Current Gene Therapy , Volume 2, No. 2, 2002
Separating Fact from Fiction: Assessing the Potential
of Modified Adenovirus Vectors for Use in Human Gene Therapy Pp. 111-133
A. Amalfitano and R.J. Parks
Adenovirus As An Integrating Vector Pp. 135-144
K. Mitani and S. Kubo
Second Generation Adeno-Associated Virus Type 2-based
Gene Therapy Systems with the Potential for Preferential Integration into AAVS1 Pp. 145-159
R.A. Owens
Advances in the Development of Non-Human Viral
DNA-Vectors for Gene Delivery Pp. 161-171
P. Lِser, A.
Hüser, M. Hillgenberg, D. Kümin, G.W. Both and
C. Hofmann
CFTR Gene Transfer to Lung Epithelium - On the Trail of
a Target Cell Pp. 173-181
S.O'Dea and D.J. Harrison
Intracellular Barriers to Non-Viral Gene Transfer Pp.
183-194
D. Lechardeur and G.L. Lukacs
Bone Marrow Stromal Cells as Targets for Gene Therapy Pp.
195-209
A.V. Damme, T. Vanden Driessche, D. Collen and M.K.L.
Chuah
The Recombinant T Cell Receptor Strategy: Insights into
Structure and Function of Recombinant Immunoreceptors on the Way Towards an
Optimal Receptor Design for Cellular Immunotherapy Pp. 211-226
A.Hombach, C. Heuser and H.
Abken
Restoration of Transgene Expression in Hematopoietic
Cells with Drug-Selectable Marker Genes Pp. 227-234
T. Licht and C. Peschel
Genetic Vaccination for the Immunotherapy of B-Cell
Malignancies Pp. 235-242
F. Benvenuti and O.R. Burrone
Oncolytic Viruses: Programmable Tumour Hunters Pp.
243-254
J.C. Bell, K.A. Garson, B.D. Lichty and D.F.
Stojdl
Cancer Gene Therapy with Tissue Inhibitors of
Metalloproteinases (TIMPs) Pp. 255-271
K. Brand
[Back to top]
Separating
Fact from Fiction: Assessing the Potential of Modified Adenovirus Vectors for
Use in Human Gene Therapy
Amalfitano
and R.J. Park
One
of the major hurdles to successful gene therapy of genetic and/or acquired
disease is the ability to efficiently introduce a foreign gene into the tissue
of interest and, in the case of some genetic diseases, achieve long-term expression
of the transgene. Due to their ability to transduce a wide variety of cell
types in a cell-cycle independent fashion, adenovirus (Ad)-based vectors have
received considerable attention in recent years as delivery vehicles for multiple
gene therapy applications. Effective use of early “first-generation” versions
of these vectors was hampered by not only the induction of strong immune
responses in the host to the Ad vector and transduced cells, but also to direct
acute and chronic toxicity caused by the vector itself. Furthermore, transgene
expression was typically transient, lasting only a few weeks. Despite these
limitations, these vectors have been used in a number of human clinical trials,
eliciting both interesting as well as controversial results, some of which are
summarized herein.
Because of these limitations, a number of advances in
adenovirus “vectorology”, manifested primarily as the development of multiply
attenuated Ads and vectors deleted of all viral protein coding sequences, has resulted
in vectors which retain all of the advantages of Ad vectors and, in addition,
do not exhibit the deleterious characteristics associated with [E1- ]deleted
Ads. This review focuses on the current state of the art regarding the potential
for human use of Ad-based vectors, and how the use of this vector continues to
offer the potential for successful use as a gene delivery tool for the treatment
of a great number of human genetic and non-genetic diseases.
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Adenovirus
As An Integrating Vector
K.
Mitani and S. Kubo
Recombinant adenoviral vectors have served as one of the
most efficient gene delivery vehicles in vivo thus far. Multiply
attenuated or completely gutless adenoviral vectors have been developed to
achieve long-term gene expression in animal models by overcoming cellular
immunity against de novo synthesized adenoviral proteins. However, since
adenovirus lacks native integration machinery, the goal of gene therapy
obtaining permanent expression cannot be realized with current adenoviral
vector systems. Recent studies have shown that replication-incompetent
adenoviral vectors randomly integrate into host chromosomes at frequencies of
0.001-1% of infected cells. To improve the integration frequencies of
adenoviral vectors, a variety of hybrid vectors combining the highly efficient
DNA delivery of adenovirus with the integrating machinery of retroviruses,
adeno-associated viruses, and transposons, have been emerging. These hybrid
vectors have shown promise, at least in in vitro systems. Furthermore,
adenoviral vectors have shown potential as gene targeting vectors. These
developments should eventually lead to more effective gene therapy vectors that
can transduce a myriad of cell types stably in vivo.
