Gene Therapy & Molecular Biology Volume 13 Issue A by Niko Boulikas

GENE THERAPY & MOLECULAR BIOLOGY

Volume 13 Number 1 June 2009 Published by Gene Therapy Press ISSN 1529-9120

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Table of contents

Gene Therapy and Molecular Biology Vol 13 Number 1, June 2009

Pages

Type of Article

Article title

Authors (corresponding author is in boldface)

1-9

Review Article

New trends in aptamer-based electrochemical biosensors

Maria N. Velasco-Garcia, Sotiris Missailidis

10-14

Research Article

Mapping of MHC class binding nonamers from lipid binding protein of Ascaridia galli

Virendra S Gomase, Somnath B Waghmare, Baba Jadhav, Karbhari V Kale

15-19

Review Article

Perspectives in vector development for systemic cancer gene therapy

Arash Hatefi, Brenda F. Canine

20-25

Research Article

Curcumin is not a ligand for peroxisome proliferator-activated receptor-!

26-35

Review Article

FAK as a target for cancer therapy

Venkata R. Narala, Monica R. Smith, Ravi K. Adapala, Rajesh Ranga, Kalpana Panati, Bethany B. Moore, Todd Leff, Vudem D. Reddy, Anand K. Kondapi, Raju C. Reddy Steven N. Hochwald, Vita M. Golubovskaya

36-52

Review Article

53-63

Review Article

Combination of immunotherapy with anaerobic bacteria for immunogene therapy of solid tumours Non-viral and local gene medicine for improvement of cutaneous wound healing

Jian Xu, Xiao Song Liu, Shu-Feng Zhou, Ming Q Wei Markus Rimann, Heike Hall

GENE THERAPY & MOLECULAR BIOLOGY Addresses of Members of the Editorial Board OPEN ACCESS www.gtmb.org Editor

Editor Assistants Boulikas, Teni, Ph.D. Chairman of the Board, Regulon, Inc. Mt View CA 94043 and Regulon AE, Athens, Greece

Koutoudi, Maria M.A. Vougiouka, Maria, B.Sc. Kruit, Adrian, Ph.D. Bellimezi, M., Ph.D Katsoupi J, Mph, Tsogas I., Ph.D, Magkos, A., Ph.D, Christofis Petros., Ph.D, Leto Tziveleka., Ph.D

Associate Editors Missailidis, Sotiris, DPhil (York) Lecturer in Chemistry and Analytical Sciences, The Open University, UK

Roberts, Michael, Ph.D., Regulon A.E., Athens Greece

Magos, Alexandros D. Ph.D. Chemist, Nanotechnology Formulations Regulon A.E., Athens Greece

Rossi, John, Ph.D., Beckman Research Institute of the City of Hope, USA

Crooke, Stanley, M.D., Ph.D., ISIS Pharmaceuticals, Inc, USA

Shen, James, Ph.D., Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China & University of California at Davis, USA.

Gronemeyer, Hinrich, Ph.D. I.N.S.E.R.M., IGBMC, France

Webb, David, Ph.D., Celgene Corporation, USA

Aguilar-Cordova, Estuardo, Ph.D., AdvantaGene, Inc., USA

Berezney, Ronald, Ph.D., State University of New York at Buffalo, USA

Editorial Board Members Akporiaye, Emmanuel, Ph.D., Arizona Cancer Center, USA

Baldwin, H. Scott, M.D Vanderbilt University Medical Center, USA

Amiji, Mansoor M. Ph.D., Professor of Pharmaceutical Sciences Northeastern University, Boston, MA Anson, Donald S., Ph.D., Women's and Children's Hospital, Australia

Barranger, John, MD, Ph.D., University of Pittsburgh, USA

Ariga, Hiroyoshi, Ph.D., Hokkaido University, Japan

Black, Keith L. M.D., Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, USA

Blum, Kenneth, Ph.D., Wake Forest University School of Medicine, USA

Eckstein, Jens W., Ph.D., Akikoa Pharmaceuticals Inc, USA

Bode, Jürgen, Gesellschaft für Biotechnologische Forschung m.b.H., Germany

Fisher, Paul A. Ph.D., State University of New York, USA

Bohn, Martha C., Ph.D., The Feinberg School of Medicine, Northwestern University, USA

Georgiev, Georgii, Ph.D., Russian Academy of Sciences, USA

Bresnick, Emery, Ph.D., University of Wisconsin Medical School, USA

Getzenberg, Robert, Ph.D., Institute Shadyside Medical Center, USA

Caiafa, Paola, Ph.D., Università di Roma “La Sapienza”, Italy

Ghosh, Sankar Ph.D., Yale University School of Medicine, USA

Cheng, Seng H. Ph.D., Genzyme Corporation, USA

Gojobori, Takashi, Ph.D., Center for Information Biology, National Institute of Genetics, Japan

Cole, David J. M.D., Medical University of South Carolina, USA

Harris David T., Ph.D., Cord Blood Bank, University of Arizona, USA

Crooke, Stanley, M.D., Ph.D. ISIS Pharmaceuticals, Inc. USA

Heldin, Paraskevi Ph.D., Uppsala Universitet, Sweden

Davie, James R, Ph.D., Manitoba Institute of Cell Biology, USA

Hesdorffer, Charles S., M.D., Columbia University, USA

DePamphilis, Melvin L, Ph.D., National Institute of Child Health and Human, National Institutes of Health, USA

Hoekstra, Merl F, Ph.D., Epoch Biosciences, Inc., USA

Hung, Mien-Chie, Ph.D., The University of Texas, USA

Kuroki, Masahide, M.D., Ph.D., Fukuoka University School of Medicine, Japan

Johnston, Brian, Ph.D., Somagenics, Inc, USA

Lai, Mei T. Ph.D., Lilly Research Laboratories USA

Jolly, Douglas J, Ph.D., Advantagene, Inc.,USA

Latchman, David S., PhD, Dsc, MRCPath

Joshi, Sadhna, Ph.D., D.Sc., University of Toronto Canada

Lavin, Martin F, Ph.D., The Queensland Cancer Fund Research Unit, The Queensland Institute of Medical Research, Australia

Kiyama, Ryoiti, Ph.D., National Institute of Bioscience and HumanTechnology, Japan

Lebkowski, Jane S., Ph.D., GERON Corporation, USA

University of London, UK

Kotoku Kurachi, Ph.D., University of Michigan Medical School, USA

Li, Liangping Ph.D., MaxDelbr端ck-Center for Molecular Medicine, Germany

Kottaridis, Stavros D., Ph.D. Regulon Inc. USA

Lu, Yi, Ph.D., University of Tennessee Health Science Center, USA

Krawetz, Stephen A., Ph.D., Wayne State University School of Medicine. USA

Lundstrom Kenneth, Ph.D., Bioxtal/Regulon, Inc. Switzerland

Kruse, Carol A., Ph.D., Sidney Kimmel Cancer Center. USA

MacDougald, Ormond A, Ph.D., University of Michigan Medical School, USA

Kuo, Tien, Ph.D., The University of Texas M. D. Anderson Cancer USA Mirkin, Sergei, M. Ph.D., University of Illinois at Chicago, USA

Malone, Robert W., M.D., Aeras Global TB Vaccine Foundation, USA

Noteborn, Mathieu, Ph.D., Leiden University, The Netherlands

Royer, Hans-Dieter, M.D., (CAESAR), Germany

Paleos, Constantinos M., Ph.D. Institute of Physical Chemistry Demokritos. Greece

Rubin, Joseph, M.D., Mayo Medical School

Perry, George , Ph.D. Dean and Professor College of Sciences University of Texas at San Antonio

Saenko Evgueni L., Ph.D., University of Maryland School of Medicine Center for Vascular and Inflammatory Diseases, USA

Pomerantz, Roger, J., M.D., Tibotec, Inc., USA

Santoro, M. Gabriella, Ph.D., University of Rome Tor Vergata, Italy

Mayo Clinic, USA

Raizada, Mohan K., Ph.D., University of Florida, USA

Salmons, Brian, Ph.D., (FSGBiotechnologie GmbH), Austria

Razin, Sergey, Ph.D., Institute of Gene Biology Russian Academy of Sciences, USA

Sharrocks, Andrew, D., Ph.D., University of Manchester, UK

Robbins, Paul, D, Ph.D., University of Pittsburgh, USA

Smythe Roy W., M.D., Texas A&M University Health Sciences Center, USA

Rosenblatt, Joseph, D., M.D, University of Miami School of Medicine, USA

Srivastava, Arun Ph.D., University of Florida College of Medicine, USA

Rosner, Marsha, R., Ph.D., Ben May Institute for Cancer Research, University of Chicago, USA

Steiner, Mitchell, M.D., University of Tennessee, USA

Tainsky, Michael A., Ph.D., Karmanos Cancer Institute, Wayne State University, USA

White, Robert, J., University of Glasgow, UK

Taira, Kazunari, Ph.D., The University of Tokyo, Japan

White-Scharf, Mary, Ph.D., Biotransplant, Inc., USA

Thierry, Alain, Ph.D., National Cancer Institute, National Institutes of Health, France

Wiginton, Dan, A., Ph.D., Children's Hospital Research Foundation, CHRF , USA

Trifonov, Edward, N. Ph.D., University of Haifa, Israel

Yung, Alfred, M.D., University of Texas, USA

Van Dyke, Michael, W., Ph.D., The University of Texas M. D. Anderson Cancer Center, USA

Zannis-Hadjopoulos, Maria Ph.D., McGill Cancer Centre, Canada

Vournakis, John N., Ph.D. Medical University of South Carolina, USA

Zorbas, Haralabos, Ph.D., BioM AG Team, Germany

Chi-Un Pae, MD, PhD, Associate Professor, Department of Psychiatry The Catholic Universoty of Korea College of Medicine

Sikorska, Marianna Ph.D. Neurogenesis and Brain Repair, Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario, Canada

Associate Board Members Falasca, Marco, M.D., University College London, UK

Hiroki, Maruyama, M.D., Ph.D., Niigata University Graduate School of Medical and Dental Sciences, Japan

Gao, Shou-Jiang, Ph.D., The University of Texas Health Science Center at San Antonio, USA

Kazunori, Aoki, M.D., Ph.D., National Cancer Center Research Institute, Japan

Gibson, Spencer Bruce, Ph.D., University of Manitoba, USA

Rigoutsos, Isidore, Ph.D., Thomas J. Watson Research Center, USA

Gu, Baohua, Ph.D., The Jefferson Center, USA

Priya, Aggarwal Ph.D., University of Pennsylvania

Morris, Kevin Vance, Assistant Professor, The Scripps Research Institute, La Jolla, CA

W. Todd Penberthy, PH.D.,

Assistant Professor, Department of Molecular Genetics,Biochemistry, and Microbiology,

Romano, Gaetano Ph.D. Research Associate Professor; Temple University, Philadelphia, U.S.A.

Yuefei Yu Ph.D. Texas Tech University Health Science Center. Research Scientist. Head of the research group.

Hongying Hao M.D./Ph.D., Instructor, Department of Surgery School of Medicine University of Louisville U.S.A. Prof. Emo Chiellini Department of Chemistry & Industrial Chemistry University of Pisa Pisa (Italy)

Robert Harrod, Ph.D. Associate Professor Department of Biological Sciences Southern Methodist University Dallas

Natesan Pushparaj, Peter, Ph.D Research Scientist Glasgow Biomedical Research Centre, University of Glasgow

Raju Reddy, M.D. Assistant Professor of Medicine University of Michigan, Ann Arbor

Hossam M Ashour, Ph.D Department of Microbiology and Immunology Faculty of Pharmacy Cairo University, Egypt

Arash Hatefi (Ph.D., Pharm.D.) Assistant Professor Department of Pharmaceutical Sciences, Center for Integrated Biotechnology, Washington State University

Selvarangan Ponnazhagan, Ph.D. Professor Department of Pathology The University of Alabama at Birmingham

Ekaterina Breous, Ph.D Postdoctoral fellow, University of Pennsylvania, Philadelphia, USA

Chittaranjan Patra Assistant Professor, Department of Biochemistry and Molecular Biology, Mayo Clinic Cancer Center, Rochester, MN, USA.

Gene Therapy and Molecular Biology Vol 13, page 1 Gene Ther Mol Biol Vol 13, 1-9, 2009

New trends in aptamer-based electrochemical biosensors Review Article

Maria N. Velasco-Garcia*, Sotiris Missailidis Department of Chemistry and Analytical Sciences, Faculty of Science, The Open University, Walton Hall, Milton Keynes, United Kingdom, MK7 6AA

__________________________________________________________________________________ *Correspondence: Maria N. Velasco-Garcia, Department of Chemistry and Analytical Sciences, Faculty of Science, The Open University, Walton Hall, Milton Keynes, United Kingdom, MK7 6AA; e-mail: m.n.velasco@open.ac.uk Sotiris Missailidis, Department of Chemistry and Analytical Sciences, Faculty of Science, The Open University, Walton Hall, Milton Keynes, United Kingdom, MK7 6AA; e-mail: s.missailidis@open.ac.uk Key words: Aptamer, Biosensor, Aptasensor, Electrochemical detection, SELEX Abbreviations: Platelet-derived growth factor BB (PDGF-BB); reverse-transcription PCR (RT PCR); self-assembled monolayers (SAMs); Systematic Evolution of Ligands by EXponential enrichment, (SELEX) Received: 28 January 2009; Revised: 6 February 2009 Accepted: 6 February 2009; electronically published: 8 February 2009

Summary The analytical characteristics of aptamers are comparable with those of antibodies for the development of biosensor technology. However, aptamers offer some crucial advantages over antibodies such as selection capability for a variety of targets, easy synthesis, improved reproducibility and stability, simple modification for immobilization to solid supports and enhanced selectivity. This article reviews aptamer technology as well as aptamer-based assay configurations and goes on to explore reported applications in electrochemical aptasensors.

applications where the later are not compatible (Tombelli et al, 2005, 2007). Despite the fact that development of aptasensors has been boosted by using optical and acoustic transducers, this review summarizes the recent developments in the design of electrochemical aptamer-based affinity sensors. In comparison with other detection systems, the electrochemical detection combines a high sensitivity, direct electronic signal production, fast response, robustness, low cost, the possibility of miniaturization and simultaneous multianalyte detection.

I. Introduction Biosensor technology holds a great promise for the healthcare market, the security sector, the food industry, environmental and veterinary diagnostic; harnessing the specificity and sensitivity of biological-based assays packaged into portable and low cost devices which allow for rapid analysis of complex samples in out-of-laboratory environments. However the application of biosensors lags far behind the fundamental research; the challenges facing this basic technology are associated with sensitive detection of specific molecules in samples, stability issues, quality assurance, instrumentation design and cost considerations (Velasco-Garcia and Tottram, 2003). The main biological sensing materials used in biosensor development are the couples enzyme/substrate and antibody/antigen. These are limited by temperature, sensitivity, stability, batch-to-batch variation, large size and difficulty in production. Recent advances and developments in the aptamer area offer a powerful alternative approach involving the use of small RNA or DNA molecules that bind to specific targets with very high affinity and specificity. Aptamer receptors are a novel entity of undeniable potential in analytical applications and can complement or substitute antibodies or offer

II. Aptamers As aptamers approach 20 years since they were originally described (Ellington and Szostak, 1990; Tuerk and Gold, 1990), they are currently receiving a wider recognition in the literature as research reagents, inhibitors, imaging or diagnostic agents (Luzi et al, 2003; Hamula et al, 2006). Aptamers are short, single stranded oligonucleotides, which inherently adopt stable three dimensional sequence-dependent structures. This intrinsic property makes them efficient binding molecules, capable of binding to an array of molecular targets ranging from small ions and organic molecules to large glycoproteins

Velasco-Garcia and Missailidis: New trends in aptamer-based electrochemical biosensors and mucins (Ferreira et al, 2006). Aptamers are a novel and particularly interesting targeting modality, with the ability to bind to a variety of targets including proteins, peptides, enzymes, antibodies and cell surface receptors, as well as small molecules ranging from glucose and caffeine, to steroids to TNT. Aptamers are single stranded oligonucleotides that vary in size between 25-90 bases long and adopt complex secondary and tertiary structures, which facilitate specific interactions with other molecules. They are derived from vast combinatorial libraries through selective targeting and competitive binding. There are two different configurations of aptamers: (i) linear and (ii) molecular beacon. Aptamers with a linear configuration maintain in certain physicochemical conditions a typical 3D conformation with specific binding sites for the target molecule. On the other hand aptamers with a molecular beacon configuration initially form a loop that changes conformation following binding to the analyte of interest. Aptamers offer unique benefits compared to other targeting agents; not only they bind specific ligands with high affinity and selectivity, but aptamers can be easily selected using in vitro techniques and are chemically synthesized, overcoming the use of animal for their production. In comparison to antibodies, aptamers are purified to a very high degree of purity, which eliminates the batch-to-batch variation found in antibodies. Aptamers have higher temperature stability (stable at room temperature) and because of their small size, denser receptor layers could be generated. The animal-free production of aptamers is especially advantageous in cases where the immune response can fail when the target molecule (e.g. a protein) has a structure similar to endogenous proteins or when the antigen consists of toxic or non-immunogenic compounds. Aptamers are relatively

stable under a wide range of buffer conditions and resistant to chemical degradation, although, due to their DNA or RNA constitution, they are sensitive to hydrolytic digestion by nucleases. Aptamers have been modified into nuclease-resistant moieties by modification of the ribose ring at the 2â&#x20AC;&#x2122;-position or by the specific modification of the pyrimidine nucleotide (Pieken et al, 1991; Heidenreich and Eckstein, 1992; Kusser, 2000). It is also possible to chemically modify aptamers to facilitate covalent conjugation to reporters and nanoparticles with 5â&#x20AC;&#x2122; or 3â&#x20AC;&#x2122; amino, biotin or thiol groups. These characteristics make them extremely attractive as alternatives to antibodies and peptides for use in assays, or as diagnostic agents.

A. The SELEX process Aptamers are typically isolated from combinatorial libraries by a process of in vitro evolution, termed SELEX (Systematic Evolution of Ligands by EXponential enrichment). This procedure is an in vitro evolutionary selection process that allows the isolation of aptamer(s), with unique binding properties, from a large library of oligonucleotides through iterative cycles of (i) interaction of a large library of aptamers with the target molecule, (ii) separation of bound from unbound aptamer species, (iii) elution of bound aptamers and (iv) PCR amplification of the binding aptamers for further selection rounds (Figure 1 for an example of the process). An aptamer library usually consists of a variable region (20-40 nucleotides) flanked by known primer sequences on either end for the amplification during the SELEX procedure. The variable region makes up to 1015 different sequences which, combined with the innate ability of oligonucleotides to form stable sequence-

Figure 1. The SELEX process

Gene Therapy and Molecular Biology Vol 13, page 3 dependent structures, provide an array of molecular shapes available for the selection process (Khan and Missailidis, 2008). In the selection steps, the library is incubated with the immobilised target. Unbound or weakbinding species are removed and bound aptamers are eluted using high salt, temperature, chaotropic agents or other such conditions that would affect molecular structure or disrupt molecular interactions. Eluted aptamers are subsequently amplified by PCR (DNA) or reversetranscription PCR (RT PCR) using primers complementary to the flanking sequences in the aptamer library. The enriched pool of binding species forms the pool for the next round of selection. Repeated selection and amplification steps allow identification of the highest binding species, through competitive binding. The selection and amplification step constitutes one round or cycle in a typical SELEX procedure, with anything between 1 and 15 cycles often described in the literature. Counter- or negative selection steps can ensure that the finally selected aptamers are very specific for their target and do not interact with homologous proteins or chemically closely-related molecular targets (Missailidis, 2008). Selected aptamers are subsequently cloned and sequenced to identify the sequence of the binding species and their interactions are usually characterised by a variety of analytical methodologies, prior to move into the various applications they were originally destined for. Selected aptamer can be easily produced by solid phase synthesis and appropriate modifications can be introduced at this stage to confer additional properties to the selected aptamers, such as nuclease resistance (Figure 2), crosslinking ability or improved pharmacokinetic properties. Although SELEX has been the initial methodology associated with aptamer selection and has remained a robust and powerful technique, which has been adapted to various systems and targets, a number of other methodologies have also emerged for the selection of aptamers. Such “non-SELEX” based methods for the selection of aptamers include capillary electrophoresis methodologies (Berezovski et al, 2005; Drabovich et al, 2005), isolation of aptamers with predefined kinetic and thermodynamic properties of their interaction with the target, without the need for amplification, allowing the use of libraries which are difficult or cannot be amplified, or computational methods, which are particularly important in selecting aptamers with inhibitory activities or sequences that undergo ligand dependent conformational changes (Ikebukuro et al, 2005). The SELEX procedure and subsequent technologies for aptamer selection have offered the tools for the designing of aptamers that have found a range of diagnostic applications (Khan and Missailidis, 2008). Such applications include Photo-SELEX (www.somalogic.com) and SELEX NADIR (Winters-Hilt, 2006) using optical probe reporting or nanopore reporting mechanisms respectively, aptamer microarrays (Cho et al., 2005), currently in the market by LC Sciences (www.lcsciences.com), fluorescent aptamers in chips and microspheres (Kirby et al, 2004; Potyrailo et al., 1998), fluorescent sensors for small molecule recognition (Ozaki

et al, 2006; Yamana et al, 2003), quantum dots (Liu et al, 2007; Levy et al, 2005; Choi et al, 2006; Ivanovic et al, 2007), colorimetric detection (Liu and Lu, 2004; Cho et al, 2006; Liu and Lu, 2006), electrochemical detection (Lai et al, 2007; Xiao et al, 2005; Papamichael et al, 2007; Mir et al, 2006) and piezoelectric quartz crystal sensors (Bini et al, 2007). The above methods, fluorescent, electrochemical and colorimetric detection, have also been used in molecular switch type sensors or modular sensor assemblies, where the aptamers usually change conformation upon binding to either emit a fluorescent signal based on an aptamer beacon on sensor, or through non-covalent interaction with the fluorescent label, triggering an electrochemical sensor or leading to change of colour (Stojanovic and Kolpashchikov, 2004; Stojanovic et al, 2001; Baker et al, 2006; Zuo et al, 2007; Stojanovic and Landry, 2002; Frauendorf and Jaschke, 2001), with particular sensitivities in the recognition of small analytes. Aptamers have also been used in enzymatic sensing, without the use of any label or signal related directly to the aptamer. These applications remain based on changes in the conformation of bifunctional aptamers that recognise the target ligand and an enzyme or ribosome. The binding of the aptamer to the ligand results in conformational changes that affect enzymatic activity or protein expression, and it is the later that is subsequently measured (Ogawa and Maeda, 2007; Yoshida et al, 2006; Yoshida et al, 2006) or utilises an enzyme to ligate proximally bound aptamers to large protein targets and allow their subsequent PCR amplification (Fredriksson et al, 2002).

III. Aptamer immobilisation Aptamers can certainly be used as molecular recognition elements in affinity sensing. The small size of aptamers provides advantages over antibodies: (i) a greater

Figure 2. An amino or fluoro modification at the 2’ position of the sugar can confer the oligonucleotide aptamer stability against nuclease degradation. An alternative to using modifications at the 2’ of the sugar (whether at the 3’ or 5’ end of the aptamer, or both) for nuclease resistance is to use a flipped base added to the end of the aptamer.

Velasco-Garcia and Missailidis: New trends in aptamer-based electrochemical biosensors surface density of receptors and (ii) multiple binding to target molecules for sandwich assays. The method of immobilization of aptamers to a solid support affects the sensitivity of the aptamer to the target molecule. Thus, the selected method should maintain the binding affinity and selectivity that the aptamers display in solution (Balamurugan et al, 2008). Aptamers can be attached to the solid support at either the 5’-end or the 3’ end. Both positions have been reported as being used for aptasensor development. However, there are very few studies looking at the effect of the two types of end attachment. Recent work suggests that it depends on the particular aptamer (Cho et al, 2006), although for biological targeting it may be that the 3’ end is more suitable, since the 3’ end is the primary target for exonucleases, and thus its coupling to the solid support would simultaneously confer resistance to nucleases. Gold is used for many electrochemical measurements. Direct attachment of aptamers to gold surfaces could be achieved by using a thiol-alkane linked to the aptamer sequence. The gold surface could also be functionalized and the type of chemistry selected is dependent on what type of terminal functional group is linked to the aptamer (amine, thiol or biotin termini; Figure 3). Gold surfaces functionalized with self-assembled monolayers (SAMs) can address the nonspecific adsorption of aptamer to the surface, which is a particular problem for long oligonucleotides with larger numbers of amine groups. Avidin-biotin technology has also been exploited for aptamer immobilization. Strepavidin can be physically adsorbed or covalently immobilized onto the support and the method mainly requires incubation of the biotin-tethered aptamer with the modified substrate. Studies of the anti-thrombin aptamer revealed this biocoating method gives best results regarding sensitivity compared to other immobilization strategies (Hianik et al, 2007).

Figure 3. Standard nucleic acid modifications used for aptamer immobilisation. Most of the common modifications are linked via the phosphate group of the oligonucleotide aptamer. Various lengths carbon chains are used that can offer higher or lower flexibility.

molecule. One aptamer is immobilized on a suitable solid support to capture the target while the other aptamer for detection is conjugated to a catalytic label. Enzymes, inorganic or organic catalysts or nanoparticles are often used for electrochemical detection. In some cases, when there is only one aptamer for the molecule of interest, antibodies have been reported to be used instead of the second aptamer (Ferreira et al, 2008). If the target protein contains two identical binding sites, the selection of a single aptamer still allows the development of a sandwich assay. Displacement assays have been also proposed to overcome the more challenging detection of small molecules. Affinity interactions between aptamers and small ligands are weaker than interaction with large molecules (with dissociation constants in the µM range, in comparison with constants for large molecules that are in the pM-nM range). The presence of the small target could induce the separation of two strands of a duplex nucleic acid (one strand being the aptamer immobilised to a solid support). Another strategy could rely on the displacement of the aptamer from its complex with the immobilised target molecule when the molecule is present in solution (De-los-Santos-Alvarez et al, 2008). Induced-fit conformational changes of the aptamer after binding to the target molecule can also be used to monitor a bio-recognition event by tagging the aptamer (Figure 4). The use of labels requires precise knowledge of the aptamer folding mechanism after binding to the target and the binding sites. In the case of a redox active marker, the accessibility of the label to the conducting support is associated with the tertiary structure of the aptamer before and after the binding event. However, for small molecules, this strategy is not always viable,

IV. Electrochemical assays In principle, aptamers can be selected for any given target, ranging from small molecules to large proteins and even cells. When aptamers bind small molecular targets, these get incorporated into the nucleic acid structure, buried within the binding pockets of aptamer structures. On the other hand, large molecules (e.g. proteins) are structurally more complicated, allowing aptamer interactions at various sites via hydrogen bonding, electrostatic interactions and shape complementarity. The use of aptamers as bio-recognition elements for small molecules has not been reported as extensively as for protein targets. Mainly two different assay configurations have been reported to transduce these target-binding aptamer events: (i) single-site binding and (ii) dual-site binding (Song et al, 2008). Small molecules are often assayed using the singlesite binding configuration. Protein targets can be assayed via both single-site and dual-site binding. The dual-site binding assay is commonly known as the sandwich assay. Normally, the target molecule is sandwiched between a pair of aptamers that bind to different regions of the large 4

Gene Therapy and Molecular Biology Vol 13, page 5 because the aptamer 3D structure could only be slightly perturbed after the ligand interaction. Redox-active reporting labels could not be covalently tethered to aptamers. Methylene blue has been intercalated into the double-stranded DNA domain of a hairpin configuration aptamer. The binding of the target with the aptamer opens the hairpin structure and releases the intercalated methylene blue. As a result, the amperometric response decreased with the addition of the analyte. This approach is known as â&#x20AC;&#x153;label-freeâ&#x20AC;? method (Figure 5). Related approaches use cationic redox-active reporting units bound to the electrode via electrostatic interactions with the DNA aptamer phosphate backbone. The binding of the target molecule with the aptamer blocked the binding of the cationic reporting units and the electrochemical response decreased. The main disadvantage of these latter approaches is a negative detection signal. Recently, nanomaterials are also providing novel electrochemical sensing approaches. Single-walled carbon nanotube field-effect transistor sensors were developed to monitor aptamer-protein binding studies. Aptamers are well suited for FET sensing due to their small size (1-2 nm) and recognition occurs inside the electrical doublelayer associated with the gate (within the Debye length). The single-walled carbon nanotubes were assembled between source and drain electrodes and the aptamers were immobilized to these nanomaterials. In this label-free approach, the binding of the target molecule to the aptamers altered conductance through the device. The ease

of miniaturization of these sensing devices opens up the feasibility of high-throughput assays in microarrays. Nanoparticles have also been reported as catalytic labels, instead of enzymes, and carriers for ultrasensitive electrochemical detection; because one nanoparticle contains a large number of aptamers, the target binding process is amplified. Impedance spectroscopy has been the most frequently used electrochemical method in the development of electrochemical aptasensors and has shown excellent sensitivity, achieving limit of detection of fM. However, despite the fact that the analytical technique is simple to perform, the data fitting remains a bit complicated. Easier data processing and faster response could be achieved with chonoamperometry, but the limit of detection will be higher and in the nM range.

