KUOPION YLIOPISTON JULKAISUJA G. – A.I.VIRTANEN –INSTITUUTTI 35 KUOPIO UNIVERSITY PUBLICATIONS G.
A.I.VIRTANEN INSTITUTE FOR MOLECULAR SCIENCES 35
TANJA HAKKARAINEN
Enhancement of Cancer Gene Therapy With Modified Viral Vectors and Fusion Genes
Doctoral dissertation
To be presented with permission of the Faculty of Natural and Environmental Sciences
of the University of Kuopio for public examination in Auditorium, Tietoteknia building, University of Kuopio, on Friday 13th May 2005, at 1 pm
Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences,
University of Kuopio
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FIN-70211 KUOPIO FINLAND
Tel. +358 17 163 430 Fax +358 17 163 410
http://www.uku.fi/kirjasto/julkaisutoiminta/julkmyyn.html Serial Editors: Professor Karl Åkerman
Department on Neurobiology
A.I.Virtanen Institute for Molecular Sciences Research Director Jarmo Wahlfors
Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences
Author’s address: Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences
University of Kuopio P.O.Box 1627 FIN-70211 KUOPIO FINLAND
Tel. +358 17 163 790 Fax +358 17 163 030
Email: tanja.hakkarainen@uku.fi
Supervisors: Docent, Research Director Jarmo Wahlfors, PhD Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences
University of Kuopio
Docent Akseli Hemminki, MD, PhD
Rational Drug Design, University of Helsinki and
Department of Oncology, Helsinki University Central Hospital Reviewers: Professor Veli-Matti Kähäri, MD, PhD
Department of Medical Biochemistry and Molecular Biology University of Turku
Docent Anu Jalanko, PhD
Department of Molecular Medicine National Public Health Institute
Opponent: Dr. Michael Blaese, MD
PreGentis, Inc.
Newtown, PA, USA
ISBN 951-781-394-5 ISBN 951-27-0098-0 (PDF) ISSN 1458-7335
Kopijyvä Kuopio 2005 Finland
Hakkarainen, Tanja. Enhancement of cancer gene therapy with modified viral vectors and fusion genes.
Kuopio University Publications G. – A.I.Virtanen Institute for Molecular Sciences 35. 2005. 63 p.
ISBN: 951-781-394-5 ISBN: 951-27-0098-0 (PDF) ISSN: 1458-7335
ABSTRACT
Even though several approaches have been developed for improvement of gene transfer efficiency, one major obstacle in to successful gene therapy is insufficient transduction efficiency and consequently poor therapeutic effect. However, several approaches have been investigated to circumvent this problem. In the following study, we evaluated the potential use of various lentiviral vectors in cancer gene therapy. In addition, we utilized CD40 for targeting adenoviral vectors to ovarian cancer cells. Further, to compensate for the low gene transfer efficiency and to enhance therapeutic outcome by spreading the therapeutic element, the feasibility of VP22 protein transduction domain (PTD) and conditionally replicative adenoviruses were studied.
Our results demonstrated that several different types of human cancer cell lines can be efficiently transduced in vitro with lentiviral vectors. These vectors also showed the potential for efficient gene transfer vehicle in vivo. Further, increased gene transfer rates were achieved in ovarian cancer cells by targeting adenoviral vectors to the CD40 receptor. In addition, the use of VP22 was able to enhance tumor cell killing under certain conditions in vitro, although no intercellular trafficking was observed. VP22 coupled therapeutic protein also significantly reduced tumor growth in vivo. However, the incorporation of VP22 did not significantly increase the antitumor effect when compared to control proteins. Finally, the use of conditionally replicative adenovirus resulted in effective oncolysis in ovarian cancer cells in vitro.
Moreover, this virus possessed a remarkably efficient antineoplastic activity in vivo. When combined with suicide gene/prodrug therapy, cell killing was further augmented in vitro, but this was not observed in vivo.
In summary, these results show that several approaches can be exploited to enhance gene transfer in tumor cells in order to achieve better therapeutic responses and simultaneously reduce the side effects of cancer gene therapy. Further, by combining various approaches more efficient tools for gene therapy purposes can be developed.
National Library of Medicine Classification: QZ 50, QZ 52, QZ 266, QW 160
Medical Subject Headings: neoplasms; gene therapy; gene transfer techniques; genetic vectors;
adenoviridae; lentivirus; viral fusion proteins; virus replication; viral structural proteins; thymidine kinase;
ganciclovir
ACKNOWLEDGEMENTS
This study was carried out in the Department of Biotechnology and Molecular Medicine, A.I.Virtanen Institute for Molecular Sciences, University of Kuopio and in the Division of Human Gene Therapy, University of Alabama at Birmingham during the years 2000-2004.
I wish to express my deepest gratitude to my principal supervisor, Docent Jarmo Wahlfors, PhD, for giving me the opportunity to become a part of the fascinating world of science and gene therapy research. I also thank him for his expert guidance, for teaching the philosophy of scientific thought and for being such a kind, fair, understanding and encouraging groupleader. I am also grateful to my second supervisor Docent Akseli Hemminki, MD, PhD, for multiple excellent ideas, his enthusiasm for research, his determination and “we are ready to rock 'n 'roll” –attitude of conducting science.
He also receives my gratitude for providing an opportunity to start working in his group while still finishing my thesis.
I am also thankful to David T.Curiel, MD, PhD, University of Alabama at Birmingham who offered me the opportunity to work in his laboratory in a truly scientifically inspiring environment.
I am indebted to Docent Anu Jalanko, PhD, and Professor Veli-Matti Kähäri. MD, PhD, for reviewing this thesis and for their constructive criticism.
My sincere and warm thanks belong to my working mates Tiina Wahlfors, MSc, Outi Rautsi (née Meriläinen), MSc, Ann-Marie Määttä, MSc, Riikka Pellinen, PhD, Anna Ketola, MD, Saara Lehmusvaara, MSc, Sanna Korja, MSc, Tuula Salonen and Katja Häkkinen for excellent team spirit, good laughs, interesting discussions, help and their friendship. It has been a great pleasure to work within this colorful, genuine and lively group. Especially I want to thank my room mate Outi, for enlightening my day with all her funny coincidences and Google’s Search -results. In addition, I want to thank “FACS-guru” Mikko Mättö, MSc, for collaboration and his priceless help with flow cytometry. I am also thankful to my collegues Terhi Pirttilä, MSc, and Eija Pirinen, MSc, for their friendship and discussions, which have enlightened my days.
I am also thankful to the whole personnel of the A.I.Virtanen Institute for creating such a pleasant working environment. My special thanks go to Riitta Laitinen and Helena Pernu for secretarial assistance, to Riitta Keinänen, PhD, for multiple advice and encouragement, to Kaija Pekkarinen for help and discussions over coffee cups, and to Pekka Alakuijala, Phil. Lic., and Jouko Mäkäräinen for technical assistance.
I thank Ewen MacDonald, PhD, for the linguistic revision of this thesis.
People at the UAB-team, Gerd Bauerschmitz, MD, Anna Kanerva, MD, PhD, Angel A Rivera, PhD, Shannon Barker, PhD, and John Lam, MD, receive my gratitude for creating a friendly, enthusiastic and unique atmosphere and working environment. Especially I want to thank Gerd, for his friendship and all the help he has provided in different circumstances.
I am also thankful to the new lab crew in Akseli’s group, Anna, Tuuli, Mari, Merja, Lotta, Maria, Tommi and Kilian for taking me to be a part of their team and for creating an energetic and fun working environment.
I express my warm gratitude to all my friends for their support, discussions, long phone calls, laughs and cries and enjoyable moments, which have made possible to temporarily forget the science and the meaning of the word “work”.
I owe my deepest thanks to my whole family. To my parents for teaching me the basic things in life, love, support, trust and just being there for me whenever I’ve needed. I want to thank my brother’s family for love and support and especially my niece Anna for being such a great and hilarious personality. My warmest thanks go to my beloved partner Markku for loving and believing in me and making me complete.
