other
physiological changes in cells, and therefore we further determined cell division rates in some of
the GCV-tested cell lines. A clear correlation between cell proliferation rate and improved cell
death induced by TAT was not observed. However, our results suggest that TAT-TK-GFP
operates more efficiently in cells which are proliferating more slowly (IV, Fig. 5). This
observation is more relevant compared to the results obtained by serum deprivation. Since the
HSV-TK/GCV system is known to operate most efficiently in rapidly dividing cells and the cellkilling capacities of TAT-TK-GFP and TK-GFP were rather similar, it is possible that such a
minor difference could not be distinguished.
Impact of membrane destabilization of the endosomes. Several studies show that TAT fusion
proteins are taken up by endocytosis, which may lead to a situation that TAT-fusion proteins
become trapped into endosomes/lysosomes. The use of lysosomotropic agents has been shown to
increase the TAT-mediated delivery (Caron et al., 2004; Wadia et al., 2004). Therefore, we
further attempted to increase GCV cytotoxicity by using chloroquine in order to enhance
endosomal escape of the TAT-fusion protein. However, those concentrations of chloroquine that
have been shown to have a significant enhancement on the TAT-mediated delivery (<100 µM)
(Wadia et al., 2004) were exceedingly toxic to our model cell lines and therefore these
experiments could not be performed (IV, unpublished data).
65
Role of type I interferon response. Since the full-length TAT protein can suppress the innate
immune response in target cells (McMillan et al., 1995; Vilcek and Sen, 1996), we evaluated if
one of the mechanisms by which TAT-TK-GFP enhances GCV cytotoxicity in certain cell lines
could be a consequence of the TAT-peptide’s property to suppress these responses as well. No
TAT-mediated enhancement on cell killing was detected in A549 cells (IV, Fig 1). In addition, it
was observed that in this cell line lentiviral vectors also failed to evoke type I IFN response (I,
Table 1). However, the type I IFN response against lentiviral vectors was not studied in A2058
cells, in which TAT-peptide enhanced GCV cytotoxicity to the greates extent (IV, Fig 1).
Nevertheless, accumulation of MxA protein was not observed in either of the cell lines after
transduction with lentiviruses carrying the TAT-TK-GFP or TK-GFP fusion genes (IV, data not
shown).
66
6 SUMMARY AND CONCLUSIONS
The aim of this study was to evaluate problems encountered in achieving therapeutic gene
transfer for cancer and further to develop methods to improve cancer gene therapy by modifying
viral vectors and the therapeutic gene.
I
Since one major limiting factor of several cancer gene therapy approaches has been the
low gene delivery rate, we evaluated the contribution of the type I IFN response to therapeutic
gene transfer. Delivery of plasmid DNA and particularly most forms of RNA appeared to be
potent inducers of the type I IFN response and furthermore this induction was cell type specific.
However, commonly used viral vectors, excluding Semliki Forest virus, were able to avoid or
suppress the response. Although the type I IFN response might represent a beneficial adjuvant in
immunotherapy for cancer, in most cancer gene therapy applications these responses are
undesirable, particularly if they represent a barrier to efficient expression of the therapeutic gene.
One considerable disadvantage of non-viral gene delivery compared to its viral counterparts is
the low efficacy. The induction of the host cell innate defense system may at least partially
contribute to this inefficiency.
II
In order to improve the gene transfer rate to tumor cells, we evaluated the properties of
cell penetrating peptides in boosting the transduction efficiency of viral vectors. Synthetic
peptides corresponding to the sequences to HIV-1 TAT protein transduction domain and
Drosophila Antennapedia homeodomain enhanced significantly adeno- and lentivirus-mediated
gene transfer to human tumor cells. However, commonly used transduction enhancers,
polybrene and protamine sulfate, turned out to be as efficient or even better boosters than these
cell penetrating peptides. Since protamine sulfate is a clinically approved drug and production of
protamine costs much less than the cationic peptides, it is unlikely that these peptides will
become widely used common transduction enhancers in clinical gene therapy applications.
III
In an attempt to enhance HSV-TK/GCV suicide gene therapy, we constructed triple
fusion protein TAT-TK-GFP in order to increase the movement of therapeutic protein between
the cells. No detectable levels of intercellular spreading were seen, although TAT-TK-GFP
rendered certain cell lines more sensitive than TK-GFP to GCV. It was noteworthy that in some
cell lines TK-GFP appeared to be more efficient, thus no overall difference in cell killing
capability between TAT-TK-GFP and TK-GFP was seen. TAT-mediated increased cell killing
67
appeared to some extent to be dependent on the cell surface heparan sulfate proteoglycans and
the cell growth rate, but was not associated with the type I IFN response. In conclusion, the
current version of TAT-TK-GFP may not be very useful tool to enhance HSV-TK/GCV
treatment, and thus further development is required to obtain more efficient TAT-containing
fusion proteins for use in cancer gene therapy.
68
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