KUOPION YLIOPISTON JULKAISUJA G. - A.I.VIRTANEN -INSTITUUTTI 46 KUOPIO UNIVERSITY PUBLICATIONS G.
A.I.VIRTANEN INSTITUTE FOR MOLECULAR SCIENCES 46
JANI RÄTY
Baculovirus Surface Modifications for Enhanced Gene Delivery and
Biodistribution Imaging
Doctoral dissertation To be presented by permission of the Faculty of Natural and Environmental Sciences of the University of Kuopio for public examination in Auditorium, Kuopio University Hospital,
on Saturday 25th November 2006, at 12 noon
Department of Biotechnology and Molecular medicine A.I. Virtanen Institute for Molecular Sciences University of Kuopio
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FI-70211 KUOPIO FINLAND
Tel. +358 17 163 430 Fax +358 17 163 410
http://www.uku.fi/kirjasto/julkaisutoiminta/julkmyyn.html Series Editors: Research Director Michael Courtney, Ph.D.
Department of Neurobiology A.I. Virtanen Institute
Research Director Olli Gröhn, Ph.D.
Department of Neurobiology A.I. Virtanen Institute
Author’s address: Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences
University of Kuopio P.O. Box 1627 FI-70211 KUOPIO FINLAND
Tel. +358 17 163 750 Fax + 358 17 163 751 E-mail: Jani.Raty@uku.fi
Supervisors: Professor Seppo Ylä-Herttuala, M.D., Ph.D.
Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences
Professor Kari Airenne, Ph.D.
Department of Biotechnology and Molecular Medicine A.I. Virtanen Institute for Molecular Sciences
Reviewers: Mikko Savontaus, M.D., Ph.D.
Turku Centre for Biotechnology University of Turku
Mikko Kettunen, Ph.D.
Department of Biochemistry University of Cambridge
Opponent: Professor Christian Oker-Blom, Ph.D.
Department of Biological and Environmental Science University of Jyväskylä
ISBN 951-27-0605-9 ISBN 951-27-0428-5 (PDF) ISSN 1458-7335
Kopijyvä Kuopio 2006 FINLAND
Räty, Jani. Baculovirus surface modifications for enhanced gene delivery and biodistribution imaging. Kuopio University Publications G. - A.I.Virtanen Institute for Molecular Sciences 46.
2006. 86 p.
ISBN 951-27-0605-9 ISBN 951-27-0428-5 (PDF) ISSN 1458-7335
Abstract
As gene therapy continues to evolve to clinical use, more interested is directed towards developing new and alternative gene delivery tools to compensate for the deficiencies of major vectors. During the last decade the research interest has turned to baculoviruses, which are ubiquitous viruses with high species specificity to intervertebrates.
In this work baculoviruses were modified to extend their properties as gene delivery vectors. A versatile avidin-displaying baculovirus, Baavi, was created by inserting avidin into a baculovirus native envelope glycoprotein, gp64. Together with the strong interaction of avidin and biotin, various ligands could then be used to further modify the properties of baculovirus.
We were able to show that possibly due to the positive charge of the avidin, Baavi had increased transduction efficiency as compared to unmodified baculovirus in various cell lines. Cell biotinylation increased the transduction efficiency a further 100-150 %. As the use of avidin display enables coating of the viral surface, we then utilized magnetic microspheres to physically target cells in vitro and showed increased binding with epidermal growth factor- ligand.
The non-invasive imaging of gene delivery vector biodistribution and kinetics in vivo after administration are essential for clinical gene therapy. Traditional biodistribution imaging, based on the transduction pattern, does not always result in accurate presentation of the viral biodistribution as baculovirus may be unable to express the transgene in all penetrated cells.
Magnetic resonance imaging has previously been utilized to image viral transgene expression, but by using 50 nm iron particles it was possible for the first time to image viral particle accumulation to rat choroid plexus cells with different time points. We were able to image the iron related signal loss up to two weeks and confirm the viral transgene expression to the same cells. We concluded that the payload could be transported to choroid plexus cells, together with the baculoviral gene delivery. By using biotinylated chelates with a technetium-label it was possible to use a microSPECT/CT device to image viral kinetics and biodistribution in vivo. Viral accumulation in different organs was analyzed after different administration routes using planar and 3D imaging as a function of time. We showed that intraperitoneal administration of Baavi resulted in signal accumulation in the spleen and kidneys and transgene expression at later time points.
We also studied the truncated version of the G-protein from Vesicular Stomatitis virus, VSV-GED and were able to show that by including it to the envelope with gp64, a significant increase in the transduction efficiency resulted in nearly all the studied cell lines. The mechanism was studied by inhibiting endosome maturation with ammonium chloride and monensin. Possibly the mechanism of action was by aiding gp64-potentiated membrane fusion.
In conclusion, the novel baculoviruses developed in this thesis result in increased transduction efficiency in vertebrate cells and provide more versatile tools to further develop gene therapy vectors for clinical use.
National Library of Medicine Classification: QZ 52, QU 470, QU 475, QW 162
Medical Subject Headings: gene therapy; gene transfer techniques; genetic vectors; Baculoviridae;
viral envelope proteins; viral fusion proteins; avidin; transduction, genetic; cell line; biotinylation;
gene targeting; microspheres; magnetic resonance imaging; tomography, emission-computed, single-photon; choroid plexus; spleen; kidney; gene expression; rats
“There are two kinds of scientific progress: the methodical experimentation and categorization which gradually extends the boundaries of knowledge,
and the revolutionary leap of genius which redefines and transcends those boundaries.
Acknowledging our debt to the former, we yearn nonetheless for the latter.”
Academician Prokhor Zakharov Address to the Faculty
“Humanity needs practical men, who get the most out of their work, and, without forgetting the general good, safeguard their own interests.
But humanity also needs dreamers, for whom the disinterested development of an enterprise is so captivating that it becomes impossible for them to devote their care to their own material profit.
Without doubt, these dreamers do not deserve wealth, because they do not desire it.
Even so, a well-organized society should assure to such workers the efficient means of
accomplishing their task, in a life freed from material care and freely concentrated to research.”
Nobel laureate Marie Curie
Acknowledgements
This study was carried out at the Department of Biotechnology and Molecular Medicine, A.I.
Virtanen Institute, University of Kuopio 2001-2006 under the guidance of professors Seppo Ylä- Herttuala, MD, PhD. and Kari Airenne, PhD.
Professor Ylä-Herttuala is acknowledged for establishing excellent research group and providing me the opportunity to be introduced into the research world. I am also deeply grateful to my main supervisor, professor Airenne, for his endless optimism, vast professional insight and severe commitment to the baculovirus research. Without the Great Baculo, this thesis would never have been created.
I am thankful to the official reviewers of this thesis, Mikko Kettunen, PhD and Mikko Savontaus, MD, PhD for the time they spent reviewing this thesis and their valuable comments. Eileen Shaw, BSc was kind enough to revise the language of this thesis.
I am very thankful to my co-author and previous graduate student Minna Kaikkonen for the fruitful collaboration during this thesis. Additionally, she is also acknowledged for kindly providing Figure 6 for this thesis. Fellowship of the baculovirus, my roommates Anssi Mähönen and Olli Laitinen earn special thanks for providing infinite support, unique humour and hours of scientific discussion about various aspects of nonintervertebrate life. Mikko Turunen and Emilia Makkonen joined us later on, but also contributed to the relaxed atmosphere of former BV-HQ.