This study was supported by The Saastamoinen Foundation, The Finnish Cultural Foundation of Northern Savo and The Cancer Society of Finland.
Kuopio, April 2005
Tanja Hakkarainen
ABBREVIATIONS
AAV adeno associated virus
ADP adenovirus death protein
APC gene adenomatous polyposis coli –gene
CAR coxsackie adenovirus receptor
CMV cytomegalovirus
Cox-2 cyclo-oxygenase-2
cPPT central polypurine tract
CRAd conditionally replicative adenovirus
EGR1 early growth response gene 1
FITC fluorescein isothiocyanate
GCV ganciclovir
GFP green fluorescent protein
HIV-1 human immunodeficiency virus type 1
HSV-1 herpes simplex virus type 1
hTERT human telomerase reverse transcriptase
ITR inverted terminal repeat
LTR long terminal repeats
MDR-1 multidrug resistance gene 1
MMR mismatch repair
MRS magnetic resonance spectroscopy
PET positron-emission tomography
PFA paraformaldehyde
pfu plaque forming unit
PSA prostate specific antigen
PSMA prostate specific membrane antigen
PTD protein transduction domain
SIN self-inactivating type vector
TK herpes simplex virus thymidine kinase
t.u. transducing unit
VEGF vascular endothelial growth factor
VEGF-R vascular endothelial growth factor receptor
VSV-G vesicular stomatitis virus G protein
VP viral particle
(W)PRE (woodchuck hepatitis virus) posttranscriptional regulatory element
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following publications referred to their corresponding Roman numerals:
I Pellinen R., Hakkarainen T., Wahlfors T., Tulimaki K., Ketola A., Tenhunen A., Salonen T. and Wahlfors J. (2004). Cancer cells as targets for lentivirus-mediated gene transfer and gene therapy. International Journal of Oncology, 25:1753-1762.
II Hakkarainen T., Hemminki A., Pereboev A., Barker S., Asiedu K., Strong T., Kanerva A., Wahlfors J. and Curiel D.T. (2003). CD40 is expressed on ovarian cancer cells and can be utilized for targeting adenoviruses. Clinical Cancer Research, 9: 619-624.
III Hakkarainen T., Wahlfors T., Meriläinen O., Loimas S., Hemminki A. and Wahlfors J.
(2005) VP22 does not significantly enhance enzyme prodrug cancer gene therapy as a part of VP22-HSVTk-GFP triple fusion construct. Journal of Gene Medicine, 9:
IV Hakkarainen T., Hemminki A., Curiel D.T. and Wahlfors J. (2005) Α conditionally replicative adenovirus that codes for a TK-GFP fusion protein (Ad5-∆24TK-GFP) for evaluation of the potency of oncolytic virotherapy combined with molecular chemotherapy, Submitted.
TABLE OF CONTENTS
1 INTRODUCTION... 13
2 REVIEW OF THE LITERATURE... 14
2.1
Cancer... 14
2.2
Ovarian cancer... 15
2.3
Cancer associated genes ... 15
2.3.1 Overview ... 15
2.3.2 Proto-oncogenes ... 16
2.3.3 Tumor suppressor genes ... 16
2.3.4 DNA mismatch repair (MMR) genes ... 17
2.4 Cancer gene therapy ... 17
2.4.1 Overview ... 17
2.4.2 Cancer gene therapy approaches... 18
2.5
Challenges of gene therapy ... 21
2.6 Improvements in cancer gene therapy ... 23
2.6.1 Overview ... 23
2.6.2 Optimal gene transfer tools for specific purposes: various viral vectors ... 23
2.6.2.1 Adenoviral vectors... 23
2.6.2.2 Retro- and lentiviral vectors... 26
2.6.3 Enhanced gene transfer and increased specificity: adenoviral targeting... 30
2.6.4 Improved therapeutic outcome by spreading the therapeutic element: oncolytic viruses and protein transduction domains ... 31
2.6.4.1 Conditionally replicative adenoviruses (CRAds)... 32
2.6.4.2 Protein transduction domains... 33
2.6.5 Combination therapies ... 34
3 AIMS OF THE STUDY... 35
4 MATERIALS AND METHODS ... 36
4.1
Fusion gene constructs (III)... 36
4.2
Viral vectors (I-IV)... 36
4.3
Cell lines and patient samples (I-IV)... 36
4.4
Protein expression levels ... 38
4.4.1 Expression of VP22-fusion proteins ... 38
4.4.2 CD40 expression (II) ... 39
4.5
Determination of lentiviral transduction efficiency by flow cytometry (I) ... 39
4.6
Gene transfer with CD40-targeted adenovirus (II)... 39
4.7
Verification of viral replication and oncolysis (IV)... 40
4.8
Ganciclovir sensitivity assays (I, III, IV)... 40
4.9
Monitoring of VP22-mediated intercellular trafficking and adenoviral spreading (III, IV) ... 41
4.9.1 Flow cytometry... 41
4.9.2 Fluorescence microscopy... 41
4.10 In vivo studies (I, III and IV)... 41
5 RESULTS AND DISCUSSION ... 43
5.1
Cancer cells are potential targets for lentiviral gene transfer (I) ... 43
5.1.1 Transduction efficiency of lentiviral vectors in human cancer cells... 43
5.1.1.1 Impact of accessory proteins... 44
5.1.1.2 Impact of WPRE and cPPT... 44
5.1.2 Lentivirus vector -mediated TK/ganciclovir gene therapy in vitro... 45
5.1.3 Lentiviral tumor transduction in vivo... 45
5.2
CD40 can be exploited for transductional targeting of adenovirus vectors (II)... 45
5.2.1 CD40 expression in ovarian cancer cells ... 46
5.2.2 Gene transfer with CD40 -targeted adenovirus... 46
5.3
VP22 does not enhance suicide gene therapy as a part of triple fusion protein (III) ... 47
5.3.1 Expression of fusion proteins ... 47
5.3.2 Monitoring of VP22 -mediated intercellular trafficking... 48
5.3.3 VP22-TK-GFP mediated cell killing in vitro... 48
5.3.4 Efficiency of VP22-TK-GFP in vivo... 49
5.4
Ad5-∆24-TK-GFP: a useful tool for evaluation of combined oncolytic virotherapy and suicide gene therapy (IV) ... 49
5.4.1 Verification of viral replication and oncolysis... 49
5.4.2 Monitoring of viral spreading ... 50
5.4.3 Effect of TK/GCV system on oncolysis in vitro... 50
5.4.4 Efficiency of Ad5-∆24TK-GFP in vivo... 51
6 SUMMARY AND CONCLUSIONS ... 52
7 REFERENCES... 54
APPENDIX: ORIGINAL PUBLICATIONS I-IV
13
1 INTRODUCTION
Cancer is a world wide health problem which was responsible for the deaths of almost 7 million people in 2002. Even though our knowledge of molecular mechanisms, diagnostic methods and traditional treatments of cancer have all improved during the last decades, most cancer types still have a poor prognosis and evoke a high mortality, this being especially the case with metastatic types. Thus, more efficient approaches and improved therapeutic strategies are needed for the treatment of cancer.
The first clinical gene therapy trial was carried out in the early 1990s. From that beginning, gene therapy has become a widely studied concept for treatment of various diseases. Even though gene therapy was initially thought to be more suitable for the treatment of inherited monogenic diseases, gene therapy has been increasingly utilized in the treatment of acquired and complex diseases such as cancer. In fact, by the year 2004, the majority of clinical gene therapy trials (66%) have focused on cancer diseases.
Even though several approaches have been successfully developed to improve the gene transfer efficiency, one major obstacle in cancer gene therapy is still insufficient transduction of the gene and consequently a poor therapeutic effect. There are several possibilities that can be utilized to circumvent this problem. First, new alternative viral vectors can be explored to find optimal gene transfer vehicles for each purpose.