IV. Applications of electrochemical aptasensors Aptamer publications have now appeared in the literature using most of the electrochemical transducers. The majority of aptamer work on electrochemical sensors is focused on amperometric transducers, but there have been references on aptamers used in impedimetric, FET and recently potentiometric sensors. Furthermore, a lot of the work on the aptamers in electrochemical sensors has been on the model protein, thrombin, which is one of the best characterised complexes in the aptamer literature.

Figure 4. Assays based on induced-fit conformational changes of aptamers.

Figure 5. Label-free electrochemical assays based on: (A) methylene blue intercalated into the DNA aptamer and (B) cationic redoxactive reporting units bound to DNA aptamer phosphate backbone.

Velasco-Garcia and Missailidis: New trends in aptamer-based electrochemical biosensors These have provided proof of principle concepts as to how aptamers could be developed in novel sensors. However, a number of other systems have also now been described, which will be presented in this review.

prepared following formation of 11-mercaptoundecanoic acid self-assembled monolayer on gold electrode. Methylene blue was intercalated on the aptamer by the interaction with two guanine bases. Binding of the thrombin is correlated with the decrease in electrical current intensity in voltammetry. The estimated detection limit of the target thrombin was 11 nM (Bang et al, 2005). The modification of antibodies is difficult, costly and time consuming; however researchers have been using conventional polyclonal antibodies as a capturing probe and labelled-aptamers as the detection probe in new sandwich approaches for protein detection. Kang and colleagues reported in 2008 a modified electrochemical sandwich model for thrombin, based on capturing antibody immobilized onto glassy carbon electrodes with nanogold-chitosan composite film and Methylene blue labelled aptamer as the electrochemical detection probe. Lu and colleagues described in 2008 an electrochemical aptasensor for thrombin that is not based on the target binding-induced conformational change of aptamers. The thrombin-binding aptamer is first assembled onto a gold electrode and then hybridized with a ferrocene labelled short aptamer-complementary DNA oligonucleotide. The binding of the thrombin to the aptamer destroys the double-stranded DNA oligonucleotide and leads to the dissociation of the label short complementary DNA oligonucleotide from the electrode surface, resulting in a decrease in the differential pulse voltammetry responses at the electrode (Lu et al, 2008). This strategy is based on the stronger binding affinity of the aptamers towards their targets rather than to the short aptamer-complementary DNA oligonucleotide labelled with electroactive moieties. The majority of the work performed on aptamerbased electrochemical biosensors is based on aptamers labelled using redox compounds, such as methylene blue, and catalysts such as horseradish peroxidase. However, nanoparticle-based materials offer excellent prospects for a new signal amplification strategy for ultrasensitive electrochemical aptasensing. Platinum nanoparticles have been reported as catalytic labels when linked to a thiolated aptamer. The nanoparticles catalysed the electrochemical reduction of H2O2 and the resulting current enabled the amplified detection of thrombin sandwiched between the aptamer on the electrode surface and the aptamer labelled with the nanoparticles (Polsky et al, 2006). Gold nanoparticles offer several advantages such as electrical conductivity, biocompatibility, ease of self-assembly through a thiol group, increase electrode surface area and amount of immobilized capturing probe. Gold nanoparticles have been used as an electrochemical sensing platform for direct detection of thrombine. The aptamer was immobilised on a screen-printed electrode modified with gold-nanoparticles by avidin-biotin technology. The gold-nanoparticles surface status is evaluated by the Au/Au oxide film formation with cyclic and stripping voltammetry. Gold nanoparticles signal changed with the deposition of biolayers due to differences in electron transfer efficacy and availability of buffer oxygen. Aptamers prefer to adopt the G-quarter structure when binding with thrombin and the

A. Electrochemical aptasensors for the model protein The thrombin-binding aptamer (15-mer, 5â&#x20AC;&#x2122;GGTTGGTGTGGTTGG-3â&#x20AC;&#x2122;) was the first one selected in 1992 by Block and colleagues and its structure has been well characterized and studied. The folded structure in solution is composed of two guanine quartets connected by two T-T loops spanning the narrow grooves at one end and a T-G-T loop spanning a wide groove at the other end (known as the G-quartet structure). This anti-thrombin aptamer has been extensively used as the model oligonucleotide by many researchers to demonstrate the wide applicability of aptamers as bio-recognition elements in biosensors. In the literature, many different electrochemical aptasensors for thrombin detection have been reported. The most straightforward configuration is based on the immobilization of a thiol terminated aptamer on a gold electrode. The aptamer-thrombin interaction is transduced by the electrochemical quantification of p-nitroaniline produced by the thrombinâ&#x20AC;&#x2122;s enzymatic reaction. Thrombin has two electropositive exosites both capable of binding the aptamer, allowing the development of an electrochemical sensor system in a sandwich manner. The thiolated aptamer was immobilized on a gold electrode and, after incubation with the thrombin, a second incubation step with an HRP labelled aptamer took place. Electrochemical detection of HRP was performed using H2O2 and a diffusional osmium based mediator. A similar aptasensor system in sandwich manner for thrombin was developed based on the aptamer for detection, labelled with pyrroquinoline quinine glucose dehydrogenase, and the electric current generated from glucose addition after the formation of the complex on a gold electrode (Ikebukuro et al, 2005). Another strategy for the thrombin sensing is the direct immobilization of the protein on the electrode surface. After the incubation with biotin-labelled aptamer and then with streptavidin-HRP, the electrochemical detection is performed using H2O2 and a diffusional osmium-based mediator. The latter approach achieved the lower limit of detection, 3.5 nM (Mir et al, 2006). Mir and colleagues also developed in 2008 a chronoamperometric beacon biosensor based on a ferrocene-labelled thiol-aptamer. The aptamer adopts a 3D conformational change after binding the thrombin, allowing the ferrocene label to approach to the gold electrode. The interaction is detected via a microperoxidase mediated electron transfer between the label and the electrode surface. The system was demonstrated with impedance spectroscopy and chronoamperometry measurements, achieving a limit of detection of 30 fM with the impedance spectroscopy (Mir et al, 2008). Methylene blue has also been used as an electrochemical marker. The beacon aptamer surface was

Gene Therapy and Molecular Biology Vol 13, page 7 conformational changes made double strand DNA zones appear and facilitated the electron transfer from solution to the electrode surface, based on the double stranded DNAâ&#x20AC;&#x2122;s ability to transport charge along the nucleotide stacking (Suprun et al, 2008). The detection limit of this novel approach is in the nM range. However, the aptasensor measured directly binding events and opened 4 orders of magnitude the operating range of protein concentration. Assays coupling aptamers with magnetic beads for the aptamer or target immobilisation before the electrochemical transduction have also been proposed (Centi et al, 2008). The use of magnetic beads improved the assay kinetics due to the beads being in suspension and also minimized matrix effect because of better washing and separation steps. An ultrasensitive electrochemical aptasensor for thrombin in a sandwich format of magnetic nanoparicleimmobilized aptamer, thrombin and gold nanoparticlelabelled aptamer was reported by Zheng and colleagues in 2007. The magnetic nanoparticle-immobilized aptamer was used for capturing and separating the target protein. The gold nanoparticle-labelled aptamer offered the electrochemical signal transduction. The signal was amplified by forming a network like thiocyanuric acid/ gold nanoparticles to cap more nanoparticles per assay, lowering the detection limit to the aM range

with an RNA aptamer and 1 pM with a DNA aptamer probe (Min et al, 2008). Electrochemical aptasensors for 17-" estradiol have also been reported. The selected biotinylated DNA aptamer was immobilized on a streptavidin-modified gold electrode. The chemical binding of the hormone to the aptamer was monitored by cyclic and square wave voltammetry. When the 17-" estradiol interacted with the aptamer, the current decreased due to the interference of the bound target molecule with the electron flow produced by a redox reaction between ferrocyanide (the mediator) and ferricyanide. The linear range of this aptasensing device was 1-0.01 nM of 17-" estradiol (Kim et al, 2007). Cocaine has been detected by an electrochemical aptasensor incorporating gold nanoparticles onto the surface of a gold electrode. The thiol-derivative aptamer was self-assembled onto the gold nanoparticles. The aptamer was also functionalized at the other termini of the strand with a redox-active ferrocene moiety. The cocaine binding to the aptamer induces the conformational change of the aptamer, bringing the redox tag in close proximity to the electrode, leading to an increase in the current (Li et al, 2008). Methylene blue tagged aptamer has been also explored for the detection of cocaine (Baker et al, 2006). A novel adenosine aptasensor was reported based on the structure change of an aptamer probe immobilized on a gold electrode. After the binding aptamer-target nucleoside, a higher surface charge density and an increasing steric hindrance were obtained that reduce the diffusion of [Fe(CN)6]3-/[Fe(CN)6]4- towards the electrode surface, resulting in a decrease of the current. The biosensing surface was easily regenerated and the aptasensor limit of detection was 10 nM (Zheng et al, 2008).

B. Other targets Aptamer have been selected against a wide range of targets with typical binding affinities in the nanomolar to picomolar range. Recently, electrochemical aptasensors have been reported to detect proteins, hormones and drugs. Papamichael and colleagues described in 2007 a disposable electrochemical aptasensor for Immunoglobulin E, a key marker of atopic diseases (such as asthma, dermatitis and pollenosis). The sensor incorporates a competitive format for IgE detection using a biotinylated form of the aptamer. A standard, indirect method was used where competition between surfacebound IgE and IgE in solution proceeded for the aptamer. The electrochemical detection is achieved by the use of an extravidin-alkaline phosphatase label. After careful optimization of conditions (buffer pH, ionic strength, additional ions and proteins), the aptasensor was performing at levels suitable for human testing (>300ng ml-1). Platelet-derived growth factor BB (PDGF-BB) is one important cytokine involved in neural inflammation and was selected as target for the development of an electrochemical aptasensor based on capacitance change induced by aptamer-protein specific binding, measured by non-faradic impedance spectroscopy. The biosensor detection limit was 40 nM. Electrochemical impedance spectroscopy is a very attractive method for in vivo diagnostics, due to its high sensitivity and label free characteristics (Liao and Cui, 2007). A similar electrochemical detection was also reported to a tuberculosis-related cytokine, the interferon-!. The aptamer-based electrochemical impedance biosensor successfully detected interferon-! to a level of 100 fM

C. Aptasensor arrays Some of the aptamer-based biosensor technology described in this review could be transferred from singleanalyte devices to electrochemical methods offering the possibility of simultaneous measurements of a panel of targets. Wang reviewed the use of metal nanoparticles as tracers for the analysis of nucleic acid hybridization. Magnetic nanoparticles were linked to different probe DNAs and incubated with samples containing different DNA targets. Semiconductor quantum dots were functionalized each with different nucleic acids complementary to the free chain of the target DNA. After dissolution of the metal nanoparticles, the identification of the metal ions by stripping voltammetry enabled the analysis of the different DNA targets (Wang, 2003). Thrombin and lysozyme were detected in parallel using a competitive assay in which thrombin and lysozyme were modified with different semiconductor quantum dots (Hansen et al, 2006). Specific aptamers were immobilized on a gold electrode and bound to the respective labelled protein. In the presence of unlabelled protein in the sample, the quantum-dot functionalized protein is displaced from the electrode into solution. The dissolution of the remaining metal ions on the surface and the electrochemical detection of the released ions enabled the quantitative detection of the proteins.

Velasco-Garcia and Missailidis: New trends in aptamer-based electrochemical biosensors Drabovich A, Berezovski M, Krylov SN (2005) Selection of Smart Aptamers by Equilibrium Capillary Electrophoresis of Equilibrium Mixtures (ECEEM). J Am Chem Soc 127, 11224-11225. Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-822. Ferreira CSM, Matthews CS, Missailidis S (2006) DNA aptamers that bind to MUC1 tumour marker: Design and characterization of MUC1-binding single stranded DNA aptamers. Tumor Biol 27, 289-301. Ferreira CSM, Papamichael K, Guilbault G, Schwarzacher T, Gariepy J, Missailidis S (2008) Design of aptamer-antibody sandwich ELISA for early tumour diagnosis. Anal Bioanal Chem 390, 1039-1050. Frauendorf C, Jaschke A (2001) Detection of small organic analytes by fluorescing molecular switches. Bioorg Med Chem 9, 2521-2524. Fredriksson S, Gullberg M, Jarvius J, Olsson C, Pietras K, Gustafsdottir SM, Ostman A, Landegren U (2002) Protein detection using proximity-dependent DNA ligation assays. Nature Biotechnol 20, 473-477. Hamula CLA, Guthrie JW, Zhang H, Li XF, Le XC (2006) Selection and analytical applications of aptamers. Trends Anal Chem 25, 681-691. Hansen JA, Wang J, Kawde AN, Xiang Y, Gothelf KV, Collins G (2006) Quantum-dot/aptamer-based ultrasensitive multianalyte electrochemical biosensor. J Am Chem Soc 128, 2228-2229. Heidenreich O, Eckstein F (1992) Hammerhead ribozymemediated cleavage of the long terminal repeat RNA of human immunodeficiency virus type 1. J Biol Chem 267, 1904-1909. Hianik T, Ostatna V, Sonlajtnerova M, Grman I (2007) Influence of ionic strength, pH and aptamer configuration for binding affinity to thrombin. Bioelectrochemistry 70, 127133. Ikanovic M, Rudzinski WE, Bruno JG, Allman A, Carrillo MP, Dwarakanath S, Bhahdigadi S, Rao P, Kiel JL, Andrews CJ (2007) Fluorescence Assay Based on Aptamer-Quantum Dot Binding to Bacillus thuringiensis Spores. J Fluoresc 17, 193199. Ikebukuro K, Kiyohara C, Sode K (2005) Novel electrochemical sensor system for protein using the aptamers in sandwich manner. Biosens Bioelec 20, 2168-2172. Ikebukuro K, Okumura Y, Sumikura K, Karube I (2005) A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm. Nucleic Acids Res 33, e108. Kang Y, Feng KJ, Chen JW, Jiang JH, Shen GL, Yu RQ (2008) Electrochemical detection of thrombin by sandwich approach using antibody and aptamer. Bioelectrochemistry 73, 76-81. Khan H, Missailidis S (2008) Aptamers in oncology: a diagnostic perspective. Gene Ther Mol Biol 12, 111-128. Kim YS, Jung HS, Matsuura T, Lee HY, Kawai T, Gu MB (2007) Electrochemical detection of 17beta-estradiol using DNA aptamer immobilized gold electrode chip. Biosens Bioelec 22, 2525-2531. Kirby R, Cho EJ, Gehrke B, Bayer T, Park YS, Neikirk DP, McDevitt JT, Ellington AD (2004) Aptamer-based sensor arrays for the detection and quantification of proteins. Anal Chem 76, 4066-4075. Kusser W (2000) Chemically modified nucleic acid aptamers for in vitro selections: evolving evolution. Rev Mol Biotechnol 74, 27-38. Lai RY, Plaxco KW, Heeger AJ (2007) Aptamer-based electrochemical detection of picomolar platelet-derived growth factor directly in blood serum. Anal Chem 79, 229233.

IV. Conclusions Aptamers have been widely used in a variety of diagnostic and sensor applications, offering a variety of possibilities for aptamer-based sensors in early disease diagnosis and prognosis, substance control, environmental measurements or national security applications on measurements of explosives or potential infectious agents. Yet, despite the advances and the huge body of literature documenting the success of the technology, the commercial application of aptamers in the field of diagnostics remains relatively undeveloped, not least due to the exclusive IP portfolio, and the fact that there is a vast antibody-based diagnostic market and a certain degree of hesitation to move to a new type of product, unless aptamers offer verifiably significant improvements on current technologies that warrant substitution of antibodies in some current assay formats. In this review, different types of electrochemical aptamer-based biosensors have been discussed. Although the optical and mass-sensitive aptasensors have been the most commonly described in the literature, electrochemical transducers have enormous potential and offer simple, rapid, cost-effective and easy to miniaturize sensing in many diagnostic fields. Emerging nanomaterials have also brought new possibilities for developing novel ultrasensitive electrochemical aptasensors.

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Maria N. Velasco-Garcia and Sotiris Missailidis

Gomase et al: Mapping of MHC class binding nonamers from lipid binding protein of Ascaridia galli Gene Ther Mol Biol Vol 13, 10-14, 2009

Mapping of MHC class binding nonamers from lipid binding protein of Ascaridia galli Research Article

Virendra S Gomase1,*, Somnath B Waghmare2, Baba Jadhav2, Karbhari V Kale1 1

Department of Computer Science and Information Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, 431004, (MS), India 2 Department of Zoology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, 431004, (MS), India

__________________________________________________________________________________ *Correspondence: Virendra S Gomase, Department of Computer Science and Information Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, 431004 (MS), India; Mobile- +91-9987770696; e-mail: virusgene1@yahoo.co.in Key words: lipid binding protein, MHC, epitope, solvent accessibility, peptide vaccine Abbreviations: Major histocompatibility complex (MHC); Position Specific Scoring Matrices, (PSSMs); Support Vector Machine, (SVM) Received: 3 March 2009; Revised: 12 March 2009 Accepted: 16 March 2009; electronically published: March 2009

Summary Ascaridia galli involved multiple antigenic components to direct and empower the immune system to protect the host from infection. MHC molecules are cell surface proteins, which take active part in host immune reactions and involvement of MHC class in response to almost all antigens and it give effects on specific sites. Predicted MHC binding regions acts like red flags for antigen specific and generate immune response against the parent antigen. So a small fragment of antigen can induce immune response against whole antigen. This theme is implemented in designing subunit and synthetic peptide vaccines. In this study, we analyzed lipid-binding protein of Ascaridia galli and is allows potential drug targets to identify active sites, which form antibodies against or infection. The method integrates prediction of peptide MHC class binding; proteosomal C terminal cleavage and TAP transport efficiency. Antigenic epitopes of lipid binding protein are important antigenic determinants against the various toxic reactions and infections.

using neural networks trained on C terminals of known epitopes. In analysis predicted MHC/peptide binding is a log-transformed value related to the IC50 values in nM units (Gomase et al, 2008b). This approach is based on the phenomenon of cross-protection, whereby a host infected with a Ascaridia galli is protected against a more severe strain of the same lipid binding protein of Ascaridia galli. The phenotype of the resistant transgenic hosts includes fewer centers of initial infection, a delay in symptom development, and low accumulation. Lipid binding protein of Ascaridia galli is necessary for new paradigm of synthetic vaccine development and target validation (Gomase, 2008a,b).

I. Introduction Ascaridia galli parasitic nematodes produce at least two structurally novel classes of small helix-rich retinoland fatty-acid-binding proteins that have no counterparts in their plant or animal hosts and thus represent potential targets for new nematicides. Nematode-specific fatty-acid family of proteins localises to the surface of the organism, placing it in a strategic position for interaction with the host. Their function as a broad-spectrum and it is thought that it is involved in the evasion of primary host plant defence systems. Prediction of peptide fragments from lipid binding protein of Ascaridia galli involved multiple antigenic components to direct and empower the immune system to protect the host from infection (Timanova et al, 1999; Jordanova et al, 2005a,b). Major histocompatibility complex (MHC) molecules are cell surface proteins, which take active part in host immune reactions and involvement of MHC class-I & II in response to almost all antigens. The predicted binding affinity is normalized by the 1% fractil. The MHC peptide binding is predicted

II. Methodology Antigenic epitopes of lipid binding protein of Ascaridia galli is determined using the Gomase in 2007, Welling, Parker antigenicity methods (Gomase et al, 2007a, b). We also found the Abraham & Leo hydrophobicity, Bull & Breese hydrophobicity, Guy hydrophobicity, Miyazawa hydrophobicity, Roseman hydrophobicity, Wolfenden hydrophobicity, scales. Theses scales

Gene Therapy and Molecular Biology Vol 13, page 11 are essentially a hydrophilic index, with polar residues assigned negative values (Gomase et al, 2008a). The MHC peptide binding of lipid binding protein is predicted using neural networks trained on C terminals of known epitopes. In analysis predicted MHC/peptide binding of lipid binding protein is a logtransformed value related to the IC50 values in nM units. MHC2Pred predicts peptide binders to MHCI and MHCII molecules from protein sequences or sequence alignments using Position Specific Scoring Matrices (PSSMs). Support Vector Machine (SVM) based method for prediction of promiscuous MHC class II binding peptides. SVM has been trained on the binary input of single amino acid sequence (Reche et al, 2002; Buus et al, 2003; Nielsen et al, 2003; Bhasin and Raghava, 2005). In addition, we predict those MHC ligands from whose Cterminal end is likely to be the result of proteosomal cleavage.

MHCII- RT1.B peptide regions, which represented predicted binders from lipid binding protein. The predicted binding affinity is normalized by the 1% fractil. We describe an improved method for predicting linear epitopes (Table 2). The region of maximal hydrophilicity is likely to be an antigenic site, having hydrophobic characteristics, because terminal regions of lipid binding protein is solvent accessible and unstructured, antibodies against those regions are also likely to recognize the native protein (Figures 1-4). It was shown that lipid binding protein is hydrophobic in nature and contains segments of low complexity and high-predicted flexibility (Figures 58). Predicted antigenic fragments can bind to MHC molecule is the first bottlenecks in vaccine design.

III. Results and Interpretation

IV. Conclusion

Lipid binding protein is 508 residues long, having antigenic MHC binding peptides. MHC molecules are cell surface glycoproteins, which take active part in host immune reactions and involvement of MHC class-I and MHC II in response to almost all antigens. BepiPrep Server antigenicity determinant shows epitopes present in the Ascaridia galli the desired immune response. PSSM based server predict the peptide binders to MHCI molecules of lipid binding protein to MHCII molecules of lipid binding protein sequence as H2_Db, I_Ab, I_Ag7, I_Ad, analysis found antigenic epitopes region in lipid binding protein (Tables 1, 2). We also found the SVM based MHCII-IAb; MHCII-IAd; MHCII-IAg7 and

Lipid binding protein of Ascaridia galli peptide nonamers are from a set of aligned peptides known to bind to a given MHC molecule as the predictor of MHCpeptide binding. MHCII molecules bind peptides in similar yet different modes and alignments of MHCIIligands were obtained to be consistent with the binding mode of the peptides to their MHC class, this means the increase in affinity of MHC binding peptides may result in enhancement of immunogenicity of lipid binding protein. These predicted of lipid binding protein antigenic peptides to MHC class molecules are important in vaccine development from Ascaridia galli.

Table 1. PSSM based prediction of MHC ligands, from whose C-terminal ends are proteosomal cleavage sites. MHC-I 8mer_H2_Db 8mer_H2_Db 8mer_H2_Db 8mer_H2_Db 8mer_H2_Db 9mer_H2_Db 9mer_H2_Db 9mer_H2_Db 9mer_H2_Db 9mer_H2_Db 9mer_H2_Db 9mer_H2_Db 9mer_H2_Db 9mer_H2_Db 9mer_H2_Db 10mer_H2_Db 10mer_H2_Db 10mer_H2_Db 10mer_H2_Db 10mer_H2_Db 10mer_H2_Db 10mer_H2_Db 11mer_H2_Db 11mer_H2_Db 11mer_H2_Db 11mer_H2_Db 11mer_H2_Db 11mer_H2_Db

POS. 254 277 64 117 338 253 53 447 157 276 408 260 420 37 427 125 370 457 276 148 253 259 369 419 445 291 325 298

N NLR QSS LLE KAL LSE ENL RDP HKT HSY KQS LKE NAI YIL IAK DHV PTK KII HLI KQS ELI ENL ENA KKI YYI FFH LEK KAM HAR

Sequence SEENAISL SYASWDTL KSPEKMDI SKGSHPTK DEHSKHDI RSEENAISL MLYDNVTKL VTFPNALHL LKDENIHAL SSYASWDTL VKAKNEKLY SLVNGFTEV FLINDHVAM KKARSFAHV AMLRRYNEL EEMTNLAKEL SSMSFYSECI QRYANTTEEY SSYASWDTLI NALFAGHSYL RSEENAISLV ISLVNGFTEV ISSMSFYSECI LFLINDHVAML KTVTFPNALHL APRSHARAVIL SAILGLLKVML AVILRDIHRCL

C VNG IAS MML EEM DAA VNG LEK IQR QEV IAS YIL CKA LRR LSK SDP SAK ITP HHQ ASL KDE NGF CKA ITP RRY IQR RDI SED VKK

MW (Da) 843.9 900.98 929.1 822.91 961.99 1000.09 1078.28 993.17 1034.18 988.06 1074.28 947.05 1041.23 1025.22 1147.37 1159.32 1135.29 1256.3 1101.22 1074.21 1099.22 1060.21 1248.45 1267.55 1222.44 1172.41 1139.5 1290.6

Gomase et al: Mapping of MHC class binding nonamers from lipid binding protein of Ascaridia galli

Table 2. SVM based prediction of promiscuous MHC class II binding peptides from lipid binding protein. MHC ALLELE I-Ab I-Ab I-Ab I-Ab I-Ad I-Ad I-Ad I-Ad I-Ag7 I-Ag7 I-Ag7 I-Ag7 RT1.B RT1.B RT1.B RT1.B

Rank

Sequence

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

PAHPVHLKR PMLYDNVTK HALAPDVKK PAEAFFHKT LINALFAGH NAISLVNGF HALQEVAAA AISLVNGFT SDPAEAFFH DIDAAIEEV QEVAAAHVH HGKPAHPAH TWARSLRTS TFPNALHLI KKAMSAILG TEVCKALKQ

Figure 1. Antigenicity plot of lipid binding protein by Welling et al, scale.

Figure 2. Antigenicity plot of lipid binding protein by HPLC / Parker et al, scale.

Residue No. 91 52 482 438 146 257 163 258 436 344 166 85 16 448 321 266

Peptide Score 1.622 1.403 1.360 1.325 0.618 0.586 0.563 0.553 1.678 1.613 1.576 1.451 1.252 1.189 0.912 0.854

Gene Therapy and Molecular Biology Vol 13, page 13

Figure 3. Hydrophobicity plot of lipid binding protein by Wolfenden et al, scale.

Figure 4. Hydrophobicity plot of lipid binding protein by Bull and Breese scale.

Figure 5. Hydrophobicity plot of lipid binding protein by Gut scale.

Figure 6. Hydrophobicity plot of lipid binding protein by Miyazawa et al, scale.

Gomase et al: Mapping of MHC class binding nonamers from lipid binding protein of Ascaridia galli

Figure 7. Hydrophobicity plot of lipid binding protein by Roseman scale.

Figure 8. Hydrophobicity plot of lipid binding protein by Abraham and Leo scale.

Bankov I, Boteva R (2005a) Conformational and functional analysis of the lipid binding protein Ag-NPA-1 from the parasitic nematode Ascaridia galli. FEBS J 272, 180-9. Jordanova R, Radoslavov G, Fischer P, Torda A, Lottspeich F, Boteva R, Walter RD, Bankov I, Liebau E (2005b) The highly abundant protein Ag-lbp55 from Ascaridia galli represents a novel type of lipid-binding proteins. J Biol Chem 280, 41429-38. Nielsen M, Lundegaard C, Worning P, Lauemøller SL, Lamberth K, Buus S, Brunak S, Lund O (2003) Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein Sci 12, 1007-1017. Reche PA, Glutting JP, Reinherz EL (2002) Prediction of MHC class I binding peptides using profile motifs. Hum Immunol 63, 701-709. Timanova A, Müller S, Marti T, Bankov I, Walter RD (1999) Ascaridia galli fatty acid-binding protein, a member of the nematode polyprotein allergens family. Eur J Biochem 261, 569-76.

