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Current
Gene Therapy
ISSN: 1566-5232
Current Gene Therapy
Volume 6, Number 3, June 2006
Contents
Herpes Simplex Virus Type 1-Based Amplicons Vectors
Guest Editor: Alberto L. Epstein
Editorial Pp. 275-276
Alberto L. Epstein
Introduction – The History of the HSV Amplicon:
From Naturally Occurring Defective Genomes to Engineered Amplicon
Vectors Pp. 277-301
Niza Frenkel
[Abstract]
DNA-Based Methods to Prepare Helper Virus-Free
Herpes Amplicon Vectors and Versatile Design of Amplicon Vector
Plasmids Pp. 303-314
Kazue Kasai and Yoshinaga Saeki
[Abstract]
Chimeric Herpes Simplex Virus/Adeno-Associated
Virus Amplicon Vectors Pp. 315-324
Daniel L. Glauser, Mathias Ackermann, Okay Saydam and
Cornel Fraefel
[Abstract]
Delivery of Large Genomic DNA Inserts >100
kb Using HSV 1 Amplicons Pp. 325-336
Olivia C. Hibbitt and Richard Wade-Martins
[Abstract]
HSV Amplicons: Neuro Applications Pp. 337-350
Carolyn M. Tyler, Charles A. Wuertzer, William J. Bowers
and Howard J. Federoff
[Abstract]
Amplicon Vectors as Outstanding Tools to Study
and Modify Cognitive Functions Pp. 351-360
Diana Jerusalinsky and Alberto L. Epstein
[Abstract]
HSV Amplicon Vectors for Cancer Therapy
Pp. 361-370
Khalid Shah and Xandra O. Breakefield
[Abstract]
HSV-1 Amplicon Vectors Are an Efficient Gene Transfer
System for Skeletal Muscle Cells Pp. 371-381
Yaming Wang
[Abstract]
Amplicons as Vaccine Vectors Pp. 383-392
Kathlyn Santos, Cindy M.P. Duke and Stephen Dewhurst
[Abstract]
HSV-1 Based Amplicon Vectors as an Alternative
System for the Expression of Functional HCV Proteins
Pp. 393-398
Eliza Tsitoura, Urania Georgopoulou and Penelope Mavromara
[Abstract]
Characterization of the Lymphotropic Amplicons-6
and Tamplicon-7 Vectors Derived from HHV-6 and HHV-7 Pp.
399-420
Niza Frenkel and Ronen Borenstein
[Abstract]
Abstracts
[Back to top]
Editorial
Alberto L. Epstein
25 Years of Amplicons!
The study by Richard R. Spaete and Niza Frenkel [Spaete et
al., 1982] that can be considered as the birth of the
concept of herpes simplex virus type 1 (HSV-1) amplicons was
published almost 25 years ago, giving us a good opportunity
to celebrate this event. That paper was actually the climax
of a series of very elegant and clever fundamental studies
aimed to identify and characterize the origins of virus DNA
replication, the packaging signals, and the packaging mechanisms
of the HSV-1 genome, as developed in the introductory review
to this special issue of Current Gene Therapy, written by
Niza Frenkel. At the same time, that paper represented the
beginning of one of the most interesting, useful, and powerful
systems of viral vectors for gene transfer and gene therapy.
After the initial demonstration, by Kwong and Frenkel in 1985,
that amplicons could be used to efficiently deliver foreign
DNA into cultured cells [Kwong et al., 1985], and
the publication, by Geller and Breakefield in 1987, of the
first study showing that these vectors could be used to express
beta-galactosidase in rat cultured peripheral neurons (Geller
and Breakefield, 1988), more than 200 papers have reported
advances in amplicon technology or applications to gene transfer
using these vectors.