Secondly, viral vectors can be re-targeted to cancer cells, which simultaneously enhance gene transfer rates in tumors and diminish undesired side effects in healthy tissue. In addition, it is possible to exploit viral replication per se to destroy cancer cells. To avoid side effects and to increase the safety of these oncolytic agents, replication can be restricted to tumor cells by partially deleting the viral genome or by using tissue specific promoters to drive the viral genes responsible for replication. Instead of modifying the gene transfer vector, one possibility is to modify the therapeutic gene so that the resulting therapeutic protein can spread to surrounding cells and thus to compensate for the low gene transfer efficiency and to enhance the therapeutic outcome.
In this study, we examined several potential approaches to enhance cancer gene therapy. First, we explored the feasibility of using lentiviral vectors in cancer gene therapy. In addition, we targeted adenoviral vectors to an alternative cellular receptor to increase the specificity and efficacy of gene transfer. Furthermore, we tested the feasibility of intercellularly trafficking protein transduction domains to achieve enhancement of the therapeutic response. Finally, we generated a novel conditionally replicative adenovirus and evaluated its oncolytic potency for cancer gene therapy purpose.
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2 REVIEW OF THE LITERATURE
2.1 Cancer
Cancer is a world wide health problem. In 2002, approximately 11 million people were estimated to have been diagnosed with cancer and almost 7 million people will die from cancer (Ferlay et al., 2004). In Finland alone, over 23 000 new cancer diagnoses were made in 2002 (Finnish Cancer Registry, www.cancerregistry.fi). The number of cancer patients has increased partly due to advances in medical sciences, such as the reduction of deaths from infectious and cardiovascular causes but also to improved diagnostic methods being better able to detect cancer. Further, many factors in the Western lifestyle may predispose to carcinogenesis. The development of a malignant tumor always involves an interaction between the environment and heredity (Tamura et al., 2004). In most cases, the causative reason for carcinogenesis is unknown, but the mechanism always involves mutations to cellular DNA. Somatic mutations can be caused by environmental factors including radiation, chemicals and viruses or by imperfection of the DNA copying machinery, which leads to abnormal cellular functions (Vogelstein and Kinzler, 1998; Zhang et al., 1995). Genetic mutations contributing to transformation of the normal cell can be also inherited in a Mendelian fashion (Knudson, 1991). These genes are typically recessive in oncogenesis. Thus, according to Knudson’s two-hit hypothesis, an inherited mutation is rarely sufficient to initiate tumorigenesis alone and an additional mutation or loss of a second allele is required (Knudson, 1971). Subsequently, Knudson classified the etiology of cancer and the role of environmental and genetic factors on tumorigenesis (Knudson, 1985). Based on that classification, most common tumors are sporadic (i.e. non-inherited) and only a small fraction (1-5%) of all cancer types belongs to the group of hereditary i.e. familial, cancers (Table 1). Currently, the familial cancer syndrome is best characterized in ovarian, breast and colorectal cancers which have the greatest heritability in their causation (Knudson, 1995).
Table 1. Knudson’s classification (modified from Tamura et al., 2004).
Even though cancer can be considered as a genetic disease, it is rarely caused by a defect in a single gene (Tamura et al., 2004). It has been estimated that three to seven mutations are required for carcinogenesis (Vogelstein and Kinzler, 1998). These mutations typically cause increased and irregular proliferation activity, lack of apopotosis, deficient cell cycle control, and ability to metastasize and create
Environment Genetic factors Etiological viewpoint % of all causes
Basic Basic Accidental 20%
Main A little Environmental related 75%
Considerable Considerable Multifactorial 75%
A little Main Hereditary 1-5%
15 vascularisation into the tumor (Hanahan and Weinberg, 2000). When these defects accumulate, the aberrant features lead to creation of cells which can proliferate unrestrictedly, form preneoplastic lesions, invade local tissues and eventually establish distant metastasis (Rieger, 2004; Zhang et al., 1995). It has been postulated that the number of mutations can influence the aggressiveness of the tumor. Benign tumors seem to contain fewer mutations than malignant, progressive neoplasms. Solid tumors (i.e. tumors in solid organs) require a greater number of mutations than diffuse tumors (i.e. leukemias), which probably explains the longer time period before solid tumors appear after the initial mutagenic stimuli when compared to diffuse tumors. (Vogelstein and Kinzler, 1998).
2.2 Ovarian cancer
Ovarian cancer is the most lethal gynecologic cancer. Worldwide it has been estimated that, over 200 000 new ovarian cancer patients are diagnosed and approximately 120 000 people died from ovarian cancer in 2002 (Ferlay et al., 2004). In Finland, there were 497 new cases in 2002, making ovarian cancer the fourth most common cancer in women (Finnish Cancer Registry, www.cancerregistry.fi). Although the 5-year survival rate in Finland is 49%, this statistic remains much lower than the overall 65% cancer survival for women (Finnish Cancer Registry, www.cancerregistry.fi).
Due to its mild symptoms, ovarian cancer is difficult to detect in its early stages. Thus, the majority of ovarian cancer cases are unfortunately diagnosed after the cancer has disseminated beyond the ovaries. In advanced ovarian cancers, the standard treatment is usually surgical cytoreduction followed by chemotherapy (Agarwal, and Kaye, 2003; DiSaia, and Bloss, 2003). Typically, a platinum/taxane combination has been considered as the post-surgical first-line chemotherapy (Coleman, 2002). Although this combined chemotherapy has been shown to result in good response rates (70-80%) in ovarian cancer patients, the majority of these patients will relapse due to the development of drug resistance to these chemotherapeutic agents (Balch et al., 2004; Stuart, 2003). Further, ovarian cancer drug resistance has been believed to result in a treatment failure and death of more than 90% of patients with metastatic disease (Balch et al., 2004).
2.3 Cancer associated genes
2.3.1 Overview
Three major gene types have been shown to be closely related to tumorigenesis: proto-oncogenes, tumor- suppressor genes and DNA mismatch repair genes (Figure 1). Since cancer is a group of neoplastic diseases involving multiple steps and genetic events, tumor development usually requires alterations in a number of cancer-associated genes. A transformed phenotype when combined with a metastatic capability typically involves both oncogene activation and tumor suppressor gene loss or inactivation (Bast et al.,
16
2000). Furthermore, it is noteworthy that the same genes causing genetic predisposition to cancer are most often associated and involved in sporadic forms of carcinogenesis (Knudson, 1991).
2.3.2 Proto-oncogenes
The proto-oncogenes encode proteins that have a crucial role in cell signaling pathways. The proto- oncogenic products (including various growth factors and their receptors, signal transducers and transcription factors) control the growth and differentiation of normal cells (Aaronson, 1991; Bast et al., 2000). These proto-oncogenes can be activated to carcinogenic oncogenes through mutation, gene amplification or chromosomal rearrangement resulting in an altered proto-oncogene structure or an increased proto-oncogene expression (Bast et al., 2000). Thus activation of oncogenes offers a growth advantage to the cell and eventually normal cells become transformed.
Figure 1. Cancer associated genes and cell cycle. Oncogene products and mutated tumor suppressor and DNA mismatch repair (MMR) proteins alter the cell cycle in various phases (in G1, S and G2) allowing abnormal and unregulated cellular growth and finally tumor cell formation.