References Bhasin M, Raghava GP (2005) P cleavage: an SVM based method for prediction of constitutive proteasome and immunoproteasome cleavage sites in antigenic sequences. Nucleic Acids Res 33, W202-207. Buus S, Lauemøller SL, Worning P, Kesmir C, Frimurer T, Corbet S, Fomsgaard A, Hilden J, Holm A, Brunak S. (2003) Sensitive quantitative predictions of peptide-MHC binding by a 'Query by Committee' artificial neural network approach. Tissue Antigens 62, 378-384. Gomase VS, Kale KV, Shyamkumar K, Shankar S (2008a) Computer Aided Multi Parameter Antigen Design: Impact of Synthetic Peptide Vaccines from Soybean Mosaic Virus. ICETET 2008, IEEE Computer Society in IEEE Xplore, Los Alamitos, California, 629-634. Gomase VS, Tagore S, Shyamkumar K (2008b) Prediction of antigenic binders from c-terminal domain Human papillomavirus oncoprotein e7. Gene Ther Mol Biol 12, 147-166. Gomase VS (2008a) Computer aided multi parameter antigen design: Impact of synthetic peptide vaccines from Latrodectus tredecimguttatus. Int J Bioinformatics 1, 53-54. Gomase VS (2008b) In silico prediction of antigenic epitope of neurotoxin M14 from Buthus eupeus. Int J Bioinformatics 1, 47-51. Gomase VS, Kale KV, Jyotiraj A, Vasanthi R (2007a) Identification of mhc ligands from alfalfa mosaic virus. Med Chem Res 15, 160. Gomase VS, Kale KV, Chikhale NJ, Changbhale SS (2007b) Prediction of MHC Binding Peptides and Epitopes from Alfalfa mosaic virus. Curr Drug Discov Technol 4, 1171215. Jordanova R, Radoslavov G, Fischer P, Liebau E, Walter RD,

Virendra Gomase and Somnath B Waghmare

Gene Therapy and Molecular Biology Vol 13, page 15 Gene Ther Mol Biol Vol 13, 15-19, 2009

Perspectives in vector development for systemic cancer gene therapy Review Article

Arash Hatefi*, Brenda F. Canine Department of Pharmaceutical Sciences, Center for Integrated Biotechnology, Washington State University, Pullman, WA, USA

__________________________________________________________________________________ *Correspondence: Arash Hatefi, Department of Pharmaceutical Sciences, Center for Integrated Biotechnology, Washington State University, P.O. Box 646534, Pullman, WA, 99164, USA; Tel: 509-335-6253; Fax: 509-335-5902; e-mail: ahatefi@wsu.edu Key words: non-viral vectors, cancer gene therapy, vector development, viral vectors Abbreviations: adenovirus, (Ad); coxsackievirus and adenovirus receptor, (CAR); fibroblast growth factor 2, (FGF2); fibroblast growth factor receptor, (FGFR); herpes simplex virus, (HSV); multiplicity of infection, (MOI); ornithine transcarbamylase, (OTC) Received: 10 February 2009; Revised: 25 March 2009 Accepted: 26 March 2009; electronically published: April 2009

Summary Gene therapy is perceived as a revolutionary technology with the promise to cure almost any disease, provided that we understand its genetic basis. However, enthusiasm has rapidly abated as multiple clinical trials have failed to show efficacy. The limiting factor seems to be the lack of a suitable delivery system to carry the therapeutic genes to the target tissue safely and efficiently. Therefore, advancements in cancer gene therapy in general depend on the development of novel vectors with maximum therapeutic efficacy at the target site and minimal toxicity to normal tissues. This mini-review highlights both the major fortes and the unique challenges associated with the state-ofâ&#x20AC;&#x201C;theart gene carriers currently being used in cancer gene therapy.

2003; Shen and Nemunaitis, 2006). What has been long desired is a technology which combines the biocompatibility, efficiency and the ability to engineer an effective gene-transfer technology. Since internalization of both viral and non-viral vectors is the first step in their transfection pathway, knowledge and understanding of their entry mechanisms is of major importance for the design of efficient viral and non-viral vehicles for cancer gene therapy.

I. Introduction Gene therapy is perceived as a ground-breaking technology with the promise to cure almost any disease, provided that we understand the genetic and molecular basis of the malady being treated. However, enthusiasm has rapidly abated as multiple clinical trials have failed to show efficacy. The limiting factor seems to be the lack of a suitable delivery system to carry the therapeutic genes safely and efficiently to the target tissue (Louise, 2006). Gene-transfer technology is still in a nascent stage owing to several inherent limitations in the existing delivery methods. While lipid-based vectors (liposomes) provide high transfection efficiency, their large scale production, reproducibility and cytotoxicity remain a major concern (Lv et al, 2006). On the other hand, cationic polymers are robust and relatively biocompatible, but they suffer from poor gene-transfer efficiency (Pack et al, 2005). Adenoviruses are the vehicles of choice for cancer gene therapy at this point particularly due to their ability to overcome the intracellular barriers and the enormous possibility for recombinant engineering. However, nonspecific binding to all cells that over-express coxsackievirus and adenovirus receptor (CAR), potential immunogenicity, high costs of production, and the fact that the majority of cancer cells do not express CAR has limited their use for cancer gene therapy (Thomas et al,

II. Strengths current vectors

and

weaknesses

A. Viral vectors for systemic cancer gene therapy Viruses have evolved to efficiently infect their host, overcome the cellular barriers and transfer their genetic material into the cellâ&#x20AC;&#x2122;s nucleus. One viral vector that has received considerable attention in cancer gene therapy is adenovirus. The basic elements of the trafficking pathway for adenovirus include high affinity binding of the capsid to receptors on the cell surface, internalization by endocytosis, lysis of the endosomal membrane resulting in escape to the cytosol, trafficking along microtubules, binding to the nuclear envelope, and insertion of the viral

Hatefi and Canine: Perspectives in vector development for systemic cancer gene therapy genome through the nuclear pore (Leopold and Crystal, 2007). Adenoviruses have high affinity for the CAR and use it to enter the cells. Although they are highly efficient in transducing cells that over-express CAR on their surface, they are considered poor gene delivery systems in cells that have low expression of CAR (Li et al, 1999). In addition, CAR is expressed on many normal cells which undermines the ability of this vector to specifically reach target cancer cells when administered systemically. Thus, adenovirus is not considered a universal efficient vehicle for cancer gene therapy as the majority of cancer cells do not over-express CAR (Shen and Nemunaitis, 2006). Another virus, Herpes Simplex Virus overcomes this deficiency by utilizing a different receptor to enter cancer cells. The initial attachment of HSV involves the interaction of viral envelope glycoproteins with the glycosaminoglycan moieties of cell surface heparan sulfates (Spear et al, 1992). However, like CAR, expression of heparin sulfates is not unique to cancer cells and can be found routinely in normal cells. As a result, systemic administration of HSV could also be problematic. Attachment of a targeting ligand to the viral capsid has been used as a means to make adenovirus specifically bind cancer cells and internalize via receptor mediated endocytosis. One example is attachment of the ligand, fibroblast growth factor 2 (FGF2) which has affinity for the basic fibroblast growth factor receptor (FGFR) (Green et al, 2008) (Figure 1). This receptor is over-expressed in subpopulations of lung, prostate and breast cancer (Chandler et al, 1999). While promising, the attachment of the ligand to the virus capsid involves chemical conjugation during which a significant portion of viruses could become inactive. As a result, obtaining high titers of active virus for delivery becomes expensive. Alternatively, retargeted viruses can be genetically engineered through the abrogation of CAR binding (e.g., Y477A mutation in adenoviral fiber protein) and insertion of a receptor-

specific binding peptide in the HI loop of the fiber protein (Piao et al, 2009). In this approach, no chemical conjugation step is involved. However, one potential problem with this approach is that targeting peptides with considerable 3D structure could interfere with the proper packaging of the viral capsid proteins and result in reduced transduction efficiency. Furthermore, such alterations in receptor targeting could impact transduction efficiency of viruses due to the change in trafficking routes and internalization pathways (Varga et al, 2000).

B. Are viral vectors highly immunogenic? There are five main classes of viral vectors which can be categorized into two groups (Table 1) according to whether their genomes integrate into host cellular chromatin (oncoretroviruses and lentiviruses) or persist in the cell nucleus predominantly as extrachromosomal episomes (AAVs, adenoviruses and herpes viruses).

Figure 1. Schematic representation of cell transfection by adenoviruses (Ad). While CAR represents coxsackie adenovirus receptor, FGFR represents fibroblast growth factor receptor (FGFR).

Table 1. Characteristics of major classes of viral vectors.

Immunogenic Potential

Specificity

Limitation

Retrovirus

Low

Dividing Cells only

Integration may induce oncogenesis

Lentivirus

Low

Broad

Integration may induce oncogenesis

AAV

Low

Broad

Herpes Simplex Virus

High

High in neurons

Adenovirus

High

Broad (CAR receptor)

Vector

Major Advantage

Integrated Persistent gene transfer in dividing cells Persistent gene transfer in most cells

Episomal Small packaging capacity Transient expression in some non-neuronal cells Capsid may induce inflammatory response

Non-inflammatory and non-pathogenic Large packaging capacity Efficient transduction of most cells

Gene Therapy and Molecular Biology Vol 13, page 17

D. Are viral vectors more efficient than non-viral vectors?

Out of these five, only herpes simplex virus (HSV) and adenovirus (Ad) have been shown to be highly immunogenic. In general, introduction of any non-self molecule, including viruses, into the body has the potential to trigger an immune response. However, the level of immune response to the foreign entity is dependent on the dose, the structure and any previous exposures. For example, a patient (Jesse Gelsinger) who suffered from a partial deficiency of ornithine transcarbamylase (OTC) took part in a gene therapy clinical trial conducted at the University of Pennsylvania in 1999. OTC is a liver enzyme that is required for the safe removal of excessive nitrogen from amino acids and proteins. Gelsinger received the highest dose of vector in the trial (3.8 ! 1013 particles). After 4 hours of treatment Gelsinger developed a high fever and within four days of treatment he died from multiorgan failure. A female patient who received a similar dose (3.6 ! 1013 particles) experienced no unexpected side effects. It has been speculated that previous exposure to a wild-type virus infection might have sensitized Gelsingerâ&#x20AC;&#x2122;s immune system to the vector (Bostanci, 2002). If a lowered dose of the adenovirus was administered, Gelsingerâ&#x20AC;&#x2122;s symptoms may not have been as catastrophic. Therefore, drawing a firm conclusion that viral vectors are highly immunogenic and deadly is premature.

1. Viral vectors versus targeted non-viral vectors From the available literature, it is apparent that the efficiency of non-viral vectors is usually compared with the adenoviral vector which arguably is the most efficient viral vector (Thomas et al, 2003). As a result of this comparison, it is generally believed that non-viral vectors are less efficient. This comparison may not be completely reliable in all situations as adenoviral vectors are targeted systems which utilize abundant CAR receptors to enter the cells (Wickham et al. 1993). When CAR receptors are not abundant the transfection levels are markedly decreased (Li et al, 1999). Targeted non-viral vectors are usually equipped with ligands that are intended to bind to overexpressed receptors. These include growth factor receptors (e.g., FGFR and HER2) and transferrin. The abundance of these receptors on the surface of the cells and their affinities towards their corresponding ligands may not be as high as CAR. Therefore, non-viral vectors that could be as efficient as adenoviruses in trasfecting dividing cells will show less efficiency when internalizing through receptors because of the difference in receptor number and binding affinity. The question then is how viral and targeted non-viral vectors can be fairly compared in terms of gene transfer efficiency. One potential solution would be evaluation of transfection efficiency normalized to the abundance of the receptor being utilized. This is to remove the bias associated with the receptor numbers. Another answer could be as simple as comparison of FGF2 targeted non-viral vector with FGF2 retargeted adenovirus. In this approach, the bias associated with receptor binding affinity and internalization pathway can be eliminated. Alternatively, adenovirus can be compared with non-viral vectors that are equipped with CAR ligands to target cells. In this way the bias associated with the number of entry gates as well as receptor binding affinity will be eliminated. It is also noteworthy that the number of viral or non-viral particles delivered needs to be kept equal to achieve the same multiplicity of infection (MOI). To date no study has been reported that has considered the abovementioned factors in order to appropriately compare viral versus targeted non-viral vectors.

C. Are non-viral vectors biocompatible? Polymeric or liposomal based non-viral vectors are utilized to complex plasmid DNA forming stable nanoparticles. This complexation protects the DNA from serum endonucleases and also condenses the DNA into nanosize particles suitable for cellular uptake. Non-viral polymeric vectors are generally believed to be nonimmunogenic mostly due to their lack of structural hierarchy. Although there has been reports on the toxicity of such vectors (e.g., PEI or liposomes) (Lv, Zhang et al, 2006), in general they are assumed to have low immunogenic potential. Polymers such as poly (ethylene glycol), for example, have been utilized to sterically stabilize the surface of particles reducing the interaction of particles with the elements of the immune system (Chekhonin et al, 2005). However, two separate groups recently reported that repeated injection of PEGylated liposomes in rats and mice elicits PEG-specific IgM/IgG (Ishida et al, 2006; Judge et al, 2006). These studies highlight the potential that even a presumably safe polymer such as PEG can invoke an immune response if injected in high doses and repeatedly. This in turn may undermine the ability of PEG to be used as surface stabilizer in drug delivery systems that need multiple injections to achieve significant therapeutic response. As a result, drawing a general conclusion that non-viral vectors are less immunogenic than viral vectors is also premature at this point. Therefore, there is a continuing need for the development of more biocompatible and bio-interactive polymers that can reduce immunogenicity. This in turn enhances blood circulation time of drug delivery systems maximizing their therapeutic efficacy at the target site.

2. Viral vectors versus non-targeted non-viral vectors For non-targeted non-viral vectors, the surface charge of the nanoparticles usually dictates the binding efficiency to the surface of the cells. Once complexed with pDNA, the nanoparticles are formulated to have a slight positive surface charge (e.g., 10-40 mV). This facilitates binding to the negatively charged phosphate groups on the surface of the cell membranes resulting in internalization via caveolae or clathrin mediated endocytosis (Midoux et al, 2008). In this scenario, comparison of viral with nontargeted non-viral vectors would not be appropriate as they utilize entirely different internalization pathways. Transfection efficiency, in this case, will be dependent on the cell type not the vector. In one cell line (e.g., CAR

Hatefi and Canine: Perspectives in vector development for systemic cancer gene therapy positive), the viral vector will be more efficient than the non-viral vector, while in another cell line (e.g., CAR negative), the non-viral vector will show higher efficiency. Therefore, drawing any conclusion regarding the efficiency of viral vectors versus non-targeted non-viral vector may not be appropriate.

Acknowledgement This work was supported in part by the NIH biotechnology training fellowship (GM008336) to Canine and Reeves

References Bostanci A (2002) Gene therapy. Blood test flags agent in death of Penn subject. Science 295, 604-5. Canine BF, Wang Y, Hatefi A (2008) Evaluation of the effect of vector architecture on DNA condensation and gene transfer efficiency. J Control Release 129, 117-123. Chandler LA, Sosnowski BA, Greenlees L, Aukerman SL, Baird A, Pierce GF (1999) Prevalent expression of fibroblast growth factor (FGF) receptors and FGF2 in human tumor cell lines. Int J Cancer 81, 451-8. Chekhonin VP, Zhirkov YA, Gurina OI, Ryabukhin IA, Lebedev SV, Kashparov IA, Dmitriyeva TB (2005) PEGylated immunoliposomes directed against brain astrocytes. Drug Deliv 12, 1-6. Dreher MR, Liu W, Michelich CR, Dewhirst MW, Yuan F, Chilkoti A (2006) Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers. J Natl Cancer Inst 98, 335-44. Furgeson DY, Dreher MR, Chilkoti A (2006) Structural optimization of a "smart" doxorubicin-polypeptide conjugate for thermally targeted delivery to solid tumors. J Control Release 110, 362-9. Green NK, Morrison J, Hale S, Briggs SS, Stevenson M, Subr V, Ulbrich K, Chandler L, Mautner V, Seymour LW, Fisher KD (2008) Retargeting polymer-coated adenovirus to the FGF receptor allows productive infection and mediates efficacy in a peritoneal model of human ovarian cancer. J Gene Med 10, 280-9. Hatefi A, Cappello J, Ghandehari H (2007) Adenoviral gene delivery to solid tumors by recombinant silk-elastinlike protein polymers. Pharm Res 24, 773-9. Hatefi A, Megeed Z, Ghandehari H (2006) Recombinant polymer-protein fusion: a promising approach towards efficient and targeted gene delivery. J Gene Med 8, 468-76. Ishida T, Ichihara M, Wang X, Yamamoto K, Kimura J, Majima E, Kiwada H (2006) Injection of PEGylated liposomes in rats elicits PEG-specific IgM, which is responsible for rapid elimination of a second dose of PEGylated liposomes. J Control Release 112, 15-25. Judge A, McClintock K, Phelps JR, Maclachlan I (2006) Hypersensitivity and loss of disease site targeting caused by antibody responses to PEGylated liposomes. Mol Ther 13, 328-37. Krajcik R, Jung A, Hirsch A, Neuhuber W, Zolk O (2008) Functionalization of carbon nanotubes enables non-covalent binding and intracellular delivery of small interfering RNA for efficient knock-down of genes. Biochem Biophys Res Commun 369, 595-602. Lacerda L, Bianco A, Prato M, Kostarelos K (2006) Carbon nanotubes as nanomedicines: from toxicology to pharmacology. Adv Drug Deliv Rev 58, 1460-70. Leopold PL, Crystal RG (2007) Intracellular trafficking of adenovirus: many means to many ends. Adv Drug Deliv Rev 59, 810-21. Li D, Duan L, Freimuth P, O'Malley BW Jr (1999) Variability of adenovirus receptor density influences gene transfer efficiency and therapeutic response in head and neck cancer. Clin Cancer Res 5, 4175-81. Liu Y, Yu ZL, Zhang YM, Guo DS, Liu YP (2008) Supramolecular architectures of beta-cyclodextrin-modified chitosan and pyrene derivatives mediated by carbon

III. Emerging new technologies In recent years there has been a great deal of interest on the development of recombinant polymers (biopolymers) with applications in tissue engineering, drug delivery and gene therapy (Dreher et al, 2006; Furgeson et al, 2006; Hatefi et al, 2006, 2007; Canine et al, 2008; Nettles et al, 2008). The major advantage of the polymers that are genetically engineered versus chemical synthetic methods is the homogeneity, control over sterotacticity and full control over the architecture (Urry, 1997). These biopolymers bear the potential to hybridize the strengths of both viral and non-viral vectors in order to overcome the extra- and intracellular barriers to efficient, safe and cost-effective gene delivery. This is due to their versatility, flexibility, unlimited capacity and most importantly ability to bioengineer at the molecular level. In addition to genetically engineered polymers with well-defined architecture, synthetic inorganic gene carriers (e.g., nanorods and tubes) are exciting, emerging technologies that would allow precise control of composition, size and multifunctionality of the delivery system (Krajcik et al, 2008; Liu et al, 2008). For example, Leongâ&#x20AC;&#x2122;s group recently reported a non-viral gene-delivery system based on multi-segment bimetallic nanorods with the ability to simultaneously bind condensed plasmid DNA and targeting ligands in a spatially defined manner (Salem et al, 2003). Although promising, there are some concerns related to the toxicity and pharmacological fate of inorganic nanocarriers (Lacerda et al, 2006). Nonetheless, synthetic inorganic gene carriers have great potential to make a significant impact on the science of cancer gene therapy.

IV. Conclusion Lack of an efficient, non-toxic and non-immunogenic gene delivery system remains the major limiting factor to advancements in cancer gene therapy. Adenovirus while efficient in some cell lines (CAR positive) raises concerns about safety as well as targetability. Non-viral vectors while potentially less immunogenic than viral vectors have not been studied thoroughly enough to reliably state that they do not trigger major immune responses. Further studies need to be done in terms of long term administration, dose scheduling, and treatment thresholds to examine these effects. The efficiency of non-viral vectors also needs to be reinvestigated taking into account the model system being used before blanket comparisons between non-viral and viral efficiency levels can be made. In both non-specific viral and non-viral vectors the use of targeting ligands is an attractive alternative to non-specific delivery particularly in cancer therapy. No matter which system, viral or non-viral, improvements in current technologies continue to be needed.

Gene Therapy and Molecular Biology Vol 13, page 19 nanotubes and their DNA condensation. J Am Chem Soc 130, 10431-9. Louise C (2006) Nonviral vectors. Methods Mol Biol 333, 20126. Lv H, Zhang S, Wang B, Cui S, Yan J (2006) Toxicity of cationic lipids and cationic polymers in gene delivery. J Control Release 114, 100-9. Midoux P, Breuzard G, Gomez JP, Pichon C (2008) Polymerbased gene delivery: a current review on the uptake and intracellular trafficking of polyplexes. Curr Gene Ther 8, 335-52. Nettles DL, Kitaoka K, Hanson NA, Flahiff CM, Mata BA, Hsu EW, Chilkoti A, Setton LA (2008) In situ crosslinking elastin-like polypeptide gels for application to articular cartilage repair in a goat osteochondral defect model. Tissue Eng Part A 14, 1133-40. Pack DW, Hoffman AS, Pun S, Stayton PS (2005) Design and development of polymers for gene delivery. Nat Rev Drug Discov 4, 581-93. Piao Y, Jiang H, Alemany R, Krasnykh V, Marini FC, Xu J, Alonso MM, Conrad CA, Aldape KD, Gomez-Manzano C, Fueyo J (2009) Oncolytic adenovirus retargeted to DeltaEGFR induces selective antiglioma activity. Cancer Gene Ther 16, 256-65. Salem AK, Searson PC, Leong KW (2003) Multifunctional nanorods for gene delivery. Nat Mater 2, 668-71. Shen Y, Nemunaitis J (2006) Herpes simplex virus 1 (HSV-1) for cancer treatment. Cancer Gene Ther 13, 975-92. Spear PG, Shieh MT, Herold BC, WuDunn D, Koshy TI (1992) Heparan sulfate glycosaminoglycans as primary cell surface

receptors for herpes simplex virus. Adv Exp Med Biol 313, 341-53. Thomas CE, Ehrhardt A, Kay MA (2003) Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet 4, 346-58. Urry DW (1997) Physical chemistry of biological free energy transduction as demonstrated by elastic protein-based polymers. J Phys Chem B 101, 11007-11028. Varga CM, Wickham TJ, Lauffenburger DA (2000) Receptormediated targeting of gene delivery vectors: insights from molecular mechanisms for improved vehicle design. Biotechnol Bioeng 70, 593-605. Wickham TJ, Filardo EJ, Cheresh DA, Nemerow GWickham TJ, Filardo EJ, Cheresh DA, Nemerow GR (1993) Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment. Cell 73, 309-19.

Arash Hatefi

Narala et al: Curcumin is not a ligand for PPAR-! Gene Ther Mol Biol Vol 13, 20-25, 2009

Curcumin is not a ligand for peroxisome proliferator-activated receptor-! Research Article

Venkata R. Narala1, Monica R. Smith1, Ravi K. Adapala1, Rajesh Ranga1, Kalpana Panati2, Bethany B. Moore1, Todd Leff3, Vudem D. Reddy2, Anand K. Kondapi4, Raju C. Reddy1,* 1

Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109, USA 2 Center for Plant Molecular Biology, Osmania University, Hyderabad 500 007, India 3 Center for Integrative Metabolic and Endocrine Research, Wayne State University School of Medicine, Detroit, MI 48201, USA 4 Department of Biotechnology, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India

__________________________________________________________________________________ *Correspondence: Raju C. Reddy M.D., University of Michigan, Division of Pulmonary and Critical Care Medicine, 109 Zina Pitcher Place, 4062 BSRB, Ann Arbor, MI 48109-2200, USA; Tel: (734) 615-2871; Fax: (734) 615-2111; e-mail: rajuc@umich.edu Key words: PPAR-!, TGF-", rosiglitazone, ciglitazone, PPRE, preadipocyte, fibroblast, turmeric, peroxisome, curcumin Abbreviations: dithiothreitol, (DTT); glutathione-S-transferase, (GST); glyceraldehyde-3-phosphate dehydrogenase, (GAPDH); isopropyl-1-"-D-galactopyranoside, (IPTG); peroxisome proliferator response element, (PPRE); peroxisome proliferator-activated receptor-!, (PPAR-!); #-smooth muscle actin, (#-SMA) Received: 24 February 2009; Revised: 14 March 2009 Accepted: 16 March 2009; electronically published: April 2009

Summary Curcumin, a compound found in the spice turmeric, has been shown to possess a number of beneficial biological activities exerted through a variety of different mechanisms. Some curcumin effects have been reported to involve activation of the nuclear transcription factor peroxisome proliferator-activated receptor-! (PPAR-!), but the concept that curcumin might be a PPAR-! ligand remains controversial. Results reported here demonstrate that, in contrast to the PPAR-! ligands ciglitazone and rosiglitazone, curcumin is inactive in five different reporter or DNAbinding assays, does not displace [3H]rosiglitazone from the PPAR-! ligand-binding site, and does not induce PPAR-!-dependent differentiation of preadipocytes, while its ability to inhibit fibroblast-to-myofibroblast differentiation is not affected by any of four PPAR-! antagonists. These multiple lines of evidence conclusively demonstrate that curcumin is not a PPAR-! ligand and indicate the need for further investigation of the mechanisms through which the compound acts.

signaling pathways. These varied beneficial effects have led to investigation of curcumin as a potential therapeutic agent in a number of disease conditions (Reddy et al, 2005; Thangapazham et al, 2006; Aggarwal et al, 2007). Peroxisome proliferator-activated receptor-! (PPAR!) is a member of the nuclear receptor family of transcription factors, a large group of proteins that mediate ligand-dependent transcriptional activation and transrepression (Issemann and Green, 1990). PPAR-! is highly expressed in adipose tissue and plays a crucial role in adipocyte differentiation (Lemberger et al, 1996). It is also expressed in a variety of other tissue and cell types, where it plays key roles in the regulation of metabolism and inflammation. Ligands for PPAR-! include a variety

I. Introduction The polyphenol curcumin (diferuloylmethane; 1,7bis(4-hydroxy-3-methoxy-phenyl)1,6-heptadiene-3,5dione) is an orange-yellow compound with limited water solubility that is obtained from the turmeric plant, Curcuma longa. Curcumin has been shown to exhibit a variety of biological effects (Maheshwari et al, 2006) such as anti-oxidant, anti-inflammatory, anti-tumor and woundhealing properties (Srivastava et al, 1995). These activities are exerted through an equally wide variety of signaling pathways, which may involve either inhibition (Chen and Tan, 1998; Gaedeke et al, 2004; Zhou et al, 2007) or activation (Hu et al, 2005) of specific intracellular

Gene Therapy and Molecular Biology Vol 13, page 21 of natural and synthetic compounds. Most of the natural ligands are fatty acids or fatty acid derivatives. Synthetic ligands include the thiazolidinediones, which are used as insulin sensitizing agents for treatment of type 2 diabetes (Berger and Moller, 2002). Curcumin has been reported to activate PPAR-! (Xu et al, 2003; Zheng and Chen, 2004; Chen and Xu, 2005; Lin and Chen, 2008). It remains unclear, however, whether this activation reflects curcumin binding to the receptor, as has been suggested (Chen and Xu, 2005; Jacob et al, 2007), or is entirely the result of indirect effects. The present study, utilizing multiple molecular and cellular assays, is the first to directly investigate the ability of curcumin to act as a PPAR-!-activating ligand.

C. Nuclear protein preparation and PPAR-!DNA binding assay CV-1 and IMR-90 cells were plated in 100 mm dishes at 70% confluence. The cells were treated with curcumin or rosiglitazone for 3 h, after which nuclear protein was isolated (Cayman nuclear protein extraction kit). Protein concentrations were estimated using the Bio-Rad (Hercules, CA) DC protein assay. PPAR-! DNA-binding activity in the nuclear protein was detected by an ELISA-based PPAR-! transcription factor assay (Cayman) that detects PPAR-! bound to PPRE-containing double-stranded DNA sequences.

