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J Gene Med 2004; 6: S164S171.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.496
ARTICLE
Christoph Volpers,1
Stefan Kochanek1,2 *
1
*Correspondence to:
Dr Stefan Kochanek, Division of
Gene Therapy, University of Ulm,
Helmholtzstr. 8/1, 89081
Ulm, Germany. E-mail:
stefan.kochanek@medizin.uniulm.de
Summary
Due to the very efficient nuclear entry mechanism of adenovirus and its
low pathogenicity for humans, adenovirus-based vectors have become gene
delivery vehicles that are widely used for transduction of different cell types,
especially for quiescent, differentiated cells, in basic research, in gene therapy
applications, and in vaccine development. As an important basis for their use
as gene medicine, adenoviral vectors can be produced in high titers, they can
transduce cells in vivo with transgenes of more than 30 kb, and they do not
integrate into the host cell genome. Recent advances in the development of
adenoviral vectors have brought considerable progress on issues like target cell
specificity and tropism modification, long-term expression of the transgene,
as well as immunogenicity and toxicity in vivo, and have suggested that the
different generations of non-replicative and replicative vectors available today
will each suit best for certain applications. Copyright 2004 John Wiley &
Sons, Ltd.
Keywords adenovirus; adenoviral vectors; nuclear entry; gene expression;
immunogenicity; gene therapy
S165
Adenoviral Vectors
Serotypes
A
B
C
D
E
F
12, 18, 31
3, 7, 11, 14, 16, 21, 34, 35, 50
1, 2, 5, 6
810, 13, 15, 17, 19, 20, 2230, 32, 33, 3639, 4249, 51
4
40, 41
CAPSID
CORE
Fiber (IV)
Penton base (III)
Hexon (II)
VIII
HexonIIIa
VI
associated
proteins
IX
DNA
V
VII
mu
TP
720 copies
60 copies
36 copies
60 copies
360 copies
130 copies
240 copies
240 trimeric units or hexon capsomers, major capsid protein, group-specific and type-specific antigenic determinants
12 pentameric units, base components of the 12 penton capsomers
12 trimeric units, spike components of the 12 penton capsomers, type-specific antigenic determinants
hexon-associated protein
hexon-associated protein, 60 hexameric units, localized to inner side of the capsid
hexon-associated protein, localized to inner side of the capsid
hexon-associated protein, partially located on the outer surface
160 copies
630 copies
100 copies
2 copies
DNA-associated protein
DNA-associated protein, arginine-rich
19-aa peptide, very basic, DNA-associated
terminal protein, covalently attached to 5 ends of the DNA, precursor (pTP) serves as primer for DNA replication
Core
V
VII
Mu (X)
TP
S166
10 000
E1A E1B
30000
20 000
MLP
nucleotides
E3
L1
L2
L4
L3
L5
Ad5 genome
E2B
E4
E2A
Transgene
E1(+/-E3)-deleted vector
(first-generation vector)
Transgene
E2
E1
E3
Transgene II
Transgene I
E4
High-capacity vector
(gutless vector)
E1B-55kD
E1B-55kD
Oncolytic vector
Figure 2. Genomic organization of Ad5 and different types of Ad5-derived vectors. Promoters are depicted by arrowheads; early
(E) and late (L) mRNAs are depicted by thin and heavy arrows, respectively
High-capacity, gutless or
helper-dependent Ad vectors
High-capacity (HC) or gutless Ad vectors are devoid
of all coding viral genes, but contain only the ITRs and
the packaging signal () as viral elements. They can
accommodate up to 36 kb of non-viral DNA so that large
cDNAs, longer tissue-specific or regulatable promoters,
several expression cassettes or even small genomic loci
can be transferred. Non-coding stuffer DNA, preferably
non-repetitive human spacer DNA, is used if necessary
to render the vector genome the packagable size of
2836 kb [22,23]. The lack of viral gene expression from
these vectors has been shown to considerably reduce their
toxicity and immunogenicity in vivo [2427], and longterm transgene expression in liver cells for more than
1 year has been observed in mice [27].
For the production of HC-Ad vectors, all viral gene
functions except E1 are provided in trans by a helper virus
(hence helper-dependent vector); cell lines expressing
all regulatory and structural viral proteins in the timely
J Gene Med 2004; 6: S164S171.
S167
Adenoviral Vectors
HC-Ad Vector
Helper Virus
xP xP
lo lo
Vector
Helper Virus
CsCl
293Cre Cells
Figure 3. Rescue and production of HC-Ad vectors. Initially,
producer cells constitutively expressing E1 functions and Cre
recombinase are transfected with recombinant HC-Ad vector
DNA carrying the transgene, inverted terminal repeats (ITRs)
as origins of replication and the packaging signal (), and
coinfected with helper virus. Due to Cre-mediated excision of
the loxP-flanked packaging signal in its genome, the helper
virus cannot be packaged in viral particles, but provides
regulatory and structural proteins required for replication and
packaging of the vector DNA. After several rounds of coinfection
of the producer cells with vector lysate and helper virus
for amplification, vector particles are purified from residual
contaminating helper virus featuring a slightly higher density by
CsCl equilibrium centrifugation
Copyright 2004 John Wiley & Sons, Ltd.
S168
Targeting strategies
In contrast to most tissues throughout the human body,
some therapeutically relevant cell types express only low
levels or completely lack expression of the primary Ad
receptor (CAR) and, therefore, are almost refractory
to Ad vector transduction, e.g., skeletal and smooth
muscle cells, endothelial cells, hematopoietic cells, and
many tumor cells. By now, quite a range of different
transductional targeting strategies have been successfully
employed in order to improve Ad vector-mediated gene
delivery to those cell types, including genetically encoded
Table 3. Strategies for tropism modification of Ad2/5-based vectors (EGF, epidermal growth factor; EGFR, EGF receptor; FGF,
fibroblast growth factor; mab, monoclonal antibody; PEG, polyethylene glycol; sCAR, soluble CAR receptor; SCF, stem cell factor;
scFv, single-chain Fv fragment)
Structural targeting to specific cellular receptors
Ref.
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
S169
Adenoviral Vectors
S170
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