A first group of studies, too large to be referred to in detail
in the scope of this short overview, aimed to improve the
production of amplicon vectors both in terms of amount of
infectious particles and of purity, in regard to the contamination
with helper virus particles [Fraefel et al., 1996,
Saeki et al., 2001, Zaupa et al., 2003],
as exemplified in particular by the review of K. Kasai and
Y. Saeki. Simultaneously, other studies focused on the possibility
of expanding the host range of amplicon application by introducing
viral or cellular genetic elements allowing vegetative replication
or maintenance of the amplicon genome in proliferating cells
[Wang et al.,1996], or by improving the stability
of the transduced transgene via its integration into targeted
loci of the cellular genome, using the adenovirus-associated
vector system (Fraefel et al., 1997, Johnston, et
al., 1997], as illustrated by the review of D. Glauser
et al., Also in the same technological chapter, we
should refer to the outstanding work developed by A. Chiocca,
Y. Saeki, R. Wade-Martins and colleagues [Wade-Martins et
al., 2001, Wade-Martins et al., 2003] to demonstrate
that it was possible to use amplicons to transfer entire genomic
loci, as developed in the review by Hibbit and Wade-Martins,
opening the way to study how the use of long native regulatory
sequences could confer physiological regulation of expression
to the transgenic sequences.
About half of the papers reporting applications of amplicon
vectors to particular experimental systems, relate to the
nervous system. Many studies have confirmed the strength of
these vectors to protect neurons against a variety of natural
or experimental injuries via expression of neurotrophins,
antiapoptotic or antioxidant molecules, heat-shock proteins
or proteins affecting neuronal metabolism. Other studies have
shown that amplicons offer a way to study and modify behavioral
traits, like anxiety, sexual behavior, learning, and memory,
while still others have focused on the possibility of using
amplicons to study and treat brain cancers or neurodegenerative
diseases, in particular Parkinson disease. It is impossible
to quote all these studies in this short introduction, but
the reviews of Tyler et al., Shah and Breakefield,
and Jerusalinsky and Epstein are here to illustrate and summarize
the particular interest of amplicons in neurobiology and neurology.
Although to a lesser extent, other works have used amplicons
as gene delivery tools to other cells or tissues, including
hepatocytes, dendritic cells, skeletal and cardiac muscle
cells, etc. The review by Y. Wang illustrates more particularly
the relevance of amplicons as tools for introducing genes
into muscle cells.
Some studies are currently exploring the ability of amplicons
to behave as heterologous vector vaccines [Hocknell et
al. 2002] and, in this regard, the ability of amplicons
to simultaneously express many different antigens, immunomodulators,
or even the whole set of structural viral proteins that could
generate empty virus-like particles (VLPs) [Savard et
al., 1997, Sena-Esteves et al., 1999] constitutes
another outstanding illustration of the significance of these
vectors as gene transfer tools. The reviews by Santos et
al., and by Tsitoura et al., exemplify these
aspects of the potentiality of amplicons.
Finally, it is clear that the amplicon concept can be extended
to other members of the herpesviridae. This is currently being
done by a small number of teams and is illustrated in this
issue by the review on HHV-6 and HHV-7 amplicons by Borenstein
and Frenkel.
The aim of this issue of Current Gene Therapy was to present
a picture of the current state-of-the-art technology of amplicon
vectors and to illustrate some of the most interesting applications
that are currently being developed using these outstanding
tools. It is clear that by adopting this point of view, and
taking into account the limited size of this journal, it was
not possible to invite other researchers for additional contributions
other reviews. We apologize for this and express our regrets,
since many of them have contributed very significant works
in the recent past and have their names engraved in the history
of amplicons.
What about the near future? It seems clear that the production
of amplicons still need, and can, be improved, in order to
generate helper-free vectors in amounts compatible with an
eventual utilization in large animals or human beings. The
stability of the vector genome or of the transgenic sequences
in the infected cells can also enhanced. We still do not understand
in detail the factors that control transgenic expression in
most helper-free amplicon-infected cell types, and only a
few studies have addressed the issue of the cell and host
responses to amplicon infection. Most probably, the number
of studies aiming to apply these vectors for functional genomics
or transgenesis, including the creation of animal models,
will increase, and perhaps we are not far from seeing the
first application of amplicon vectors to human beings. The
steady state progress in the development and applications
of amplicon vectors, allows us to be optimistic. We hope that
most or all of these improvements will be achieved rather
soon, and in any case before we celebrate the 50 years of
amplicon vectors…
REFERENCES
Fraefel, C., Song, S., Lim, F., Lang, P., Yu, L., Wang, Y.,
Wild, P., Geller, A.I. Helper virus-free transfer of herpes
simplex virus type 1 plasmid vectors into neural cells. J.