The good people of the SYH group, past and present, are acknowledged for the help they provided, drifting discussions, friendship and for creating the mind-boggling numbers of computer problems.
Especially Pauliina Lehtolainen, Johanna Tietäväinen, Jonna Koponen, Sanna-Kaisa Häkkinen, Mervi Riekkinen, Tommi Heikura, Johanna Markkanen, Elisa Vähäkangas, Antti Kivelä, Marja Hedman, Anniina Laurema, Juha Rutanen, Tuomas Rissanen and Tiina Tuomisto are likely to find themselves in at least one of the categories.
I wish also to acknowledge those people specially participating in my thesis studies and experiments; Sanna Turpeinen, Hanna Lesch, Thomas Wirth, Jere Pikkarainen and Haritha Samaranayake.
Without the excellent technical assistance from Tarja Taskinen, Erik Peltomaa, Mervi Nieminen, Aila Seppänen, Riina Kylätie, Anneli Miettinen, Anne Martikainen, Seija Sahrio, Tiina Koponen, Jaana Pelkonen, Mari Supinen, Joonas Malinen, Riikka Eisto and Maarit Pulkkinen this study would not have been possible. Marja Poikolainen and Helena Pernu, the dynamic duo, are acknowledged for being the cornerstones for the group. People at AIV and LYT, especially Raili Rytöluoto-Kärkkäinen, Pekka Alakuijala, Riitta Keinänen and Jouko Mäkäräinen are also acknowledged for the help they provided during the years.
The people at Ark Therapeutics have influenced me greatly during the years spent with this research. Anne Kainulainen, Ville Harjulampi, Eero Paananen, Saija Paukkunen, Johanna Konttinen, Kaisa Ikonen, Outi Närvänen, Minna Karvinen, Minna Nokelainen and Maiju Jääskeläinen have been excellent collegues with lots of fun moments. The rest of the RD department with Sari Kukkonen, Miia Roschier, Diana Schenkwein, Pyry Toivanen, Tytteli Turunen, Tiina Nieminen and Hanna-Riikka Kärkkäinen have created an inspiring and supporting environment, both in and out of office.
I wish to thank my friends and business partners, Jarkko Surakka and Päivi Turunen for the moments we have shared in work and private life. The long-lasting friendship with Sami, Arto, Janne and Aki has spawned from the carefree days of past. Similarly, my friends Satu, the Korjamo family and Tuomas shan’t be forgotten. The exchange of bruises with my friends Olli, Markku, Elina, Pirkko, Mikko, Jatta, Jarimatti and Jari from UKU TKD has kept me in shape and lively during these studies.
Thanks go out to my kin, mother Riitta and aunts Tiina and Hilkka for support.
Finally, my wife Piia shall be acknowledged for always being there when needed mostly.
Once again, thanks.
Kuopio, 2006
Abbreviations
aa amino acids
AAV Adeno associated virus
AcMNPV Autographa californica multicapsid nucleopolyhedrovirus
Ad Adenovirus
AIDS Aqcuired immunodeficiency syndrome ATP Adenosine triphosphate BAAVI Baculovirus displaying avidin BBB Blood-brain-barrier
BV Budded virus
CAR Coxsackie-Adenovirus receptor CCD Charge-coupled device
cDNA Complementary deoxyribonucleic acid
CMV Cytomegalovirus
CNS Central neural system
CP Chroroid plexus
CSF Cerebrospinal fluid
CT Computer tomography DAB 3,3’-diaminobenzidine DNA Deoxyribonucleic acid EGF Epidermal growth factor
FCS Fluorescence correlation spectroscopy FDG Fluorodeoxyglucose
FGCV 18F-labeled-9[(1,3-dihydroxy-2-propoxy)methyl] guanine FHPG 18F-labeled 9-[(3-fluoro-1-hydroxy-2propoxy)methyl]guanine FHBG 18F-labeled 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine
FIAU 131I-labeled2'-fluoro-2'-deoxy-1-ß-D-arabinofuranosyl-5-iodouracil FMAU 2-fluoro-5-methyl-1-beta-D-arabifuranocyluracil
GV Granulosis virus
GFP Green fluorescent protein
HIV-1 Human Immunodeficiency Virus, strain 1 HSV-tk Herpes simplex virus thymidine kinase ICV Intracerebroventricular IF Intrafemoral
IM Intramuscular
IP Intraperitoneal
IV Intravenous
kb kilobase
kbp kilobasepair
Kd Dissociation constant
lacZ a reporter gene coding for beta-galactosidase MLV Murine leukaemia virus
moi Multiplicity of infection mRNA Messenger ribonucleic acid MRI Magnetic resonance imaging
MRS Magnetic resonance spectroscopy NdFeB Neodymium-iron-borate magnetic alloy, Nd2Fe14B NMR Nuclear magnetic resonance
NPV Nucleopolyhedrovirus
OB Occlusion body
ODV Occlusion derived virus
PBS Phosphate buffered saline PCR Polymerase chain reaction
PEG Polyethyleneglycol pfu Plaque forming units p.i. Post infection
pI Isoelectric point
PET Positron emission tomography
pKa Negative logarithm of acid dissociation constant polh Polyhedrin promoter
Q-PCR Quantitative polymerase chain reaction RGD arginine-glysine-aspartate RNA Ribonucleic acid
RT-PCR Reverse transcriptase polymerase chain reaction ssRNA Single stranded ribonucleic acid
SCID Severe combined immunodeficiency SD Standard deviation SEM Standard error of mean
SF Spodoptera frugiperda
SFV Semliki Forest virus
SPECT Single photon emission computer tomography SPIO Small iron oxide paramagnetic particle
SV40 Simian vacuolating virus 40 TU Transduction units
USPIO Ultrasmall paramagnetic iron oxide particle VEGF Vascular endothelial growth factor
VSV Vesicular stomatitis virus VSV-G VSV G-protein VSV-GS VSV-G stem region
VSV-GED VSV- GS with transmembrane and cytoplasmic domains WPRE Woodchuck hepatitis virus post-transcriptional element X-Gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside ZZ-domain IgG binding domain from protein A
List of Original Publications
This Study is based on the following articles, which are referred to in the text body by the corresponding Roman numerals (I-IV):
I
Räty JK, Airenne KJ, Marttila AT, Marjomäki V, Hytönen VP, Lehtolainen P, Laitinen OH, Mähönen AJ, Kulomaa MS and Ylä-Herttuala S.Enhanced gene delivery by avidin-displaying baculovirus.
Molecular Therapy. 2004 Feb;9(2):282-91.
II
Räty JK, Liimatainen T, Wirth T, Airenne KJ, Ihalainen TO, Huhtala T, Hamerlynck E, Vihinen-Ranta M, Närvänen A, Ylä-Herttuala S* and Hakumäki JM*,Magnetic resonance imaging of viral particle biodistribution in vivo Gene Therapy. 2006 Oct;13(20):1440-6.