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Second
Generation Adeno-Associated Virus Type 2-based Gene Therapy Systems with the
Potential for Preferential Integration into AAVS1
R.A.
Owens
Adeno-associated virus type 2 (AAV-2) is a non-pathogenic
human parvovirus that is being developed as a gene therapy vector for the
treatment of numerous diseases. One property of wild-type AAV-2, that is highly
desirable in a gene therapy vector, is its ability to preferentially integrate
its DNA into a 4 kilobase region of human chromosome 19, designated AAVS1. One
disadvantage of AAV-2 is its relatively small packaging capacity, approximately
4.7 kilobases. Because of this size limitation, the AAV-2 rep and cap
genes were removed from first-generation AAV-2-based gene therapy vectors
to make room for the therapeutic or marker gene. It was later discovered that
the rep gene, or at least one of its products, the Rep68 or Rep78
protein, is required for preferential integration of AAV-2. Recent developments
in AAV-2 gene therapy vector construction allow the inclusion of the rep gene
into a second generation of AAV-2-based gene therapy systems. These new systems
fall into four major categories: plasmid-based systems, co-transduction with multiple
AAV-2 vectors, incorporation of the AAV-2 vector into a larger virus, and in
vitro packaging. These systems not only allow the inclusion of the rep gene,
they also allow the delivery of larger therapeutic genes.
[Back to top]
Advances
in the Development of Non-Human Viral DNA-Vectors for Gene Delivery
P.
Lِser, A. Hüser, M. Hillgenberg, D.
Kümin, G.W. Both and C. Hofmann
Within the last two decades, various vectors based on
human viruses have been developed as gene transfer vehicles for gene therapy
applications and vaccination. However, one yet unresolved problem connected to the
use of viral vectors in humans is the pre-existing immunity to most of these
vectors in the vast majority of the population which can result in impaired
gene transfer efficiency and increased secondary toxicity. One approach to solve
this problem is the development of recombinant viruses of non-human origin as
vectors for gene transfer. The major rationale for using such vectors is the
avoidance of vector neutralization by pre-existing antibodies directed against
the virus on which the vector is based. Use of vectors based on non-human
viruses may therefore allow the use of lower initial vector doses to achieve
efficient gene transfer. Side-effects caused by interactions between vectors
derived from human viruses with a primed immune system or with blood components
could also be reduced. Furthermore, these vectors might show new cell type
tropisms and could therefore infect tissues and organs that are not accessible
to current viral vectors. This review outlines some of the problems inherent in
the human origin of current viral vectors and describes features and progress
with non-human adenovirus and baculovirus-derived vectors that may provide
alternatives.
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CFTR
Gene Transfer to Lung Epithelium - On the Trail of a Target Cell
S.O'Dea
and D.J. Harrison
Cystic fibrosis (CF) is a lethal inherited disease that
afflicts up to 1 in 2,500 people in the western world. Since 1989, when
mutations in the cystic fibrosis transmembrane conductance regulator (CFTR)
gene were identified as responsible for the disease, intense effort has been
applied to the development of replacement gene therapy strategies to cure CF.
Problems with basic gene delivery techniques along with limited knowledge of
the pathogenesis of CF have hindered progress so far. However, recent insights
into the expression patterns and functions of CFTR in developing and adult
lungs are now advancing our understanding of this disease. It is becoming
apparent that progress in gene delivery to cure CF may be best served by
identification of a target cell(s) around which gene transfer strategies can be
specifically tailored to most closely reproduce the effects of normal CFTR expression.
In fact, accurate restoration of endogenous expression patterns may be crucial,
not only for disease reversal, but also to avoid potentially deleterious
effects of inappropriate expression. This approach is in turn confounded
however, by ill-defined stem and progenitor cell pathways within the lung
epithelium. Nonetheless, studies to date suggest that these pathways are relatively
plastic and may respond differently during homeostasis compared with repair
following injury. It may therefore be feasible to target the lung epithelium in
a non-cell specific manner and allow endogenous differentiation pathways to
subsequently establish correct CFTR distribution patterns. In this review,
emerging information on CFTR expression and function in developing and adult
lungs is discussed in the context of putative stem cell populations and their
potential for current gene delivery approaches.