Virol., 1996; 70: 7190-7197
.
Fraefel, C., Jacoby, D.R., Lage, C., Hilderbrand, H., Chou,
J.Y., Alt, F.W., Breakefield, X.O. and Majzoub, J.A. Gene
transfer into hepatocytes mediated by helper virus-free HSV/AAV
hybrid vectors. Mol. Med., 1997; 3:
813-825.
Geller, A.I. and Breakefield, X.O. A defective HSV-1 vector
expresses Escherichia coli beta-galactosidase in cultured
peripheral neurons. Science, 1988; 241:
1667-1669.
Hocknell, P.K., Wiley, R.D., Wang, X., Evans, T.G., Bowers,
W.J., Hanke, T., Federoff, H.J., Dewhurst, S. Expression of
human immunodeficiency virus type1 gp120 from herpes simplex
virus type 1-derived amplicons result in potent, specific,
and durable cellular and humoral immune responses. J.
Virol., 2002; 76: 5565-5580.
Johnston, K.M., Jacoby, D., Pechan, P.A., Fraefel, C., Borghesani,
P., Schuback, D., Dunn, R.J., Smith, F.I. and Breakefield,
X.O. HSV/AAV hybrid amplicon vectors extend transgene expression
in human glioma cells. Hum. Gene Ther., 1997; 8:
359-370.
Kwong, A.D. and Frenkel, N. The herpes simplex virus amplicon.
IV. Efficient expression of a chimeric chicken ovalbumin gene
amplified within defective virus genomes. Virology,
1985; 142: 421-425.
Saeki, Y., Fraefel, C., Ichikawa, T., Breakefield, X.O. and
Chiocca EA. Improved helper virus-free packaging system for
HSV-1amplicon vectors using an ICP27-deleted, oversized HSV-1
DNA in bacterial artificial chromosome. Mol. Ther.,
2001; 3: 591-601.
Savard, N., Cosset, F.-L. and Epstein, A.L. Use of defective
HSV-1 vectors harbouring Gag, Pol and Env genes to induce
the rescue of defective retroviral vectors. J. Virol.,
1997; 71: 4111-4117.
Sena-Esteves, M., Saeki, Y., Camp, S.M., Chiocca, E.A. and
Breakefield, X.O. Single-step conversion of cells to retrovirus
vector producers with herpes simplex virus-Epstein-Barr virus
hybrid amplicons. J. Virol., 1999; 73:
10426-10439.
Spaete, R.R. and Frenkel, N. The herpes simplex virus amplicon:
A new eucaryotic defective-virus cloning-amplifying vector.
Cell, 1982; 30: 295-304.
Wade-Martins, R., Saeki, Y. and Chiocca, E.A. Infectious delivery
of a 135-kb LDLR genomic locus leads to regulated complementation
of low-density lipoprotein receptor deficiency in human cells.
Mol. Ther., 2003; 7: 604-612.
Wade-Martins, R., Smith, E.R., Tyminski, E., Chiocca, E.A.
and Saeki, Y. An infectious transfer and expression system
for genomic DNA loci in human and mouse cells. Nat. Biotech.,
2001; 19: 1067-1070.
Wang, S., Voss, J.-M.H. A hybrid herpesvirus infectious vector
based on Epstein-Barr virus and herpes simplex virus type
1 for gene transfer into human cells in vitro and
in vivo. J. Virol., 1996; 70: 8422-8430.
Zaupa, C., Revol-Guyot, V. and Epstein, A.L. Improved packaging
system for generation of high-level non-cytotoxic HSV-1 amplicon
vectors using Cre-loxP site-specific recombination to delete
the packaging signals of defective helper genomes. Hum.
Gene Ther., 2003; 14: 1049-1063.