D. Ligand binding by PPAR-!-GST The ligand binding domain of PPAR-! was introduced into the pGEX-2T bacterial expression vector (Amersham Pharmacia; Buckinghamshire, UK). Expression of glutathione-S-transferase (GST)-tagged PPAR-! in Escherichia coli strain BL21-DE3 (Novagen; San Diego, CA) was induced by the addition of 1 mM isopropyl-1-"-D-galactopyranoside (IPTG) to the growth medium. Bacterial extracts were prepared using standard methods and the fusion proteins were purified using Glutathione Sepharose 4B (GE Healthcare; Piscataway, NJ). GST-PPAR-! protein induction and receptor binding was assessed as described (Fu et al, 2003). Briefly, 5 %g of GST-PPAR-! protein, [3H]rosiglitazone (specific activity, 5 Ci/mmol), and various concentrations of curcumin or unlabeled rosiglitazone were incubated for 2 h at 25°C in a buffer containing 10 mM Tris HCl (pH 8.0), 50 mM KCl, and 10 mM dithiothreitol (DTT). Bound [3H]rosiglitazone was separated from free [3H]rosiglitazone by centrifugation at 8000 rpm for 1 min. The radioactivity of the bound [3H]rosiglitazone fraction was determined by liquid scintillation counting.

II. Material and Methods A. Reagents DMEM and DMEM/F12 were purchased from Gibco-BRL Life Technologies (Grand Island, NY). High purity curcumin was obtained from Sigma Chemical Co. (St. Louis, MO), Bioprex (Pune, Maharashtra, India), and Alfa Aesar (Ward Hill, MA); all experiments were repeated using each formulation. Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT). PPAR-! antagonists GW9662 and BADGE were purchased from Cayman Chemical (Ann Arbor, MI), while PPAR-! Antagonist III (G3335), and T0070907 were purchased from Calbiochem (La Jolla, CA). The PPAR-! agonists ciglitazone and rosiglitazone were purchased from Cayman. Aliquots of agonists and antagonists were dissolved in DMSO (SigmaAldrich, St. Louis, MO) at 100 mM and stored at -30°C until use. [3H]rosiglitazone was obtained from American Radiolabeled Chemicals (St. Louis, MO). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody was obtained from Abcam (Cambridge, UK), while anti-#-smooth muscle actin (#-SMA) mouse antibody, clone 1A4, was obtained from Dako Automation (Carpentaria, CA), and TGF-"1 was obtained from R&D Systems (Minneapolis, MN). GAL4-PPAR! plasmid was a kind gift from YE Chen, University of Michigan, Ann Arbor. The aP2-luc plasmid (Camp et al, 2001) and the FATP-PPRE-luc plasmid (Monajemi et al, 2007) were constructed as previously described.

E. 3T3-L1 differentiation and Oil Red O staining 3T3-L1 preadipocytes were grown and maintained in DMEM containing 10% FBS. Differentiation of preadipocytes was studied in cells 2 days following confluence (designated day 0). These cells were cultured for 14 d in DMEM containing 10% FBS and either curcumin or rosiglitazone. The medium was changed every 2 d. The differentiated adipocytes were stained by Oil Red O (Sigma) as described previously (Song et al, 2007). Briefly, cells were washed with PBS and fixed in 4% paraformaldehyde for 1 h, followed by rinsing with PBS and with water. After the rinsing, cells were stained with 0.1% Oil Red O for 1 h. Plates were rinsed with water and images of cells on the plate were taken in water.

B. Cell culture and transfection CV-1 and 3T3-L1 cells were obtained from American Type Culture Collection (Manassas, VA). IMR-90 cells were obtained from the Coriell Institute for Medical Research (Camden, NJ). CV-1 cells were grown to 70% confluence in DMEM/F12 supplemented with 10% FBS and 1% penicillinstreptomycin. Cells were then transiently co-transfected with pRL-SV40 and a PPAR-dependent luciferase reporter, pFATPluc. In separate experiments, cells were co-transfected with pRLSV40 plus a luciferase gene under the control of four Gal4 DNAbinding elements (UASG $ 4 TK-luciferase) and a plasmid containing the ligand-binding domain for PPAR-! fused to the Gal4 DNA-binding domain. All transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Twenty-four h after transfection, cells were treated with test compounds and incubated for an additional 24 h in medium with 10% FBS. The resulting luciferase activity was measured with reporter luciferase assay kits (Promega; Madison, WI) employing a Modulus 9201 luminometer (Turner Biosystems; Sunnydale, CA) and normalized by comparison to Renilla luciferase.

F. RNA isolation and real-time PCR Total RNA was extracted using TRI-Reagent (Sigma) according to the manufacturer’s instructions. cDNA was generated from 1 %g of total RNA and real-time quantitative PCR was performed using Sybr Green PCR Master Mix (Applied Biosystems; Foster City, CA) according to the manufacturer’s protocol. Quantitative changes were expressed relative to "-actin. Primers used were: PPAR-!: (F) 5'-ATTCTGGCCCACCAACTTCGG-3' (R) 5'-TGGAAGCCTGATGCTTTATCCCCA-3' "-actin: (F) 5'-GTGGGGCGCCCCCAGGCACCA-3' (R) 5'-GCTCGGCCGTGGTGGTGAAGC-3'

G. Western immunoblotting Cells were lysed in radioimmunoprecipitation (RIPA) buffer and whole-cell protein was quantified. Ten %g of protein was subjected to 12% Tris-glycine SDS-PAGE (Invitrogen).

Narala et al: Curcumin is not a ligand for PPAR-! After transfer to a polyvinylidene fluoride membrane (Millipore), #-SMA and GAPDH were detected using appropriate dilutions of primary mouse monoclonal antibodies followed by a horseradish peroxidase-conjugated anti-mouse IgG. Protein was visualized using the ECL chemiluminescent detection system (Amersham Pharmacia).

We then examined the ability of curcumin to stimulate binding of PPAR-! to DNA using a commercially available transcription factor assay that measures binding of PPAR-! to double stranded DNA probe containing a PPRE sequence. Cells were treated with curcumin (10-40 %M), rosiglitazone (10 %M), or vehicle (DMSO) for 3 h, after which nuclear extracts were prepared and subjected to PPAR-! binding assay. In order to investigate the possibility that curcumin up-regulates PPAR-! expression, we employed IMR-90 as well as CV1 cells. Curcumin gave results similar to those with vehicle, demonstrating no activation of PPAR-! in either CV-1 cells (Figure 2B) or IMR-90 cells (Figure 2C). Rosiglitazone (10 %M), as expected, increased PPAR-! binding.

H. Statistical analysis Data are represented as mean Âą SE and were analyzed with the Prism 5.0 statistical program (GraphPad Software Inc.; San Diego, CA). Comparisons between experimental groups were performed using one-way ANOVA followed by Dunnettâ&#x20AC;&#x2122;s post hoc test. All data shown are averages from at least 3 independent experiments. Differences were considered significant if P was less than .05.

III. Results A. Curcumin does not activate PPAR reporter constructs Previous studies have reported that curcumin activates PPAR-!. To test this, we transfected CV-1 cells with FATP-PPRE-luc plasmid in which the peroxisome proliferator response element (PPRE) from fatty acid transport protein controls expression of firefly luciferase. After 24 h, cells were treated with curcumin at different concentrations (1-20 %M) and following an additional 24-h incubation, cells were lysed and luciferase activity was measured. Curcumin did not increase the relative transcriptional activity of PPAR-! in CV-1 cells at any dose tested (Figure 1A). By contrast, the positive control ciglitazone (10 %M) increased transcriptional activity ~7fold. To increase the robustness of the reporter assay, CV1 cells were co-transfected with a PPAR-! expression plasmid (TR100-PPAR-!) in addition to FATP-PPRE-luc. Curcumin (1-20 %M) did not induce detectable PPAR-! activation even in the presence of elevated amounts of receptor, whereas transcriptional activity induced by ciglitazone (10 %M) was greater than that observed in the absence of the expression plasmid (Figure 1B). Similar results were obtained with curcumin and rosiglitazone in NIH/3T3 cells with an aP2-PPRE-luc reporter plasmid in the presence of TR100-PPAR-! (data not shown). We also performed reporter assays using the highly specific Gal4-luc system, in which the PPAR-! ligandbinding domain is fused to the Gal4 DNA-binding domain and a luciferase reporter gene is under the control of four Gal4 DNA-binding elements. In this case also, we did not observe activation of PPAR-! by curcumin (Figure 1C).

B. Curcumin does not bind to the ligandbinding domain of PPAR-! or stimulate binding of PPAR-! to DNA

Figure 1. Curcumin is inactive in reporter assays. CV-1 cells were transiently transfected with pRL-SV40 and with one of the following constructs: (A) PPAR-dependent luciferase reporter, pFATP-luc; (B) PPAR-! expression plasmid, pTR100-PPAR-!, along with pFATP-luc; (C) PPAR-! GAL4 reporter system, UASG $ 4 TK-luciferase + GAL4-PPAR-!. Cells were then incubated with vehicle (DMSO), curcumin (Cur; 1-20 %M) or ciglitazone (Cig; 10 %M). After 24 h, the relative luciferase activity was calculated by normalizing firefly luciferase activity to that of Renilla luciferase. *P < 0.05 vs. vehicle.

To directly determine whether curcumin binds to the PPAR-! activating site, we quantified displacement of bound [3H]rosiglitazone by unlabeled rosiglitazone or curcumin. The Ki for rosiglitazone was found to be ~50 nM, consistent with reported values (Schopfer et al, 2005). By contrast, curcumin displayed no competition for the binding site at concentrations up to 10 %M (Figure 2A) or even as high as 40 %M (data not shown).

Gene Therapy and Molecular Biology Vol 13, page 23

D. PPAR-! antagonists do not block curcumin inhibition of TGF-"-induced fibroblast-to-myofibroblast differentiation As a further test of the extent to which biological effects of curcumin may be mediated by PPAR-! activation, we examined inhibition of the TGF-"-induced differentiation of human lung fibroblasts into myofibroblasts. PPAR-! activation has been shown to inhibit this differentiation, signaled by appearance of #smooth muscle actin (#-SMA) (Burgess et al, 2005; Milam et al, 2008). We treated serum-starved IMR-90 fibroblasts with curcumin (10 %M) for 1 h followed by TGF-" (2 ng/ml), finding that curcumin inhibited the expression of #-SMA. To determine whether this inhibition is mediated through PPAR-!, we added one of four different PPAR-! antagonists 1 h prior to curcumin. #-SMA expression was assessed by Western immunoblotting and quantified by densitometric scanning of the blots (Figure 3C). None of the antagonists reduced the ability of curcumin to inhibit myofibroblast differentiation.

IV. Discussion Previous studies have suggested that certain curcumin effects involved an increase in PPAR-! activity. Some investigators have suggested that this increased activity may represent direct ligand-binding activation of the receptor by curcumin, although this remains controversial. Our results conclusively address this issue utilizing a variety of rigorous assays. At the molecular level, ligand-induced activation of PPAR-! is reflected in increased binding to its response elements. We find, however, that incubation with curcumin does not increase binding to the consensus PPRE in a transcription factor assay, nor does it increase transcriptional activity in any of four different reporter assays. Furthermore, definitively, curcumin does not displace a standard synthetic PPAR-! ligand from the receptorâ&#x20AC;&#x2122;s binding site. At the cellular level, we investigated the ability of curcumin to induce PPAR-!mediated differentiation of preadipocytes to adipocytes. Whereas synthetic PPAR-! ligands induced differentiation, as expected, curcumin did not. Furthermore, although curcumin reduces the ability of TGF-" to induce fibroblast differentiation, as do PPAR-! ligands, a variety of different PPAR-! antagonists have no effect on curcuminâ&#x20AC;&#x2122;s inhibitor activity. Thus, at both the molecular and cellular levels, our results support the conclusion that the known biological activities of curcumin do not involve binding to, and activation of, the nuclear transcription factor PPAR-!. Studies in hepatic stellate cells (Xu et al, 2003; Zheng and Chen, 2004; Lin and Chen, 2008), in a rodent model of sepsis (Siddiqui et al, 2006), and in Moser colon cancer cells (Chen and Xu, 2005) have suggested that PPAR-! signaling is required for curcumin to exert the effects observed. In Moser cells, it was found that curcumin reduced phosphorylation and consequent inactivation of PPAR-! (Chen and Xu, 2005).

Figure 2. Curcumin does not bind to or activate PPAR-!. (A) Competitive binding assay was performed using GST-PPAR-! ligand-binding domain and [3H]rosiglitazone in the presence of unlabeled curcumin (Cur) or rosiglitazone (Rosi). In a separate experiment, PPAR-! activation was analyzed by DNA-binding assay in (B) CV-1 and (C) IMR-90 cells. *P < 0.05 vs. vehicle.

C. Curcumin does not induce differentiation of 3T3-L1 preadipocytes To investigate PPAR-!-mediated biological effects of curcumin, we employed a well established model of adipocyte differentiation. PPAR-! plays an essential role in the differentiation of adipocytes (Tontonoz et al, 1994), with selective disruption of PPAR-! resulting in impaired development of adipose tissue (Evans et al, 2004). 3T3-L1 preadipocytes were treated with curcumin (5 and 10 %M) or rosiglitazone (5 %M) for 2 weeks. Adipocyte differentiation was assessed both morphologically and by means of Oil Red O staining, which reveals the accumulation of intracellular lipids (Figure 3A). Expression of PPAR-!, which is up-regulated during differentiation, was also assessed (Figure 3B). On both assessments, vehicle and curcumin did not induce differentiation, while rosiglitazone treatment produced the expected PPAR-!-dependent differentiation.

Narala et al: Curcumin is not a ligand for PPAR-!

Figure 3. Curcumin has no effect on preadipocyte differentiation and effects on fibroblast differentiation are not blocked by PPAR-! antagonists. (A, B) 3T3-L1 preadipocytes were treated with curcumin (Cur; 5 and 10 %M) or rosiglitazone (Rosi; 5 %M) for 2 weeks. Adipocyte differentiation was assessed (A) both morphologically and via oil red O staining and (B) by relative expression of PPAR-! mRNA. The MDI differentiation protocol (isobutylmethylxanthine + dexamethasone for 48 h, followed, after their removal, by insulin + the test compound) was used in all experiments. *P < 0.05 vs. vehicle. (C) Confluent, serum-deprived human fetal lung fibroblasts (IMR-90) were pretreated with PPAR-! antagonists (GW: GW9662, T007: T0070907, and Ant. III: Antagonist III) for 1 h, then with curcumin for 1 h, after which cells were stimulated with TGF-" (2 ng/ml). After an additional 24 h, cell lysates were subjected to SDSPAGE and Western blotting. Membranes were probed first with antiâ&#x20AC;&#x201C;#-SMA antibody, then reprobed with anti-GAPDH antibody to confirm equal protein loading. The blots were scanned densitometrically. *P < 0.05 vs. vehicle.

Up-regulation of PPAR-! expression has been demonstrated in hepatic stellate cells (Cheng et al, 2007; Lin and Chen, 2008; Xu et al, 2003; Zheng and Chen, 2004; Zhou et al, 2007), in a macrophage cell line (Siddiqui et al, 2006), and in colonic mucosal cells from a rodent model of colitis induced by trinitrobenzene sulfonic acid (Zhang et al, 2006). One study found that this upregulation of PPAR-! expression was secondary to inhibition of PDGF and EGF signaling pathways (Zhou et al, 2007). Furthermore, in the rat model of colitis induced by trinitrobenzene sulfonic acid, curcumin was observed to increase levels of the endogenous PPAR-! ligand 15dPGJ2 (Zhang et al, 2006). None of these studies directly examined possible binding of curcumin to the PPAR-! ligand-binding site, however. Although the reported increases in amount of receptor, and possibly of its endogenous ligands, appear to be plausible explanations for the results obtained, the possibility that curcumin also bound to and directly activated PPAR-! had been suggested (Chen and Xu, 2005; Jacob et al, 2007). In direct contrast to our results, one group has specifically asserted that curcumin is a PPAR-! ligand (Kuroda et al, 2005; Nishiyama et al, 2005). This group reported increased activity in a GAL4-PPAR-! chimeric assay in CV-1 cells. These researchers also noted that curcumin induced differentiation of preadipocytes, which we did not observe, although these were primary human preadipocytes rather than the standard 3T3-L1 cells that were employed in this study. Furthermore, while we repeated all experiments with three different commercially

available high-purity curcumin formulations (data not shown), this group conducted preadipocyte differentiation studies and some ligand-binding studies with ethanolic extracts of turmeric. Other ligand-binding studies were performed with curcumin purified in their laboratories. Because these curcumin preparations were not standardized, the possible role of other compounds present in these formulations cannot be ruled out. Recently, it has also been shown that curcumin down-regulates PPAR-! expression in preadipocytes, thus actively inhibiting their differentiation (Lee et al, 2009). This observation further supports our conclusions. In summary, our results conclusively show that curcumin is not a PPAR-! ligand. Thus, any observed PPAR-!-mediated effects of curcumin must be indirect and mediated through effects of receptor expression or levels of endogenous ligands that are mediated through other pathways. Since we have now ruled out one suggested mechanism for curcumin, further study of alternative mechanisms is warranted.

Acknowlegements Supported by National Institutes of Health grants HL070068 and AI079539, a University of Michigan Global REACH International Grant, and the Martin E. Galvin Fund and Quest for Breath Foundation (all to R.C.R.).

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Hochwald and Golubovskaya: FAK and cancer therapy Gene Ther Mol Biol Vol 13, 26-35, 2009

FAK as a target for cancer therapy Review Article

Steven N. Hochwald*, Vita M. Golubovskaya Department of Surgery, University of Florida College of Medicine, Gainesville, Florida

__________________________________________________________________________________ *Correspondence: Steven N. Hochwald MD, Department of Surgery, University of Florida College of Medicine, 1600 SW Archer Road, P.O. Box 100109, Gainesville, FL 32609, USA; Tel: 352-265-0761, Fax: 352-265-0262, e-mail: Steven.Hochwald@surgery.ufl.edu Key words: ocal Adhesion Kinase; malignancy; cancer; Y15 Abbreviations: FAK, (Focal Adhesion Kinase); FERM, (Focal Adhesion Kinase Ezrin/Radixin/Moesin; FRNK, (FAK-related non kinase) This work was supported by the following NIH grant: CA113766 (S.N.H.) Received: 20 March 2009; Accepted: 24 March 2009; electronically published: April 2009

Summary We have learned that malignant cells are similar to normal cells in the signaling pathways that they use. However, cancer cells acquire aberrations that favor their growth in the complex environments of living tissues. This includes their ability to invade and metastasize and their ability to grow and divide indefinitely. The progression of human cancer is characterized by a process of tumor cell motility, invasion, and metastasis to distant sites, requiring the cancer cells to be able to survive the apoptotic pressures of anchorage-independent conditions. One of the main tyrosine kinases that are linked to this malignant phenotype is the Focal Adhesion Kinase (FAK). FAK is overexpressed in many types of tumors and recently has been proposed to be a target for anti-cancer therapy. In this review, we will review the FAK structure, its role in signaling, and FAK targeted therapy approaches in malignancy.

surface receptor) signaling. The FAK gene was first isolated from chicken embryo fibroblasts transformed by v-src (Schaller et al, 1992). Subsequently, the FAK gene was identified in human tumors, and FAK mRNA has been shown to be up-regulated in invasive and metastatic human breast and colon cancer samples as compared to normal tissues (Weiner et al, 1993). This was the first evidence that FAK might be regulated at the level of gene transcription. Subsequently, up-regulation of FAK has been demonstrated at the protein level in a wide variety of human tumors, including breast cancer, colon cancer, ovarian cancer, thyroid cancer, melanoma, and sarcoma (Owens et al, 1995, 1996; Judson et al, 1999; Cance et al, 2000). Recently, the regulatory promoter region of the FAK gene was cloned and confirmed transcriptional upregulation in cancer cell lines (Golubovskaya et al, 2004).

I. Introduction Despite recent advances in surgery, chemotherapy and radiation treatment, survival of patients with advanced malignancy remains suboptimal. Fortunately, our understanding of the origins of cancer has changed dramatically over the last twenty-five years, owing in large part to the revolution in molecular biology that has changed all biomedical research. Powerful experimental tools are available to cancer biologists and have made it possible to uncover and dissect the complex molecular machinery operating inside normal and malignant cells. In addition, these tools have allowed researchers to pinpoint the defects that cause cancer cells to signal and proliferate abnormally. Focal Adhesion Kinase (FAK) was discovered about 15 years ago as a tyrosine phosphorylated protein kinase. Investigations in several laboratories have shown that this protein plays a critical role in intracellular processes of cell adhesion, motility, survival, and cell cycle progression. The FAK gene encodes a non-receptor tyrosine kinase that localizes at contact points of cells with extracellular matrix and is activated by integrin (cell

II. Molecular structure of focal adhesion kinase The human FAK (also known as PTK2a) gene has been mapped to chromosome 8 (Fiedorek, Jr. and Kay, 1995; Agochiya et al, 1999), and there appears to be a high

Gene Therapy and Molecular Biology Vol 13, page 27 degree of homology between vertebrate species. Human complete FAK mRNA sequence (NCBI Accession number: L13616) is 3791 bases long and includes a 5’untranslated 233 base pair region (Whitney et al, 1993). Human FAK cDNA was first isolated from primary sarcoma tissue and increased FAKmRNA was seen in tumor samples compared with normal tissue samples (Weiner et al, 1993). Subsequently, Xenopus laevis FAK cDNA (Zhang et al, 1995) and rat FAK cDNA (Burgaya and Girault, 1996) were identified. Recently, Drosophila FAK cDNA (Dfak56) was isolated (Fujimoto et al, 1999). FAK cDNA is closely related to the homologous prolinerich calcium dependent tyrosine kinase (45% amino-acid identity) that is also located on human chromosome 8, locus p21.1, named PYK2 (RAFTK (related adhesion focal tyrosine kinase), CADTK (calcium-dependent tyrosine kinase), CAK (cell adhesion kinase) b, PTK 2b (protein tyrosine kinase 2b) (Avraham et al, 1995; Lev et al, 1995; Sasaki et al, 1995). The gene coding FAK contains 34 exons (NCBI Gene ID: 5747), and genomic sequence spans 230 kb (Corsi et al, 2006). The FAK gene contains four 5’ noncoding exons and 34 coding exons and has been shown to have multiple alternatively spliced forms. Comparison of the mouse and human FAK genes detected conservative and non-conservative 5’-untranslated exons that suggests a complex regulation of FAK expression. Exons (Sasaki et al, 1995; Burgaya and Girault, 1996; Fujimoto et al, 1999; Golubovskaya et al, 2002) are highly conserved among vertebrate species, suggesting their critical function in gene regulation (Corsi et al, 2006). It is known that alternative splicing often occurs and plays an important role in cancer (Caballero et al, 2001; Venables, 2006). Alternative splicing most often results from different exon inclusion, but can also occur from intron retention or alternative choice between two splice sites leading to changes in protein localization, structure, removal of phosphorylation sites, or proteasomal degradation (Venables, 2006). There were several cases of alternatively spliced genes that are involved in invasion and metastasis (Rac 1, !-catenin, Crk) or angiogenesis (VEGFR-2, VEGFR-3 (Flt-4)). Thus, detailed study of alternatively spliced forms of FAK that are overexpressed in pre- and metastatic cancers will be critical for understanding mechanisms and regulation of FAK expression in carcinogenesis, either by changes in mRNA, by changes in the coding sequence (exon inclusion/exclusion), or by changes in protein levels (stability, etc.). The human FAK promoter regulating FAK expression contains 600 base pairs and includes many transcription binding sites, such as AP-1, AP-2, SP-1, PU.1, GCF, TCF-1, EGR-1, NF-!B and p53 (Golubovskaya et al, 2004). Interestingly, two transcription binding sites for p53 have been identified in the FAK promoter, and p53 can block FAK promoter activity (Golubovskaya et al, 2004). Recently, the mouse promoter has been cloned and found to be highly homologous to the human promoter and contains the same binding sites (Corsi et al, 2006). In addition, the FAK gene has an internal FRNK promoter or C-terminal, FAK-CD

promoter that has been recently cloned by Parsons group (Hayasaka et al, 2005), regulating expression of autonomously expressed FRNK protein.

A. FAK protein structure The FAK protein is a 125 kDa tyrosine kinase (p125FAK) with a large amino-N-terminal domain, exhibiting homology with a FERM (protein 4.1, ezrin, radixin and moesin) domain with an autophosphorylation site (Y-397), a central catalytic domain, and a large carboxy-C-terminal domain that contains a number of potential protein interacting sites, including two prolinerich domains and FAT domain (Schaller and Parsons, 1994; Schaller et al, 1994; Hanks and Polte, 1997) (Figure 1).

B. The kinase domain The central kinase domain of FAK (amino acids 424676) contains the Y576 and Y577, major phosphorylation sites, and also K454, which is the ATP binding site (Figure 1). Phosphorylation of FAK by Src on Y576 and Y577 is an important step in the formation of an active signaling complex and is required for maximal FAK enzymatic activity (Calalb et al, 1995). The crystal structure of the FAK kinase domain reveals an open conformation similar to other kinases (Nowakowski et al, 2002). The FAK kinase domain structure has an unusual bisulphite bond between the conserved cysteines 456 and 459, suggesting a possible role in protein-protein interactions and kinase function (Nowakowski et al, 2002). The ATP binding site of protein kinases is the most common target for the small-molecule inhibitors, although the design and specificity of these inhibitors can be complicated by structural similarities between kinase domains. Thus, finding small structural differences between the ATP binding site of kinases is crucial in the design of small molecule kinase inhibitors. For example, the side chain of glutamic acid, E506 forms a bifurcated hydrogen bond to the 2’ and 3’ hydroxyl groups of the ribose (Nowakowski et al, 2002). The corresponding side chains in EphA2 and Aurora-A kinases are smaller and do not contact with sugar (Nowakowski et al, 2002).

C. The N-terminal domain The first function of the N-terminal, homologous to FERM domain was linked to the binding of integrins, via their ! subunits (Schaller et al, 1995). The N-terminal domain of FAK protein contains the major autophosphorylation site Y397-tyrosine, that in its phosphorylated form becomes a binding site of the SH-2 domain of Src, leading to its conformational changes and activation (Hanks and Polte, 1997). Tyrosine phosphorylation of FAK and binding of Src leads to tyrosine phosphorylation of other tyrosine phosphorylation sites of FAK: Y407; Y576,Y577- major phosphorylation sites in the catalytic domain of FAK; Y861 and Y925 (Hanks and Polte, 1997; McLean et al, 2005), and to phosphorylation of FAK binding proteins, such as paxillin and Cas (Schaller et al, 1999). This leads to subsequent cytoskeletal changes and activation of RAS-MAPK (mitogen-activated protein kinase) signaling pathways 27

Hochwald and Golubovskaya: FAK and cancer therapy (Hanks et al, 2003; McLean et al, 2005). Thus, the FAKSrc signaling complex activates many signaling proteins involved in survival, motility and metastatic, invasive phenotype in cancer cells (Figures 1 and 2). Phosphorylated Y397 FAK is able to recruit important signaling molecules, p85 PI3-kinase (phosphoinositide 3kinase), growth factor receptor bound protein Grb 7, phospholipase C" (PLC") and others. Crystal structure of the N-terminal domain of avian FAK, containing the FERM domain, has been recently reported (Ceccarelli et al, 2006). Of note, negative regulation of FAK function by FERM domain was revealed (Cooper et al, 2003), where the N-terminal domain had an autoinhibitory effect through interaction with the kinase domain of FAK.

Recently, several novel binding partners in cancer cells of the FAK N-terminus, such as EGFR ( Sieg et al, 2000; Golubovskaya et al, 2002), RIP (Kurenova et al, 2004) and p53 (Golubovskaya et al, 2005) have been reported (Figure 1). The N-terminal domain of FAK has been shown to cause apoptosis in breast cancer cells (Beviglia et al, 2003) and can be localized to the nucleus (Lobo and Zachary, 2000; Jones et al, 2001; Stewart et al, 2002; Jones and Stewart, 2004). Thus, the N-terminal domain of FAK binds to the extracellular matrix receptors, integrins, growth factor receptors, and important cytoplasmic, cytoskeletal and nuclear proteins, mediating signaling from the extracellular matrix to the cytoplasm and nucleus and controlling cytoskeletal changes, survival, motility, and invasion.

Figure 1. Structure of FAK molecule with multiple interacting partners. FAK has multiple important functions including an impact on cell survival pathways and apoptosis.