2.3.3 Tumor suppressor genes
Tumor suppressor genes, or antioncogenes can prevent cell growth in many ways e.g. by blocking the cell cycle or by promoting cell cycle arrest and apoptosis (McCormick, 2001). In cancer cells, these genes are inactivated and this blocks the control of normal cell cycle and thus increases the probability of tumor
M
G1
S G2
Restriction point
MMR genes
Oncogenes
Oncogenes
Tumor Suppressor genes
Tumor Suppressor genes
G0
17 formation (Knudson, 2001). The best characterized and most widely studied tumor suppressor genes are RB1 (Francke and Kung, 1976; Friend et al., 1986; Knudson et al., 1976), WT1 (Call et al., 1990; Gessler et al., 1990), TP53 (Finlay et al., 1989), colon cancer associated APC (Ichii et al., 1992; Nishisho et al., 1991) and breast- and ovarian cancer associated BRCA1 and 2 (Miki et al., 1994; Steichen-Gersdorf et al., 1994; Wooster et al., 1995).
2.3.4 DNA mismatch repair (MMR) genes
The DNA mismatch repair (MMR) system is one of the three cellular mechanisms involved in DNA repair (Charames and Bapat, 2003). MMR proteins recognize and eliminate potential misincorporated nucleotides on the newly synthesized DNA strand during DNA replication (Fedier and Fink, 2004). Hence, MMR machinery is a crucial mechanism for maintaining genome integrity during cell proliferation. Mutations in genes encoding MMR proteins attenuate or inactivate the DNA repair machinery and thus interfere with genetic stability and increase the incidence of further mutations during the DNA synthesis (Fedier and Fink, 2004). This increased mutation frequency thus augments the susceptibility to undergo cellular transformations and tumor formation (Eshleman and Markowitz, 1996). Mutated MMR proteins have been identified and associated with various cancer types including hereditary nonpolyposis colon cancer (Bronner et al., 1994; Fishel et al., 1993; Risinger et al., 1993), breast (Balogh et al., 2003; Seitz et al., 2003) and bladder cancer (Kassem et al., 2001) as well as gliomas (Wei et al., 1997). In addition, tumor cells with a defective MMR system have been shown to display reduced sensitivity to the cytotoxic, DNA damaging chemotherapeutic drugs such as cisplatin (Brown et al., 1997), doxorubicin (adriamycin) (Drummond et al., 1996) and topotecan (Fedier et al., 2001).
2.4 Cancer gene therapy
2.4.1 Overview
Conventionally cancer is treated with chemo- and radiotherapy, hormonal therapies or surgery. In addition to the growth advantage, the genetic mutations responsible for carcinogenesis may increase the resistance of malignant cells to various conventional treatments, thus ensuring survival of cancer cells and uninterrupted tumor growth. Cancer cells have been shown to develop resistance to chemotherapeutic agents in a variety of ways (Fojo and Bates, 2003). Furthermore, cancer cells are often relatively resistant to radiation. One fundamental mechanism mediating resistance to radiation involves activation of NF- KappaB (Orlowski and Baldwin, 2002). In normal cells, a lethal radiation dose activates p53, which in turn down-regulates NF-KappaB-controlled survival circuits, resulting in apoptosis (Chen et al., 2002; Rocha et al., 2003). However, most advanced tumors feature a defective p53/p14ARF pathway (Sherr, 1996), which inhibits apoptosis. In addition to failure due to therapeutic resistance, another disadvantage of conventional treatments is their lack of selectivity. Especially chemo- and radiation therapies also damage healthy tissue,
18
leading to severe side effects and decreased therapeutic response. Surgical treatment of cancer has its own limitations: tumors have to be solid, well confined and accessible. However, conventional therapies have developed enormously due to technological improvements and increased knowledge of cancer biology and genetics. In radiation therapy, the development of novel biological imaging systems (mainly positron- emission tomography (PET) and magnetic resonance spectroscopy (MRS) have provided tools to obtain information about tumor cell proliferation, the presence of specific tumor markers, metabolism and vascularisation of the tumor (Ling et al., 2000). These advanced imaging techniques allow radiation doses to be calculated more precisely and to be tumor cell specific avoiding damage to healthy tissue (Bernier et al., 2004). In addition, knowledge of genetic mutations has been exploited to re-sensitize resistant cancer cells to radiation in various cancer types including prostate, breast and lung cancer (Cowen et al., 2000; Mu et al., 2004; Sakakura et al., 1997; Coleman, 1999; Shinohara et al., 2004). Further, more specific chemotherapeutic drugs have been designed including trastuzumab (Herceptin), an antibody against HER- 2, which is known to be over expressed in breast cancer cells (Wong, 1999). Despite the development of conventional therapies, most disseminated cancers are unfortunately relatively resistant to these treatments resulting in relapse and mortality. Thus, more efficient approaches and tools are needed for the treatment of cancer.
Increased knowledge of the molecular basis of cancer has also provided the possibility to exploit gene therapy as a novel strategy to treat cancer. The principle of gene therapy is rather straightforward: i.e. one introduces a therapeutic gene, whose product should stop or slow down the progression of disease, into a target cell (Mountain, 2000). Therapeutic genes can be transferred to target cells using modified viruses as vectors (Somia and Verma, 2000) or with non-viral methods including electroporation, gene gun and cationic lipid or polymer coating (Li and Huang, 2000). Gene transfer technology theoretically make it possible to treat the source of the disease whereas many conventional therapies are mainly palliative i.e.
treat the symptoms. This makes gene therapy an attractive and potentially revolutionary tool for modern medicine. The era of gene therapy was initiated in the early 1990s with the first somatic gene therapy clinical trial for the treatment of inherited genetic disease (Blaese et al., 1995). During the last fifteen years, there have been dramatic advances in the modern medicine and biosciences. Since 1990 nearly 990 clinical trials have been approved, the vast majority (~97%) of them being phase I or II trials (http://www.wiley.co.uk/genetherapy/clinical/). More than half (66%) of the approved trials have focused on cancer diseases (http://www.wiley.co.uk/genetherapy/clinical/).
2.4.2 Cancer gene therapy approaches
Since cancer is a group of complex neoplastic diseases caused by multiple genetic factors, it is not feasible to correct all of the defective genes. Despite the deficiency of several genes in tumor cells, inactivation of one particular oncogene or restoration of a single tumor suppressor gene has been shown to be adequate to significantly inhibit tumor growth. It has been reported that blockade of oncogenic HRAS –protein
19 expression induced tumor regression in animal models (Barrington et al., 1998; Chin et al., 1999).
Furthermore, Druker et al. demonstrated that inhibition of BCR-ABL -protein exhibited antineoplastic activity in a phase I clinical trial in patients with chronic myeloid leukemia (Druker et al., 2001).
Restoration of tumor suppressor protein p53 has been shown to reduce tumor growth both in patients with lung and advanced head and neck cancers (Roth et al., 1996; Schuler et al., 1998; Clayman et al., 1998;
Clayman et al., 1999).
Instead of correcting mutated genes or suppressing active oncogenes, a more widely utilized approach has been to introduce exogenous therapeutic genes into tumor cells. The inserted therapeutic genes can be immunotherapeutic, anti-angiogenetic, chemoprotective or so called suicide genes.
In immunotherapy, tumor cells are destroyed via the immune response raised against tumor specific antigens (Pardoll, 2000). Tumor antigens are typically tissue specific proteins (i.e. tyrosinase), unique mutated proteins (i.e. CDK4 variant proteins), over-expressed proteins (i.e. prostate specific antigen, PSA) or proteins from viral origin (i.e. human papilloma virus E6 and E7) (Pardoll, 2002; Vakkila and Pihkala, 1999; van der Bruggen et al., 1991). An immune response has been raised in humans against a tumor by inserting genes encoding stimulatory proteins such as cytokines into the cancer cells (Nemunaitis et al., 1999; Stingl et al., 1996) or by introducing tumor antigens to dendritic or other antigen presenting cells (Brossart et al., 2000; Simons et al., 1999).
