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 Distributor : Kuopio University Library P.O. Box 1627 FI-70211 KUOPIO FINLAND Tel. +358 17 163 430 Fax +358 17 163 410 http://www.uku.fi/kirjasto/julkaisutoiminta/julkmyyn.html 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 22.214.171.124 BYSTANDER EFFECT ............................................................................................. 22 126.96.36.199 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. 188.8.131.52 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). 184.108.40.206 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) 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 ), 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. 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