III
Räty JK, Liimatainen T, Huhtala T, Kaikkonen MU, Airenne KJ, Hakumäki JM, Närvänen A, Ylä-Herttuala S, SPECT/CT imaging of baculovirus biodistribution in rat(submitted manuscript)
IV
Kaikkonen MU*, Räty JK*, Airenne KJ, Wirth T, Heikura T and Ylä-Herttuala S.Truncated Vesicular Stomatitis virus G-protein improves baculovirus transduction efficiency in vitro and in vivo, Gene Therapy. 2006 Feb;13(4):304-12.
*Equal contribution
TABLE OF CONTENTS
1 INTRODUCTION... 15
2 REVIEW OF THE LITERATURE... 16
2.1 GENE THERAPY... 16
2.2 GENE TRANSFER VECTORS... 17
2.2.1 Baculoviruses... 18
2.2.1.1 General properties of baculovirus...18
2.2.1.2 Structure of the virion...18
2.2.1.3 Major envelope glycoprotein Gp64 ...19
2.2.1.4 Baculovirus life cycle ...20
2.2.1.5 Use as a gene therapy vector...22
2.2.2 Adenoviruses... 25
2.2.3 Adeno-associated viruses (AAVs) ... 25
2.2.4 Retro- and lentiviruses... 26
2.2.5 Other viruses... 27
2.2.6 Non-viral vectors ... 28
2.3 DEVELOPMENT OF TARGETED VECTORS... 29
2.3.1 Targeting gene therapy vectors ... 29
2.3.1.1 Physical targeting ...29
2.3.1.2 Viral surface modifications...30
2.3.1.3 Targeting at genetic level...31
2.3.2 Vesicular stomatitis virus G-protein ... 32
2.3.3 (Strept)avidin – biotin technology... 33
2.3.3.1 Avidin and streptavidin...34
2.3.3.2 Biotin...34
2.3.3.3 (Strept)avidin-biotin technology in gene therapy...35
2.4 IMAGING IN GENE THERAPY... 36
2.4.1 Transduction and biodistribution imaging... 37
2.4.1.1 MRI ...38
2.4.1.2 PET / SPECT...39
2.4.1.3 Optical imaging ...40
3 AIMS ... 41
4 MATERIALS AND METHODS... 42
5 RESULTS AND DISCUSSION... 45
5.1 ARTICLE I... 45
5.1.1 Gp64-avidin fusion protein was incorporated in the baculovirus surface... 45
5.1.2 Titering of baculovirus... 47
5.1.3 Baavi resulted in enhanced transduction in vitro ... 47
5.1.4 Targeting the Baavi ... 48
5.1.5 Magnetically targeted transduction in vitro ... 49
5.1.6 Additional in vivo data... 51
5.2 ARTICLE II... 53
5.2.1 Contrast agent selection ... 53
5.2.2 Atomic force microscopy... 54
5.2.3 In vitro transduction ... 54
5.2.4 Detection of viral particles by MRI... 55
5.2.5 Iron detection by Prussian blue staining ... 56
5.2.6 Viral transgene expression ... 56
5.2.7 Choroid plexus as targets for gene therapy ... 57
5.3 ARTICLE III ... 59
5.3.1 Intravenous administration ... 59
5.3.2 Intraperitoneal and intramuscular administration ... 60
5.3.3 Intracerebroventricular administration ... 60
5.3.4 Other administration routes and conclusions ... 61
5.4 ARTICLE IV ... 62
5.4.1 VSV-GED displaying virus ... 62
5.4.2 Enhanced transduction efficiency in vitro... 62
5.4.3 Mechanism for transduction enhancement ... 63
5.4.4 Cytotoxicity ... 64
5.4.5 Transduction in vivo ... 64
6 SUMMARY ... 66
REFERENCES ... 67 APPENDIX: ORIGINAL PUBLICATIONS I-IV
1 Introduction
Gene therapy provides methods to treat the genetic reasons underlying behind the diseases with efficient methods and effect a cure instead of merely relieving the symptoms. After the initial discoveries and clinical trials, extensive research and development has been taken place to chart the full clinical and commercial possibilities of gene therapy.
As there has been success with treating some diseases, there has also been acknowledgement of the boundaries in the knowledge concerning the complex mechanisms in a living body. Some of the drawbacks have raised concern to design novel gene delivery vehicles for safer and more efficient treatments for gene therapy.
During evolution viruses have developed elaborate mechanisms to penetrate cells and take over the cellular machinery, each virus developing its preferred cell type and transduction mechanisms. For gene delivery use, the viral vectors are extensively modified for increased safety and selective transduction of target cells. Novel vectors, such as baculoviruses, provide an alternative method to treat diseases and provide new information about the processes in the path to a therapeutic result.
In this work, baculovirus surface modifications have provided ways to introduce new properties for the baculovirus and create prototypes for next generation enveloped viral vectors. The use of the avidin-biotin system has enabled flexibility and compability resulting in new data on viral behaviour and the use of modified envelope glycoproteins from other viruses has increased the efficiency of the baculovirus.
2 Review of the literature
2.1 Gene Therapy
Gene therapy has been defined as a method to treat disease by replacing, manipulating, or supplementing genes (Boulikas, 1998). Therapeutic gene therapy has been defined as the transfer of nucleic acids to somatic cells of a patient to result in therapeutic effect (Ylä-Herttuala and Alitalo, 2003). As compared to traditional medicine, gene therapy offers unique possibilities to treat the genetic causes of diseases. Special hope has been set on treatments for the monogenic diseases.
The treatment of adenosine deaminase enzyme lacking patients with gene therapy showed positive effects (Blaese et al., 1995). This encouraged more research towards gene therapy and resulted in numerous clinical trials. Since the first clinical gene transfer was made in 1989 (Blaese et al., 1995) the number of clinical trials per year increased steadily, peaking at 116 trials during 1999, but decreased to 77 trials in 2005 (Figure 1). The death of a patient in a gene therapy trial in 1999 (Raper et al., 2003; Couzin and Kaiser, 2005) and induction of leukaemia in some patients in another trial (Hacein-Bey-Abina et al., 2003) have caused the regulatory authorities to tighten the rules for clinical research and directed research to increase the safety of the treatment.
Meanwhile, the use of gene therapy as a supportive method along with traditional treatments has gained promising results for example in the treatment of malignant glioma (Immonen et al., 2004). As gene therapy modifies a complex cellular system with a method consisting of many steps such as delivery, cellular entry, transcription and expression, the limits in the current knowledge are bound to hinder the transfer of this treatment to everyday clinical use.
Number of Gene T herapy T rial Approved Worldwide 1989-2005
67 51
68 116
94 106
91 94 87 79
82
37 38
8 14 1 2 0
20 40 60 80 100 120 140
1989 1990
1991 1992
1993 1994
1995 1996
1997 1998
1999 2000
2001 2002
2003 2004
2005 Year
Trials
Figure 1. Number of gene therapy trials worldwide 1989-2005 (Journal of Gene Medicine, http://www.wiley.co.uk/genmed/clinical).
2.2 Gene transfer vectors
A gene transfer vector is, by definition, a vehicle which delivers genetic material to cells. These vectors can be divided into two categories: viral and non-viral. As viruses have evolved to be efficient in gene delivery they surpass non-viral vectors in many aspects, especially in efficiency (Kootstra and Verma, 2003). Currently the most popular vectors are adeno- and retroviruses, together representing 49% of vectors used in clinical trials (Figure 2).