Figure 2. FAK expression in human pancreatic cancers. (A, top) Immunohistochemical staining of FAK in human pancreatic adenocarcinomas. Intensity of FAK staining is higher in metastases than in primary tumor. (Mean±SE: 3.5±0.2 vs 4±0, p=0.001). (B, bottom) FAK staining in pancreatic cancer. Representative example demonstrating staining of FAK in primary and metastatic pancreatic cancer.

Gene Therapy and Molecular Biology Vol 13, page 29 now there are total of 30 sites of phosphorylation of FAK, including those reported before, requiring detailed analysis of their biological functioning in vivo.

D. The C-terminal domain Different proteins can bind to the C-terminal domain of FAK (amino acids 677-1052), including paxillin, p130cas, PI3-kinase, and GTP-ase-activating protein Graf, leading to changes in the cytoskeleton and to activation of the Ras-MAP kinase pathway (Schaller and Parsons, 1994; Windham et al, 2002; Hanks et al, 2003; Parsons, 2003). The carboxy-terminal domain of FAK contains sequences responsible for its targeting to focal adhesions, also known as the FAT domain. Alternative splicing of FAK results in autonomous expression of the C-terminal part of FAK, FAK-related non-kinase (FRNK) (Richardson and Parsons, 1995). The crystal structure of the C-terminal domain of FAK, FAT, has been determined recently by several groups (Hayashi et al, 2002; Prutzman et al, 2004) and structure analysis demonstrates that it can exist as a dimer or monomer, allowing binding of several binding partners.

III. FAK functioning in cells Attachment to the underlying extracellular matrix provides cells with both a means of anchorage needed for traction during migration via a link to the actin cytoskeleton and also with intracellular structures that house membrane-associated signaling proteins. This leads to the transmission of biochemical signals into the cell interior to induce multiple biological responses. Loss of regulation of the process of adhesion formation or turnover, or of downstream signaling is likely to contribute to primary tumor development and/or tumor dissemination. Signaling via adhesion-associated kinases controls the changes that are necessary for cell migration including regulation of cell-matrix adhesion turnover and coordination of remodeling of the actin cytoskeleton network (Cance et al, 2000). FAK has numerous functions in cell survival, motility, metastasis, invasion, and angiogenesis. FAK has also been shown to be important for cell motility (Hauck et al, 2001; Schaller, 2001; Hanks et al, 2003; Schlaepfer and Mitra, 2004). FAK-null embryos exhibit decreased motility in vitro (Ilic et al, 1995). Furthermore, forced expression of FAK stimulated cell migration (Hildebrand et al, 1993; Sieg et al, 1999). Cell migration is initiated by protrusion at the leading edge of the cell, by the formation of peripheral adhesions, exertion of force on these adhesions, and then the release of the adhesions at the rear of the cell (Tilghman et al, 2005). FAK has been shown to be required for the organization of the leading edge in migrating cells by coordinating integrin signaling in order to direct the correct activation of membrane protrusion (Tilghman et al, 2005). SH2 domain of Src, targeting Src to focal adhesions and Y397 activity has been shown to be important for motility (Yeo et al, 2006). PI3 kinase has been also shown to be critical for FAK-mediated motility in Chinese hamster ovary (CHO) cells (Reiske et al, 1999). Tumor suppressor gene PTEN, encoding phosphatase has been shown to interact with FAK, causing its dephosphorylation and blocked motility (Tamura et al, 1998). Moreover, Y397FAK was important for PTEN interaction with FAK (Tamura et al, 1999). Overexpression of FAK reversed the inhibitory effect of PTEN on cell migration (Tamura et al, 1998). Activation of FAK is linked to invasion and metastasis signaling pathways. FAK was important in Erb2/Erb3-induced oncogenic transformation and invasion (Benlimame et al, 2005). Inhibition of FAK in FAKproficient invasive cancer cells prevented cell invasion and metastasis processes (Benlimame et al, 2005). In addition, FAK has been shown to be activated in invading fibrosarcoma and regulated metastasis (Hanada et al, 2005). Inhibition of FAK with dominant-negative FAKCD disrupted invasion of cancer cells (Hauck et al, 2001). We have also shown high FAK expression in breast cancers associated with an aggressive tumor phenotype (Lark et al, 2005). Subsequently, we analyzed FAK expression in pre-invasive ductal carcinoma in situ, DCIS

E. Post-translational protein modifications FAK function is altered by post-translational modifications including phosphorylation of tyrosines or serines. FAK has numerous tyrosine phosphorylated sites: Y397, Y407, Y576/Y577, Y861 and Y925. Phosphorylation of Y397, creates a binding site for Src, PI3K, PLC-g, Grb-7 and Grb-2-SOS. Phosphorylation of tyrosine 407, as well as Y397, correlated with differentiation and with the level of gastrin-releasing peptide and its receptor in colon cancer cells (Matkowskyj et al, 2003). Phosphorylation of Y576 and Y577 correlated with maximal activity of FAK (Calalb et al, 1995). Srcdependent phosphorylation of Y861 was induced by VEGFR in HUVEC endothelial cells (bu-Ghazaleh et al, 2001). FAT domain mediates signaling through Grb-2 binding to Y925 site of FAK (Arold et al, 2002). Inhibition of FAK that resulted in decreased Y925 phosphorylation of FAK resulted in decreased FAK-Grb2MAPK signaling and VEGFR-induced tumor growth of 4T1 breast carcinoma cells (Mitra et al, 2006). In addition to tyrosine phosphorylation, several serine phosphorylation sites have been reported to play a major role in FAK function, such as serines 722, 732, 843 and 910. The role of serine phosphorylation is less described than phosphorylation of tyrosines but was suggested to play a role in binding/stability of proteins (Parsons, 2003). In addition, recent mass spectrometry analysis of chicken FAK revealed 19 new sites of phosphorylation with some sites reported before: 15 serine, 5 threonine, and 5 tyrosine residues (Grigera et al, 2005). The authors suggested that coordinated phosphorylation of FAK by tyrosine and serine/threonine-specific kinases may be critical a step in regulation of FAK function (Grigera et al, 2005). Some of the sites were present only in chicken FAK, such as S386, T388 and T393, but several chicken phosphorylation sites were conserved in human, mouse, and frog species, such as S29, Y155, S390, S392, T394, Y397, T406, Y407, Y570, T700, S708, S722, S725, S726, S732, S766, S845 (S843 in human), S894, Y899 and S911 (S910 in human and mouse) (Grigera et al, 2005). Thus, 29

Hochwald and Golubovskaya: FAK and cancer therapy tumors and detected protein overexpression in preinvasive tumors (Lightfoot, Jr. et al, 2004), suggesting that FAK survival function occurs as an early event in breast tumorigenesis. FAK plays a major role in survival signaling and has been linked to detachment-induced apoptosis or anoikis (Frisch et al, 1996). It has been shown that constitutively activated forms of FAK rescued epithelial cells from anoikis, suggesting that FAK can regulate this process (Frisch et al, 1996; Frisch and Ruoslahti, 1997; Frisch, 1999; Frisch and Screaton, 2001; Windham et al, 2002). Similarly, both FAK antisense oligonucleotides (Xu et al, 1996; Smith et al, 2005), as well as dominant-negative FAK protein (FAK-CD), caused cell detachment and apoptosis in tumor cells (Xu et al, 1996, 1998, 2000; van de et al, 2001; Golubovskaya et al, 2002, 2003; Beviglia et al, 2003; Gabarra-Niecko et al, 2003; Park et al, 2004b). The anti-apoptotic role of FAK was also demonstrated in FAK-transfected FAK/HL60 cells that were highly resistant to apoptosis induced with etoposide and hydrogen peroxide compared with the parental HL-60 cells or the vector-transfected cells (Sonoda et al, 2000; Kasahara et al, 2002). HL-60/FAK cells activated the AKT pathway and NF-!B survival pathways with the induction of inhibitor-of-apoptosis proteins, IAPs (Sonoda et al, 2000). We have demonstrated that EGFR and Src signaling cooperate with FAK survival signaling in colon and breast cancer cells (Golubovskaya et al, 2002, 2003; Park et al, 2004a,b). We have also demonstrated that simultaneous inhibition of Src and FAK or EGFR and FAK pathways was able to increase apoptosis in cancer cells (Golubovskaya et al, 2002, 2003). Thus, cancer cells use the cooperative function of kinases and growth factor receptor signaling to increase survival. Vascular endothelial growth factor (VEGF) is one of the known angiogenic growth factors, stimulating formation of new blood vessels or angiogenesis. FAK has been shown to play a major role in vasculogenesis. It has been shown that VEGF induced tyrosine phosphorylation of FAK in human umbilical vein endothelial cells (HUVEC) and other endothelial cell lines (Abedi and Zachary, 1997). VEGF-induced stimulation of FAK phosphorylation was also demonstrated in cultured rat cardiac myocytes that was accompanied by subcellular translocation of FAK from perinuclear sites to the focal adhesions and increased association with the adaptor proteins Shc, Grb-2 and c-Src (Takahashi et al, 1999). VEGF-induced phosphorylation of FAK was inhibited by the tyrosine kinase inhibitors tyrphostin and genistein (Takahashi et al, 1999). VEGF-induced phosphorylation of FAK was induced in human brain microvascular endothelial cell (HBMEC) (Avraham et al, 2003). Furthermore, inhibition of FAK with the dominantnegative inhibitor FRNK (FAK-related non-kinase) or the C-terminal FAK (FAK-CD) significantly decreased HBMEC spreading and migration (Avraham et al, 2003, 2004). In addition, angiogenic inhibitor endostatin blocked VEGF-induced activation of FAK (Kim et al, 2002). Recently, we have shown that FAK binds to VEGFR-3 (Flt-4) protein in cancer cell lines (Garces et al, 2006), suggesting an important role of FAK in lymphogenesis in

addition to angiogenesis. We have shown that the Cterminal domain of FAK binds to VEGFR-3. Disruption of this binding with VEGFR peptides caused apoptosis in breast cancer cells, allowing novel therapeutic approaches in breast tumors (Garces et al, 2006). The detailed interaction of FAK and VEGFR signaling and its mechanisms remain to be discovered in the future.

IV. FAK as a target for therapy Recently, several reports describe the properties of FAK inhibitors in vitro and in vivo. FAK has been proposed to be a new therapeutic target (McLean et al, 2005). Initial studies which evaluated the effects of FAK inhibition in preclinical models focused on dominant negative mutants of FAK, antisense oligonucleotides and siRNAs (Parsons et al, 2008). More recently, scientists at Novartis Pharmaceuticals designed and synthesized a series of 2-amino-9-aryl-7H-pyrrolo[2,3-d]pyrimidines to inhibit FAK using molecular modeling in conjunction with a co-crystal structure (Choi et al, 2006). Chemistry was developed to introduce functionality onto the 9-aryl ring, which resulted in the identification of potent FAK inhibitors. We and others have published reports on the use of such FAK inhibitors that have targeted the ATP binding site in the kinase domain. In human pancreatic cancer, we have shown widespread expression of FAK in primary pancreatic adenocarcinoma. In addition, we have shown significant upregulation of FAK protein expression in metastatic lesions (Figure 2, unpublished data). In human pancreatic cancer cells, we have identified that the FAK kinase inhibitor, TAE226, decreases viability, increases cell detachment and increases apoptosis (Liu et al, 2008). Other studies have shown that TAE226 readily induced apoptosis in human breast cancer cells with overexpressed Src or EGFR. Of note, these cells were resistant to adenoviral FAK dominant negative treatment, indicating that kinase inhibition was important for downregulation of FAK function and the observed phenotypic changes (Golubovskaya et al, 2008b). Subsequent studies have studied the in vivo effects of TAE226. The expression status of FAK in Barrett’s esophageal adenocarcinoma has been recently reported. FAK expression was studied in frank adenocarcinoma, areas of Barrett’s epithelia, squamous epithelia, and gastric epithelia. FAK expression was increased in cancerous parts compared to non-cancerous areas and strong expression (>50% positive staining cells per area) were observed in 94% of Barrett’s esophageal adenocarcinoma compared with 18% of Barrett’s epithelia. In a subcutaneous model of human esophageal cancer, TAE226 given orally at 30 mg/kg significantly decreased tumor volume and weight compared with placebo (Watanabe et al, 2008). Similar results from in vivo studies have confirmed the ability of TAE226 to decrease the growth of ovarian and glioma xenografts (Shi et al, 2007). While initial results with kinase inhibition of FAK has shown anti-neoplastic effects, TAE226 has been shown to also inhibit the activity of IGF-1R at nanomolar concentrations (Liu et al, 2007). Therefore, the activities against multiple tumor types likely reflect its dual inhibition of adhesion and growth promoting pathways. 30

Gene Therapy and Molecular Biology Vol 13, page 31 Recently, Pfizer pharmaceuticals have published results on an ATP competitive reversible inhibitor of FAK that has bioavailability suitable for preclinical animal and human studies. PF-562,271 was shown to exhibit >100 fold selectivity for FAK when assayed against a panel of unrelated kinases. Treatment of cancer cells lines showed a dose dependent decrease in FAK phosphorylation at the Y397 site. The IC50 for FAK phosphorylation was reported to be 5 nmol/L. Anti-tumor efficacy was observed in multiple human subcutaneous xenograft models with minimal weight loss or mortality (Parsons et al, 2008; Roberts et al, 2008). PF-562,271 is currently in phase 2 clinical trials. Phase 1 study results with this drug in patients with advanced solid malignancy have been reported in abstract form (Siu ll et al, 2008). Studies have been performed in 2 centers in the United States and one center in Canada and Australia with oral dosing as a single agent. Thirty two patients received from 5 mg up to 105 mg twice a day. Adverse events possibly related to the drug in over 10% were nausea, vomiting, fatigue, anorexia, abdominal pain, diarrhea, headache, sensory neuropathy, rash, constipation, and dizziness. Adverse events were generally grade 1-2 and reversible. Doses over 15 mg twice a day produced

steady state plasma concentrations exceeding target efficacious levels predicted from preclinical models. Prolonged disease stabilization was observed in several tumor types. Phase 1 results indicated good tolerability of this drug with favorable pharmacokinetics and pharmacodynamics (Siu ll et al, 2008). This drug represents the sole FAK inhibitor being tested in humans to date. Another approach to inhibit FAK function can be to target protein-protein interactions between FAK and its binding partners such as p53, VEGFR-3 or EGFR or targeting sites of FAK phosphorylation (Golubovskaya et al, 2008a). Tyrosine 397 is an autophosphorylation site of FAK that is a critical component in downstream signaling, providing a high-affinity binding site for the SH2 domain of Src family kinases (Figure 3). Y397 is also a site of binding of PI3 kinase, growth factor receptor binding Grb7, Shc and other proteins. Thus, the Y397 site is one of the main phosphorylation sites that can activate FAK signaling in cells. We recently demonstrated that computer modeling and screening can be performed to identify novel small molecules that inhibit protein-protein interactions at the Y397 site (Golubovskaya et al, 2008a).

Figure 3. The Y397 autophosphorylation site of FAK has several binding proteins and is critical for survival signaling.

Figure 4. (A, Left) Molecular modeling of Y15 compound in the Y397 pocket of FAK. Y15 is shown in purple and the FAK pocket in green. (B, right) Structure of Y15. Reproduced from Golubovskaya et al, 2008 with kind permission from Journal of Medicinal Chemistry.

Hochwald and Golubovskaya: FAK and cancer therapy

Figure 5. Y15 significantly blocks tumor growth in vivo and its effects are synergistic with gemcitabine treatment. Mice (n=5/group) were subcutaneously injected with Panc-1 cells. The day after injection, mice were treated with daily intraperitoneal PBS, intraperitoneal Y15 (30mg/kg), intraperitoneal gemcitabine alone (30mg/kg) or Y15 (30mg/kg) + gemcitabine (30mg/kg). The combination of Y15 + gemcitabine significantly decreased tumor volume compared to Y15 or gemcitabine (Gen) alone. *p<0.05 vs. Y15 or gemcitabine alone.

focal adhesions of focal adhesion kinase and paxillin in endothelial cells. J Biol Chem 272, 15442-15451. Agochiya M, Brunton VG, Owens DW, Parkinson EK, Paraskeva C, Keith WN, Frame MC (1999) Increased dosage and amplification of the focal adhesion kinase gene in human cancer cells. Oncogene 18, 5646-5653. Arold ST, Hoellerer MK, Noble ME (2002) The structural basis of localization and signaling by the focal adhesion targeting domain. Structure 10, 319-327. Avraham HK, Jiang S, Lee TH, Prakash O, Avraham S (2004) HIV-1 Tat-mediated effects on focal adhesion assembly and permeability in brain microvascular endothelial cells. J Immunol 173, 6228-6233. Avraham HK, Lee TH, Koh Y, Kim TA, Jiang S, Sussman M, Samarel AM, Avraham S (2003) Vascular endothelial growth factor regulates focal adhesion assembly in human brain microvascular endothelial cells through activation of the focal adhesion kinase and related adhesion focal tyrosine kinase. J Biol Chem 278, 36661-36668. Avraham S, London R, Fu Y, Ota S, Hiregowdara D, Li J, Jiang S, Pasztor LM, White RA, Groopman JE (1995) Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain. J Biol Chem 270, 27742-27751. Benlimame N, He Q, Jie S, Xiao D, Xu YJ, Loignon M, Schlaepfer DD, aoui-Jamali MA (2005) FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion. J Cell Biol 171, 505516. Beviglia L, Golubovskaya V, Xu L, Yang X, Craven RJ, Cance WG (2003) Focal adhesion kinase N-terminus in breast carcinoma cells induces rounding, detachment and apoptosis. Biochem J 373, 201-210. bu-Ghazaleh R, Kabir J, Jia H, Lobo M, Zachary I (2001) Src mediates stimulation by vascular endothelial growth factor of the phosphorylation of focal adhesion kinase at tyrosine 861, and migration and anti-apoptosis in endothelial cells. Biochem J 360, 255-264. Burgaya F, Girault JA (1996) Cloning of focal adhesion kinase, pp125FAK, from rat brain reveals multiple transcripts with

In this approach, more than 140,000 small molecule compounds were docked into the N-terminal domain of the FAK crystal structure in 100 different orientations. Those compounds with the greatest energy of interaction based on van der Waals and electrostatic charges were identified as lead compounds. One compound, 1,2,4,5benzenetetraamine tetrahydrocholoride (Y15) significantly decreased viability in most cancer cells and specifically and directly blocked phosphorylation of Y397-FAK in a dose and time dependent manner (Figure 4). Furthermore, it inhibited cell adhesion and effectively caused breast tumor regression in vivo (Golubovskaya et al, 2008a). Finally, we have shown that it inhibits pancreatic cancer growth in vivo both alone and in combination with gemcitabine chemotherapy (Figure 5, unpublished data). One potential advantage of this approach utilized to identify small molecules through in silico screening is increased target specificity. Y15 did not affect phosphorylation of the FAK homologue, Pyk-2, which can be explained by only 43% amino acid identity between Nterminal domains of FAK and Pyk-2. Other kinase inhibitors of FAK have shown inhibition of Pyk-2 autophosphorylation and likely are less specific for inhibition of FAK function.

V. Conclusions FAK is an emerging target for therapy. A FAK inhibitor is currently in Phase II clinical trials in cancer patients. Novel approaches to FAK inhibition are needed and offer directed molecular therapy. This work was supported by NIH grant number CA113766.

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Steven N. Hochwald and Vita M. Golubovskaya

Gene Therapy and Molecular Biology Vol 13, page 36 Gene Ther Mol Biol Vol 13, 36-52, 2009

Combination of immunotherapy with anaerobic bacteria for immunogene therapy of solid tumours Review Article

Jian Xu1, Xiao Song Liu 2*, Shu-Feng Zhou3, Ming Q Wei1* 1

Division of Molecular and Gene Therapies, Griffith Institute for Health and Medical research, School of Medical Science, Griffith University, Gold Coast campus, Southport, Queensland 4215 2 Diamantina Institute for Cancer, Immunology and Metabolic Medicine, University of Queensland, Princess Alexandra Hospital, Wollongabba, Queensland 410 3 School of Health Sciences, RMIT, Victoria 3083, Australia *Correspondence: A/Prof Ming Q Wei, Director of Division of Molecular and Gene Therapies, Griffith Institute for Health and Medical research, School of Medical Science, Griffith University, Gold Coast campus, Qld 4215, Australia. Tel: 617 5678 0745; Mobile: 61 422888780; Email: m.wei@Griffith.edu.au Dr Xiao Song Liu, Diamantina Institute for Cancer, Immunology and Metabolic Medicine, University of Queensland, Princess Alexandra, Hospital, Wollongabba, Qld 4102, Australia, Email: X.liu1@uq.edu.au Key words: Tumour microenvironment, Immunotherapy, Anaerobic bacteria, Hypoxia, Clostridial spores Received: 16 December 2009; Revised 2009; Accepted: 14 April 2009; electronically published: 26 April 2009

Summary Solid tumours possess unique microenvironment characterised by defective vessels, heterogeneous tumour cell, hypoxic regions, and anaerobic metabolisms. These often become intrinsic and acquired barriers to current therapeutical approaches, but they also create an ideal condition for the growth of anaerobic bacteria, which have shown specificity in their germination and multiplication. Spores from the strictly anaerobic clostridial had demonstrated ability in tumour specific colonisation and induction of tumour lysis following intravenous delivery. Clostridial strains genetically modified to act as â&#x20AC;&#x153;Trojan horseâ&#x20AC;? gene therapy vectors have been developed. Similarly, recent development in immunotherapy strategies for cancer also utilizes gene transfer to facilitate a dormant host immune response directed against the tumour. Combination of anaerobic bacteria for cancer gene therapies with immunotherapy will probably be the most promising approach that can potentially generate a prolonged anti-tumour effect beyond the immediate treatment period of gene therapy, allowing for treatment of advanced primary tumours and disseminated disease. In this review, we introduce the recent understanding of tumour microenvironment and detail the advances in the use of anaerobic bacteria for cancer gene therapies and recent studies in immuno therapy for cancers. We believe that the use of combined treatment modalities of such will provide a rational paradigm to improve upon the clinical efficacy of cancer therapy. in developing countries where diagnostic facilities are suboptimal. Conventional therapies include surgical operation, radiation and chemotherapy. Single or a combination of methods may be used, depending on various factors such as the type and location of the cancer. Unfortunately, current cancer treatments are limited to effect. Furthermore they also cause severe side effects. The search for new cancer therapies is one of the most pressing tasks of medical science. Cancer development results from constant battle between tumour cells and host defence system. Once it establish by itself. Its microenvironments are hostile to therapeutic including immunotherapy as well as gene

I. Introduction Cancer is one of the major health problems of mankind, accounting for 7.6 million of death world wide. Cancer mortality is expected to increase further, with an estimated 9 million people dying from cancer in 2015. This figure will rise to 11.4 million in 2030 (WHO 2006) (Cho, 2007). Of all cancer diagnosed, 90% of these are solid tumours. As they do not have particular noticeable symptom or signs for early detection, a significant percentage of the patients with newly diagnosed disease have regional or advanced, inoperable disease, especially

Xu J et al: immunotherapy with anaerobic bacteria for immunogene therapy of solid tumours therapy. In this paper, we review current understanding of tumour microenvironments and recent advances in therapy of solid tumour and explore potential combinations of immunization and anaerobic bacteria for cancer management.

tumour vessels leaky, although their permeability varies both within and among tumours.

C. Tumour hypoxia and acidity Most solid tumours contain regions of hypoxia (Wu et al, 2006). The limited vasculature of tumours results in insufficient blood supply and chronic or diffusion-limited hypoxia. Tumour cells in hypoxic regions may be viable, but they are often adjacent to regions of necrosis. Tumour cells in regions proximal to blood vessels can migrate into hypoxic areas and become necrotic, presumably because of nutrient deprivation. If cells close to blood vessels are killed by treatment, the nutrient supply to previously hypoxic cells may improve, allowing those cells to survive and regenerate the tumour (Trédan et al, 2007). Transient hypoxia is also common in tumours and results from the temporary shutdown of blood vessels. Hypoxic regions of tumours are likely to have a decreased supply of nutrients such as glucose and essential amino acids (Pouysségur et al, 2006). The presence of hypoxia in tumours is known to lead to the activation of genes associated with angiogenesis and cell survival that is mediated by the transcription factor hypoxia-inducible factor 1(Bos R et al, 2004). Expression of these genes may result in the expansion of populations of cells with altered biochemical pathways that may have a drug-resistant phenotype. Transient hypoxia has been reported to cause amplification and increased expression of the genes encoding Pglycoprotein and dihydrofolate reductase, which induce drug resistance to substrates of P-glycoprotein and to folate antagonists, respectively. Transient hypoxia that is associated with glucose deprivation can also disrupt protein folding in the endoplasmic reticulum; this effect may confer resistance to topoisomerase II–targeted drugs and enhance P-glycoprotein expression and multidrug resistance (Chen et al, 2003). The pH in the tumour microenvironment can influence the cytotoxicity of anticancer drugs (Philip et al, 2005). Molecules diffuse passively across the cell membrane most efficiently in the uncharged form. The extracellular pH in tumours is low and the intracellular pH of tumour cells is neutral to alkaline, weakly basic drugs that have an acid dissociation constant of 7.5–9.5 are protonated and display decreased cellular uptake. Alkalinization of the extracellular environment enhances the uptake and cytotoxicity of some of these drugs (Trédan et al, 2007). By contrast, weakly acidic drugs concentrate some in the relatively neutral intracellular space. The acidic microenvironment may also inhibit active transport of some drugs (Mahoney et al, 2003).

II. The unique microenvironment of solid tumours A. Overview All solid tumours, when they grow more than 2 mm diameter in size, undergo angiogenesis that results in biological changes and adaptive metabolisms, i.e.: formation of defective vessels, appearance of hypoxic areas, and emergence of heterogeneous tumour cell population. Thus, solid tumours are organ-like structures that are heterogeneous and structurally complex, consisting cancer cells and stromal cells (i.e., fibroblasts and inflammatory cells) that are embedded in an extracellular matrix and nourished by a vascular network; each of these components may vary from one location to another in the same tumour. Compared with normal tissues, the tumour stroma is associated with an altered extracellular matrix and an increased number of stromal that synthesize growth factors, chemokines, and adhesion molecules (Aznavoorian et al, 1990). The extracellular matrix can vary greatly among tumours, both in amount and in composition (Ohtani, 1998). Also the tumour stroma can influence malignant transformation (Tlsty 2001) plays an important role in the ability of tumours to invade and metastasize, and affects the sensitivity of tumour cells to drug treatment. The amount composition and structure of stromal components in tumours also contribute to an increase in interstitial fluid pressure, which hinders the penetration of macromolecules through tissue (Croker, 2008). Also, the three-dimensional structure of tissue itself can influence the sensitivity of constituent cells to both radiation and chemotherapy (Shicang 2007).

B. Tumour vasculature and blood flow Solid tumours at advanced stages have abnormal vasculature, which influences the sensitivity of the tumour to therapies. Anticancer drugs gain access to tumours via the blood and limited supply of nutrients in tumours leads to metabolic changes (including hypoxia) and gradients of cell proliferation that influence drug sensitivity (Tatum et al, 2006). Also, blood vessels in tumours are often dilated and convoluted. Compared with normal tissues, tumour blood vessels have branching patterns that feature excessive loops and arteriolar–venous shunts, in some tumours they are not organized into arterioles, capillaries, and venules but instead share features of all of these structures. The walls of tumour vessels may have fenestrations, discontinuous or absent basement membranes that may lack perivascular smooth muscle (Hallmann et al, 2005) and fewer pericytes than walls of normal vessels. In addition, cancer cells may be integrated into the vessel wall. These abnormalities tend to make

D. Tumour immunosuppression During the constant battle between tumour and immune system, tumour cells developed multiple ways to fight back the immune system.