Alberto L. Epstein
Guest Editor
Current Gene Therapy
Centre de Génétique Moléculaire et Cellulaire
CNRS UMR 5534
Université Claude Bernard Lyon 1
16 rue Raphaël Dubois, 69100 Villeur-banne
France
E-mail: epstein@cgmc.univ-lyon1.fr
[Back to top]
The History of the HSV Amplicon: From Naturally Occurring
Defective Genomes to Engineered Amplicon Vectors
Niza Frenkel
The HSV amplicon vector was derived in 1981/1982 after
elaborate experience with "defective viruses", arising
spontaneously in viral stocks propagated at high multiplicities
of infection (m.o.i.). The defective viruses were found to
contain large concatemeric genomes with repeat units of limited
complexity. We employed cloned defective genome repeats to
generate the "amplicon" vectors, which in the presence
of helper virus replicate to produce packaged large concatemeric
genomes, transmissible to uninfected cells. The cloned amplicons
were then employed to fine map and analyze the signals essential
for amplicon propagation: (i) A DNA replication origin, producing
concatemeric genomes by rolling circle replication. Three
DNA replication origins were identified in the HSV genome.
(ii) Signals termed pac-1 and pac-2, directing a measuring
function for coordinate cleavage of the concatemeric genomes
and their packaging as full-size (150 kb) genomes. Using amplicons,
foreign genes of large sizes could be linked to less than
1 kb of the cis-acting HSV DNA sequences and become amplified
in packaged defective genomes, transmissible to new cells.
The transgenes are expressed efficiently, due to sequence
reiterations. Large quantities of vectors can be produced
in vitro. The amplicons are attractive vectors for
use as non-integrating gene delivery vectors. The packaging
signals pac-1 and pac-2 are well conserved in different herpesviruses
and amplicons with a DNA replication origin and cleavage and
packaging signals have been produced in additional herpesviruses.
Depending on amplicon-host cell combination, the vectors can
be employed with and without mutated helper virus(es) to obtain
high gene expression, and desired effect on the target cell.
In the absence of helper virus, the defective virus produced
is limited for spread in the targeted cells. We expect that
new vectors employing state of the art transgenes, will be
developed to generate amplicon based concatemeric defective
viruses capable of efficient expression of these genes.
[Back to top]
DNA-Based Methods to Prepare Helper Virus-Free Herpes
Amplicon Vectors and Versatile Design of Amplicon Vector Plasmids
Kazue Kasai and Yoshinaga Saeki
The herpes simplex virus (HSV) amplicon vector is a versatile
plasmid-based gene delivery vehicle with a large transgene
capacity (up to 150 kb) and the ability to infect a broad
range of cell types. The vector system was originally developed
by Frenkel and her colleagues in 1980. Ever since, a great
deal of effort by various investigators has been directed
at minimizing the toxicity associated with the inevitable
contamination by helper virus. In 1996, Fraefel and his colleagues
successfully devised a cosmid-based packaging system that
was free of contamination by helper virus (so-called helper
virus-free packaging), which utilized as helper a set of 5
overlapping cosmid clones that covered the entire HSV genome,
which lacked the DNA packaging/cleavage signals. With the
helper virus-free system, broader applications of the vector
became possible. Cloning of the entire HSV genome in bacteria
artificial chromosome (BAC) plasmids enabled stable maintenance
and propagation of the helper HSV genome in bacteria. It also
allowed for the development of BAC-based helper virus-free
packaging systems. In this article, we review various versions
of DNA-based methods to prepare HSV amplicon vectors free
of helper virus contamination. We also examine recent advances
in vector design, including methods of vector construction,
hybrid amplicon vectors, and the infectious BAC system. Future
directions in improving packaging systems and vector designs
are discussed.
[Back to top]
Chimeric Herpes Simplex Virus/Adeno-Associated
Virus Amplicon Vectors
Daniel L. Glauser, Mathias Ackermann, Okay Saydam and
Cornel Fraefel
Chimeric or hybrid herpes simplex virus type 1/adeno-associated
virus amplicon vectors combine the large transgene capacity
of HSV-1 with the potential for site-specific genomic integration
and stable transgene expression of AAV. These chimeric vectors
have been demonstrated to support transgene expression for
significantly longer periods than standard HSV-1 amplicons.