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Intracellular
Barriers to Non-Viral Gene Transfer
D.
Lechardeur and G.L. Lukacs
Non-viral vector mediated gene transfer, compared to viral
vector mediated one, is a promising tool for the safe delivery of therapeutic
DNA in genetic and acquired human diseases. Although the lack of specific
immune response favor the clinical application of non-viral vectors, comprising
of an expression cassette complexed to cationic liposome or cationic polymer,
the limited efficacy and short duration of transgene expression impose major
hurdles in the widespread application of non-viral gene therapy. The trafficking
of transgene, complexed with chemical vectors, has been the subject of
intensive investigations to improve our understanding of cellular and extracellular
barriers impeding gene delivery. Here, we review those physical and metabolic
impediments that account, at least in part, for the inefficient translocation
of transgene into the nucleus of target cells. Following the internalization of
the DNA-polycation complex by endocytosis, a large fraction is targeted to the
lysosomal compartment by default. Since the cytosolic release of heterelogous
DNA is a prerequisite for nuclear translocation, entrapment and degradation of
plasmid DNA in endo-lysosomes constitute a major impediment to efficient gene transfer.
Only a small fraction of internalized plasmid DNA penetrates the cytoplasm.
Plasmid DNA encounters the diffusional and metabolic barriers of the cytoplasm,
further decreasing the number of intact plasmid molecules reaching the nuclear
pore complex (NPC), the gateway of nucleosol. Nuclear translocation of DNA
requires either the disassembly of the nuclear envelope or active nuclear
transport via the NPC. Comparison of viral and plasmid DNA cellular trafficking
should reveal strategies that viruses have developed to overcome those cellular
barriers that impede non-viral DNA delivery in gene therapy attempts.
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Bone
Marrow Stromal Cells as Targets for Gene Therapy
A.V.
Damme, T. Vanden Driessche, D. Collen and M.K.L. Chuah
The bone marrow (BM) is composed of the non-adherent
hematopoietic and adherent stromal cell compartment. This adherent BM stromal
cell fraction contains pluripotent mesenchymal stem cells (MSCs) and differentiated
mesenchymal BM stromal cells. The MSCs self-renew by proliferation while
maintaining their stem-cell phenotype and give rise to the differentiated
stromal cells which belong to the osteogenic, chondrogenic, adipogenic, myogenic
and fibroblastic lineages. A more primitive adherent stem cell was recently identified,
the multipotent adult progenitor cell (MAPC) or mesodermal progenitor cell,
which co-purifies with MSCs. These MAPCs differentiate into MSCs, endothelial,
epithelial and even hematopoietic cells. BM stroma cells, including the
primitive pluripotent MSCs and MAPCs, are attractive targets for cell and gene
therapy. The BM stromal cell population and its multipotent stem cells can be
engineered to secrete a series of different proteins in vitro and in
vivo that could potentially treat a variety of serum protein deficiencies
and other genetic or acquired diseases, including bone, cartilage and BM
stromal disorders or even cancer.
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The
Recombinant T Cell Receptor Strategy: Insights into Structure and Function of Recombinant
Immunoreceptors on the Way Towards an Optimal Receptor Design for Cellular
Immunotherapy
Hombach,
C. Heuser and H. Abken
A promising approach in adoptive immunotherapy is based on
the induction of a specific cellular anti-tumor response by antigen-specific,
cytolytic T cells. Due to difficulties in isolating tumor-specific T cells in
sufficient amounts, it was proposed to graft cytolytic T cells with an antigen-specific,
recombinant T cell receptor. The antigen binding domain of the receptor
consists of a single-chain antibody fragment (scFv) that is derived from a
monoclonal antibody and binds to a tumor associated antigen. The intracellular
signalling domain is derived from the cytoplasmic part of a membrane bound
receptor to induce cellular activation, e.g., the FceRI receptor g-chain or the
CD3 z-chain. By use of this type of recombinant receptor, the strategy combines
the advantages of MHC-independent, antibody-based antigen binding with
efficient T cell activation upon specific binding to the receptor ligand. The
modular composition of the receptor, moreover, facilitates modification of both
the antigen binding and signalling properties. Accordingly, we and others have generated
a panel of recombinant T cell receptors with specificities for malignantly or
virally transformed cells. Receptor grafted effector cells were demonstrated to
mediate a highly efficient immune response towards antigen expressing target cells.
