gene therapy & molecular biology volume 13 issue a

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-!

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

26-35 Review

Article

FAK as a target for cancer therapy

Steven N. Hochwald, Vita M.

Golubovskaya

36-52 Review

Article

Combination of immunotherapy with

anaerobic bacteria for immunogene

therapy of solid tumours

Jian Xu, Xiao Song Liu, Shu-Feng

Zhou, Ming Q Wei

53-63 Review

Article

Non-viral and local gene medicine for

improvement of cutaneous wound

healing

Markus Rimann, Heike Hall

GENE THERAPY & MOLECULAR BIOLOGY Addresses of Members of the Editorial Board

OPEN ACCESS www.gtmb.org

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

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

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

University of London, UK

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 Human-

Technology, Japan

Lebkowski, Jane S., Ph.D.,

GERON Corporation, USA

Kotoku Kurachi, Ph.D.,

University of Michigan

Medical School, USA

Li, Liangping Ph.D., Max-

Delbrück-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

Mayo Clinic, USA

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

Raizada, Mohan K., Ph.D.,

University of Florida, USA

Salmons, Brian, Ph.D., (FSG-

Biotechnologie 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.

Robert Harrod, Ph.D.

Associate Professor

Department of Biological

Sciences

Southern Methodist

University

Dallas

Prof. Emo Chiellini

Department of

Chemistry & Industrial

Chemistry

University of Pisa

Pisa (Italy)

Chittaranjan Patra

Assistant Professor,

Department of Biochemistry

and Molecular Biology,

Mayo Clinic Cancer Center,

Rochester, MN, USA.

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

Gene Therapy and Molecular Biology Vol 13, page 1

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: [email protected]

Sotiris Missailidis, Department of Chemistry and Analytical Sciences, Faculty of Science, The Open University, Walton Hall, Milton

Keynes, United Kingdom, MK7 6AA; e-mail: [email protected]

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.

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

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.

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

2

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 3-

D 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 2’-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 5’ or 3’

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


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 weak-

binding 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 reverse-

transcription 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), cross-

linking 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.


4

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).

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 single-

site 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

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,


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 “label-free” 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 double-

layer 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 redox-

active reporting units bound to DNA aptamer phosphate backbone.


6

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.

A. Electrochemical aptasensors for the model

protein The thrombin-binding aptamer (15-mer, 5’-

GGTTGGTGTGGTTGG-3’) 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 thrombin’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 3-

D 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

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 aptamer-

based 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


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 DNA’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 nanoparicle-

immobilized aptamer, thrombin and gold nanoparticle-

labelled 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

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 surface-

bound 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

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).

C. Aptasensor arrays Some of the aptamer-based biosensor technology

described in this review could be transferred from single-

analyte 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.


8

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

10


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 1Department of Computer Science and Information Technology, Dr. Babasaheb Ambedkar Marathwada University,

Aurangabad, 431004, (MS), India 2Department 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: [email protected]

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.

I. Introduction Ascaridia galli parasitic nematodes produce at least

two structurally novel classes of small helix-rich retinol-

and 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

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).

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


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 log-

transformed 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 C-

terminal end is likely to be the result of proteosomal cleavage.

III. Results and Interpretation 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

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 5-

8). Predicted antigenic fragments can bind to MHC

molecule is the first bottlenecks in vaccine design.

IV. Conclusion 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 MHC-

peptide binding. MHCII molecules bind peptides in

similar yet different modes and alignments of MHCII-

ligands 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 POS. N Sequence C MW (Da)