One prerequisite for tumor formation is neovascularization (i.e. angiogenesis) of the tumor. (Vile et al., 2000). Reduced oxygen levels and certain cellular factors up-regulate the production of vascularisation stimulating factors and their receptors (such as VEGF/VEGF-R) in cancer cells (Kong and Crystal, 1998), which make these molecules attractive targets for gene therapy. By means of gene therapy, the expression or function of angiogenetic proteins can be inhibited (Im et al., 2001; Kong et al., 1998) or anti- angiogenetic proteins (such as angiostatin) can be introduced to cancer cells (Sacco et al., 2000; Tanaka et al., 1997; Tanaka et al., 1998).
In several cases, the efficacious treatment of cancer would require the use of high-dose chemotherapy, which is impracticable due to the cytotoxicity of these drugs to normal cells, especially to hematopoietic cells. To enable the use of larger and more efficient drug doses, chemoprotective genes can be transferred to hematopoietic stem cells to increase their resistance to chemotherapy (D'Hondt et al., 2001). The most extensively studied genes have been multidrug resistance 1 (MDR-1) and multidrug resistance-associated protein genes (Abonour et al., 2000; Cowan et al., 1999; Machiels et al., 1999).
Suicide genes encode enzymes that can convert a harmless, separately administered prodrug into a cytotoxic molecule (McCormick, 2001). The most widely used enzyme-prodrug combination is herpes simplex virus type 1 thymidine kinase (TK)/ganciclovir (GCV). TK phosphorylates, in concert with other cellular kinases, GCV into its triphosphate form, which can then be incorporated into DNA during cell
20
Table 2. An overview of clinical gene therapy trials.
Cancer Type Therapeutic approach/gene Delivery method Phase Reference
Brain cancer
suicide gene therapy/ thymidine kinase adenovirus I (Eck et al., 1996; Smitt et al., 2003)
immunotherapy/IFN-β adenovirus I (Eck et al., 2001)
immunotherapy/ IL-12 semliki forest virus I/II (Ren et al., 2003) tumor suppressor gene therapy/ p53 adenovirus I (Lang et al., 2003)
virotherapy herpes simplex virus I (Markert et al., 2000) Colon cancer
(metastatic)
suicide gene therapy/ cytosine deaminase adenovirus I (Crystal et al., 1997) suicide gene therapy/ thymidine kinase adenovirus I (Sung et al., 2001)
immunotherapy/HLA-B7 liposomes I (Rubin et al., 1997)
virotherapy adenovirus I (Reid et al., 2002)
Lung cancer
tumor suppressor gene therapy/ p53 adenovirus I (Roth et al., 1998; Schuler et al., 1998) Melanoma
suicide gene therapy/ thymidine kinase adenovirus I (Morris et al., 2000a)
immunotherapy/ IL-2 adenovirus I (Stewart et al., 1999)
immunotherapy/IFN-γ retrovirus I (Nemunaitis et al., 1999)
tumor suppressor gene therapy/ p53 adenovirus I (Dummer et al., 2000) Ovarian cancer
suicide gene therapy/ thymidine kinase adenovirus I (Alvarez et al., 2000) immunotherapy/ BRCA-1 retrovirus I, II (Tait et al., 1997; Tait et al., 1999) tumor suppressor gene therapy/ p53 adenovirus I/II (Buller et al., 2002)
virotherapy adenovirus I (Vasey et al., 2002)
Prostate cancer
suicide gene therapy/ thymidine kinase adenovirus I, I/II (Herman et al., 1999; Miles et al.,2001)
immunotherapy/ IL-2 DNA-lipid complex,
adenovirus I (Belldegrun et al., 2001;
Trudel et al., 2003) immunotherapy/MUC1 and IL-2 vaccinia virus I (Pantuck et al., 2004)
virotherapy adenovirus I (Freytag et al., 2002)
21 division and blocks DNA replication, preventing cell proliferation (Moolten, 1986). In addition, triphosphorylated GCV can diffuse into neighboring cells via gap-junctions and spread the cytotoxic effect, creating the so called bystander effect (Mesnil et al., 1996; van Dillen et al., 2002). The efficiency of TK/GCV system has been demonstrated successfully in patients with malignant glioma; the mean survival of patients treated with adenovirus mediated TK/GCV therapy after surgical tumor resection was prolonged by 81% when compared to the conventionally treated control group (Immonen et al., 2004; Sandmair et al., 2000). Several other combinations have been also evaluated in clinical trials including cytosine demaminase/5-Fluorocytosine (Crystal et al., 1997; Pandha et al., 1999) and nitroreductase/CB1954 (Palmer et al., 2004).
One strategy which has been extensively studied for cancer gene therapy purposes during the last ten years is so called virotherapy. This approach does not necessary involve therapeutic gene transfer since it is based on tumor cell killing caused by viral replication and it will be discussed later (see chapter 2.5.4.1 Conditionally replicative adenoviruses). An overview of clinical cancer gene therapy trials and the used gene therapeutic approaches is presented in table 2.
2.5 Challenges of gene therapy
Although the theory laying the foundations for gene therapy is rather simple and good safety and promising efficiency data have been achieved from preclinical research and clinical trials, there are still several problems encountered with this technique. The key to successful gene therapy is efficient and specific gene transfer, which is a sum of multiple factors. Due to inefficiency of non-viral gene transfer, the following review will focus on viral vector mediated gene therapy.
In order to deliver genes into target tissue, gene transfer vectors first have to escape destruction by the host immune system. It has been shown that 55% of adult humans have pre-existing neutralizing antibodies against adenovirus serotype 5, which is one of the most frequently used gene transfer vectors (Chirmule et al., 1999). In contrast, retroviruses, another group of viral vectors, do not elicit host immune response but they can be rapidly degraded by the complement system (Takeuchi et al., 1994). In addition, the route of viral vector administration plays an important role in gene delivery to target tissues. It has been shown in several preclinical studies that irrespective of the administration route, adenoviral vectors almost invariably evoke neutralizing antibodies directed against adenoviruses (Gahery-Segard et al., 1997; Setoguchi et al., 1994; Smith et al., 1996; Van Ginkel et al., 1995). However, the proteins of the viral capsid have been reported to be differentially recognized depending on the route of administration (Gahery-Segard et al., 1997). In contrast, in humans adenoviral vectors can cause variable, administration route dependent, humoral immune responses (Harvey et al., 1999). In cancer gene therapy, viral vectors are often administered directly into tumor tissue (intratumorally) to achieve therapeutically relevant gene transfer rates. In fact, the majority of reported clinical studies have been carried out using intratumoral
22
administration. However, the intratumoral route is possible only if tumor mass is local and accessible.
Some tumors are located primarily within specific body cavities which enables the intracavitary administration (e.g. intraperitoneal) of gene transfer vectors. Intracavitary administration has been used in several clinical trials e.g. for ovarian cancer (intraperitoneal) (Alvarez et al., 2000; Buller et al., 2002) and for malignant mesothelioma (intrapleural) (Sterman et al., 1998). Nevertheless, if one wishes to treat metastatic cancer, then systemic administration of the gene transfer vector is invariably required.
Intravenous and intra-arterial administrations have been used in clinical studies e.g. for metastatic osteosarcoma (Benjamin et al., 2001) and hepatic metastases for colorectal cancer (Reid et al., 2002), respectively. Further, for immunotherapeutic purposes, viral vectors have been delivered intramuscularly in humans in order to achieve systemic humoral immune response against prostate cancer cells (Pantuck et al., 2004).
The second challenge is to reach the target cells and deliver the therapeutic genes into these cells. Since viral vector uptake requires the binding of the vector to cellular receptors, one limiting factor is the expression levels of viral receptors on the target cell. For example, the expression level of coxsackie- adenovirus receptor (CAR), which mediates adenoviral attachment to their target cells, has been shown to be variable and often very low in tumor cells (Asaoka et al., 2000; Li et al., 1999a; Li et al., 1999b). Thus, this receptor deficiency makes cancer cells rather refractory to adenoviral mediated gene transfer. Further, viral receptors are often widely expressed in normal cells and this means that, healthy tissue can also be susceptible to gene transfer. This naturally leads to the appearance of side effects and decreased gene transfer into target cells.