1. Avoidance of effectors T cell killing

Gene Therapy and Molecular Biology Vol 13, page 38 One of well established strategies is down regulation of antigen presentation by tumour cells, especially through MHC class I restricted antigen presentation pathway. Tumour cells can down regulation, even loss of MHC class I molecules on their cell surface (Frey, 2006), mutation of proteins associated with this pathway, such as TAP and LMP2 and LMP7. Tumour or stromal cells also secrete factors that damp immune responses. TGF (tumour growth factor), IL10 are two cytokines with immune suppressive functions usually found with high levels within tumour. TGF levels are associated with poor prognoses of cancers including prostate, gastric and bladder carcinoma (Biswas et al, 2007). TGF inhibits T cell activation and differention of cytotoxic T cells and promotes NKT cells mediated inhibition of CTL responses together with IL-13 (Biswas et al, 2007). IL-10 down regulate antigen presentation by dendritic cells and promote the generation of Tr1 regulatory T cell generation (Suciu-Foca et al, 2003) and Inhibit CTL response in antigen experienced host (Tamada et al, 2002). High levels of prostaglandin E2 (PGE2) have been shown in colorectal, lung and bladder cancer (Akasaki et al, 2006). It has been demonstrated that PGE2 promotes the generation of IL-10 secreting CD4 T cells through the induction of IL-10 secreting dendritic cells (Cools et al, 2007). Different tumour types have also been expressed PDL1, an immune suppressive molecule. Tissue histology study showed that freshly isolated carcinomas of human lung, ovarian, colon, melanoma, head and neck cancers, and breast cancers can express PD-L123. PD-L1 a suppressive molecule, engagement of PD-L1 with PD-1 of effector T cells causes T cell apoptosis (Yang et al, 2008). B7-H1 positive melanoma cells were also more resistant to specific CTL, while nearly all B7-H1 negative tumour cells were eliminated in the cultures (Dong and Chen, 2003), these results suggest that expression of suppressive molecule is another strategy used by tumour cells to avoid from killing by effector cells.

regulatory cells were boosted from existing T regulatory cells or vaccine induced. However, immunotherapy has shown to amplify tumour specific T regulatory cells, thus impede effective immunotherapy in a mouse tumour model (Reilly et al, 2000); moreover, similar results were also observed clinically. Patients with resected HPV16-positive cervical cancer were vaccinated with an overlapping set of long peptides comprising the sequences of the HPV16 E6 and E7 oncoproteins emulsified in Montanide ISA-51. The vaccine-induced responses were dominated by effector type CD4(+)CD25(+)Foxp3(-) type 1 cytokine IFN gamma-producing T cells but also included the expansion of T cells with a CD4(+)CD25(+)Foxp3(+) phenotype (Welters et al, 2008).

3. Abnormal antigen presentation cells Antigen presentation cells include dendritic cells (DC), macropaghes and B cells. Matured DCs play key roles for the priming of naive T cells, including CD8+ T cells, which is critical for the killing of tumour cells. Tumour microenvironments usually have less functional competent matured but more immature DCs, which can not effectively activate T cells. Furthermore, it has been reported that in tumour tissues, there are subset of DCs that suppress T cell function. This T cell suppression has been shown in cancer patients as well as animal tumour models. Immune cells in the tumour microenvironment are dysfunctional, generally fail to control tumour growth and may even promote its progression. Molecular mechanisms responsible for tumour-induced local and systemic immune suppression are currently under intense discussed. It appears that tumours can deregulate recruitment, effector functions and survival of immune cells, interfering with all stages of antitumour response. Suppressive mechanisms targeting key signalling pathways in immune cells have been identified. Strategies for reversal of tumour-mediated immunsuppression are being developed. Confirmation of multiple and varied mechanisms used by tumours to escape immune surveillance is crucial for the future design in antitumour therapies.

2. Regulation of immunoresponses by regulatory T cells Regulatory T cells are groups of T cells that regulatory immune response, different compartments of T regulatory cells including CD4+, CD8+ and NKT cells have been identified. CD4+CD25+ Foxp3+ thymus derived T regulatory cells and antigen induced IL-10 secreting CD4 T cells are the 2 main types identified. NKT cells have also been shown to have regulatory function during tumour development (Berzofsky et al, 2008). However, the number of T regulatory cells with human ovary cancer is related to poor prognosis of cancer (Koido et al, 2005). Also, it has been shown that myeloma cells promote the generation of IL-10 secreting Tr1 T cells (Battaglia et al, 2006). Tr1 cells can be isolated from tumour infiltrating lymphocytes in B16 tumour model (Seo et al, 2001). Human bladder cancer tissues contain high number of Foxp3+ cells and mRNA level of IL-10 (Petrulio et al, 2006). It is not clear whether the T

III. Current cancer gene therapy and immunotherapy approaches A. Current development in gene therapy of solid tumour Cancer is, at present, the disease most frequently targeted by gene therapy because its promise of potential for selective potency. To achieve this aim, cancer gene therapy strategies attempt to exploit the biological uniqueness of each particular tumour. Cancer gene therapy may be defined as the transfer of recombinant DNA into human cells to achieve an anti-tumour effect. Gene therapy will have a major impact on the healthcare of our population only when vectors are developed that can

Xu J et al: immunotherapy with anaerobic bacteria for immunogene therapy of solid tumours safely and efficiently be injected directly into patients as drugs. One of the most strategies of vector development is that of non-viral vectors, which consist of liposomes, molecular conjugates, and naked DNA delivered by mechanical methods. The modifying viral vectors should be focused to reduce toxicity and immunogenic, increasing the transduction efficiency of non-viral vectors, enhancing vector targeting and specificity, regulating gene expression, and identifying synergies between gene-based agents and other cancer therapeutics. A universal gene delivery system has yet to be identified, but the further optimization of each of these vectors should result in each having a unique application.

linked to the promoter to allow for assessment of expression. In preliminary experiments, this vector was able to transfect ovarian cancer cells isolated from ascites fluid, and confer tumour-specific expression of betagalactosidase. This method creates the possibility of targeting expression of certain genes in specific tissues

3. Herpes simplex virus thymidine kinase gene To broaden the effect of gene therapy, vectors employing both the thymidine kinase gene and the genes for immunomodulatory cytokines such as IL-2 or granulocyte-macrophage colony-stimulating factor (GMCSF) have been developed (Iwadate et al, 1997). In mice, injection of these vectors into tumours and treatment with ganciclovir had both a direct anti-tumour effect in the liver, as well as a systemic effect in generating tumourspecific immune responses. As a result, these mice are resistant to subsequent tumour challenge. This system establishes the principle that localized gene therapy might ultimately have systemic protective or therapeutic effect by stimulating immune mechanisms which can act throughout the organism. A phase I trial for patients that would include treatment with a thymidine kinase and cytokine (IL-2) vector is being planned. The principle endpoint of the study will be the determination of an antitumour immune response.

1. Pro-Drug activation vectors Several experimental models relying on pro-drug activation vectors (Kanai et al, 2008). One such a model involves local injection of gene therapy vectors into tumour sites. This model may benefit from the so-called "bystander effect," a reflection of the biological observation that pro-drug activation to 5-fluorocysteine (5FU) releases this chemotherapeutic not just in the tumour cells, but in the surrounding cell environment as well. In fact, using in vitro systems, it has been found that only 5% of tumour cells need to be infected by the delivery vector for anti-tumour effect to be seen throughout the whole tumour cell population. An adenovirus vector expressing the cytosine deaminase enzyme will be injected into the prostate bed using similar techniques as those now used for radiation implants. These patients will then be given the pro-drug, which in principle will be activated to 5-FU in the prostate gland. This should allow localized cytotoxic therapy to the prostate and possible synergistic benefit between 5-FU and the concurrent radiation therapy. The other model system which is used in clinical trials deals with autologous transplantation for metastatic breast cancer. In this system, harvested bone marrow is exposed to the viral vector, which infects the epithelial tumour cells efficiently, but normal marrow stem cells less efficiently. After intensive chemotherapy, patients are then given this modified marrow population. Once engrafted, patients are treated with the pro-drug 5-FC, which in principle should be toxic only to the infected tumour cells. This trial is open to women with known marrow

4. Dendritic cells as targets for cancer gene therapy DCs are the most potent APCs in the immune system and are central to the success of these genetically engineered tumour vaccine strategies. Activated DCs can present prostate tumour vaccine-associated antigens; they have processed to both CD4 (helper) and CD8 (cytolytic) T cells in the draining lymph node of the vaccination sites, activating a systemic tumouricidal immune response. The possibility of obtaining large numbers of DCs in vitro has boosted research on their ontogeny and functions. The unique ability of DCs to take up, process, and present antigens, and to activate naive CD4+ and CD8+ T cells, makes them appropriate candidates for the immunotherapeutic approach. In a mouse model, DCs are harvested and then transfected with adenoviral vectors. These vectors expressed a foreign protein, beta-galactosidase. The dendritic cells were then injected into mice, and served to prime an immune response against that protein. This ex vivo gene therapy has many potential human applications. Three major myeloid DC populations have been identified in vivo: (1) epidermal Langerhansâ&#x20AC;&#x2122; cells (LC); (2) interstitial (or dermal) immature DC; and (3) mature interdigitating DC, found in secondary lymphoid organs. In the early stages of DC research, the limited accessibility of these cells in vivo as well as their difficult ex vivo culture hampered attempts to study this particular cell type in more detail. In the 1990s, this problem was solved by the efforts of various research teams which revealed the hematopoietic lineages through which DC differentiate,

involvement by tumour cells, and who are therefore not candidates for standard high-dose therapy. 2. Tumour-specific gene promoters The L-plastin gene (Akbulut et al, 2003), as another means of conferring tumour-specific expression which encodes an actin-binding protein, show the new vector model with a tumour specific gene promoter. The estrogen-dependent tissues such as ovary and breast were selectively expressed in ovarian and breast cancer. The promoter for this gene is added to the adenoviral vector, and a reporter enzyme, such as beta-galactosidase, is

Gene Therapy and Molecular Biology Vol 13, page 40 and established in vitro expansion protocols to obtain sufficient quantities of DC for clinical use (Caux et al, 1992; Sallusto, 1994). The unique ability of DC to stimulate primary immune responses stems from several factors. The immature DC type uses elegant systems, including macropinocytosis, mannose receptor-mediated uptake, Fcg receptor III (FcgRIII)-mediated uptake and phagocytosis to efficiently take up exogenous antigens, either self or non-self, from the periphery (Steinman et al, 1999). After antigen capture, DC leaves the peripheral tissue and migrates via blood or lymphatic vessels to the draining lymph nodes where they activate T cells Given their central role in controlling immunity and their link with the innate immune system, DC are often called natureâ&#x20AC;&#x2122;s adjuvant. Therefore, DC is logical targets for immunotherapy of cancer. The fact that tumours do not elicit a therapeutic T cell response may be due to the absence of competent DC at the tumour site.

principle' demonstration of the potential power of gene therapy to combat cancers. To establish efficient and safe gene delivery in vivo, a number of new techniques and concepts have been introduced with improvements in targeted or controlled delivery of genes. But we have come a long way in understanding the cellular barriers which prevent proper delivery of DNA or viral vectors. Cancer gene therapy has still a long way to go in the basic and clinical sciences.

C. Anaerobic bacteria for cancer treatment Interest in microbe-based approaches to cancer therapy has recently re-emerged with the development of methods to genetically engineer bacteria, reducing their toxicity and arming them with genes encoding pro drugmetabolizing enzymes.

B. Cancer gene therapy existing problems

1. Anaerobic bacteria as tumour target vector

Currently, there are many different approaches to fight cancer with gene therapy. Morgan et al report has revealed encouraging results for the use of gene therapy as a treatment for cancer (Morgan et al, 2006). However; two principal obstacles continue to limit further advances in gene therapy. The first is a technical problem, the development of an appropriate delivery system -- a reliable, safe, and effective means for introducing genetic material into the target cells or tissues. The second problem is a biological one -- developing an understanding of the molecular basis underlying cancer in order to determine where single alterations in genetic expression might allow effective anti-cancer therapy. In viral vector, the efficiency of transduction is not sufficient for therapeutic measures (Marina et al, 2003). One important parameter is whether the genetic alteration has to be lasting or temporary (stable or transient transfection). Of overall importance is the question of biological safety, which means that the vector itself does not create a novel threat to the patient's health. The key to a successful gene therapy is the vector system. Various vectors have been developed with unique features, including viral and nonviral based therapy systems (Wagner, 2007). However, due to the complex nature of cancers, these vectors suffer from several deficiencies: firstly the majority of vectors currently in use require intratumoural injection to elicit an effect, far from ideal as many tumours are inaccessible and spread to other areas of the body making them difficult to locate and treat. Second, most vectors do not have the capacity to efficiently enter and kill every tumour cell. The emerging challenges of cancer gene therapy: i) which better route of administration is best for improving gene delivery; iii) optimizing new vector best suited to the target type of tissue and reducing toxicity, Although as with many gene-therapy approaches, considerable barriers will need to be overcome to make the technique more reliable and widely applicable - achieving long-term expression of therapeutic genes is a particular problem these results are nevertheless a heartening 'proof-of-

The unique solid tumour micro-milieu, though, provides a haven for anaerobic bacteria. Anaerobic and facultative anaerobes tested so far fell into three classes. (1) the lactic acid, Gram-positive anaerobic bacteria; (2) the intracellular, Gram-negative facultative anaerobes, and (3) the strictly anaerobic, Gram-positive saccharolytic/ proteolytic Clostridia. At the molecular level, bacterial infections like those of Clostridia novyi (C. novyi) are associated with the release of pathogen-associated molecular patterns (PAMPs) from bacteria and Hsp70 from necrotic cells (Gelman, 2003). Hsp70 induces maturation of DCs, professional antigen-presenting cells that are essential for the production of potent immune responses. PAMPs interact with Toll-like receptors, leading to up-regulation of costimulatory molecules such as CD40 and proinflammatory cytokines such as IL-12. These in turn induce the production of IFN-! and initiate a Th1-dependent cell-mediated response, primarily affected by CD8+ cytolytic T cells (Kay, 2001). The demonstration that CD8+ T cells from C. novyi-NT-cured mice can confer adoptive immunity in a tumour-specific fashion is consistent. Clostridium is strictly anaerobic, sporulating Grampositive bacteria. This genus is one of the largest genera comprising of about 80 species. Up to 10 species of Clostridia have been studied and as strictly anaerobic bacteria they all require an anaerobic environment to grow but their oxygen tolerance and biochemical profile varies considerably among different species. Clostridial spores had been used to induce tumour lysis following intravenous delivery and shown a distinct advantage over Bifidobacterium and Salmonella in terms of easy production, hardy storage and impressive oncolytic effects. Both proteolytic and saccharolytic Clostridia have been tested for cancer therapy. When C. novyi-NT spores are injected intravenously into immunodeficient mice bearing human xenografts, the spores quickly germinate within necrotic regions of the tumours. Hypoxic and

Xu J et al: immunotherapy with anaerobic bacteria for immunogene therapy of solid tumours necrotic regions are generally localized within the central parts of tumours, with well perfused tumour cells occupying the rim. Because of the exquisite sensitivity of C. novyi-NT to oxygen (Dang et al, 2001), bacterial germination and spread halt when the bacteria reach the well oxygenated rim. It was shown that conventional chemotherapy and radiation therapy could be used to destroy the well oxygenated cells in this rim, and that the combination of C. novyi-NT provided substantial antitumour activity in several xenograft models.

Clostridia to produce anti-cancer drugs (Jennifer et al, 2006). The strictly anaerobic clostridia, on the other hand, have been shown to selectively colonise in solid tumours when delivered systemically and has resulted in high percentage of "cures" of experimental tumours. A phase I clinical trial combining spores of a non toxic strain (C. novyi-NT) with an antimicrotubuli agent has been initiated. The recombinant DNA technology reignited the field, enabling genetic improvement of Clostridia’s innate oncolytic capability. It provides a possible alternative to overcome the hitch of using wild type strains Anaerobic bacteria, such as Clostridia have now been convincingly shown to selectively colonise and regerminate in the hypoxic/necrotic regions of solid tumours and can be delivered systemically. Furthermore, existing plasmidbased gene modification strategy harbours several safety concerns regarding possible horizontal plasmid transfer and spread of plasmid-associated antibiotic resistant genes.

2. Anaerobic bacteria and immune response C. novyi is well known for its capacity to induce massive leukocytosis and inflammation (Agrawal et al, 2004), whereas many other species of Clostridia do not induce this level of response. The inflammatory reaction is classic in many ways, including the observed increase in neutrophil-directed cytokines in serum and the cellular nature and time course of the infiltrate. The antitumour effects of inflammation are well documented. Systemically administered C. novyi-NT spores are distributed throughout the body, but due to their strict anaerobic growth requirements, germinate only within anoxic or markedly hypoxic regions of tumours. Once germinated, the bacteria destroy adjacent cancer cells through the secretion of lipases, proteases, and other degradative enzymes. At the same time, the host reacts to this localized infection, producing cytokines such as IL-6, MIP-2, GCSF, TIMP-1, and KC that attract a massive influx of inflammatory cells, initiated largely by neutrophils and followed within a few days by monocyte and lymphocyte infiltration. The inflammatory reaction restrains the spread of the bacterial infection, providing a second layer of control in addition to that provided by the requisite anaerobic environment. The inflammation may also directly contribute to the destruction of tumour cells through the production of reactive oxygen species, proteases, and other degradative enzymes. Moreover, it stimulates a potent cellular immune response that can subsequently destroy residual tumour cells not lysed by the bacteria. The cure rate is determined by the balance between bacteriolysis, angiogenesis, regrowth of residual tumour cells, and the rate of development of the immune response. During these years, bacteriological research on tumour associated anaerobic spore forming bacteria has accumulated a considerable amount of information and a variety of new concepts in experimental and clinical oncology (Agrawal et al, 2004). Of great importance was the systematic elucidation which convincingly demonstrated that the growth of anaerobes can be strictly interconnected with tumour growth. A whole series of experimental studies have been performed to elucidate the mechanisms which governed the selective, temporarily unrestricted clostridial growth and which formed the basis for the liquefaction of tumour tissue. Since tumour lysis with Clostridium oncolyticum spores is incomplete and, possibly, subject to non-specific systemic incompatibility [‘acute tumour lysis syndrome’]. Clostridia became significant in pursuing the concept of engineered

IV. Current approaches for immunotherapy of cancer A. Overview The aim of cancer immunotherapy is to activate patient’s immune system to eradiate tumour cells. It was expected that when appropriately primed, the activated host immune cells, especially tumour antigen specific CD4+ and CD8+ T cells, can specifically kill tumour cells. Tumour antigens are usually self antigens, both central and peripheral tolerance apply to tumour antigens. Central tolerance occurs in the thymus, T cells with strong self reactivity are eliminated. Peripheral tolerance make tumour specific T cells anergy or suppressive. Cancer vaccine will activate T cells purged of strong activity and influenced by different peripheral tolerance mechanisms. Different approaches have been employed to overcome the tolerance, in order to achieve better T cell responses, including immunization with different routs and with different adjuvant, providing co-stimulating signals while inhibiting signals such as CTLA-4. Neutralizing IL-10 at the same time of immunization has been show to generate better CTL response in antigen experienced host, which is important for cancer immunotherapy; as patients with cancer are tumour antigens experienced.

B. Combining immunostimulation with gene-silencing by siRNA The innate immune system recognizes pathogens by means of germ line-encoded pattern recognition receptors (PRRs) (Gro F, 2006). A subfamily of PRRs is the Tolllike receptors (TLRs), which is important for initiation of an immune response. siRNAs can activate innate immunity through the activation of Toll-like receptor (Sioud et al, 2007). These findings suggest potential prophylactic and therapeutic use of immunostimulatory siRNAs as adjuvant. In addition, to immune stimulation,

Gene Therapy and Molecular Biology Vol 13, page 42 gene-silencing through RNAi is another potency of immunostimulatory siRNAs. RNAi is a widely conserved post-transcriptional gene-silencing mechanism where double-stranded (ds) RNAs trigger the degradation of homologous mRNA sequences and certain siRNA sequences can activate immune cells to secrete proinflammatory cytokines and type I interferons in immune cells. As a consequence of these findings any therapeutic siRNA should be tested in human blood cells prior to use in (Gelman, 2003). However, if we view the activation of innate immunity by siRNAs as beneficial for cancer therapy and infectious diseases, then immuostimulatory siRNAs could emerge as useful agents to knockdown gene expression and activate innate and adaptive immunity against tumour cells. This observation prompted us to design bifunctional siRNAs, which combine gene-silencing and immunostimulation in one single siRNA molecule (Gro F, 2006).

specific immune response both in vitro to a viral antigen and in vivo to a tumour-associated antigen in patients with cancer. Current efforts in cancer immune therapy and bacteria therapy are largely aimed at stimulating antitumour immune responses by using various tumour antigens and adjuvants. The involvement of TLR-activated pathways in immune response is supported by the induction of DC maturation and secretion of various cytokines (Palucka et al, 2007), leading to the induction of innate and adaptive immunity.

E. Targeting cancer stem/progenitor cells for anticancer therapy The cancer recurrence phenomenon has been associated with the accumulating genetic or epigenic alterations in cancer cells which may contribute to their uncontrolled growth, survival and invasion as well as their intrinsic or acquired resistance to clinical treatments (Lowenberg et al, 2003; Mimeault et al, 2005). Recent investigations have revealed that the most aggressive cancers may originate from the malignant transformation of embryonic or adult stem/progenitor cells into cancer progenitor cells (Mimeault, 2006). The cancer progenitor cells can provide critical functions in cancer initiation and progression into metastatic and recurrent disease states. Numerous investigations have provided evidence that the genetic and/or epigenic alterations occurring in the multipotent tissue-specific adult stem cells, the most cancers may arise from the malignant transformation of multipotent tissue-specific adult stem cells and/or their early progenitors into cancer progenitor cells, the accumulation of different genetic and/or epigenic alterations in cancer progenitor cells during cancer progression also seems to be associated with the occurrence of highly aggressive cancer subtypes. The functional properties of cancer progenitor cells may be influenced through external signals mediated by other further differentiated cancer cells and host stromal cells including activated fibroblasts and infiltrating immune cells, such as macrophages and endothelial cells (Kopp et al, 2006). Among the diverse growth factors, chemokines and angiogenic substances released by stromal cells (Kopp, 2006). All these soluble factors can influence, of autocrine or paracrine manner, the tumour cell behaviour and neovascularization process during cancer progression. The intrinsic or acquired resistance of poorly differentiated and tumourigenic cancer progenitor cells to current clinical therapies may lead to their persistence in primary and secondary neoplasms after treatments, and thereby contribute to cancer recurrence (Mimeault, 2007; de Jonge-Peeters et al, 2007). The cancer stem/progenitor cell model of carcinogenesis may also explain the differences of response of distinct cancer subtypes to current therapies as well as the dormancy phenomenon and disease relapse, which may be associated with a higher resistance of cancer progenitor cells to conventional therapies under specific conditions prevalent in primary and/or secondary neoplasms relative to their further differentiated progeny

C. Development of strategies to promote effector cell recruitment into tumour One strategy is to promote effector cell recruitment into metastases when it fails spontaneously (Shakhar, 2003). Intratumoural introduction of chemokines through the use of viral vectors would serve as a proof of concept. Transduction of tumour cells to express specific chemokines has shown benefit in some experimental murine models. Similarly, introduction of the TNF superfamily member LIGHT (homologous to lymphotoxins, inducible expression, competes with HSV glycoprotein D for HVEM, a receptor expressed on T lymphocytes) has been expressed at tumour sites with dramatic results (Kunz M et al, 1999). However, direct intratumoural injection of recombinant viral vectors will only serve as a proof of concept, and development of agents that can be delivered systemically yet target tumour metastases would have to be pursued for practical application.

D. Modulating tumour cell biology to alter the tumour microenvironment Once the oncogenic signals present in tumour cells that determine the nature of the tumour microenvironment are defined, then it should be possible to target those pathways directly to eliminate the underlying basis for immunosuppression at tumour sites. For example, Stat can drive the expression of vascular endothelial growth factor (VEGF) (Burdelya et al, 2005), which in addition to promoting neoangiogenesis has been reported to be inhibitory for dendritic cell generation in vivo (Della et al, 2005). The interface between tumour biology and the creation of the immunosuppressive tumour microenvironment is an area ripe for additional research. Another strategy in the immunotherapy of tumours is the use of mRNA-encoding tumour antigens to induce Tand B-cell immunity to the encoded antigens. In vivo application of mRNA induced cytotoxic T-cell activity and specific antibodies in mice. Furthermore, human DCs transfected ex vivo with mRNA induced an antigen42

Xu J et al: immunotherapy with anaerobic bacteria for immunogene therapy of solid tumours (Mimeault, 2007). Based on these observations, the new cancer therapeutic strategies should be based on targeting of different oncogenic cascades activated in tumourigenic cancer progenitor cells, and which must now be considered for improving the current therapeutic treatments. The molecular targeting of tumourigenic cancer progenitor cells must be considered for improving the efficacy of the current cancer therapies.

associated with immunologic memory. Such a response potentially allows for treatment of disseminated disease and a prolonged anti-tumour effect that persists beyond the immediate treatment period. Immunogene therapy strategies involve both ex vivo and in vivo approaches (Glick et al, 2006). Increasing the capacity of the immune system to mediate tumour regression has been a major goal for tumour immunologists. Progress towards tumour vaccines has been recently made by the molecular identification of novel tumour-associated antigens (TAA) and by a better understanding of cellular signals required for efficient T cell activation (Pule et al, 2002). Cancer vaccination is of therapeutic rather than prophylactic nature, involving attempts to activate immune responses against TAA to which the immune system has already been exposed. To date, advances in gene delivery technology have led to the development of immuno-gene therapy strategies to augment host-immune responses to tumours. These approaches include (1) the use of tumour cells genetically modified with genes encoding costimulatory ligands, cytokines or HLA molecules to enhance their immunogenicity and (2) the genetic modification of immune-competent cells with TAA in order to enhance their anti-tumour response. Despite the continuous increase in clinical gene therapy protocols for immunotherapy of cancer, many aspects of gene transfer are still far from ideal. A basic requirement, not yet adequately and routinely fulfilled, is to introduce the gene of interest with sufficient efficiency into the target cells in order to achieve therapeutic benefit in cancer patients.

F. Gene-based tumour immunization For any gene therapy application including genetic immunization, the goal is to deliver genes into therapeutically-relevant cells while avoiding other cells that cannot contribute to immunization or therapeutic effects. While this is the goal, particularly for in vivo gene therapy, current gene delivery vectors cannot specifically deliver genes to the cells we want and frequently deliver genes into non-target tissues reducing therapy and increasing dangerous side effects. Generally, the level of gene transfer into tumour cells and immune effector cells determined the level of immunogenetics, they have been shown to be limited, and this has been thought to account for the poor results obtained by cancer gene immunotherapy. Therefore, vector design is one of the most critical areas for future research (Logan et al, 2002). Gene delivery vectors thus are required fall into three areas: 1) identification of celltargeting ligands using random peptide-presenting phage libraries; 2) engineering viral and non-viral gene delivery vectors to accept cell-targeting ligands; and 3) developing effective methods to image gene and vector delivery in vivo to determine the efficacy of targeted vectors in the complex tumour environment. The different vector systems can have strengths or weaknesses, depending on their use. For ex vivo gene delivery and clinical use in cancer protocols, design of optimized transduction protocols and development of improved vectors, exhibiting improved gene transfer efficiency and stability for large-scale production, have just begun to be evaluated. Nonviral gene delivery systems are cost- and time-effective and large-scale manufacturing of clinicalgrade plasmid vectors is logistically simple. The major disadvantages are the low transfection efficiency and the transient expression in target cells. As already mentioned earlier, one of the attractive features of immunological gene therapy approaches is that they capitalize on the ability to amplify the outcome of the gene transfer (â&#x20AC;&#x2DC;genetic immunopotentiationâ&#x20AC;&#x2122;). Consequently, high efficiency gene transfer may not be an essential requirement in these protocols. Given this problem, we are interested in developing gene delivery with recombinant engineer bacteria vectors that can be tuned to target specific cells in vivo for gene therapy and immunization applications. As recombinant engineer bacteria are so far the best characterized bacteria vectors, they are most frequently used vectors for immuno-gene therapy of cancer. Immunogene therapies have the theoretical advantage of inducing a systemic anti-tumour response

G. Breakdown of immune tolerance to tumours The current rationale lies in the local recruitment of inflammatory cells that can destroy a fraction of the tumour cells directly or indirectly, thereby releasing tumour antigens. These antigens can be taken up in the form of peptides, proteins or apoptotic bodies by professional antigenpresenting cells (APC) by a process known as cross-priming (i.e. indirect presentation of tumour antigens to the immune system by a host-derived APC), that travel to the draining lymph nodes where they will activate naive antigen-specific T cells and initiate a primary cellular immune response. The new approach enlists the help of the immune system to target and kill tumour blood vessel cells, through an unprecedented recruitment of the immune system; they were able to generate a strong anti-tumour effect by targeting the central component of what tumours need most-a blood supply (Niethammer et al, 2002). According to the classical paradigm in tumour immunology, immune responses are believed to follow a model of discrimination between self and non-self. Consequently, tumours should be considered as non-self, like viruses or bacteria. Therefore, an important task of the immune system is to search for and destroy tumour cells as they arise, in concordance with the original proposals of Burnetâ&#x20AC;&#x2122;s immunological surveillance hypothesis.