8mer_H2_Db 254 NLR SEENAISL VNG 843.9

8mer_H2_Db 277 QSS SYASWDTL IAS 900.98

8mer_H2_Db 64 LLE KSPEKMDI MML 929.1

8mer_H2_Db 117 KAL SKGSHPTK EEM 822.91

8mer_H2_Db 338 LSE DEHSKHDI DAA 961.99

9mer_H2_Db 253 ENL RSEENAISL VNG 1000.09

9mer_H2_Db 53 RDP MLYDNVTKL LEK 1078.28

9mer_H2_Db 447 HKT VTFPNALHL IQR 993.17

9mer_H2_Db 157 HSY LKDENIHAL QEV 1034.18

9mer_H2_Db 276 KQS SSYASWDTL IAS 988.06

9mer_H2_Db 408 LKE VKAKNEKLY YIL 1074.28

9mer_H2_Db 260 NAI SLVNGFTEV CKA 947.05

9mer_H2_Db 420 YIL FLINDHVAM LRR 1041.23

9mer_H2_Db 37 IAK KKARSFAHV LSK 1025.22

9mer_H2_Db 427 DHV AMLRRYNEL SDP 1147.37

10mer_H2_Db 125 PTK EEMTNLAKEL SAK 1159.32

10mer_H2_Db 370 KII SSMSFYSECI ITP 1135.29

10mer_H2_Db 457 HLI QRYANTTEEY HHQ 1256.3

10mer_H2_Db 276 KQS SSYASWDTLI ASL 1101.22

10mer_H2_Db 148 ELI NALFAGHSYL KDE 1074.21

10mer_H2_Db 253 ENL RSEENAISLV NGF 1099.22

10mer_H2_Db 259 ENA ISLVNGFTEV CKA 1060.21

11mer_H2_Db 369 KKI ISSMSFYSECI ITP 1248.45

11mer_H2_Db 419 YYI LFLINDHVAML RRY 1267.55

11mer_H2_Db 445 FFH KTVTFPNALHL IQR 1222.44

11mer_H2_Db 291 LEK APRSHARAVIL RDI 1172.41

11mer_H2_Db 325 KAM SAILGLLKVML SED 1139.5

11mer_H2_Db 298 HAR AVILRDIHRCL VKK 1290.6


12

Table 2. SVM based prediction of promiscuous MHC class II binding peptides from lipid binding protein.

MHC

ALLELE

Rank Sequence Residue

No.

Peptide

Score

I-Ab 1 PAHPVHLKR 91 1.622

I-Ab 2 PMLYDNVTK 52 1.403

I-Ab 3 HALAPDVKK 482 1.360

I-Ab 4 PAEAFFHKT 438 1.325

I-Ad 1 LINALFAGH 146 0.618

I-Ad 2 NAISLVNGF 257 0.586

I-Ad 3 HALQEVAAA 163 0.563

I-Ad 4 AISLVNGFT 258 0.553

I-Ag7 1 SDPAEAFFH 436 1.678

I-Ag7 2 DIDAAIEEV 344 1.613

I-Ag7 3 QEVAAAHVH 166 1.576

I-Ag7 4 HGKPAHPAH 85 1.451

RT1.B 1 TWARSLRTS 16 1.252

RT1.B 2 TFPNALHLI 448 1.189

RT1.B 3 KKAMSAILG 321 0.912

RT1.B 4 TEVCKALKQ 266 0.854

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.


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.


14

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

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

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Virendra Gomase and Somnath B Waghmare


15


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: [email protected]

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-of–the-

art gene carriers currently being used in 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, non-

specific 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,

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.

II. Strengths and weaknesses of

current vectors 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 cell’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

16

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.

Vector Immunogenic

Potential Specificity Limitation Major Advantage

Integrated

Retrovirus Low Dividing Cells only Integration may induce

oncogenesis

Persistent gene

transfer in dividing

cells

Lentivirus Low Broad Integration may induce

oncogenesis

Persistent gene

transfer in most

cells

Episomal

AAV Low Broad Small packaging

capacity

Non-inflammatory

and non-pathogenic

Herpes

Simplex Virus High High in neurons

Transient expression in

some non-neuronal cells

Large packaging

capacity

Adenovirus High Broad (CAR

receptor)

Capsid may induce

inflammatory response

Efficient

transduction of

most cells


17

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 Gelsinger’s immune system to the vector

(Bostanci, 2002). If a lowered dose of the adenovirus was

administered, Gelsinger’s symptoms may not have been as

catastrophic. Therefore, drawing a firm conclusion that

viral vectors are highly immunogenic and deadly is

premature.