Ideally, a gene therapy vector would target a specific tissue with high transduction efficiency and sustain a stable, regulated gene expression without any side effects or immunogenic response. As none of the currently used vectors directly match the ideal vector profile, there is an ongoing search for new vectors and the development of vectors combining properties from different viruses and artificial virus-like-particles. Currently each vector system has its own characteristic benefits, drawbacks and preferred applications. The next chapters will briefly introduce some major viral vectors with a special focus on baculovirus.
Vectors Used in Gene Therapy Trials 1989-2005
Others; 2.4 Adeno-
associated virus; 3.4
Adenovirus;
26.0
Retrovirus; 24.0 Naked/plasmid
DNA; 17.0 Lipofection; 8.3
Poxvirus; 6.9 Vaccinia virus;
6.5
Herpes simplex virus; 3.4
RNA transfer;
1.3 Unknown; 3.1
Figure 2. Vectors used in clinical gene therapy trials 1989- 2005 (Journal of Gene Medicine, http://www.wiley.co.uk/genmed/clinical).
2.2.1 Baculoviruses
Baculoviruses are a virus family which probably originated 400 to 450 million years ago and are ubiquitous in the modern environment (Heimpel et al., 1973). Apart from ancient Chinese literature, the earliest evidence of baculoviruses in Western literature can be traced to the sixteenth century by Marco Vida of Cremona describing gory liquefaction of silk worms, (reviewed in Benz, 1986).
Starting from the 1930’s baculoviruses were used and studied widely as biopesticides in crop fields (Miller, 1997) and a specific baculovirus from Finland was successfully introduced to Canada to abolish spruce sawfly, Gilpinia hercyniae (Balch and Bird, 1944; Arif, 2005). Since the late 80’s and 90’s they have been utilized for production of complex eukaryotic proteins in insect cell cultures (Kost and Condreay, 1999) and later on for viral display (Oker-Blom et al., 2003).
In 1985 it was discovered that a baculovirus with suitable promoter was able to transduce mammalian cells (Carbonell et al., 1985), an observation not confirmed until 1995 (Hofmann et al., 1995). Even though baculoviruses were discovered to be degraded by the classical complement pathway of blood (Hofmann and Strauss, 1998), successful ex vivo experiments soon led to successful in vivo experiments in 2000 (Airenne et al., 2000). Since then, there have been several publications using baculoviruses with various targets in vitro and in vivo (Huser and Hofmann, 2003; Kost et al., 2005).
2.2.1.1 General properties of baculovirus
Baculoviridae are a family of rod-shaped viruses which can be divided into two genera:
granuloviruses (GV) and nucleopolyhedroviruses (NPV). While GVs contain only one nucleocapsid per envelope, NPVs contain either single (SNPV) or multiple (MNPV) nucleocapsids per envelope in the occlusion body. The enveloped capsids of GV’s are occluded in granulin matrix and NPVs in polyhedrin. Moreover, GV have only a single virion per granulin occlusion body whilst the polyhedra of NPV contain multiple embedded virions. Baculoviruses have a circular double-stranded genome ranging from 80-180 kbp with the potential to encode about 100-200 proteins (Possee and Rohrmann, 1997; Theilmann et al., 2005).
Baculoviruses have very species-specific tropism among the intervertebrates with over 500 host species (Herniou et al., 2004) and they are not known to replicate in mammalian cells (Carbonell and Miller, 1987). However there is a report of detected early viral gene expression in human and rat cells (Kenoutis et al., 2006).
2.2.1.2 Structure of the virion
The most studied baculovirus is Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). This virus was originally isolated from lepidoptheran Alfalfa looper and contains a 134 kbp genome with 154 open reading frames (Ayres et al., 1994). The major capsid protein VP39 together with some minor proteins forms a nucleocapsid (21 nm x 260 nm, (Fraser, 1986) which encloses the DNA with p6.9 protein. The virus appears in two distinctive forms depending on the stage of its lifecycle; a single budded virus (BV) and an occlusion particle containing multiple virions, called occlusion derived virus (ODV) (Figure 3).
BV obtains its envelope from the cell membrane and requires glycoprotein GP64 to be able to spread systemic infection. This protein is not found on ODV whereas several other proteins are only associated to ODV. Some differencies are also seen in the lipid composition of the viral envelope (Braunagel and Summers, 1994).
Figure 3. Baculovirus structural protein comparison between a) budded virus (BV), b) occlusion derived virus (ODV) and c) polyhedra embedded virions (OB) (based on Blissard, 1996)
2.2.1.3 Major envelope glycoprotein Gp64
Gp64 is a homotrimeric membrane glycoprotein which is polarly present on the rod-shaped virion.
It consists of 512 amino acids (aa) with five glycosylation sites at asparagine residues (Jarvis et al., 1998) and has a 20 aa long N-terminal signal sequence, oligomerization and fusion domains and a 7 aa hydrophobic transmembrane domain near the C-terminus (Oomens and Blissard, 1999; Blissard and Rohrmann, 1989). The gp64 is produced in both early and late phases of the infection cycle with a maximal rate of synthesis occurring in 24-26 h post infection (p.i.).
Trimerization with intermolecular cysteine-bonds seems to be a crucial step for protein transport, since only 25% of synthesized protein reaches the cell surface as monomeric gp64 is degraded within the cells (Oomens et al., 1995).
There is an estimation that a virion contains ~1000 gp64 peplomers (Dee and Shuler, 1996). Gp64 causes the pH-mediated envelope fusion to the endosome (van Loo et al., 2001). It is also essential for efficient budding of the virion (Oomens and Blissard, 1999), cell-to-cell transmission during the infection cycle (Keddie et al., 1989; Mangor et al., 2001; Monsma et al., 1996) as well as binding to the cell surface i.e. causing viral tropism (van Loo et al., 2001).
Although gp64 has a variety of essential functions, it has been reported that gp64-null baculoviruses can be substituted with other viral glycoproteins such as ld130 or G-protein of Vesicular stomatitis virus (VSV-G) to produce a functional virus (Mangor et al., 2001; Lung et al., 2002).
During the viral evolution the baculoviral envelope glycoproteins have undergone changes. Ld130, also known as baculovirus F-protein, from Lymantria dispar is suggested to be an ancestral envelope fusion protein which has been replaced by gp64 in AcMNPV, Bombyx mori and Orgyia pseudotsugata, while they still retain the original ld130 gene (Pearson et al., 2001; Galperin
and Koonin, 2000). Possibly gp64 provides an advantage in the infection cycle of the virus, therefore outperforming the ancestral glycoprotein.
2.2.1.4 Baculovirus life cycle
The baculovirus life-cycle involves two distinct viral forms of baculovirus. The occlusion derived virus (ODV) is derived from the occlusion matrix embedded virions and is responsible for the primary infection of the host (Figure 4). The budded virus (BV) is released from the infected cells during the secondary infection (Figure 5)(Williams and Faulkner, 1997).
Primary infection begins by the host feeding on plants contaminated with virions embedded in the polyhedra-matrix. This matrix is dissolved in the alkaline environment of the host midgut, releasing ODV to fuse with the columnar epithelial cell membrane of the host intestine (Keddie et al., 1989). Nucleocapsids are then transported to the nucleus, a step possibly mediated by actin filaments (Charlton and Volkman, 1993), via nuclear pores. Viral transcription and replication occur in the nucleus and new BV particles are budded out from the basolateral side to spread the infection systemically. During the budding BVs acquire loosely fitting host cell membrane with expressed and displayed viral glycoproteins (Monsma et al., 1996).