Moreover, HSV/AAV hybrid vectors can mediate integration at
the AAVS1 pre-integration site on human chromosome 19 at a
relatively high rate, although random integration has also
been observed. One major remaining hurdle of HSV/AAV hybrid
vectors is the low packaging efficiency and titers when AAV
rep sequences are included in the amplicon vector.
In the conditions prevalent during the replication/packaging
of HSV/AAV hybrid amplicons into HSV-1 virions, in particular
the presence of HSV-1 replication factors and AAV Rep protein,
at least three different viral origins of DNA replication
are active: the HSV-1 ori, the AAV inverted terminal repeats
(ITRs), and the p5 promoter/ori driving expression of the
AAV rep gene. A detailed understanding of the properties
of these origins of DNA replication and the molecular mechanisms
of interactions between them, may allow designing novel hybrid
vectors that allow the efficient and precise integration of
large transgenes in the human genome.
[Back to top]
Delivery of Large Genomic DNA Inserts >100
kb Using HSV 1 Amplicons
Olivia C. Hibbitt and Richard Wade-Martins
The principal aim of gene therapy for recessive genetic
diseases is to supplement the loss of function of an endogenous
gene. For the treatment of many diseases regulation of transgene
expression at physiological levels, expression of multiple
splice variants, and correct tissue specificity are of utmost
importance for effective therapy. We therefore believe the
use of a complete genomic locus, in which the native promoter
and regulatory regions drive and control expression, is an
elegant and effective alternative to traditional complementary
DNA (cDNA) vectors utilising heterologous promoters. Viral
vectors have proved, over the years, to be an effective means
of gene delivery in vitro and in vivo, but
the size of complete genomic loci precludes their use in most
viral systems. One notable exception comprises the amplicon-type
vectors based on human herpesviruses, such as the herpes simplex
virus type I (HSV-1) amplicon vector. The large genome of
HSV-1 (152 kb) confers upon HSV-1 amplicons a very large transgene
capacity sufficient to accommodate approximately 95% of human
genomic loci. The combination of the large transgene capacity,
a broad range of cell tropism, and the ability to infect dividing
and non-dividing cells makes HSV-1 amplicons an excellent
vector system to develop for the delivery of large genomic
loci. Here we review recent work which has shown that HSV-1
amplicons can be used for the delivery and expression of large
genomic inserts >100 kb to cells in culture to rescue phenotypes
in cellular models of genetic disease. We then discuss applications
for high capacity HSV-1 amplicons in vivo and their
potential to facilitate the use of large genomic inserts in
gene therapy treatment regimes.
[Back to top]
HSV Amplicons: Neuro Applications
Carolyn M. Tyler, Charles A. Wuertzer, William J. Bowers
and Howard J. Federoff
Strategies that employ HSV amplicon vectors in the prevention
and/or amelioration of pathogenic states afflicting the central
nervous system (CNS) have been extensively documented in preclinical
disease models. The versatility of the HSV amplicon platform
allows for the implementation of therapeutic approaches that
require expression of genes ex-hibiting neuroprotective or
neuroplastic activities, or even applications that necessitate
the elaboration of antigen-specific immune responses to pathogenic
proteins/structures harbored within the CNS. This discourse
highlights the successes and challenges encountered using
HSV amplicon vectors as tools for the dissection of neural
network function and as therapeutics directed against a variety
of neurologic disorders.
[Back to top]
Amplicon Vectors as Outstanding Tools to Study
and Modify Cognitive Functions
Diana Jerusalinsky and Alberto L. Epstein
This review summarizes recent data on the use of HSV-1–based
amplicon vectors for in vivo gene delivery to the
brains of rats and mice to study and modify behaviour. Here
we describe studies that have focused on cognitive functions
like learning and memory. In addition, the use of amplicons
in other behavioural studies, like addiction, social interaction,
anxiety and stress, will be briefly updated. Several remarkable
findings have been achieved, thanks to the use of these very
efficient and non-toxic naturally neurotropic vectors, most
particularly the consistent observation that genetic manipulation
of a rather limited number of neurons in restricted regions
of the brain, could result in significant behavioural changes,
a notion that is therefore emerging as a common unifying hypothesis,
thanks to these works.