The final critical step after viral uptake into the target cell is transgene expression. In order to achieve an adequate therapeutic response, the transgene expression has to be at a sufficient level for a sufficient time.
When using non-integrating vectors such as adenoviral vectors, transgene is maintained extrachromosomally in the nucleus, resulting in only transient expression (Somia and Verma, 2000). Long term expression is typically required for a successful therapeutic outcome, especially when correcting inactive or partially functioning genes. Even though there are various viral vectors which integrate their transgene containing DNA into the host cell genome (including retroviral- and AAV-vectors), the use of these vectors does not necessarily ensure long term gene expression (Kay et al., 2001). Since integration into the host cell genome is a random process, the therapeutic gene may become integrated into an inactive part of the genome, resulting in silenced transgene expression (Chen and Townes, 2000). The integrated transgene may also inactivate host genes, which are crucial for normal cellular function or activate harmful genes such as proto-oncogenes (Bushman and Miller, 1997; Shiramizu et al., 1994). In addition, the host immune system may recognize transgene -encoded proteins as foreign and evoke an immune response, which is likely to suppress the transgene expression (Tripathy et al., 1996).
23 2.6 Improvements in cancer gene therapy
2.6.1 Overview
Although there still are many obstacles for successful cancer gene therapy, several approaches have been investigated to circumvent the major problems. One advance has been the characterization of different viruses and their utilization and further modification into safe and efficient gene transfer vehicles (Kootstra and Verma, 2003). Furthermore, development of various targeting techniques means that it has been possible to modify the viral vectors to recognize cancer cells and thus cause minimal transduction to healthy, non-target tissues (Nettelbeck et al., 2000; Peng and Russell, 1999). Thus, targeting can achieve more efficient and accurate gene transfer. In addition, the natural replication capability of various viruses including adeno, herpes simplex and alpha viruses has been exploited in cancer gene therapy (Alemany et al., 2000; Post et al., 2004; Lundstrom, 2001). These oncolytic, replicative viruses can destroy tumor cells via replication which can be limited into target cells by genetic modification. Instead of viral vector modification, also transgenes can be modified so that the resulting therapeutic protein can spread to surrounding cells and thus compensate for the initially low gene transfer efficiency. Several so called translocatory proteins are currently known and their features have been characterized and evaluated for cancer gene therapy purposes (Leifert and Whitton, 2003).
2.6.2 Optimal gene transfer tools for specific purposes: various viral vectors
Viruses need to transfer their genomes efficiently into host cell in order to replicate. Thus, viruses are evolutionarily optimized gene transfer machines. In order to use viruses as safe gene transfer vehicles, virulence genes and genes responsible for viral replication have to be eliminated. In addition to increasing the safety of the viral vector, partial genomic deletions also enable the insertion of foreign genetic material e.g. transgenes.
Viral vectors can be divided into different categories based on their genome (DNA vs. RNA), structure (enveloped vs. non-enveloped) or integration (Fields et al., 1996). The most frequently used viral vectors in human clinical gene therapy trials are based on adeno- and retroviruses and the less common vectors on adeno associated-, herpes simplex-, pox- and alphaviruses (http://www.wiley.co.uk/genetherapy/clinical/).
2.6.2.1 Adenoviral vectors
Adenoviruses are a family of viruses (over 50 serotypes) that most commonly cause benign respiratory illnesses in humans (Volpers and Kochanek, 2004). Adenoviruses are nonenveloped, double stranded DNA viruses, whose genome is surrounded by an icosahedral protein capsid comprising of three major proteins, hexon, penton base and knobbed fiber (Russell, 2000). The linear virus genome is about 36 bp in size and consists of immediate early (E1A), early (E1-E4), intermediate and late genes (L1-L5) (Figure 2).
24
Transcription of these genes can be divided into early and late phases, i.e. those occurring before and after DNA replication (Kootstra and Verma, 2003).
Figure 2. Adenoviral genome. Adenoviral genome contains early (E1-4), intermediate (IX and IVa2) and late (L1-5) genes flanked by left and right inverted terminal repeats (LITR and RITR, respectively). MLP:
major late promoter, Ψ packaging signal.
In order to penetrate inside the host cell, adenoviruses first attach to their primary cellular receptor, the coxsackie- adenovirus receptor (CAR) (Bergelson et al., 1997) followed by interaction with cellular integrins resulting in internalization of the virus via receptor-mediated endocytosis (Wickham et al., 1993) (Figure 3). In the endosomes, the viral genome is released from the viral capsid and thereafter transported into the nucleus. The adenoviral replication cycle is initiated by transcription of the E1A gene followed by transcription of other early genes (Volpers and Kochanek, 2004). Early gene products interfere with antiviral host cell defense mechanism, alter the cell cycle and modulate cellular metabolism in favor of viral replication (Russell, 2000). The linear DNA is flanked by ITRs, which contain the sequences required for the DNA replication (Hay et al., 1995). After the onset of DNA replication mediated by E2 and E4 gene products, intermediate genes are expressed at high levels followed by the expression of late genes driven by the major late promoter (Kay et al., 2001; Russell, 2000). Late genes encode for structural viral proteins that assemble together with viral genomes in the nucleus followed by cell lysis and release of newly synthesized virions (Volpers and Kochanek, 2004).
E1A
E2A E3
E4 E1B
E2B IVa2 IX
LITR Ψ Adenoviral genome RITR
L1 L2 L3
L4 L5
MLP
25 Figure 3. Adenoviral replication cycle. Viruses first attach to the coxsackie- adenovirus receptor (CAR) followed by an interaction with cellular integrins resulting in internalization of the virus via receptor- mediated endocytosis. In the endosomes, the viral genome is released from the viral capsid and thereafter transported into the nucleus for DNA replication. Structural viral proteins assemble together with viral genomes in the nucleus followed by cell lysis and release of newly synthesized virions.
Since their first description in the 1950s, adenoviruses have been studied extensively and they have become one of the most widely used gene transfer tools in human gene therapy. From the gene therapy standpoint, adenoviruses offer numerous advantages: 1) good characterization and reasonable understanding of their biology, 2) low pathogenicity in humans, 3) capability to infect both dividing and quiescent cells, 4) capacity to accommodate relatively large transgenes, 5) low risk for insertional mutagenesis due to an inability to integrate into the host cell genome and 6) relatively easy manipulation and high-titer production (Danthinne and Imperiale, 2000).
The most frequently used adenoviral vectors are based on serotype 5 virus. Adenoviruses can be modified to viral gene transfer vehicles by partial deletion of the viral genome. The first generation adenoviral vectors were made by deleting the E3 and E1 region, which is responsible for initiation of viral replication.
These deletions enabled insertion of ~8 kbp of foreign DNA into the first generation vectors (Danthinne and Imperiale, 2000). To increase the safety and transgene capacity of the adenoviral vectors, additional deletions have yielded second generation adenoviral vectors (E1-4 regions deleted) (Armentano et al.,
CAR Fiber knob
6. Viral protein production
Cytoplasm
Nucleus Adenoviral genome
Penton base
αυβ integrins 1. Binding 2. Internalization
3. Endosytosis
4. Release of
viral genome 5. DNA replication
8. Cell lysis and release of new adenoviruses 7. Viral assembly
26
1995; Gorziglia et al., 1996) and so called gutless vectors (all viral genes deleted) (Kochanek et al., 1996).
Although deletion of various viral proteins have decreased the immunogenicity and toxicity of adenoviral vectors and prolonged the persistence of transgene expression (Engelhardt et al., 1994; Kochanek et al., 1996; Wang et al., 1997), the existing viral structural proteins still elicit an immune response, which prevents repeated administration of these vectors (Somia and Verma, 2000). One possibility to circumvent the immune system would be to use vectors based on various human adenovirus serotypes (Barouch et al., 2004; Mack et al., 1997) or animal adenoviruses (Moffatt et al., 2000; Rasmussen et al., 1999) for readministration. Another disadvantageous feature of adenoviral vectors is their propensity to accumulate into liver and cause hepatotoxicity. This problem can be partially circumvented by targeting the viral vectors to cancer cells (see chapter 2.5.3 Enhanced gene transfer and increased specificity: adenoviral targeting).