Gene Therapy and Molecular Biology Vol 13, page 44 However, the limited successes of cancer immunotherapy approaches based on these concepts, prompted a revision of tumour immunology (Luis et al, 2005). Ultimately, it appears that the immune response at the T cell level is based on the presence of the appropriate costimulatory molecules on APC that promote T cell activation. DCs (DC) form a complex network of antigen-capturing and presenting cells (APC) defined by morphological, phenotypical and functional criteria which distinguish them from monocytes and macrophages (Elke et al, 2002). Immunity against cancer is necessary if gene transfer is going to be applied in a clinically relevant way. Instead of exploiting the increasing knowledge on cytokines and their plethora of actions in the immune response, immunology may provide a more fundamental mechanism to explain the immunological unresponsiveness to cancer than the classical self/non-self paradigm. At a later stage, we will focus on a new gene-based tumour immunization that seems to fit within this conceptual framework.

be derived from a natural or genetically engineered initiating cell. Moreover, the TCR of cytotoxic T cells can be substituted with an immunoglobulin-like surface molecule, which allows the binding to tumour-specific surface molecules not presented by MHC molecules (Keith et al, 2002). These more elaborate forms of adoptive transfer of killer cells are being studied in ongoing clinical trials. A second approach in preclinical development involves genetic modification of DCs with the gene for interleukin-7 (IL-7). IL-7 stimulates cytotoxic Tlymphocyte responses and down-regulates tumour production of the immunosuppressive growth factor, TGF!.

V. Cancer vaccine A. Overview In the past two decades, adoptive immunotherapy, based on tumour-infiltrating lymphocytes or lymphokineactivated killer cells, has been used in clinical trials (Rosenberg et al, 1986; Rosenberg et al, 1987). These early results gave first evidence that the manipulation of the immune system represents a promising tool in cancer immunotherapy. The main rationale of genetic immunopotentiation protocols is the possibility of enlisting the immune system for a potentially vast amplification of gene therapy, thereby enhancing therapeutic benefit. The recognition that most tumours encode TAA and are capable of inducing protective immunity in preclinical models has reinvigorated the field of cancer immunotherapy (Pule et al, 2002). It has been hypothesized that the immune system of tumour patients, characterized by tolerance, can be modified to mount an immunological response against the tumour and thus facilitate tumour rejection. This ‘cancer vaccination’ is to be accomplished through exposure of TAA in a more favourable context to the immune system (Christian et al, 2006). Despite ongoing efforts to define and characterize TAA and, more importantly, clinically relevant TAA, little is known about TAA for the majority of human cancers and the largest part of clinical experience with tumour vaccines has been obtained in melanoma patients. Therefore, most cancer vaccines, to date, use tumour cells as a source of TAA. The molecular identification of antigens expressed by tumour cells that can be recognized by specific CD8+ cytotoxic T lymphocytes (CTLs) has provided a means by which to explore anti-tumour T-cell parameters in patients and also to develop antigen-specific immunotherapies.

H. Stimulation to illicit an active immunoresponse in a solid tumour environment Van Pel and Boon (1982) demonstrated that a protective immune response could be generated against a ‘non-immunogenic’ murine tumour, providing the first experimental evidence that the lack of tumour immunity was not due to the absence of TAA but rather to the inability to stimulate the immune system. Factors that can explain the failure of the immune system in tumourbearing hosts are numerous, and it is not clear which of them are critical in the clinical context. We all know that tumour cells are poor stimulators of immune responses and capable of inducing immune tolerance. Alternatively, it may well be that the lack of costimulatory molecules (e.g. CD80, CD86) on the surface of tumour cells accounts for the immune tolerance which keeps the tumour from being rejected. Deficiency of the immune system could be responsible for the lack of immunity and induction of T cell tolerance (von Euler et al, 2008). In this case; the tumour actively suppresses host antigen presentation and immune effect or functions by expression of a variety of local inhibitory molecules, such as VEGF and IL-10, especially when large tumour burdens are involved. Antigen-specific cytotoxic cells that do specifically recognize tumour cells can be generated by cell cloning techniques ex vivo or can be genetically engineered by the stable transfection of a TCR that specifically recognizes a certain MHC-tumour antigen complex (Keith et al, 2002). This has been made possible by the use of defined tumour antigens to stimulate lymphocytes in vitro, and the ability to clone lymphocytes derived from a single, antigenspecific T cell (Pule et al, 2002). Adoptive transfer of clonally expanded lymphocytes to lymphopenic hosts after nonmyeloablative conditioning chemotherapy has resulted in cell proliferation and persistent clonal repopulation correlated with tumour regressions in patients with melanoma (Keith et al, 2002). Ex vivo–expanded clonal populations of tumour antigen–specific lymphocytes can

B. Current vaccines 1. Antigen Presentation to the Immune System The immune system responds to intracellular events in target cells by the recognition of intracellularly derived protein fragments presented on the cell surface by major histocompatibility complex (MHC) molecules. Circulating T lymphocytes can potentially engage these peptide-MHC complexes through their T-cell receptors (TCR). This 44

Xu J et al: immunotherapy with anaerobic bacteria for immunogene therapy of solid tumours mechanism allows the immune system to differentiate abnormal intracellular processes from normally functioning cells expressing so-called self proteins. The key steps in the generation of an immune response to cancer cells include loading of tumour antigens onto antigen-present cells in vitro or in vivo (Figure 1).

contain the relevant tumour antigens; however, the logistic drawback is that it is difficult to obtain and individually prepare vaccines for each patient. To avoid this problem, other tumour cell vaccines have been formulated as lysates of allogeneic laboratory cell lines containing shared tumour antigens (Sondak et al, 2002).

2. Intratumoral bacillus Calmette-GuĂŠrin (BCG)

5. Naked DNA and gene-modified tumour vaccines

This strategy may be one of the earliest forms of cellular immunotherapy tested by the Intratumoral injection of the BCG in cancer (Mathe et al, 1973). The immunologic basis is that BCG generates an inflammatory process ideal for the attraction of APCs, which pick up tumour antigens released by the tumour cells, damaged by the bacterial infection and cross-present them in a socalled danger environment. This form of treatment generates occasional antitumor immune responses.

Intramuscular injection of naked DNA sequences results in gene expression and the generation of immune responses (Wolff et al, 1990; Kumar et al, 1996). These DNA plasmids, which consist of an antigen gene regulated by a promoter with constitutive activity can be conjugated with gold particles and propelled into the skin using a helium gas gene gun. The protein antigen produced by the target cells is taken up by host APCs, processed, and crosspresented to the immune system in the draining lymph nodes. Gene-modified tumour vaccines have been tested in clinical trials for many years, the paracrine expression of cytokines such as IL-2 or IFN!, would allow the tumour cell to provide all of the signals for direct cytotoxic T cell activation, bypassing the need for host APCs and CD4+ T lymphocyte assist (Fearon et al, 1990). However, comparison of the antitumor capacity of gene-modified tumour vaccines in preclinical models was surprising in that the introduction of GM-CSF into tumour cells produced the most active vaccine (Dranoff et al, 1993). Bone marrow chimeras were used to show that the GMCSF gene-modified tumour vaccines attracted host APCs, which picked up tumour antigens and cross-presented them to the host immune system (Huang et al, 1994).

3. Intratumoral HLA-B7 The intratumoral injection of BCG, the recognition of a powerful alloantigen by cells with NK activity allows the recruitment of APCs, among other inflammatory cells, which will pick up tumour antigens released by the HLAB7â&#x20AC;&#x201C;transfected cells and cross-present them to cytotoxic effector cells. These tumours antigen-specific CD8+ CTLs would then be permitted to attack other tumour cells without the requirement of the presence of the alloantigen HLA-B7 on tumour cells.

4. Whole-cell tumour vaccines Whole-cell autologous tumour vaccines are personalized vaccines, and it can be assumed that they

Figure1: Cross-presentation of tumour antigens derived from cancer vaccines. Several immunologic manipulations lead to a common pathway of cross presentation of proteins derived from tumour antigens. a) in vivo APC-Based Vaccines; b) ex Vivo APC-Based Vaccines; c) augment the number of APC; d) non-T cell-DC. These host antigen-presenting cells (APCs), the most powerful of which are the DCs, circulate through the afferent lymphatic vessels to the T-cell areas of lymph nodes. There they cross-present the tumour antigen to T lymphocytes.

Gene Therapy and Molecular Biology Vol 13, page 46 As can be noted by the mechanism of action of most of the prior immunologic maneuvers, the common pathway of anticancer immune activation is the recruitment and activation of host APCs to cross-present tumour antigens to effector CD8+ cytotoxic T cells (Figure1). Cytokines such as GM-CSF have been used as vaccine adjuvants with the hope of attracting and activating DCs locally at the site of vaccination. Other strategies are aimed at systemically expanding the dendritic cell pool in the hosts, which may be achieved by the administration of cytokines such as the combination of GM-CSF and IL-4 (Roth et al, 2000). In retrospective studies of tumour biopsies, a greater number of APCs infiltrating the cancer have been correlated with improvements in survival of patients (Lotze, 1997). This increase in the availability of intratumoral APCs may allow more efficient cross-presentation of tumour antigens.

6. Microbe-based vaccines A variety of microbiology vectors have been adapted to cancer immunotherapy. Tumour antigen DNA sequences can be inserted into attenuated pox viruses that are unable to replicate in mammalian hosts or tumour antigen gene segments have been introduced into bacteria such as Salmonella and Listeria, resulting in protective immunity in animal models (Huang et al, 1994). Other vectors include recombinant replication-incompetent viral vectors (adenovirus, retrovirus, lentivirus), which are modified viruses that have been specifically mutated to be incapable of self-replication into infectious progeny virions after infection of a single target cell, but that efficiently express the foreign gene inserted in the vector. This form of genetic immunization has also resulted in weak immunologic responses in humans (Rosenberg et al, 1998), enhancing the immune potency of viral vector. Immunization can be achieved by the coexpression of cytokines or costimulatory molecules in the viral vector because these viral vectors usually have a large capacity to carry and express multiple genes (Rosenberg et al, 1998). Several anaerobic bacteria vectors are testing in lab now. Advantages may include the ability to use the oral route for immunization and the strong inflammatory milieu created by bacterial products, leading to the attraction of APCs, and a preferential Th1 cytokine polarizing pattern stimulated by certain bacteria such as Listeria.

C. Ex vivo APC-based vaccines 1. DCs and exosomes The crucial role of DCs was discovered for the induction of primary T-cellâ&#x20AC;&#x201C;dependent immune responses. DCs are now considered to be the best adjuvant for antitumor immunity. Different antigen loading procedures have been used for dendritic cell antigen presentation. For well-characterized antigens, synthetic HLA-binding peptide epitopes or the complete DNA sequence in a viral vector can be used to load the dendritic cell vaccines. DCs pulsed with peptide epitopes and genetically-modified with recombinant viral or bacteria vectors are conceptually similar to the vaccination with peptides in immunologic adjuvants or the direct administration of recombinant viruses, respectively, in which the DCs should be perceived as powerful immunologic adjuvants for the tumour antigen. Also, DCs can be loaded with defined antigens to take advantage of antigen uptake surface receptors, such as FC receptors to take up immune complexes carrying a tumour antigen (Rafiq et al, 2002). The nanometer vesicles derived from late endosomes are released differentiated in vitro by DCs , which contain most of the appropriate molecules to adequately present MHC-antigen complexes to the immune system (Wolfers et al, 2001; Zitvogel et al, 1998). These exosomes can be isolated by filtration of dendritic cell culture media and then loaded with custom antigens. Their use alone as vaccines or as vehicles to transfer back preassembled MHC-peptide complexes to DCs is under clinical investigation

7. The prime-boost strategy The sequential administration of naked DNA and a viral vector has resulted in synergistic immune activation; it is a potent method of generating immune responses to tumour antigens in what is now known as the prime-boost strategy. The initial injection of a plasmid allows the activation of infrequent T cells without other immune cells competing for the antigen because the naked DNA has a limited inflammatory potential. After a rest period, these antigen-specific high-avidity lymphocytes are boosted by the re-exposure to the same antigen, now in a more inflammatory milieu generated by the highly immunogenic viral proteins from the recombinant viral vector. Preclinical murine and primate models have shown that this heterologous prime-boost regimen induces 10- to 100-fold higher frequencies of T cells than do naked DNA or recombinant viral vectors alone (Ramshaw et al, 2000). A modification of this strategy is the sequential administration of two different viral vectors carrying the same tumour antigen gene, which bypasses the limitation of the development of neutralizing antibodies to the viral backbone by boosting with a different vector without shared viral epitopes (Mincheff et al, 2000; Marshall et al, 2000). These strategies, which avoid the need of cell culture common to the majority of highly immunologically active vaccine strategies, are rapidly undergoing clinical testing for infectious disease and cancer.

2. Nonâ&#x20AC;&#x201C;T-cellâ&#x20AC;&#x201C;directed cancer vaccines Monoclonal antibodies to surface receptors, such as trastuzumab or rituximab, have complex mechanisms of action leading to effective tumour regressions. One such mechanism is the stimulation of antibody-dependent cellmediated cytotoxicity. This immune-based effect, together with the recognized ability of immune complexes to allow antigen cross-presentation in DCs (Clynes et al, 2000), may contribute to their antitumour effects by a coordinated humoral and cellular response. Several other cancer vaccines are in different phases of clinical testing. Most of

8. Augmentation of the number of APCs

Xu J et al: immunotherapy with anaerobic bacteria for immunogene therapy of solid tumours

removed but who have a high risk of relapse. In these categories of patients, disease stabilisation, frequency of relapse, time-span to relapse and length of survival are the most rational parameters for evaluating cancer immunization effectiveness. Even if optimal gene delivery is achieved, the success of gene therapy, like conventional therapy, may be impeded by tumour cell resistance and intratumoural cell heterogeneity. The use of combined treatment modalities provides a rational paradigm to improve upon the clinical efficacy of cancer gene therapy (Klencke et al, 2002). Within the modality of gene therapy itself, multiple therapies may be combined in an attempt to benefit from additive or synergistic efficacy. Multi-gene therapy approaches already under evaluation include the transduction of dual immunostimulatory molecules for immunotherapy, and anaerobic bacteria therapy (Figure 2). A major limitation in the use of gene therapy in solid tumours in vivo is the diffusion-limited tissue penetration into the target tissue. The ability of immunotherapy and anaerobic bacteria therapy has been observed in vitro and in vivo. The effects we observed in animals are contingent on both bacteriolysis and immunity. There are three reasons to believe that systemic injection of Clostridium. Novyi-NT (C. novyi-NT) into humans would lead to bacteriolysis of tumours. First, C. novyi-NT germinates within the tumours of all three species tested (rabbits, rats, and mice), whether the tumours are s.c., intramuscular, or intrahepatic. Second, C. novyi-NT can germinate within human tumour xenografts in the nude mouse host (although complete regressions and cures are not generally observed as there is minimal T cell-mediated immunity). And third, there are many case reports of C. novyi germination and gangrene developing in penetrating wounds or after illicit drug injection. These reports demonstrate that the parental strain of C. novyi, differing from C. novyi-NT only in that the latter is devoid of the lethal "-toxin gene, can proliferate within hypoxic regions in humans. C. novyi-NT infection of cancers in humans will induce tumour immunity is more difficult to predict (Dang et al, 2004). There are many studies indicating that human tumours are immunogenic, as assessed by the presence of specific antibodies or reactive T cells in untreated patients. Furthermore, it has been shown that stronger immune responses can be elicited through the administration of various vaccines in several clinical trials. But there are also many studies indicating that human tumour cells can protect themselves against potential immune responses through a variety of direct and indirect mechanisms.

these strategies rely on the activation of humoral (antibody) responses to a peptide or nonpeptide antigen. Resultant tumour cell damage and cross-presentation of antigen by host APCs may allow the transfer of the immunologic stimulus to cellular immune responses. Advances in the understanding of the mechanisms of action of cellular antitumour immune responses have allowed the development of new generations of cancer vaccines, in which the key step is the recognition of the need for professional APCs to cross-present the antigen to the host immune system. The most immunologically active vaccines usually require costly and laborious ex vivo cellular cultures, whereas the cell-free vaccines that can be directly administered from an easily stored and transported vial are usually less immunologically active but more suitable for widespread clinical testing. New advances in the formulation of cancer vaccines brought by a more precise knowledge of the requirements for the generation of cellular immune responses to tumour antigens, together with the current ability to closely monitor cellular immune responses, will likely provide powerful, nonindividualized, cell-free vaccines in the near future.

VI. Combined multi-modality therapy: immunization with anaerobic bacteria therapy for tumour Immunotherapy strategies for cancer gene therapy utilize gene transfer to facilitate a dormant host immune response directed against the tumour. Evasion of autologous host cellular immunity is a common feature of tumour cell neoantigens. Tumour cells are poor antigen presenting cells. â&#x20AC;&#x2DC;Cancer vaccineâ&#x20AC;&#x2122; strategies are based on optimization of the context in which tumour antigens or tissue-specific antigens are presented to the host immune system (Sobol et al, 1995). Utilizing gene therapy to optimize tumourantigen presentation is through the targeted expression of cytokines in tumour cells. Targeted paracrine expression eliminates the toxicities associated with systemic cytokine administration. The transduced cytokines result in a combination of improved tumour cell vaccine antigen presentation, and activation of APCs, both essential for effective priming of the cellular immune response. The vector-induced inflammatory/immune response functions as an adjuvant to the transduced antigen, resulting in local release of cytokines and influx of APCs to the vaccine site. The immunotherapy of cancer is now being assessed in the clinics. An immune response has a potentially long-term clinical impact on the course of the disease by stabilising the condition and thus prolonging survival rather than by performing massive tumour elimination, those with minor tumour burden or patients who have had their tumour surgically 47

Gene Therapy and Molecular Biology Vol 13, page 48

Figure 2: Anaerobic bacteria-mediated immunologic therapy for solid tumour Anaerobic bacteria therapy has been observed these effects in treatment of solid tumour: a) nonspecific immunologic therapy which the characterization of cytokines produced by immune system cells and their production by genetic recombinant techniques, such as IL-2 and IFN, the significant toxicity of high-dose systemic cytokine therapy is the major drawback; b) specific immunisation represent which allow the stimulation of an immune response while avoiding the high toxicity of systemic administration of recombinant anaerobic bacteria vectors and gene modification of tumour cells, which allows an initial direct cytotoxic effect on the cancer cell by antibody dependent cellular cytotoxicity, thereby releasing tumour antigens; c) the adoptive transfer of immune effector cells from the immune system, T cell, DCs pulsed with genetically-modified with recombinant anaerobic bacteria vectors are conceptually similar to the vaccination with peptides in immunologic adjuvant.

way for the design of gene-immune therapies (Ribas et al, 2000). To this end, three cellular sources can be envisaged for genetic modification: tumour cells, effector T cells and DCs. However, before ex vivo immuno-gene therapy can become a realistic treatment modality for cancer, several barriers have yet to be overcome. First, improved (bacteria) vectors should lead to higher gene delivery rates and transgene expression. Therefore, carefully designed clinical studies are necessary to assess gene transfer efficiency, safety and toxicity, and eventually to establish the clinical efficacy of the tumour immunization. With regard to gene-modified tumour cells, another major issue still unsolved at the clinical level is to determine what is the best cytokine the tumour cells to release in order to recruit the immune system. Second, it will be imperative to break down the immunological tolerance against the tumour through reversal of T cell ignorance, anergy or tumour-induced immunosuppression in order to achieve a therapeutic outcome. Use of DCs, whether gene-modified or not, in the context of danger signals could provide a means to initiate a cellular immune response against the tumour. An additional general feature to be considered when designing immuno-gene therapy of cancer is the complex redundancy of the immune system. Its effectiveness in protecting the body from harmful infections demands a sophisticated network to control the pathways of activation and termination of an immune response, as well as maintenance of life-long tolerance. This suggests that a combination of multiple strategies,

As similar observations, both with respect to the potential of tumours to elicit an immune response and their ability to evade such responses, have been recorded in animals, there is reason to hope that the immune therapeutic effects stimulated by C. novyiNT germination might be obtainable in carefully selected patients. In experimental setting, the strictly anaerobic Clostridia have demonstrated several advantages over others as clostridial spores specifically colonise and germinate into vegetative cells in the hypoxic regions of solid tumours, causing tumour lysis and destruction. Early trials in the 70's of non pathogenic strains in human had shown plausible safety (Carey et al, 1976).

VII. Conclusions Current innovative approaches for cancer therapy hold significant potentials for effective cancer management; bacteria therapies and immunotherapies will probably be the most promising, especially when genetic manipulation of bacteria to improve its potential have applied. Recent understanding of tumour microenvironment, detailed characterization of tumour antigens and the increased revealing of the immunological pathways involved in tumour immunity have paved the

Xu J et al: immunotherapy with anaerobic bacteria for immunogene therapy of solid tumours gene-based or not, acting at different levels may be advantageous to boost the immune system against the tumour. Moreover, it is believed that the breakdown of tolerance to tumours will require, in addition to the strategies discussed in this review, complementary strategies that specifically counteract the active tumourinduced immunosuppression.

VIII. Future directions The challenges facing the implementation of successful gene therapeutic strategies will be better understood as the early clinical trials for cancer gene therapy begin to return more results. Vector development with increased transgene size capacity, optimized immunogenic properties, and improved gene transfer efficiency and targeting will facilitate the next generation of gene therapy strategies (Kanai et al, 1998). The burgeoning field of genomics provides an exciting new resource for the design of tumour-specific gene therapy strategies. Harnessing these tumour gene products and others for use as immunization offers exciting prospects for a whole new class of cancer gene therapy strategies. As the diversity of molecular lesions underlying tumourigenesis is better characterized, new targets for corrective and cytoreductive approaches will emerge. Effective anticancer gene therapy may ultimately require individualized molecular profiles. Solid tumours meet their demands for nascent blood vessels and increased glycolysis, to combat hypoxia, by activating multiple genes involved in angiogenesis and glucose metabolism. Hypoxia inducible factor-1(HIF-1) is a constitutively expressed basic helix-loop-helix transcription factor, formed by the assembly of HIF-1alpha and HIF-1beta, which is stabilized in response to hypoxia, and rapidly degraded under normoxic conditions (Kanai et al, 1998). It activates the transcription of genes important for maintaining oxygen homeostasis but failed to stimulate systemic T-cell-mediated antitumour immunity, and synergized with B7-1-mediated immunotherapy. This approach holds promise to form the foundation for the transition between the traditional anticancer therapies and the molecular antineoplastic gene therapy of the future. Other approaches are to develop new gene therapy vectors whose expression is selectively activated by hypoxia (Rosenberg et al, 1998). As VEGF is upregulated by hypoxia, such regulatory mechanisms would enable us to achieve hypoxia-inducible expression of therapeutic genes. The unique pathophysiology of solid tumours presents a huge problem for the conventional therapies. Thus, the outcomes of current therapies are so far disappointing. Several new approaches aiming at developing effective treatments are on the horizon. These include a variety of bacteria-based therapy systems. Amongst all these, anaerobic bacteria vector-mediated cancer therapy is most promising and expected to generate new data and new protocols for cancer gene therapy.

Acknowledgements

This work is partly supported by a project grant from the NHMRC/Cancer Council Queensland (Grant ID No. 401681) and the Dr. Jian Zhou smart state fellowship from the State Government of Queensland to MQW.

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Gene Therapy and Molecular Biology Vol 13, page 53 Gene Ther Mol Biol Vol 13, 53-63, 2009

Non-viral and local gene medicine for improvement of cutaneous wound healing Review Article

Markus Rimann1, Heike Hall1* 1

Cells and BioMaterials, Department of Materials, ETH Zurich, Zurich, Switzerland

__________________________________________________________________________________ *Correspondence: Heike Hall, ETH Zurich, Department of Materials, HCI E415, Cells and BioMaterials, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich, Switzerland; Tel: +41 44 633 69 75; Fax: +41 44 632 10 73; email: heike.hall@mat.ethz.ch Key words: wound healing, local gene therapy, gene medicine, non-viral gene delivery systems, matrix-mediated gene delivery, PLL-gPEG nanoparticles Abbreviations: adeno-associated viruses, (AAV); early endosome antigen-1, (EEA-1); extracellular matrix, (ECM); hypoxia-inducible factor, (HIF); HIF-1! lacking the oxygen-sensitive degradation domain (HIF-1!!ODD); Low-level laser therapy, (LLLT); matrix metalloproteinases, (MMPs); negative pressure wound therapy, (NPWT); platelet-derived growth factor, (PDGF); poly(ethylene glycol), (PEG); poly(lactide-co-glycolide), (PLGA); polyethylenimine, (PEI); poly-L-lysine, (PLL); transferrin receptor, (TFR); US Food and Drug Administration, (FDA); vacuum-assisted closure, (VAC); vascular endothelial growth factor-A, (VEGF-A) Received: 23 March 2009; Revised: 01 April 2009 Accepted: 03 April 2009; electronically published: April 2009

Summary Deficient vascularisation is a major clinical incidence and affects wound healing especially in elderly people as well as in diabetes patients. Many studies and different technologies aim to locally increase blood perfusion and improve the endogenous wound healing capacity and thereby ameliorate the patientâ&#x20AC;&#x2122;s life quality. Gene therapy has gained a lot of attention for treatment of chronic diseases, cancer and genetic disorders. It is also considered as a valuable alternative for conventional protein therapy, since it overcomes inherent problems that are associated with administration of protein drugs in terms of bioavailability, systemic toxicity, in vivo clearance rate and manufacturing costs. For this reason safe and efficient delivery systems for therapeutic DNA are developed. Polycationic substances have been shown to form complexes with DNA and are widely used as an attractive alternative to viral vectors in gene therapy. One promising approach consists in the usage of grafted copolymers of poly-L-lysine (PLL) and poly(ethylene glycol) (PEG) that forms stable complexes with plasmid DNA, which are highly transfection-efficient and are suitable to deliver DNA from 3D-fibrin wound healing matrices. A gene of interest to be delivered should stimulate endogenous wound healing and may consist of a stabilized form of hypoxiainducible factor-1! (HIF-1!!ODD), a transcription factor that ultimately leads to the increase in vascular endothelial growth factor-A (VEGF-A) expression that in turn activates angiogenesis followed by wound healing. Local administration of a matrix-mediated DNA delivery system on cutaneous wounds will be a big step in the direction of specific gene medicine and might represent a powerful tool in clinical wound therapy.

al, 2004; Anscher and Vujaskovic, 2005), (for review: Branski et al, 2007; Eming et al, 2007; Jensen, 2007). Since these risk factors affect large proportions of the aging population, the need of an adequate approach to treat impaired wound healing e.g. by locally increasing the blood perfusion seems essential. Worldwide approximately twenty million people suffer from chronic wounds caused by diabetes (alone > 7 million diabetic ulcers), circulatory problems and many other conditions such as surgical site infections that generate huge demands on the health care systems (http://www.prlog.org /10076809-wound-types-and-advanced-wound-products-

I. Introduction For most people, wound healing is a natural process of repair, which follows injuries of the skin and other soft tissues. For diseased individuals, however, it becomes a complex medical problem requiring specialized treatment and care. Together with many local factors that impede the healing process such as trauma, edema and infections, many systemic factors also contribute to impair wound healing processes. Among them are age, chronic diseases, such as diabetes mellitus, vascular insufficiencies, immunosuppressant and radiation therapy (Gosain and DiPietro, 2004; Hausman and Rinker, 2004; Jeffecoate et 53

Rimann and Hall: Gene therapy in wound healing market-worldwide). In Europe only, diabetic patients exceed 30 million people and cause 5-10 % of the total health care costs (www.idf-europe.org). Therefore, therapeutic improvements of wound healing especially by increasing the patientsâ&#x20AC;&#x2122; endogenous wound healing potentials are highly appreciated by the patients themselves and by the entire society.

platelet-derived growth factor (PDGF). Therefore the chronic wound displays a destructive environment that is not favorable for wound healing.