[Back to top]
HSV Amplicon Vectors for Cancer Therapy
Khalid Shah and Xandra O. Breakefield
HSV amplicon vectors provide a unique tool in the armamentarium
of weapons for treatment of cancer. Their large capacity (up
to 150 kb) allows incorporation of multiple and large transgenes,
including whole gene loci, as well as components of other
viruses to control the fate of transgenes in the host cells.
Means have been developed to achieve heritable transmission
of transgenes in tumor cells by episomal replication or genomic
integration. Therapeutic transgenes incorporated into amplicon
vectors have included anti-angiogenic agents, immune enhancing
proteins, prodrug activating enzymes, and apoptosis-inducing
factors, as well as inhibitory RNAs for tumor-associated messages.
Perks of this vector system include the ability to combine
amplicon vectors with oncolytic HSV recombinant vectors to
extend the therapeutic range and to target non-dividing as
well as dividing tumor cells. Tumor vaccination is favored
by the high infectivity of dendritic antigen-presenting cells
with HSV vectors, and the vectors themselves appear to have
intrinsic immune enhancing properties. Promoter manipulation
can be used to target therapeutic gene expression to specific
tumor cell types and to achieve drug regulated transgene expression.
Further, amplicon vectors can be used to convert tumor cells
into packaging cells for retrovirus and adeno-associated virus
vectors, thus generating vectors on site. Amplicon vectors
have also proven to be a versatile tool to explore imaging
modalities to monitor gene delivery and tumor responses to
therapeutic intervention.
[Back to top]
HSV-1 Amplicon Vectors Are an Efficient Gene Transfer
System for Skeletal Muscle Cells
Yaming Wang
HSV-1 amplicon vectors have been considered as a promising
gene delivery system for gene therapy of skeletal muscle diseases,
due to the ability to infect non-dividing cells such as differentiated
muscle cells, and to accommodate large transgenes such as
the 14-kb dystrophin cDNA. Studies revealed that HSV-1 amplicons
can transduce cultured differentiated and undifferentiated
muscle cells with high efficiency. Studies also revealed that
HSV-1 amplicons are capable of delivering at least 23-kb transgene
DNA, including the full-length dystrophin cDNA into muscle
cells. The combination of high transduction efficiency, the
ability to accommodate large constructs and ease of manipulation
makes HSV-1 amplicons an ideal gene delivery tool for the
study of muscle ion channels in which gene transduction is
frequently employed in cultured muscle cells that are resistant
to all the transfecting reagents. However, intramuscular injection
of HSV-1 amplicons has been proven inefficient in mature muscles.
Evidence has shown that this is mainly because the basal membrane
that sheaths each myofibers blocks HSV-1 virions from myofiber
cell surface receptors. This result led to the conclusion
that HSV-1 amplicons are more suitable for ex vivo
manipulation of diseased muscle progenitors or stem cells
for autologous cell therapy than in vivo intramuscular
injection. Efforts to confer stable transduction ability on
amplicons have made progress. A new generation of HSV/AAV
hybrid amplicons has been shown to be capable of integrating
large transgenes into the AAVS1 site of the human genome,
thus, holding potential to achieve a safe and lasting gene
transduction in human muscle cells.