Due to the nonintegrating nature of adenoviruses, transgene expression from adenoviral vectors is only transient. Therefore, use of these vectors has mainly focused on treatment of cancer types, where high level, short-term gene expression is sufficient to evoke a therapeutic response. In humans, adenoviral vectors have been widely used in various cancer types including lung-, ovarian-, prostate- and breast cancer and malignant glioma (Tursz et al., 1996; Alvarez et al., 2000; Herman et al., 1999; Stewart et al., 1999;
Immonen et al., 2004).
2.6.2.2 Retro- and lentiviral vectors
Retroviruses are lipid-enveloped, single stranded RNA viruses, which can be divided into oncoretro-, lenti- and spumaviruses (Fields et al., 1996). The enveloped viral particle contains the viral genome consisting of two copies of 8-12 kbp -sized RNA strands surrounded by the nucleocapsid (Kootstra and Verma, 2003).
The retroviral genome flanked by long terminal repeats (LTR) contains three essential genes: gag encoding viral structural protein, pol encoding reverse transcriptase and integrase and env encoding viral envelope glycoprotein, which mediates virus entry (Figure 4). In the lentiviral genome, there are additional accessory genes: for example HIV-1 has vif, vpr, vpu, tat, rev and nef genes that encode for the proteins necessary for efficient viral replication and persistence of infection in the natural target cells of this virus (Kootstra and Verma, 2003).
27 Figure 4. Genome structure of oncoretrovirus and HIV-1 lentivirus. Both genomes contain gag, pol and env genes flanked by long terminal repeats, LTRs. The HIV-1 genome contains six additional genes encoding vif, vpr, vpu, tat, rev and nef. Ψ Packaging signal.
In the early onset of retroviral replication cycle (Figure 5), the retrovirus binds to its receptor followed by membrane fusion and release of RNA genome from the viral capsid (Fields et al., 1996). In the cytosol, the viral genome is copied to double-stranded DNA by the viral reverse transcriptase (Jolly, 1994). The viral DNA is then translocated to the nucleus (retroviruses with passive migration and lentiviruses with active transport), where it becomes integrated into the host-cell genome by its own integrase enzyme to yield a provirus. The cellular machinery is then utilized to make viral RNA, using the provirus as a template. The viral RNA also serves as mRNA, which is translated into viral proteins (Kootstra and Verma, 2003). For viral particle formation, translated viral proteins or their precursors assemble together with two viral RNA strands followed by budding from the plasma membrane. During the budding process, the virus acquires its lipid-coated envelope by incorporating env -glycoproteins from the host cell membrane (Jolly, 1994).
Oncoretrovirus
Lentivirus
TAT VIF ENV
NEF
REV GAG
LTR POL LTR
ψ
VPR GAG
LTR POL ENV LTR
ψ
VPU
28
Figure 5. Retroviral replication cycle. Retrovirus binds to its receptor followed by membrane fusion and release of the RNA genome from the viral capsid. In the cytosol, the viral genome is copied into double- stranded DNA by viral reverse transcriptase. The viral DNA is then translocated to the nucleus where it integrates into the host cell genome. Cellular machinery is then utilized to make new viral genomes and proteins. Translated viral proteins assemble together with two viral RNA strands followed by budding from the plasma membrane. During the budding process virus acquires a lipid-coated envelope (with incorporated env–glycoproteins) from the host cell membrane.
Retroviruses were the first viral vectors used in clinical studies (Blaese et al., 1995). At present, retroviruses have become the most widely used gene transfer vectors in gene therapy. Several features have lead to the wide use of retroviral vectors. First, their genome is rather simple, making the genetic modification required for vector production relatively straightforward. Second, retroviral vectors are able to integrate into the host cell genome, enabling long-term expression of the transgene in target cells and also in their progeny. However, integration does not necessarily ensure stable transgene expression, in fact it may increase the risk for insertional mutagenesis (Bushman and Miller, 1997; Kay et al., 2001;
Shiramizu et al., 1994). Third, retroviral vectors do not elicit an immune response, which minimizes their cytotoxicity and allows the readministration of these vectors. On the other hand, retroviral vectors are susceptible to rapid degradation by the complement system (Takeuchi et al., 1994). The main factor limiting the use of most retroviral vectors (including the most widely used Moloney murine leukemia virus based vectors) is their inability to transduce into non-dividing cells (Barquinero et al., 2004). In contrast, vectors based on lentiviruses are capable of transducing both into dividing and quiescent cells (Delenda, 2004). Another limiting factor for retroviral vectors is their inefficient production at high titers (Romano et
Retroviral receptor
8. Viral protein production Cytoplasm
Nucleus Retroviral
genome (RNA)
1. Binding
2. Fusion of plasmamembranes
3. Endocytosis
7. Production of viral RNA
9. Viral assembly
4. Release of viral RNA genome
5. Reverse transcription Retroviral genome
(dsDNA)
6. Integration Retroviral genomes
29 al., 1999). However, by replacing the region responsible for initiation of transcription (U3 –region) from the 5’ LTR with the CMV promoter, higher vector titers have been obtained (Finer et al., 1994). In addition, modification of viral glycoproteins has generated more stable viral particles allowing them to be concentrated to higher titers (Burns et al., 1993). Nevertheless, retroviral titers still lag far behind, for example adenoviral titers; adenoviruses can be routinely produced in titers 2-3 orders of magnitude higher than the best retroviral-/lentiviral titers.
Due to the relatively small size of the retroviral genome, their transgene capacity is a mere 8 kbp (McCormick, 2001). If one wishes to utilize retroviruses as gene transfer vectors, then the viral genes are completely replaced with the desired transgene and in many cases also with an internal promoter. The viral proteins required for functionality of the vector are produced from separate packaging constructs, which minimize the probability for generation of replication competent viruses and thus increase the safety of these vectors (Romano et al., 1999). Instead of using the packaging cell lines, transient transfection can be used to deliver the required constructs to the producer cells to obtain efficient virus production (Pear et al., 1993; Soneoka et al., 1995). Self-inactivating type vectors (SIN) have been developed to further increase the safety of retroviral vectors (Yu et al., 1986). These vectors contain a deletion on the 3’ LTR, which inactivates the functionality of both enhancer and promoter. When the vector genome is reversely transcribed, this deletion is transferred to the 5‘LTR, abolishing transcriptional activity of the integrated provirus. In the context of HIV-1 based lentiviral vectors, safety aspects have to be more carefully addressed. Thus, all the HIV-1 accessory genes have been deleted and the virus production components have been divided into 3-4 separate parts (Zufferey et al., 1997). Additionally, self-inactivating deletions have been introduced into the vector backbones (Zufferey et al., 1998).
Various approaches have been developed to enhance the transduction rates of retroviral vectors. Retroviral particles have been pseudotyped to broaden the host cell tropism, e.g. the env-glycoprotein has been replaced by other viral proteins such as glycoprotein from vesicular stomatitis virus (VSV-G), a technique which has also been shown to stabilize the vector particles (Yee et al., 1994). Furthermore, oncoretroviral vectors have been retargeted by fusing polypeptides into the envelope glycoproteins (Peng et al., 1999;
Peng et al., 2001). Transduction efficiency and transgene expression of lentivirus vectors has also been enhanced by incorporating central polypurine tract (cPPT) and posttranscriptional regulatory elements (PRE) into the vector constructs. The cPPT has been reported to act by increasing nuclear transport of the viral preintegration complex (Follenzi et al., 2000; Zennou et al., 2000) but it may also facilitate nuclear import of viral RNA species and in their way improve the lentiviral transduction efficiency (Van Maele et al., 2003). Further, PREs from human or woodchuck hepatitis viral origin have been shown to stabilize viral vector RNA improving transgene expression (Patzel and Sczakiel, 1997; Zufferey et al., 1999).