The baculovirus infection can be divided to three distinct phases, early (0-6 hours p.i.), late (6-24 h p.i.) and very late phase (18-24 to 72 h p.i.). While BV is produced in the late phase, the ODV form is produced in the very late phase acquiring the envelope from the host cell nucleus and embedded in the matrix of occlusion body protein. These occlusion bodies are released when cells lyse to further spread baculovirus infection to the next host. To adapt to survival in the wild, occlusion body (OB) particles are resistant to heat and light inactivation, whereas BV is more sensitive to the environment (Williams and Faulkner, 1997).
Figure 4. Baculovirus primary infection, from OB to BV in intervertebrate host midgut.
Figure 5. Baculovirus secondary infection, from BV to OBs in intervertebrate host cells.
2.2.1.5 Use as a gene therapy vector
Baculoviruses have several advantages as a gene therapy vector. Viruses have a long history with extensive studies on safety and viral structure (Black et al., 1997). They can easily be produced in high titers (up to 1012 pfu/ml), easily manipulated and quickly produced without animal serum (Luckow, 1993). Baculovirus transduction is not restricted to dividing cells only, but includes also G1/S arrested cells (van Loo et al., 2001). Most importantly, since viruses are derived from an insect host, they do not replicate in vertebrate cells, however there is a contradictory report on expression of baculoviral immediate-early genes (Kenoutis et al., 2006). Still, the safety of the occluded viruses has been studied with various methods including intravenous, oral, intracerebral and intramuscular administrations in experimental animals and with feeding tests on voluntary humans without any signs of toxicity (Ignoffo and Heimpel, 1965; Heimpel and Buchanan, 1967).
Even so, very limited information is available on the effects of high dose of budded viruses in vivo.
The rod-shape capsid enables high transgene capacity, without known limits (O'Reilly et al., 1994; Fipaldini et al., 1999; Cheshenko et al., 2001). However, the drawbacks include the production of the virus in insect cells which results in the display of foreign glycoproteins, thus increasing possible immunogenic responses and inactivation by the blood complement system by classical pathway (Hofmann and Strauss, 1998; Huser et al., 2001). This problem has been avoided by using complement-protecting factors (Huser et al., 2001; Hofmann et al., 1999; Pieroni et al., 2001), avoiding exposure to the complement (Sandig et al., 1996; Airenne et al., 2000) or using the virus in immunopriviledged areas such as the eye (Haeseleer et al., 2001) and the brain (Sarkis et al., 2000; Lehtolainen et al., 2002). Currently the baculovirus has produced the most promising results in vivo in the CNS gene delivery, for example inhibiting glioma cell growth in an animal model showing higher transduction rate in glioma as compared to surrounding brain tissue (Wang et al., 2006). Additionally, it has been reported baculoviruses may be able to utilize axonal transport to cell bodies (Li et al., 2004). While the later observation may be regarded as safety risk, together these studies may promote focusing the research to brain and CNS.
As compared to adenoviruses, the overall transduction efficiency of baculoviruses is somewhat lower and while baculoviruses are less cytotoxic as compared to adenoviruses (Airenne et al., 2000; Lehtolainen et al., 2002), the baculovirus envelope is still of insect origin, possibly creating immunoresponse with repeated administrations as suggested by cytokine eliciting interaction with Kupffer cells (Beck et al., 2000). Additionally, the large genome of baculovirus shows signs on instability, hindering the production of established clinical gene therapy vectors (Pijlman et al., 2004).
Some of these drawbacks related to baculovirus properties can be avoided by enhancing transduction efficiency by pseudotyping (Park et al., 2001) or by inserting enhancing elements to the viral transgene cassette or by using histone deacetylase inhibitors (Condreay et al., 1999; Sarkis et al., 2000).
It is not currently known if the baculoviruses have a specific receptor for their attachment and cell entry. However there are reports on specific requirements for their transduction (van Loo et al., 2001), involving heparin sulphate residues and electrostatic interactions.
Additionally, the interaction of gp64 and cell surface phospholipids has been shown to play a role in the viral entry (Tani et al., 2001; Ernst et al., 2006). Interestingly, disruption of the cell-cell junctions with chelator EGTA has resulted in increased transduction efficiency (Bilello et al., 2001) achieved also by transient calcium depletion (Bilello et al., 2003). As it has been shown that basolateral surface is important for the baculoviral transduction, the loosening of cell-cell junctions might enable the entry from the basolateral side.
While the baculovirus receptor remains unknown, a large number of susceptible cell types (Table 1) suggests the viral tropism to result from very universal interactions. However, when examining the difference between highly permissive and less permissive cell lines, it has been reported that the difference was in the presence or absence of baculovirus DNA in the nucleus
(Barsoum et al., 1997). This indicates that while the attachment might be universal, the later steps with nuclear entry and viral disassembly are likely to affect the baculovirus transduction and tropism (Kukkonen et al., 2003). Interestingly, baculoviruses are one of the few viruses which carry their capsid to the nucleus (van Loo et al., 2001), enabling the transport of therapeutic proteins to the nucleus by using capsid display (Kukkonen et al., 2003).
The transgene expression of baculovirus is transient, peaking levels are 2-5 days in vitro (Liang et al., 2004) and from 3-5 days to one week in vivo (Airenne et al., 2000; Lehtolainen et al., 2002), but without complement lasting to nearly 200 days (Pieroni et al., 2001). However these results are with universal promoters and the transgene expression length and strength or tropism can be modified by using different promoters (Park et al., 2001; Wang and Wang, 2006;
Wang et al., 2006).
Table 1. Different mammalian cell lines transduced with baculoviruses (Dai et al., 1995; Shoji et al., 1997;
Airenne et al., 2000; Kost and Condreay, 2002; Ghosh et al., 2002; Song et al., 2003; Airenne et al., 2004; Cheng et al., 2004).
Human cell lines Other mammalian cell lines
Huh7 (Hepatoblastoma cell) Monkey
HepG2 (Hepatoblastoma cell) COS-7 (Kidney cell) sk-Hep-1 (Hepatoblastoma cell) CV-1 (Kidney cell) FLC4 (Hepatocarcinoma cell) Vero (Kidney cell) primary hepatocytes
293 (Kidney) Porcine
HeLa (Cervical carcinoma cell) CPK (Kidney cell)
primary neural cells FS-L3 (Kidney cell)
IMR32 (Neuroblastoma cell) PK-15 (Kidney cell) CHP212 (Neuroblastoma cell)
SK-N-MC (Neuroblastoma cell) Rodent
A 549 (Lung) RAASMC (Rabbit aortic smooth muscle
WI-38 (Lung fibroblast) cell)
Ramos (B-cell) RGM1 (Rat gastric mucosal cell)
Jurkat (T-cell) PC12 (Rat adrenal cell)
MT-2 (T-cell) Primary rat hepatocytes
W12 (Keratinocyte) BT4C (Rat glioma cell)
primary foreskin fibroblasts BHK (Baby hamster kidney)
HL-60 (Promyelocyte) CHO (Hamster ovary cell)
K-562 (Myelocyte) Mouse pancreatic β-cells
KATO-III (Gastric carcinoma cell) MKC (Mouse kidney cell)
Pancreatic β-cells NIH3T3 (Mouse embryo fibroblast)
Bone marrow fibroblasts C2C12 (Mouse muscle)
Saos-2 (Osteosarcoma cell) N2a (Mouse neuroblastoma cell) 143TK- (Osteosarcoma cell) L929 (Mouse fibrosarcoma cell) MG63 (Osteoblast-like cell)
DLD-1 (Colon carcinoma cell) Bovine
SKOV3 (Ovarian carsinoma) BT (Trophoblast) MRC5 (Lung fibroblast)
ECV-304 (Vascular endothelial cell) Ovine
FLL-YFT (Lamb primary foetal lung cell)
2.2.2 Adeno
Human adenoviruses belong to the Adenoviridae family and cause mild infections in membranes of the respiratory inary tract. Viruses contain a linear double-stranded 36 kbp DNA g non-enveloped icosahedral capsid of 70-1 iameter. Today, over 50 serotypes, divided into six subgroups (A-F), have been identified (Douglas, 2004).