However, little is known about the impact of the recombinant receptor modules
on recognition of highly heterologous target antigens and on cellular
activation in a complex immunological context. This review summarizes the current
knowledge about the generation and function of recombinant immunoreceptors and
discusses the limitations and perspectives of the methodology for use in
cellular immunotherapy.
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Restoration
of Transgene Expression in Hematopoietic Cells with Drug-Selectable Marker
Genes
T.
Licht and C. Peschel
Somatic gene therapy is supposed to cure life-threatening
hematopoietic disorders but is limited by unstable transgene expression.
Efficient gene transfer to hematopoietic progenitor cells does not ensure
long-term gene expression. It would therefore be advantageous if the expression
of transgenes could be restored in bone marrow. Transfer of drug resistance
genes such as the multidrug resistance (MDR1) or mutated dihydrofolate
reductase (DHFR) genes to hematopoietic cells protects them from the toxicity
of anticancer drugs. In addition, transduced cells obtain a selective growth
advantage in the presence of anticancer drugs. This can be used to introduce
and enrich otherwise non-selectable genes by cotransfer to target cells.
Bicistronic vectors have been constructed for coexpression of drug resistance
genes and non-selectable, therapeutic genes with the use of an internal
ribosomal entry-site (IRES). With the use of bicistronic vectors, expression
and function of therapeutic genes have been increased in tissue culture and in
animal models. Further preclinical investigations are needed to identify
optimal conditions for selection.
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Genetic
Vaccination for the Immunotherapy of B-Cell Malignancies
F.
Benvenuti and O.R. Burrone
Vaccination protocols based on targeting of the idiotype
expressed on malignant B cells have so far provided encouraging results in
clinical trials. The essential requirement to induce an immune response is the
inclusion of carriers to overcome T-cell tolerance. Chemical cross-linking of
idiotypic protein is so far the method of choice to induce protective responses
in human studies. Meanwhile, a flurry of alternative strategies to simplify
vaccine production is being tested in murine model. Thanks to the advance in
antibody engineering the two relevant antigenic domains of the lymphoma
immunoglobulin can be assembled into an appropriate format, genetically linked
to molecules that act as immunological adjuvants and directly delivered as
plasmid DNA. Upon immunization, rejection of tumor cells may depend on cellular
or humoral mechanisms, whose relative importance has not been entirely
estimated. We have recently analyzed the specificity of anti-idiotypic
antibodies induced by DNA vaccination and characterised the elements contributing
to optimal anti-idiotypic responses.
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Oncolytic
Viruses: Programmable Tumour Hunters
J.C.
Bell, K.A. Garson, B.D. Lichty and D.F. Stojdl
Despite significant improvements in early detection and
refinements of therapeutic protocols over the last several decades, cancer
remains one of the leading causes of death in North America. In particular,
treatment of metastatic cancers is a highly desirable and yet still elusive
goal of the oncologist. One strategy which holds promise is the use of self
replicating viral strains with the ability to specifically kill tumour but not
normal cells. These so-called “oncolytic viruses” are in general, attenuated
for growth in normal cells but are able to exploit tumour specific, genetic
defects to gain a growth advantage. In this review, we will discuss the
virus:host cell interactions which help form the niche occupied by oncolytic
viruses. The current and potential clinical applications/limitations will be
discussed for oncolytic viruses from the herpesvirus, adenoviruses,
picornavirus, rhabdovirus, and paramyxovirus families.
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Cancer
Gene Therapy with Tissue Inhibitors of Metalloproteinases (TIMPs)
K.
Brand
Matrix metalloproteinases (MMPs) are of crucial importance for the invasive behavior of primary tumors and their metastases. MMP activity is regulated by the four naturally occurring tissue inhibitors of metalloproteinases (TIMPs). It has been shown that overexpression of TIMPs in tumors of various origins leads to reduced tumor growth and formation of metastases. More recently, antitumor efficacy by in vivo gene transfer of TIMPs has been reported in several clinically relevant animal models. This review analyses the therapeutic potential of the TIMPs from a cancer gene therapeutic point of view with particular emphasis on cell culture and in vivo data.