[Back to top]
Amplicons as Vaccine Vectors
Kathlyn Santos, Cindy M.P. Duke and Stephen Dewhurst
HSV-1 amplicon vectors efficiently transduce cultured
antigen-presenting cells (APC), including both human and murine
dendritic cells as well as primary human chronic lymphocytic
leukemia (CLL) B cells. Helper-free amplicons have been shown
to be especially well-suited for this purpose, since they
do not impair the antigen-presenting functions of these target
cells. In vivo, amplicon vectors have been used in preclinical
studies aimed at the development of therapeutic cancer vaccines,
as well as vaccines for Alzheimer's disease, and selected
microbial pathogens. Studies in small animal model systems
have shown that ex vivo transduction of irradiated tumor cells
with amplicon vectors encoding immunomodulatory cytokines
such as IL-2 or GM-CSF can elicit protective responses against
a tumor challenge. In an experimental model for cancer immunotherapy,
direct transduction of preformed tumors with vectors encoding
CD40L resulted in slowed tumor growth or tumor eradication.
Other studies have examined the ability of amplicons to elicit
immune responses against encoded antigens, and have shown
that strong cellular immune responses can be generated against
amplicon encoded HIV-1 antigens in mice. Thus, amplicon vectors
have shown significant promise as vaccine vectors in a range
of settings. These promising initial findings highlight the
need to perform additional studies, including experiments
to evaluate the immunogenicity of amplicon vectors in additional
animal models, possibly including nonhuman primates. Overall,
amplicon vectors offer compelling advantages when compared
to other vaccine-delivery platforms, which include the capacity
to incorporate a very large transgene payload and the potential
to efficiently transduce mucosal surfaces. It will be important
to design future studies to directly test and exploit these
features of the amplicon system. The next few years therefore
promise to be an exciting and important period in the development
of amplicons as vaccine vectors.
[Back to top]
HSV-1 Based Amplicon Vectors as an Alternative
System for the Expression of Functional HCV Proteins
Eliza Tsitoura, Urania Georgopoulou and Penelope Mavromara
The lack of efficient systems for the propagation of
the hepatitis C virus in vitro, in the past decade, led to
the development of several heterologous expression systems
for the study of the HCV proteins and the HCV life cycle.
HSV-1 amplicon vectors encoding the HCV structural and some
of the non structural proteins were generated initially for
the expression of high levels of these proteins into mammalian
cells. The recent developments in the production of amplicon
vectors, allowing the elimination of the contaminating helper
HSV-1 virus have given a novel impulse in the study of these
vectors as possible vaccine candidates. In this review, an
extensive list of the existing amplicon vectors expressing
HCV proteins is provided, together with a brief overview of
the results obtained by these studies.
[Back to top]
Characterization of the Lymphotropic Amplicons-6 and
Tamplicon-7 Vectors Derived from HHV-6 and HHV-7
Niza Frenkel and Ronen Borenstein
Amplicon-6 and Tamplicon-7 are novel non-integrating
vectors derived from the lymphotropic Human Herpesviruses
6 and 7 (HHV-6 and HHV-7). In the presence of helper viruses
the amplicon vectors replicate to yield packaged defective
genomes of size approximately 150 kb and consisting of multiple
repeat units containing (i) the oriLyt DNA replication origin
(ii) the pac-1 and pac-2 cleavage and packaging signals (iii)
bacterial plasmid DNA sequences (iv) the chosen transgene(s).
Employing CD46 as a receptor HHV-6 gains entry into varied
cells, including lymphocytes and dendritic cells, whereas
HHV-7 employs the CD4 receptor to target CD4+ cells. The amplicon-based
vectors have facilitated the characterization of viral DNA
replication and packaging. Following electroporation and helper
virus superinfection, the vectors can be transmitted as cell
associated and as cell-free virions secreted into the medium.
Analyses by flow cytometry have shown good cell spread and
efficient gene expression. Exemplary transgenes have included:
(i) The Green Fluorescence Protein (GFP) (ii) Genes for potential
use in anti-viral vaccination e.g., the HSV-1 glycoprotein
D (gD) with and without the trans-membrane region, expressed
intracellularly, at the cell membrane or as secreted proteins.
(iii) Tumor cell antigens. (iv) Apoptotic genes for development
of oncolytic vectors. Due to their cell tropism, their structure
as concatemeric genomes, with less than 1.5 kb of viral DNA
sequences, the HHV-6 and 7 amplicons have the potential to
become unique vectors for immunization and lymphotropic gene
therapy.
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