Gene therapy utilises repli ich are non-pathogenic and can
transiently transduce a variety of both dividing and non-dividing cell iency without integration to the genom romosomal position of the v sults in loss of the viral DNA during cell divisions and explains the expression peak tim
2004).
Adenoviruses are dominating gene therapy vectors with 301 finished or ongoing
clinical trials d ley.com/genmed/clinic dvantages include
production to nd a packaging capac kb with so-called
gutless vectors. However, as they belong to harmful viruses, pre- or acquired immune
response again ay cause severe immuno oblems (Chen et al.,
2000; Zoltick et al., 2001; Youil et al., 2002). Major adenoviral vector clinical trials belong
to the serotype 996). These serotype rmined to be
the most com exposed (Parks et al., 1999), possibly causing
problems for their repeated use for the same individual (Bessis et is issue can be addressed for erotype (Parks et al., 1999) ating chimeric vectors (Hedley et al.,
The efficiency of the vector is closely related to the cellular c ovirus receptor (CAR), which is the primary adenovirus receptor. As in the tumor cells, the amount of CAR is often low, therefore creating a need to modify the interaction of cell and adenovirus fibre knob (Noureddini and Curiel, 2005). However, adenovirus also binds, to some extent, to integrins αVβ3
and αVβ5 (Wickham et al., 1993).
An interesting approach has been the development of conditionally replicating adenoviruses, which result to destructive viral replication but limit it selectively to onabend et al., 2006).
2.2.3 Adeno-associated viruses (AAVs)
Adeno-associated viruses belong to the Parvoviridae family and are non-pathogenic to humans.
They contain a single-stranded 4.7 kb DNA genome within a small 20 n-enveloped capsid.
The number of known AAV serotypes increases constantly and is cu ever when including different hybrid capsids, the number is within hundreds (DiMattia et al., 2005). AAV transduces a wide variety of dividing and non-dividing cells in muscle r, brain and vasculature through heparin sulphate proteoglycans, integrin αVβ5 or fibroblast growth factor receptor with long-term expression (Grieger and Samulski, 2005).
Since AAV belong to the dependoviruses, it needs adeno-, herpes-, papilloma- or vaccinia virus to facilitate efficient and fully permissive infection and replication. Without a helper
irus or a wild-type virus AAV causes a latent infection and integrates into a unique site in human osome 19 (Kotin et al., 1990). The virus has a small capacity for foreign DNA (<5 kbp), but the packaging capacity has been expanded by utilizing trans-splicing and overlapping vectors resulting in intact protein in dual-transduced cells (Duan et al., 2001).
The most commonly used serotype in gene therapy is AAV-2, to which the human population has a high prevalence of antibodies (Zaiss and Muruve, 2005). To solve this problem, cross-packing AAV serotypes have been utilized (Choi et al., 2005). One of the major drawbacks in
viruses
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AAV is the purification and concentration of viruses. Together with a low transgene capacity these drawbacks severely limit its use (Blouin et al., 2004). However, with the transduction peak time of le expression for years, AAV offers long-term possibilities to treat
in a stable expression.
genes, the retroviral transgene capacity is ~8-10 kbp. The production of retrovir
f species. Viral pseudotyping is invariably used to broaden and modify
of limitations in crossing the nuclear membrane (Roe et al., 1993) MLV is capable of ansducing only dividing cells, whereas lentiviruses are also capable of transducing quiescent cells.
The integration of the viral genome is not totally random (Wu and Burgess, 2004), and it has been s are targets for retroviral integration (Schroder et 3-6 weeks and sustainab
metabolic diseases in a non-integrating manner (Wang and Herzog, 2005; Flotte, 2005; Auricchio and Rolling, 2005).
2.2.4 Retro- and lentiviruses
The family of Retroviridae comprises a group of enveloped 80-100 nm viruses containing linear single stranded-RNA (ssRNA). Retroviruses have a common property of reverse transcription of the viral RNA to linear double stranded DNA and subsequent integration of this DNA to host genome resulting
Retroviruses can be further divided into simple and complex retroviruses where orthoretrovirinae Murine Leukaemia Virus (MLV) represent the former and lentivirinae Human Immunodeficiency Virus strain 1 (HIV-1) the latter. Spumaretrovirinae include several non-human viruses such as Simian Foamy virus (Mergia and Heinkelein, 2003).
Among the lentiviruses, HIV-2, FIV (Feline), SIV (Simian), EIAV (Equine Infectious Anaemia Virus), BIV (bovine) based vector systems have been designed and used for gene delivery (Romano, 2005). Being the most studied among the lentiviruses, HIV has currently advanced first clinical trial for treating AIDS (Manilla et al., 2005).
A viral genome consists of genes for viral structural proteins: gag, pol, env and in the complex retroviruses additional regulatory and accessory genes like tat, rev, vif, nef, vpr and vpu.
After deletion of viral
uses requires packaging cells where removed viral proteins are expressed in trans (Coffin, 1996).
The tropism of retroviruses is highly dependable on envelope glycoprotein composition dividing viruses into ampho-, eco-, and polytrophic subsets transducing accordingly mammalian, rodent or a selection o
the retroviral tropism (Sandrin et al., 2003).
Murine retroviruses were the first viral vectors used for gene therapy (Selkirk, 2004).
Because tr
suggested that actively read genes in the genome
al., 2002) and in addition, several loci associated with leukaemia have been detected as integration sites (Calmels et al., 2005; Hematti et al., 2004; Wu et al., 2003). Possibly due to this, insertional mutagenesis in primitive multipotent progenitor cells has been detected as a result of lentiviral SCID-X1 gene therapy trial, resulting in leukaemia but also in restoration of normal enzyme levels (Schmidt et al., 2005). In gene therapy, retroviruses are widely used (Figure 2) and have a selection of applications which rely on their special properties (Barquinero et al., 2004).
2.2.5 Other viruses
As the limitations of dominant viral vectors have became obvious, interest has risen to develop new viral vectors or transfer their properties to current vectors. There has been some interest in research to naturally oncolytic viral vectors and to utilize the mechanisms causing the selective replication in tumor cells (Thorne et al., 2005).
e key features of some gene therapy vectors and Table 3 some in
Table 2 summarizes th
naturally oncolytic viruses. Hybrid vectors such as chimeric vaccinia/ retrovirus (Falkner and Holzer, 2004) and retrotransposon-adenovirus (Soifer and Kasahara, 2004) combine selected features from different viruses with a rational design, perhaps resulting in a more effective outcome (Tomanin and Scarpa, 2004).