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 non-

immunogenic 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.

D. Are viral vectors more efficient than

non-viral vectors? 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 over-

expressed 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.

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 non-

targeted 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

18

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.

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,

Leong’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.

Acknowledgement This work was supported in part by the NIH

biotechnology training fellowship (GM008336) to Canine

and Reeves

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Arash Hatefi

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

20


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,* 1Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical

Center, Ann Arbor, MI 48109, USA 2Center for Plant Molecular Biology, Osmania University, Hyderabad 500 007, India 3Center for Integrative Metabolic and Endocrine Research, Wayne State University School of Medicine, Detroit, MI

48201, USA 4Department 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: [email protected]

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 DNA-

binding 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.

I. Introduction The polyphenol curcumin (diferuloylmethane; 1,7-

bis(4-hydroxy-3-methoxy-phenyl)1,6-heptadiene-3,5-

dione) 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 wound-

healing 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

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


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.

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 (Sigma-

Aldrich, 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.

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% penicillin-

streptomycin. Cells were then transiently co-transfected with

pRL-SV40 and a PPAR-dependent luciferase reporter, pFATP-

luc. In separate experiments, cells were co-transfected with pRL-

SV40 plus a luciferase gene under the control of four Gal4 DNA-

binding 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.

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.

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.

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).


22

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).

H. Statistical analysis Data are represented as mean ± 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 Dunnett’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 ~7-

fold.

To increase the robustness of the reporter assay, CV-

1 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-! ligand-

binding 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 ligand-

binding domain of PPAR-! or stimulate

binding of PPAR-! to DNA 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).

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 CV-

1 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.

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.


23

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.

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

receptor’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 curcumin’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).


24

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 SDS-

PAGE and Western blotting. Membranes were probed first with anti–#-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 up-

regulation 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 15d-

PGJ2 (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.).


25

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Hochwald and Golubovskaya: FAK and cancer therapy

26


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:

[email protected]

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.

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

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 up-

regulation in cancer cell lines (Golubovskaya et al, 2004).

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


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 proline-

rich 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’ non-

coding 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 proline-

rich 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 424-

676) 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


28

(Hanks et al, 2003; McLean et al, 2005). Thus, the FAK-

Src 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 3-

kinase), 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 auto-

inhibitory 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.


29

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.

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). Src-

dependent 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-Grb2-

MAPK 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,

now there are total of 30 sites of phosphorylation of FAK,

including those reported before, requiring detailed analysis

of their biological functioning in vivo.

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 Erb-

2/Erb3-induced oncogenic transformation and invasion

(Benlimame et al, 2005). Inhibition of FAK in FAK-

proficient 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 FAK-

CD 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


30

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 dominant-

negative 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 C-

terminal 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.


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 Grb-

7, 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.


32

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.

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, 1,2,4,5-

benzenetetraamine 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 N-

terminal 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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36


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*

1Division of Molecular and Gene Therapies, Griffith Institute for Health and Medical research, School of Medical Science, Griffith University, Gold Coast campus, Southport, Queensland 4215 2Diamantina Institute for Cancer, Immunology and Metabolic Medicine, University of Queensland, Princess Alexandra Hospital, Wollongabba, Queensland 410 3School 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: [email protected]

Dr Xiao Song Liu, Diamantina Institute for Cancer, Immunology and Metabolic Medicine, University of Queensland, Princess

Alexandra, Hospital, Wollongabba, Qld 4102, Australia, Email: [email protected]

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 “Trojan horse” 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.

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

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

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

37

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.

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

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 P-

glycoprotein 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).