Although the use of retroviral vectors has mainly focused on inherited genetic disease where stable, long- term transgene expression is required, also several clinical studies have also been reported for cancer diseases. Due to the inability of retroviral vectors to transduce non-proliferating cells (e.g. neurons),
30
retrovirally mediated suicide gene therapy has been proposed for the treatment of malignant brain tumors (Culver et al., 1994). Retroviral gene delivery has also been exploited in studies, where extended gene expression is required for therapeutic outcome including introducing wild-type tumor suppressor gene into lung tumors (Roth et al., 1996). In preclinical studies, lentiviral vectors have been evaluated against several cancer types including ovarian (Indraccolo et al., 2002), prostate (Bastide et al., 2003; Zheng et al., 2003) and bladder cancer (Kikuchi et al., 2004). In addition, a cancer gene therapy approach based on lentiviral vector targeting to tumor endothelium has been introduced (De Palma et al., 2003).
2.6.3 Enhanced gene transfer and increased specificity: adenoviral targeting
Adverse side effects caused by unspecific gene transfer to non-target organs can be avoided by targeting the viral vectors and/or the transgenes into cancer cells. Additionally, such maneuvers allow enhancement of gene transfer rates in tumor tissue resulting in an enhanced therapeutic outcome. Especially in adenoviral gene transfer, the biggest obstacle is the variable and often low expression level of CAR in the tumor cells, making these cells rather refractory to adenoviral gene transfer. Further, it would be also important to minimize the adverse side effects by targeting the vectors to avoid the liver. Targeting strategies can be based on transductional or transcriptional approaches.
Transductional targeting is based on altered viral tropism by modifying the viral proteins mediating receptor binding. In adenoviral vectors, the re-targeting moieties allowing CAR- independent delivery can be linked physically to fiber knob or introduced genetically by incorporating the necessary changes into the viral genome (Figure 6). Simultaneously, the binding to the primary viral receptor is blocked. Alternative receptors are typically expressed at high levels in cancer cells but to a lesser extent in normal cells, and this is a one way to improve the tumor cell specificity of the gene transfer. There are several reports of successful adenoviral targeting in vitro and in animal models which have evaluated many alternative cellular receptors including αv integrins (Wickham et al., 1996), CD3 (Wickham et al., 1997), CD40 (Tillman et al., 1999), adenovirus serotype 3 receptor (Kanerva et al., 2002) and prostate specific membrane antigen, PSMA (Kraaij et al., 2005). Further, several studies have shown that adenoviral vector mediated liver toxicity can be reduced by targeting the vectors to cancer cells (Einfeld et al., 2001; Printz et al., 2000).
31 Figure 6. Transductional targeting of adenoviruses. Transductional targeting can be used to re-route the adenoviral to alternative cellular receptors instead of its primary coxsackie-adenovirus receptor (CAR).
Targeting can be based on genetically modified knobs or bi-specific ligands, which bind both to viral knob and alternative receptor.
In addition to re-routing the viral vectors to alternative receptors, expression of therapeutic genes can be limited into tumor tissue. The expression of transcriptionally targeted genes is driven by tissue specific promoters, which are activated in target cells by tissue specific transcription factors. Several tissue specific promoters have been recently characterized and studied for cancer gene therapy purposes including α- fetoprotein for hepatomas (Kanai et al., 1997), cyclo-oxygenase-2 (Cox-2) for ovarian and gastric cancer (Casado et al., 2001; Yamamoto et al., 2001) and osteocalcin for metastatic prostate cancer (Koeneman et al., 2000). Additionally, radiation or drug inducible promoters have been exploited for transcriptional targeting such as early growth response gene 1 (EGR-1) promoter (Manome et al., 1998).
2.6.4 Improved therapeutic outcome by spreading the therapeutic element: oncolytic viruses and protein transduction domains
One possibility to circumvent the initially low gene transfer efficiency is to spread the therapeutic element inside the tumor tissue. One approach is based on oncolytic adenoviruses, which replicate in tumor cells, killing the host cell and spreading to the neighboring cells, eventually throughout the tumor. Another possibility is to exploit protein transduction domains (PTD), which can spread the fused therapeutic protein from one cell to another throughout the tumor.
Integrins
Genetically targeted adenovirus CAR
Untargeted adenovirus
Alternative cellular receptor
Alternative cellular receptor Physically targeted adenovirus
32
2.6.4.1 Conditionally replicative adenoviruses (CRAds)
The use of conditionally replicative adenoviruses (CRAds) is based on their ability to spread throughout the tumor theoretically as long as tumor cells persist by virtue of viral replication and concomitant cell lysis (Alemany et al., 2000; Post et al., 2003).
To minimize adverse side effects and to increase the safety of these anti-cancer agents, replication can be limited to tumor tissue by genetically modifying the CRAd genome. This can be achieved either by partial deletions in the E1 region or by using tissue specific promoters to drive the genes responsible for viral replication (Alemany et al., 2000). Partial viral genome deletions allow virus to replicate selectively in cells with defective p53/p14ARF (Bischoff et al., 1996) or Rb-p16 pathway (Fueyo et al., 2000) that are hallmarks of many cancer cells. Replication has also been limited by using tissue specific promoters such as human telomerase reverse transcriptase (hTERT) (Irving et al., 2004), hepatocellular carcinoma specific α-fetoprotein promoter (Hallenbeck et al., 1999) and melanoma specific tyrosinase promoter (Nettelbeck et al., 2002) to drive the E1A region expression.
To increase the specificity and antitumoral activity of these oncolytic agents, several targeting approaches have been developed. CRAds have been successfully targeted to alternative cellular receptors, e.g. the adenovirus serotype 3 receptor and integrins (Kanerva et al., 2003; Suzuki et al., 2001). In addition, antitumoral activity of CRAds has been improved by exploiting existing viral genes. It has been reported that retaining the adenovirus E3 region (Suzuki et al., 2002) and overexpression of its adenovirus death protein (ADP) (Yun et al., 2004) can increase the oncolytic activity of CRAds. Also, deletion of unnecessary viral genes such as the gene encoding the apoptose inhibitor E1B-19 kDa protein, can enhance oncolysis (Liu et al., 2004).
The most widely studied and first clinically tested CRAd was ONYX-015 (dl1520), from which the p53- inhibitory protein encoding E1B-55 kDa gene had been deleted (Bischoff et al., 1996). It was hypothesized that due to this deletion, ONYX-015 would only be able to replicate in those tumor cells which have lost p53 function, a common occurrence in many cancer cells. In initial preclinical studies, ONYX-015 was reported to replicate selectively in p53-deficient tumor cells (Bischoff et al., 1996) and in addition, it was shown not to replicate in normal epithelial and endothelial cells (Heise et al., 1997). However, subsequent studies showed that there was no correlation between the viral replication and p53-status of the tumor cells and the mechanism of selectivity was far more complex than initially proposed (Goodrum, and Ornelles, 1998; Rothmann et al., 1998). ONYX-015 has also been tested extensively in humans; over 10 clinical trials (phase I-III) have enrolled approximately 300 patients with head and neck cancer (Ganly et al., 2000;
Nemunaitis et al., 2000; Nemunaitis et al., 2001a), metastatic colorectal cancer (Reid et al., 2002), pancreatic cancer (Mulvihill et al., 2001) and ovarian cancer (Vasey et al., 2002). ONYX-015 has also been studied in patients with lung metastasis (Nemunaitis et al., 2001b). The results from these studies showed that although ONYX-015 does seem to be a safe, well tolerated vector that can be administered by various