Table 2. Characteristics of some unconventional viral gene transfer vectors
Viral vectors Advantages Limitations Reference
Alphaviruses/
Semliki Forest (SFV), Sindbis (SIN) and Venezuelan Equine Encephalitis (VEE)
High titer, rapid production, broad host range and extreme transgene expression levels
Low transgene capacity (<7 kb), highly cytotoxic,
(Liljestrom and Garoff, 1991;
Xiong et al., 1989;
short term expression Davis et al., 1989;
Yamanaka, 2004) Spumaretroviruses /
Foamy viruses (FV) Broad tropism, high efficiency, high capacity (>9 kbp), resistant to complement
Low titer, few (Mergia and studies made Heinkelein, 2003)
Hepadnaviridae/
Hepatitis B (HBV) High liver specificity, High
titers, transduces quiescent cells Very low transgene (Untergasser and capacity (<1,6 kb) Protzer, 2004) Polyomaviridae/
Simian virus 40 (SV40)
Stable integration to dividing and non-dividing cells, long term expression
Low transgene capacity (< 4,7)
(Strayer et al., 2005)
Poxviridae/ Vaccinia, Western reserve strain
Replicates in tumor cells, safety with cytosolic transcription and replication, high capacity ( <25 kb). Tumor vaccination.
Pathogenic, possible previous vaccination, highly immunogenic
(Guo and Bartlett, 2004)
Alphaherpesvirinae/
Herpes Simplex 1 (HSV-1)
Broad range, high titers, large capacity
Pathogen, latent wild (Martuza et al., type-virus activation, 1991; Post et al.,
antigenic 2004)
Bacteriophages Non-pathogenic, easy to produce, easy manipulation
Immunogenic, large (Larocca et al., particles, very low 2002)
gene delivery efficiency
Table 3. Some naturally oncolytic viruses (Wildner, 2003).
Oncolytic viruses Advantages Limitations Reference
Paramyxoviridae/
Measles virus (MV)
Oncolytic Pathogenic (Fielding, 2005)
Paramyxoviridae/
Newcastle disease virus (NDV)
Non-pathogenic in humans, moderate efficiency, oncolytic
Unclear mechanism, not (Lorence et al., well studied, non-
recombinant viruses used
2003) Paramyxoviridae/
Mumps virus Oncolytic Pathogenic (Russell, 2002)
Parvoviridae/
Rat virus H1 and Minute virus of mice (MVM)
Tumor tropism,
autonomous replication, low immunogenity
Low transgene capacity, (Cornelis et al., low titres, replication 2004)
competent viruses Reoviridae/
Respiratory enteric orphan virus (reovirus)
Mild pathogen, specific
oncolytic activity Previous antigens exist (Norman and Lee, 2005)
Picornaviridae/
poliovirus
Oncolytic Narrow tropism, (Gromeier et al., pathogenic, difficult 2000)
manipulation Rhabdoviridae/ Vesicular
Stomatitis virus (VSV)
Relatively non- pathogenic, oncolytic
Difficult manipulation, (Barber, 2004) VSV-G inactivation
2.2.6 Non-viral vectors
Non-viral vectors offer a bypass to some of the fundamental draw
vectors. As compared to viral vectors, non-viral approaches have a theoretically better safety profile, cheaper and scalable production, fewer restrictions to the size of the transgene and no risk of insertional mutagenesis. However, due to the poor uptake DNA to cells their transduction efficiency is low and expression is transient (Glover et al., 2005; Kootstra and Verma, 2003). The most i ligonucleotides, naked DNA, cationic lipids or polycationic carriers and different formulations of lipids and other components, such as nanoparticles (
Systemic delivery of naked DNA suffers from rapid clearance from the blood or degradation by restriction nucleases (Brown et al., 2001). Method ectroporation (Wells, 2004), hydrodynamic injections (Al-Dosari et al., 2005) and ultrasound (Hosseinkhani et al., 2003) have been used to increase the transfection efficiency. While the increasing tissue damage may prevent the use of electroporation and hydrodynamic pressure, ultrasound may provide an alternative me gene transfer efficiency in clinical use. Naked DNA has already proven to be efficient enough in genetic vaccination (Rodriguez, 20
To shield and package DNA, several different c such as monovalent cationic lipids, polyvalent cationic lipids, cationic polymers, guanidine containing compounds, cationic peptides and cholesterol containing compounds have bee d (Rodriguez, 2004).
backs related to the use of viral
of plasmid mportant non-viral vectors are o
Schatzlein, 2001).
s, such as el
thod to increase
04).
ompounds n develope
When formulated together with DNA they form lipoplexes and enhance the stability of the complex in vivo (Dass,
An interesting approach is to develop methods integrating viral proteins or artificial particles to sy an artificial gene transfer vect Wolf and Schmidt- Wolf, 2003). These virus-like-particles or nanoparticles might o ise between the benefits and drawbacks of viral and non-viral systems.
2.3 Development of targeted vectors
The route and interval of repeated administrations, the dose of ve the surface moieties of the vector, length of the expression and the type of promoter contribu ficiency and immune response in a subject of gene therapy. To balance between effective dose and minimal immune response - the concept of “magic bullets”- specific homing gene therapy vectors are pursued by the gene therapy researchers.
After system nistration gene therapy vectors are diluted to the surrounding fluids, moving nd Brownian motion. The vectors attach according to their interaction wit To concentrate the vector to th e in such quantities that a therapeu , the interactions between the vector and target cells need to be modified to increase the affinity to cellular receptors. These interac be influenced either by pseudotyping a virus with other viral proteins to alter its tropism, as often used with enveloped retroviruses (Sandrin et al., 2003) or by including targeting moieties to viral surface proteins, an approach used often with non-enveloped viruses such as adenovirus (Mizuguchi and Hayakawa, 2004). The nex troduce some key concepts of vecto eting.
2.3.1 Targeting gene therapy vectors
During the path of viral gene delivery, there are several possible targeting. The primary targeting consists of selection of the injection method and a method to retain the viral particles in the area of interest, by physical means. To further target the vector ecific manner, viral surface modifications provide the means to affect the attachment of the virus to the cell surface.
Following the penetration of the virus to the cell, some viruses, such as baculoviruses and parvoviruses, continue to travel to the nucleus with intact capsids, possibly enabling the alteration of viral kinetics by capsid display.
After the viral genomic material is released to cells, various tissue-specific promoters can be used to restrict the expression almost exclusively to the targeted tissue. Finally, specific areas of the host genome can be targeted to specifically integrate the transgene to safe positions or to selectively replace a defective gene. Many of these techniques have been published individually;
extensive integration of these methods to a single vector system still remains to be achieved.
2.3.1.1 Physical targeting
With the ex vivo approach, target cells are removed from the patient, transduced with viral vectors and re-introduced to the patient (Cavazzana-Calvo et al., 2000; Harrington et al., 2002). This method increases the physical proximity and limits the target population thus increasing the vector moi (multiplicity of infection). The body’s immunogenic response can also be reduced even with high mois. However, the targeting is limited to the cells which are available either by extracting the selected population from patient’s blood by cell sorting or by growing from the stem cells in vitro.