III. Treatment of chronic wounds Treatment of wounds can be divided into physical and biological methods. The physical treatments include surgical debridement, vacuum-assisted closure (VAC) therapy and low level laser treatments. Surgical debridement involves the removal of necrotic tissue out of the wound bed. This may eventually lead to a reset of the disturbed sequence of wound healing processes (Falanga, 2004, 2005). Clinical success of these methods is assigned to a reduction of excess of wound fluid, edema and exudate. Furthermore the putative bacterial burden and phenotypically abnormal cells are removed. In vacuumassisted closure therapy also termed negative pressure wound therapy (NPWT), a controlled level of negative pressure of -80 to -125 mmHg is applied on the wounds leading to accelerated debridement and promotion of healing in many different types of wounds (Saxena et al, 2004; Lindstedt et al, 2006; Jones et al, 2005; Kanakaris et al, 2007; KĂśrber et al, 2008; Labanaris et al, 2008). The underlying mechanisms of NPWT suggest mechanical deformation of cells in and around the wound resulting in increased matrix synthesis, which ultimately leads to an improved wound healing (Saxena et al, 2004; Wilkes et al, 2007; Eneroth and van Houtum, 2008; Ennis et al, 2008; Jacobs et al, 2008). Low-level laser therapy (LLLT) has been introduced by Mester and colleagues in 1968 and uses a single, coherent, monochromatic wavelength of light. The power varies from 5 to 500 mW and the emission wavelength is between 600 to 1000 nm. It has been shown that LLLT led to increased production of procollagen by human skin fibroblasts, increased fibroblast and keratinocyte proliferation, increased angiogenesis, tension resistance of scars and improved epithelialization (Sobanko and Alster, 2008). Another way to treat wounds and improve their healing capacities is to use specialized and bioactive wound dressings either made of molecules from the ECM or of synthetic polymers. Many dressings are already commercially available and are composed of collagen, hyaluronic acid, amelogenins, chondroitin-6-sulphate and fibrin. The compositions, functionalities and their applications have been recently reviewed in (Agren and Werthen, 2007). Some of the dressings combine ECM molecules with exogenously applied cells, such as human fibroblasts, autologous human keratinocytes and allogenic human fibroblasts. These bioactive dressings have been recently reviewed in (Boateng et al, 2008). Today more and more dressings are composed of polymeric molecules either artificial or of natural origin. The idea is to simulate the native ECM of the wound site and adjacent tissue using water swollen, gas permeable and fibrillar polymer structures that allow gas and nutrient exchange. Often used polymers are poly(lactide-co-glycolide) (PLGA), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyurethane foams, hydrocolloid and alginate dressings (reviewed in (Boateng et al, 2008)). Other hydrogel dressings are made of native polymers such as hyaluronic acid, collagen,

II. Wound healing Wound healing is a highly dynamic process related to growth and tissue regeneration and involves complex interactions of extracellular matrix (ECM) molecules, soluble mediators, various resident cells and infiltrating cells to reachieve tissue integrity (Singer and Clark, 1999; Baum and Arpey, 2005; Gurtner et al, 2008). Wound healing comprises four overlapping phases: hemostasis and inflammation, migration, proliferation and maturation (Gurtner et al, 2008). Details about the overlapping phases of wound healing are available in excellent recent reviews (Baum and Arpey, 2005; Barrientos et al, 2008; Gurtner et al, 2008) and will not be repeated here. The focus of this review will be on elucidating impaired wound healing that results when the well-orchestrated sequence of events is disturbed or stopped and non-healing or chronic wounds develop.

A. Impaired wound healing Wounds can be categorized into two different types distinguished by their healing properties: i) The acute wound follows the well-orchestrated phases of inflammation, new tissue formation and remodeling leading to tissue repair and scar formation, whereas ii) chronic wounds fail to heal within the expected time frame, which arises from the disruption of the orderly sequence of events at one or more stages in the wound healing process. In order to ensure an effective wound repair, interfering factors such as diseases (e.g. diabetes mellitus), drug therapies (e.g. growth factor delivery) and patient circumstances (e.g. pressure sores because of neuropathy), wounds in immunocompromised people (after systemic chemotherapy and/or radiation therapy, chronic steroid use) must be all taken into considerations (Boateng et al, 2008). In addition, aged people often show slowed or impaired wound healing even without an underlying disease (Swift et al, 2001). On the molecular level chronic wounds display a deficiency of endogenous growth factors (Pierce et al, 1995; Jeffecoate et al, 2004; Whitney, 2005) or an excessive production of exudate and expression of high levels of tissue-degrading proteases creating a destructive non-healing-promoting wound environment (Fahey et al, 1991; Loots et al, 1998). Often prolonged inflammation, impaired neovascularization, decreased synthesis of collagen, increased levels of proteases and defective macrophage function are observed (Fahey et al, 1991; Loots et al, 1998; Branski et al, 2007; Bao et al, 2008). In the case of prolonged inflammation the upregulation of neutrophils leads to increased secretion of matrix metalloproteinases (MMPs) that are imbalanced because of the lack of their natural inhibitors. Furthermore the mitogenic activity of cells is suppressed because of missing growth factors that promote proliferation such as 54

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A. Gene delivery systems

chitosan and fibrin (reviewed in (Boateng et al, 2008)). Hydrogel dressings can be loaded with therapeuticallyactive substances in order to achieve a controlled and sustained release thereby avoiding multiple interventions by changing the wound dressing several times. Commonly used are bactericides such as silver ions, antibiotics, or antimicrobial peptides and different growth factors. In order to support physical wound therapies significant efforts have been made to develop protein growth factors as wound healing therapeutics. First clinical trials were performed with exogenous application of growth factors like platelet-derived growth factor (PDGF) and others (Robson et al, 2001; Steed, 2006; Viswanathan et al, 2006). So far only PDGF-BB has received approval by the US Food and Drug Administration (FDA) but solely for the treatment of diabetic foot ulcers (Margolis et al, 2004; Robson et al, 2001; Steed, 2006; Viswanathan et al, 2006). Unfortunately, these efforts have not produced clinically significant improvements. The overall lack of success with protein growth factors has been attributed in part to short persistence of the growth factor in the protease-rich environment of the wound bed as only 1-9 % of the applied growth factor dose reached a depth of 1-3 mm (Trengove et al, 1999; Yager and Nwomeh, 1999). This required repeated applications of growth factors that are very costly to produce. Further difficulties are associated with the wound healing process itself as it is very complex and involves many different growth factors acting in concert and need to succeed one after the other thus being very difficult to simulate by application of single therapeutics.

Viral gene delivery systems use recombinant viruses, such as retroviruses (including lentiviruses), adenoviruses and adeno-associated viruses (AAV) containing therapeutic DNA (Breckpot et al, 2007; Flotte, 2007; Stender et al, 2007). Due to their inherent cell infection ability these gene delivery systems are very efficient and transduce dividing and some of them also non-dividing cells. Thus most of the clinical trials used viral delivery systems for gene therapy (http://www.wiley.co.uk/genetherapy/clinical/). Recent reviews summarize applications of viral vectors for cutaneous wound healing in animal and human studies (Branski et al, 2007; Eming et al, 2007; Jensen, 2007). Unfortunately viruses have several drawbacks such as high immunogenicity, packaging size limitations and some of them show random integration into the host genome, which leads to non-controllable side effects (Wu and Burgess, 2004). Therefore, many non-viral gene delivery vehicles have been designed to overcome the inherent limitations of viral vectors. Non-viral gene delivery systems may consist of naked DNA transfer, lipid-mediated, peptidemediated and polymer-mediated condensation of therapeutic DNA that lead to an improved cellular uptake (Panyam and Labhasetwar, 2003; Wells, 2004; Trentin et al, 2005; Park et al, 2006; Jeon et al, 2006; Gao et al, 2007; Shigeta et al, 2007). The major limits of these nonviral vectors are their poor in vivo transfection efficiencies resulting in low protein production, as well as their transient gene expression profile, which in some cases such as in wound healing, is desirable. The benefits of non-viral gene delivery vehicles are their safety and their unlimited gene size transportation capacity (Tal, 2000). Currently three different strategies for non-viral applications of therapeutic DNA are pursued. The simplest way is the use of naked DNA, which is either injected directly into the target tissue (Liu et al, 2007), applied via electroporation or ultrasound (Kusumanto et al, 2007) or loaded onto nano-sized particles of heavy metal and brought into the cell by gene gun applications (Kuriyama et al, 2000). Alternatively, microseeding delivers DNA directly into target cells by solid microneedles (Eriksson et al, 1998). However, enzymatic degradation of the unprotected DNA and poor cell transfection efficiencies are the major drawbacks (Liu et al, 2007). Another approach is the use of lipoplexes that are lipid-based DNA vehicles, entering the cytoplasm by cell membrane fusion (Felgner et al, 1987; Lv et al, 2006). Many different modifications for specific cell targeting and intracellular routing have been developed (Kawakami et al, 2000; Vandenbroucke et al, 2007). However, the primary drawback of lipid-based DNA delivery systems is their rapid clearance from the blood stream and their short-term stability (Lai and van Zanten, 2002). Another group of DNA-complexing substances consists of polycationic molecules such as PLL, poly-Lornithine or polyethylenimine (PEI) that have been previously demonstrated to be used as gene delivery vehicles (Ramsay et al, 2000; Pichon et al, 2001; Davis, 2002; Zaitsev et al, 2004) for review see: (Park et al,

IV. Gene therapy/gene medicine to improve wound healing In recent years, gene therapy has been evaluated as an alternative approach in wound therapy (Chandler et al, 2000; Petrie et al, 2003; Eriksson and Vranckx, 2004; Keswani et al, 2004; Theopold et al, 2004; Yla-Herttuala et al, 2004; Glover et al, 2005). Two different strategies are distinguished concerning the introduction and time of foreign gene expression: gene therapy that refers to the permanent substitution of a defect or missing gene whereas gene medicine leads to transient transformation and short term expression of a gene product (Morgan and Anderson, 1993; Khavari et al, 2002). As genes encoding for a growth factor or a defective protein could be placed into the wound milieu (reviewed by: (Hirsch et al, 2007; Davidson, 2008)), sustained local production of the growth factor might yield improvements over purely proteinbased therapies. Advantages of gene-based as compared to protein-based therapies are longer life times of applied genes and therefore prolonged expression of the therapeutic protein, immunological tolerance and faster and easier production and storage of the components that might finally lead to a reduction in costs for the health care systems. Gene therapy and gene medicine use a number of different DNA delivery systems that can be divided into two major groups: namely viral and non-viral delivery systems.

Rimann and Hall: Gene therapy in wound healing 2006). Although polymers with high cationic charge density condense the DNA into structures amenable to cellular internalization via endocytosis, the high charge density is one factor that contributes to their cytotoxicity (Wagner et al, 1998; Lee et al, 2002). To reduce cytotoxicity different block-copolymers between PEG and PLL, PEG and PEI as well as PEG and poly-aspartic acid were used to form DNA-vehicles (Choi et al, 1998; Ramsay et al, 2000; Davis, 2002; Lee et al, 2002; Mishra et al, 2004; Zaitsev et al, 2004; Dhanikula and Hildgen, 2006; Park et al, 2006; Walsh et al, 2006). Moreover, peptide-based DNA-vectors or covalent complexes between PEG-peptides and PEG-glycopeptides were developed (Pichon et al, 2001; Kwok et al, 2003; Trentin et al, 2006; Chen et al, 2007). On the other hand, low cationic charge density can reduce or eliminate DNA condensation capability. The balance between cationic charge density and DNA condensation is complicated even further when endosomal escape moieties and nuclear membrane translocation sites are considered. However, inherent cytotoxicity of polycationic PLL-DNA condensates can be circumvented by forming polymerDNA nanoparticles using grafted copolymers of PLL and PEG to increase biocompatibility and stealth properties. PLL-g-PEG-DNA nanoparticles were demonstrated to be a promising tool for effective transport and delivery of therapeutic DNA as they show long-term stability, a hydrodynamic diameter of 80-90 nm and high transfection efficiency of ~ 40 % combined with low cytotoxicity (> 95 % of cell viability) in COS-7 cells (Rimann et al, 2008) (Figure 1).

Currently, however, the greatest hurdle to actual realization of in vivo gene therapy is the development of efficacious delivery systems. Gene expression only results when DNA is transported inside the nucleus of the target cell. On its way the DNA needs to cross several biological barriers beginning with the plasma membrane, followed by intracellular pathways, escaping endosomal degradation and finally entering the nucleus to be at the location where mRNA-transcription takes place in eukaryotic cells. PEGylation of PLL-g-PEG-DNA nanoparticles contributes to DNA-nanoparticle uptake as cellular uptake into COS-7 cells was found to be strongly dependent on PEG-grafting. PLL-g-PEG-DNA nanoparticles entered COS-7 cells by an energy-dependent mechanism in the first 2 h of transfection and later the nanoparticles accumulated in the perinuclear region preceding nuclear uptake (Figure 2a, b). Furthermore, PLL-g-PEG-DNA nanoparticles were found within the cytoplasm at least for 24 h and no colocalization with endosomal compartments, as indicated by fluorescence staining against early endosome antigen-1 (EEA-1) or by colocalization with markers for known endocytotic pathways such as GM1, transferrin receptor (TFR) and caveolin-1 was observed (Figure 2c; Luhmann et al, 2008). These experiments indicate that PLL-g-PEG-DNA nanoparticles translocate efficiently to the nucleus and eventually enter to express the gene of interest. However, the exact uptake mechanism and intracellular pathway(s) remain still unclear. In spite of that PLL-g-PEG-DNA nanoparticles are considered as fast and

Figure 1. (a) Schematic of a PLL-grafted with PEG side chains used to form DNAcontaining nanoparticles; (b) Negative staining transmission electron micrograph of PLL20-g5PEG5-DNA nanoparticles; (c) Left: Transfection efficiency of PLL20-g5-PEG5-DNA nanoparticles in COS-7 cells, middle: Cell viability of COS-7 cells that were transfected with PLL20-g5-PEG5-DNA nanoparticles and right: Hydrodynamic diameter of PLL20-g5-PEG5-DNA nanoparticles with time. Adapted from Rimann et al, 2008 with kind permission.

Gene Therapy and Molecular Biology Vol 13, page 57

Figure 2. (a) Relative transfection efficiency of PLL20-g5-PEG5-DNA nanoparticles in COS-7 cells. The uptake is temperaturedependent. (b) Colocalization of PLL20-g5-PEG5-FITC and CX-rhodamine-labeled pEGFP-N1 (DNA-CX-rh) in COS-7 cells. Blue: Hoechst-stained nuclei, green: PLL20-g5-PEG5-FITC and red: DNA-CX-rh, yellow: Colocalization of PLL20-g5-PEG5-FITC and DNACX-rh (c) Colocalization of PLL20-g5-PEG5-DNA nanoparticles with GM1, TFR, caveolin-1 or EEA-1, respectively. Nanoparticles were prepared and applied on COS-7 cells between 30 min and 24 h as indicated. Later cells were fixed and analyzed by confocal microscopy. Blue: Hoechst-stained nuclei, green: different endocytosis markers and red: DNA-CX-rh, Scale bars are 10 Âľm, Adapted from Luhmann et al, 2008 with kind permission.

efficient delivery vehicles of plasmid DNA combined with low cytotoxicity and might be used to deliver relevant therapeutic DNA to improve local wound healing.

matrices such as fibrin, chitosan, hyaluronan, gelatine or collagen were used in various applications to increase wound repair and angiogenesis by releasing growth factors and other bioactive molecules (Zisch et al, 2003; Ishihara et al, 2006b; Masayuki et al, 2006a; Pike et al, 2006) reviewed in: (Ruszczak and Friess, 2003; Wallace and Rosenblatt, 2003; Young et al, 2005; Ishihara et al, 2006a). 3D-Fibrin matrices are among the most often used native hydrogels to induce angiogenesis and/or as drug delivery systems. Although fibrin is derived from human blood, it is FDA-approved because of its very favourable wound healing-inductive capacities (Zilla, 1991; Zilla et al, 1994; Currie et al, 2001; Horch et al, 2001). In the healthy body, fibrinogen circulates as an inactive precursor in the blood stream and is recruited to the site of the injured vasculature where it leaks out into the surrounding tissue. Fibrin clots are formed by initial physical association followed by covalent cross-linking through the concerted activity of thrombin and factor XIIIa (Weisel et al, 1985; Ariens et al, 2002; Lorand and Graham, 2003; BlombĂ¤ck and Bark, 2004; Mosesson, 2005). The fibrin clot is a complex network, composed of fibrils with different diameter and strength and provides a natural wound healing matrix that is remodelled through cellular activities to form the tissue-specific mature ECM. Because of its favourable wound healing-inducing properties and its clinical availability fibrin has been used

B. Matrix-released gene delivery Hydrogel matrices are highly swollen threedimensional cross-linked structures. They are mechanically flexible and can simulate the natural ECM to a certain extent. These matrices provide a versatile platform for molecular interactions with target tissues since they are composed of native or synthetic monomers that can be covalently modified with biologically active signals such as adhesion sequences or growth factors (Zisch et al, 2003; Pike et al, 2006). Moreover, hydrogel matrices are usually composed of soluble precursor solutions that can be applied at the site of injury by minimal invasive methods. They are induced to polymerize in situ under very mild conditions. In addition to their structural similarity to the native ECM, hydrogel matrices can be used as depots for drugs that are released by hydrolytic degradation of the hydrogel or on specific cellular demand (Drury and Mooney, 2003; Zisch et al, 2003; Ehrbar et al, 2005) reviewed in (Lutholf and Hubbell, 2005). Hydrogel release systems have been explored for delivery of bFGF from peptide amphiphiles to increase subcutaneous neovascularization (Hosseinkhani et al, 2006). Moreover, native hydrogel 57

Rimann and Hall: Gene therapy in wound healing as a drug delivery matrix. Different forms of VEGF alone or in combination with bFGF have been included into fibrin sealant products and were examined for their potential to induce neovascularization in vitro and in vivo (Wong et al, 2003). Growth factor release from fibrin hydrogels was controlled by using different fibrin concentrations, various cross-link densities, precipitation of growth factors by heparin or growth factor-containing heparin-conjugated poly(L-lactide-co-glycolide) nanospheres or other polymer microspheres (Keshet and Ben-Sasson, 1999; Royce et al, 2004; Jeon et al, 2005, 2006). Moreover, fibrin matrices were also used as adenoviral gene transfer and controlled delivery matrices (Breen et al, 2008a, b, 2009). Here, PLL-g-PEG-DNA nanoparticles were included into 3D-fibrin matrices and released over 7 days. The released PLL-g-PEG-DNA nanoparticles were collected and used for transfection of COS-7 cells (Figure 3). Transfection efficiency with released PLL-g-PEG-DNA nanoparticles was very similar to freshly prepared PLL-g-PEG-DNA nanoparticles suggesting that inclusion and release of these nanoparticles did not affect functionality.

PLGF, angiopoietins (ANGPT1, ANGPT2), and plateletderived growth factor B (PDGF-B; (Kelly et al, 2003; Pugh and Ratcliffe, 2003; Paul et al, 2004; Patel et al, 2005; Mace et al, 2007). Heterodimers of HIF-1! and HIF-1" subunits are constitutively expressed. HIF-1" is translocated into the nucleus, whereas HIF-1! possesses an oxygen-sensitive degradation domain (ODD), spanning from residues 401 to 603 (Huang et al, 1998). This domain is prolyl-hydroxylated in an oxygen-dependent manner (Bruick and McKnight, 2001) leading to binding of the von Hippel-Lindau protein, which then targets HIF-1! for ubiquitination and degradation in the proteosome (Huang et al, 1998). As such, under normoxia, HIF-1! is rapidly degraded in the cytoplasm and its nuclear localization is competitively inhibited, whereas under hypoxia, the factor is free to enter the nucleus and dimerizes with HIF-1" to induce gene expression leading to induction of proangiogenic proteins. Interference with the process of HIF-1! degradation under normoxia can induce effects related to hypoxia. HIF-1! expression is induced during wound healing (Albina et al, 2001; Elson et al, 2001) and is impaired in dermal fibroblasts and endothelial cells exposed to increased glucose concentrations (Catrina et al, 2004). HIF-1! expression was impaired during the healing of large cutaneous wounds in young db/db mice and HIF-1! gene therapy accelerated wound healing and angiogenesis in this model (Mace et al, 2007). Based on the important role of HIF-1! in expression of proangiogenic proteins, plasmid DNA encoding a stabilized variant HIF-1!"ODD (HIF-1! lacking the oxygen-sensitive degradation domain) was cloned and was shown to stimulate production of

C. Transcription factor HIF-1! to induce wound healing Until now most approaches used physical entrapment of bioactive molecules to be delivered from 3D-fibrin matrices whereas our laboratory included cellular activity for local and controlled release of DNA-nanoparticles directly into the wound site. Transcription factor hypoxiainducible factor (HIF) plays a central role in the induction of angiogenesis since it is primarily responsible for the detection of hypoxia and induces production of VEGF-A,

Figure 3. PLL-g-PEG-DNA nanoparticle release from 3D-fibrin wound healing matrices. (a) 2 mg/ml 3D-fibrin matrices were produced and visualized by confocal microscopy using Oregon-green-conjugated fibrinogen in a ratio of 1:50. The scale bar is 8 Âľm. In green, schematics of PLL-g-PEG-DNA nanoparticles included into such matrices (not to scale); (b) PLL20-g5-PEG5-DNA nanoparticle release over time as compared to release of naked plasmid DNA; (c) Transfection efficiency of PLL20-g5-PEG5-DNA nanoparticles released from fibrin matrices. Reproduced from Masters Thesis, Yanhong Wen, ETH Zurich, HS08.

Gene Therapy and Molecular Biology Vol 13, page 59 VEGF-A from HEK 293T cells in vitro (Trentin et al, 2006). Another study used a different variant of HIF-1! encoding a constitutively active form, designated HIF1!CA5, which induces HIF-1-regulated gene expression also under non-hypoxic conditions (Kelly et al, 2003; Patel et al, 2005; Mace et al, 2007). The study demonstrated that transfection with HIF-1!CA5 by electroporation into cutaneous wounds corrected the age-dependent reduction of HIF-1 expression, angiogenic cytokine expression, and the number of circulating angiogenic cells that contribute to the age-dependent impairment of wound healing in db/db mice (Liu et al, 2008). When HIF-1!!ODD was complexed by peptides that contained an N-terminal transglutaminase substrate sequence (TG-peptide) the entire TG-peptide-DNA condensate could be covalently incorporated into fibrin matrices through the activity of transglutaminase factor XIIIa. For covalently-immobilized TG-peptide-DNA condensates prolonged release profiles were observed as compared to released naked HIF1!!ODD plasmid DNA (Trentin et al, 2006). Moreover, when TG-peptide-DNA condensates were applied to full thickness dermal wounds on normal mice, 50 % more newly formed blood vessels as compared to native 3Dfibrin matrices, were observed and nearly 50 % of these vessels were surrounded by smooth muscle cells indicating a high degree of differentiation and maturation (Figure 4, Trentin et al, 2006). These experiments suggest that depot and release of angiogenesis-stimulating substances from modified 3D-fibrin matrices are indeed able to affect the number and the quality of newly formed blood vessels in vivo. As formation of new differentiated blood vessels are a prerequisite for successful wound healing, this approach might be a potential avenue to go towards improvement of wound healing.

transfection efficiencies combined with low cytotoxicity and long shelf live, which is a requirement for potential clinical use. The use of naked DNA is mainly hampered because of the need of special equipment to introduce it into the wound site, such as electroporation and ultrasound devices, gene guns and special needles for microseeding, furthermore naked DNA is not stable in the destructive wound environment and often degrades very fast. Most of the non-viral gene delivery systems also suffer from the short persistence in the wound environment due to fast clearance as long as they are not protected and/or embedded in a 3D-hydrogel matrix thus mimicking the native ECM. Therefore several approaches combine matrix-mediated delivery of naked DNA, DNA-containing condensates or of viral delivery systems. The idea is to obtain a sustained and controlled release of DNA over an extended period of time within the destructive environment of the chronic wound. Such matrix-mediated gene delivery systems might be a solution to overcome the very short live span of directly applied protein growth factors as well as the problem of dosage. When therapeutic DNA is released only by matrix degradation an initial burst-release can be avoided and might not lead to overshooting initial responses, which have been shown for burst-released therapeutics. Moreover, several rodent animal models were developed to test normal and impaired wound healing in vivo (reviewed by (Branski et al, 2007; Eming et al, 2007). The success seems encouraging and might lead to transfer into human clinical trials. However, more general considerations have to be made when turning to the bedside of human patients. Preclinical models often rely on young, healthy animals or artificially induced diseased animals, which might have a biological response that is fundamentally different from that in elderly human patients with advanced stages of arteriosclerosis, diabetes mellitus or other kinds of

V. Conclusions Many studies are ongoing in developing numerous non-viral gene delivery systems that gain more and more complexity. In vitro and in vivo studies show increased

Figure 4. Non-viral delivery of HIF-1#!ODD plasmid DNA increases formation and differentiation of newly formed blood vessels in cutaneous wounds in the back skin of normal mice. Wounds were placed and filled with 3D-fibrin matrices containing either TGpeptide-condensates with HIF-1#!ODD plasmid DNA or VEGF-A165 as a protein. Native fibrin was used as a control. The wounds were left for healing for 1 week prior to histological analysis. (a) Quantity of newly formed blood vessels by assessing the number of CD31positive vascular structures; (b) Differentiation of vascular structures was assumed when vascular structures were both: positive for CD31 and !-smooth muscle actin (!-sma). Adapted from Trentin et al, 2006 with kind permission.

Rimann and Hall: Gene therapy in wound healing underlying systemic diseases. In addition, the question remains whether in vitro models can be compared with in vivo experiments in rodent animal models and if the results obtained can then be transferred to human patients. When a comparison between different DNA delivery vehicles in vitro and in vivo was performed, Lipofectamine 2000 and DOTAP/Chol lipoplexes showed significantly enhanced gene transfer in vitro, whereas no transfection was detected for naked DNA. In contrast, naked DNA was found to be most efficient in gene transfer in experimental burn wounds in rats (Steinstraesser et al, 2007). Therefore, it has to be taken into consideration that in vitro test systems offer very limited predictability for subsequent in vivo gene therapy/gene medicine approaches especially when diseased human tissues are addressed. Moreover, transfer from small animal models where cardiovascular diseases such as diabetes mellitus or old age can only be simulated by genetic manipulation, medication or specific inbreeding of several strains, the etiology of impaired wound healing might look comparable to human wounds but the underlying mechanisms can not be compared so easily.

regulatory factors need to be activated at what time. Moreover, it requires joining forces from interdisciplinary researchers coming from medicine, life sciences, pharmaceutical sciences and engineering.

Acknowledgments This study was supported by Gebert R체f Foundation (GRS-053/05), Switzerland.

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VI. Future perspectives It is well understood that one single growth factor gene therapy/gene medicine cannot stimulate all interlinked phases of wound healing in an orchestrated manner as required. In order to address the complexity of succeeding active factors (growth factors, cytokines and enzymes) acting in normal wound healing processes the strategies must go towards the direction of multiple gene delivery or address key control genes that stimulate entire cascades of complex processes. It was demonstrated in a partial thickness wound healing model that the combination of PDGF and IGF-I was more effective than either growth factor alone (Lynch et al, 1987). Moreover a combination of PDGF and FGF-2 increased the DNA content of wounds in the rat more than any single growth factor alone (Sprugel et al, 1987) and transfection of KGF cDNA in combination with IGF-I cDNA compared to the same genes individually seems to be more efficient than the single genes (Jeschke and Klein, 2004). Alternatively, it was described that specific and local transfection of single key-transcription factor genes such as HIF-1! at strategic time points of wound healing might substitute a sequential growth factor therapy (Trentin et al, 2006; Liu et al, 2008). This single transcription factor might be able to switch on the entire cascade of angiogenesis followed by proper wound healing such that a self-regulating system is activated. Upon such stimulation ideally the patients' endogenous regulatory system would take over and a natural healing response would follow. Another very important aspect for future gene therapy and gene medicine will be to formulate safe and efficient delivery systems that can be controlled by endogenous regulation such as by cellular demand, in addition it might be therapeutically interesting to be able to support the release of therapeutic DNA by external stimuli e.g. by slight temperature or pH change or light of specific wavelengths. However, in order to define such key-genes a lot of basic research is still necessary to find out which 60

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Heike Hall and Markus Rimann