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


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), IL-

10 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 PD-

L1, 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.

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

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.

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


39

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.

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 (5-

FU) 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 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

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 beta-

galactosidase. 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 (GM-

CSF) 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 tumour-

specific 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 anti-

tumour immune response.

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 Langerhans’ 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,


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

nature’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.

B. Cancer gene therapy existing problems

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 non-

viral 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-

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 drug-

metabolizing enzymes.

1. Anaerobic bacteria as tumour target

vector

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 Gram-

positive 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


41

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.

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, G-

CSF, 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

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 plasmid-

based gene modification strategy harbours several safety

concerns regarding possible horizontal plasmid transfer

and spread of plasmid-associated antibiotic resistant

genes.

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 Toll-

like 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,


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).

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 T-

and 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 antigen-

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 anti-

tumour 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 multi-

potent tissue-specific adult stem cells, the most cancers

may arise from the malignant transformation of multi-

potent 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


43

(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.

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 cell-

targeting 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 clinical-

grade 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

(‘genetic immunopotentiation’). 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

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.

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

Burnet’s immunological surveillance hypothesis.


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.

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 tumour-

bearing 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, antigen-

specific 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

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 T-

lymphocyte 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 lymphokine-

activated 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.

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


45

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).

2. Intratumoral bacillus Calmette-Guérin

(BCG)

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 so-

called danger environment. This form of treatment

generates occasional antitumor immune responses.

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 HLA-

B7–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

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).

5. Naked DNA and gene-modified tumour

vaccines

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 cross-

presented 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 GM-

CSF 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).

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.


46

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.

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.

8. Augmentation of the number of APCs

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.

C. Ex vivo APC-based vaccines

1. DCs and exosomes

The crucial role of DCs was discovered for the

induction of primary T-cell–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

2. Non–T-cell–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 cell-

mediated 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


47

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. ‘Cancer

vaccine’ 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

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.


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.

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. novyi-

NT 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

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,


49

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 tumour-

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

Markus Rimann1, Heike Hall1* 1Cells 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: [email protected]

Key words: wound healing, local gene therapy, gene medicine, non-viral gene delivery systems, matrix-mediated gene delivery, PLL-g-

PEG 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 patient’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 hypoxia-

inducible 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.

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

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-

Rimann and Hall: Gene therapy in wound healing

54

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 patients’ endogenous wound healing

potentials are highly appreciated by the patients

themselves and by the entire society.

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

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 vacuum-

assisted 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; Körber 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,


55

chitosan and fibrin (reviewed in (Boateng et al, 2008)).

Hydrogel dressings can be loaded with therapeutically-

active 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.

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 protein-

based 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.

A. Gene delivery systems 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, peptide-

mediated 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 non-

viral 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-L-

ornithine 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,


56

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 polymer-

DNA 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 DNA-

containing nanoparticles; (b)

Negative staining transmission

electron micrograph of PLL20-g5-

PEG5-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.


57

Figure 2. (a) Relative transfection efficiency of PLL20-g5-PEG5-DNA nanoparticles in COS-7 cells. The uptake is temperature-

dependent. (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 DNA-

CX-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 µm, 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.

B. Matrix-released gene delivery Hydrogel matrices are highly swollen three-

dimensional 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

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; Blombäck 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


58

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.

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 hypoxia-

inducible 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,

PLGF, angiopoietins (ANGPT1, ANGPT2), and platelet-

derived 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

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 µm. 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.


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 HIF-

1!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 HIF-

1!!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 3D-

fibrin 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.

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

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

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 TG-

peptide-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 CD31-

positive 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.


60

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.

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

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 Rüf Foundation

(GRS-053/05), Switzerland.

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Heike Hall and Markus Rimann

gene therapy & molecular biology volume 13 issue a

Documents

fields of gene therapy

scope gene therapy

scope of gene therapy

gene therapy of cancer

aspects of gene therapy

gene therapy approach

gene discovery

gene targeting