Excluding the ex vivo approach, the viruses need to be injected to the patient by using a method suitable to the disease. The selection of the injection method enables primary targeting, as used with intracranial injections for treatment for brain tumours (Sandmair et al., 2000; Tyynela et al., 2002). As compared to multiple injections into solid tissue using a needle and a syringe, gene
2004).
stemically engineer or (Schmidt-
ffer a comprom
ctor, te to the ef
ical admi with liquid flow a h cellular surfaces.
tic window is reached
e target tissu tions can
t chapters will in r targ
points for in a tissue-sp
gun ejected gold particles carrying plasmid DNA pen
and with high velocity (Chen et al., 2002) but the transfectio
etrate directly to individual cells in a wide area n depth remains only a few millimetres (Wells, 2004). preparation of the ammuniti can only be used for plasmid DNA. The gene gun method has been used with success to transfer plasmids to foetuses in utero (Yoshizawa et al., 2004) and in vaccination trials (Chen et al., 2002). However, to access internal organs, such as the heart, catheters provide an alternative to surgery with a limited invasion and high targetability (Rutanen et al., 2004).
In addition, viruses can be delivered and retained in the target area with silicon collars (Bhardwaj et al., 2005), or biodegrable polymer encapsulation to also reduce immunogenicity (Sailaja et al., etain the viruses, magnetic ta
practice (Lubbe et al., 2001) as well as to enhance viral infectio al., 2005; Haim et al., 2005). Similar features such as synovial capsules can be used to retain the viruses (Schopf et al.,
2.3.1.2 Viral surface modifications
In order to influence the interactions between the virus and the cell surface, viral surface proteins can be modified, removed or replaced, leading to targeted transduction. Targeting can be either direct or inver disables binding of the viruse rget tissue and increases the concentrat lls. This can be achieved by including a protease-cleavable blocking domain to the virus targeting protein. Specific proteases in the target tissue will remove the blocking domain and allow attachment of the virus to the target cells (Fielding et al., 1998).
Retrovirus tropism has been broadened by direct targeting with other viral glycoproteins, such as Vesicular Stom rotein VSV-G (Cronin et al., 2005
While changing viral glycoproteins is feasible, non-enveloped viruses require different methods. Adenovirus type 5 transduction is dependant on the adenovirus fibre knob binding to the CAR expressed on the target tissue (Mizuguchi and Hayakawa, 2004; Noureddini and Curiel, 2005). Methods to redirect or retarget adenovirus modificati
peptides to the HI loop (Work et al., 2004), binding moieties to the capsid (Parrott et al., 2003), chemical alteration of the capsid proteins (Turunen et al., 2002) or combinations of several techniques (Kreppel et al., 2005). However, with the genetic m the major limiting factor for recovering viable adenovirus is the correct folding of modified f lar cytoplasm. Incorrect folding of modified surface protein results in a dramatic decre iral titers and therefore reduced transduction efficiency (Magnusson et al., 2002). Adenovirus chemically coupled with polyethylglycol (PEG) resulted in reduced binding of neutralizing antibodies (Chillon et al., 1998) and increased circulation time after systemic increase (Ogawara et al., 2004). PEGylated baculovirus however resulted in decreased total transduction efficiency, but increased transduction in lungs and brain (Kim et al., 2006). Adenoviruses have also ied to block binding to native targets, lower toxicity and to prepare the virus for targeting (Koizumi et al., 2006).
As a compromise between the flexible chemical surface modification and robust genetic modification, display systems utilizing various binding moieties have emerged. Baculo- and adenoviruses displaying a synthetic immunoglobulin G (IgG) binding domain (ZZ domain) of protein A have been constructed and shown to be functionall ottershead et al., 2000;
Volpers et al., 2003). These vectors enable a display on antibodies on the viral surface enabling the use of a wide selection of tissue-specific antibodies. Metabo ylated vectors further widen the selection to different molecules by using avidin as g reagent (Purow and Staveley-O'Carroll, 2005; Parrott et al., 2003). Another me
reagents, such as bispecific antibodies to provide a molecular bridge between the ector and targeting ligand (Choi et al., 2005).
Due to limitations in on this method
2002). To further r ly, anatomical 2005).
rgeting has been utilized in clinical n (Kadota et
se. Inverse targeting ion to target ce
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2.3.1.3 Targeting at genetic level
There are several proteins associated with the transport of nucleic acids to the nucleus, several of which are of viral origin. Nuclear targeting can be used to increase the transduction efficiency for example by using SV40 T-antigen nuclear localization signal (Nakanishi et al., 2001). After the genetic material has been delivered to the nucleus, the nucleic acid sequence will determinate the transduction efficiency.
Tissue-specific promoters and enhancer elements can be used to express transgene only in selected tissues (Sadeghi and Hitt, 2005). This method is often used as such to compensate for the lack of viral vector cell specificity, allowing the vector to penetrate to various cell types and yet express the desired gene in only a few. A drawback of tissue-specific promoters is that they are often weaker than constitutive promoters, such as cytomegalovirus (CMV) immediate/early promoter. Therefore use of weaker promoters would require either the use of enhancer elements or a higher viral dose, the latter leading to stronger immuno response (Gerdes et al., 2000). However, tissue-specifi
in hybrid promoter resulted in 90 day transgene expression, by which time CMV immed
cal), the use of conditionally replicative denoviruses might offer required specificity after systemic administration. However the replication increases the potential side-effects and cytotoxicity of the viruses (Kruyt and Curiel, 2002;
c promoters can be modified to result in stronger protein expression, for example by introducing a positive feed-back loop by using transcriptional activators (Nettelbeck et al., 1998) or by adding stabilization elements for labile mRNA, such as Woodchuck hepatitis virus posttranscriptional enhancer (WPRE) (Lee et al., 2005). Baculoviruses with tissue specific glial fibrillary acidic prote
iate/early promoter derived expression was already undetectable (Wang and Wang, 2006).
Since the majority of the gene therapy research (67%) is cancer-related (Journal of Gene Medicine, http://www.wiley.co.uk/genmed/clini
a
Sonabend et al., 2006).
In addition to promoters and enhancers, codon optimization and specific signal sequences together with traditional control elements, such as Kozak sequence, 3’ and 5’
untranslated region signals, polyadenylation sequences and introns (Makrides, 1999) should be used when optimizing the vector for clinical use. However, due to the long approval process, vectors may consist of decades old technology when finally arriving to clinical use.
Transposon systems such as Sleeping Beauty (Izsvak and Ivics, 2004) provide methods to affect to the possible viral integration and long-term expression of the transgene. As integration of the retroviral transgene has been determined to be associated with leukaemia after treatment for SCID (Schmidt et al., 2005; Woods et al., 2006), episomally maintained transgenes (Kreppel and Kochanek, 2004) might offer a method to avoid the drawbacks of uncontrolled genetic targeting. Another possibility to avoid the adverse effects deriving from gene integration to harmful sites could be targeting the retroviral integration to those actively transcribed sites in the genome, which are often related to fundamental cellular processes (Bushman, 2003), but exist with a high copy number and use insulator elements (Brasset and Vaury, 2005) to restrict the viral promoter to viral transgene expression only.
