THE JOURNAL OF GENE MEDICINE

REVIEW
J Gene Med 2004; 6: S164S171.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.496

ARTICLE

Adenoviral vectors for gene transfer and therapy

Christoph Volpers,1
Stefan Kochanek1,2 *
1

Center for Molecular Medicine

(ZMMK) and Institute for Genetics,
University of Cologne, Kerpener Str.
34, 50931 Cologne, Germany
2
Division of Gene Therapy, University
of Ulm, Helmholtzstr. 8/1, 89081
Ulm, Germany

*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

Adenovirus structure and biology

Since the first isolation of adenovirus (Ad) from adenoid tissue in 1953,
more than 50 different human serotypes have been identified (Table 1).
These serotypes were initially classified on the basis of serological
tests and their ability to hemagglutinate red blood cells from rhesus
monkey or rat, later on the basis of restriction and sequence analysis.
Human adenoviruses cause acute respiratory infections, pharyngitis and
conjunctivitis, some types are responsible for epidemic keratoconjunctivitis,
and adenovirus has been associated with gastroenteritis and pneumonia in
young children and, rarely, with pancreatitis and urinary tract infections in
immunocompromised individuals. Most human adenoviruses do not produce
proliferative infections or recognizable disease in animals; few serotypes
(e.g., Ad12) are oncogenic in rodents after inoculation of large amounts.
Non-human adenovirus has been isolated from many species as diverse as
non-human primates, mouse, dog, pig, chicken, duck, turkey, frog, snake,
and others [13].
Adenovirus is a non-enveloped, icosahedral virus of 6090 nm in diameter with a linear, double-stranded DNA genome of 3040 kb. The capsid
is composed of 240 hexon capsomeres forming the 20 triangular faces of
the icosahedron, and 12 penton capsomeres with spike-shaped protrusions
located at the 12 vertices (Figure 1). In addition to the major capsid protein
(hexon) and the penton base and fiber proteins associated to form the penton
capsomere, several hexon- and penton-base-associated proteins are stabilizing components of the capsid. Histon-like viral core proteins are associated
with the DNA packaged within the capsid (Figure 1, Table 2). Altogether,
the adenoviral (Ad) particle has a molecular weight of about 150 MDa [3,4].
Copyright 2004 John Wiley & Sons, Ltd.

S165

Adenoviral Vectors

Table 1. Human Ad serotypes

Group

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

Figure 1. Structure of the adenovirus particle (modified from

[1])

The terminal globular domain or knob region of

the homotrimeric protruding fibers of the Ad capsid
is responsible for the primary virus attachment to
the cellular receptor, the coxsackie- and adenovirusreceptor (CAR) [5,6]. All serotypes except group B
adenoviruses have been shown to use CAR as a primary
docking receptor [7]. Following the initial attachment,
the interaction between an RGD-motif exposed in a
protruding loop of the penton base protein with a cell
surface integrin molecule (v 1 , v 3 , or v 5 ) serving
as secondary or internalization receptor triggers the
virus uptake by clathrin-dependent receptor-mediated
endocytosis [8,9]. The endosomal uptake of the virus and
release into the cytoplasm is accompanied by a stepwise
dismantling of the capsid, leading to the microtubuliassisted transport and finally very efficient delivery of
the core protein-coated viral genome to the nucleus
[10].

The structural organization of the Ad genome is

depicted in Figure 2. The first Ad gene to be expressed is
the immediate early E1A gene encoding a transactivator
for the transcription of the early genes E1B, E2A, E2B,
E3 and E4, as well as protein functions involved in
cellular transformation, together with an E1B protein.
The E2 region encodes for proteins required for viral
DNA replication: DNA polymerase, DNA-binding protein
and the precursor of the terminal protein. Replication of
the viral genome, which depends on inverted terminal
repeats (ITRs) of 100140 bp in length at both ends
as cis-acting elements (origin of replication) and one
copy of the terminal protein (TP) covalently attached
to each 5
end as initiation primer, starts about 56 h
after infection. Core and capsid proteins and a cysteine
protease important for proteolytic trimming of TP and
other structural proteins are expressed from a common
major late promoter after sophisticated splicing of long
precursor transcripts. Intranuclear virion assembly starts
about 8 h after infection and leads to the production of 104
to 105 progeny particles per cell which can be released
after final proteolytic maturation by cell lysis 3040 h
post-infection, completing the viral life cycle [1,3].

First- and second-generation Ad

vectors
The concept of deriving gene delivery vectors from
adenovirus (usually Ad2 or Ad5) especially exploits the
high nuclear transfer efficiency, the broad tissue tropism,
and the low pathogenicity of this virus. Conventional
replication-deficient so-called first-generation vectors
are based on the substitution of the E1 gene region
by the transgene or therapeutic gene to be delivered to
the target cell. Since the transactivating and transforming
E1 functions are therefore not present in the transduced
target cell, the vector does not replicate in these cells
under regular conditions. For vector production, the E1
functions have to be provided in trans by a complementing
producer cell line, like the well-known HEK293 cell line
prepared by Frank Graham in 1977 [11], or others

Table 2. Structural proteins of the Ad particle

Capsid
Hexon (II)
Penton base (III)
Fiber (IV)
IIIa
VI
VIII
IX

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

Copyright 2004 John Wiley & Sons, Ltd.

J Gene Med 2004; 6: S164S171.

S166

C. Volpers and S. Kochanek

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

E1(+/-E3) + E2/4-deleted vector

(second generation vector)

High-capacity vector
(gutless vector)

Non-coding stuffer DNA

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

[12,13]. The recombinant vector genome was initially

constructed by homologous recombination in 293 cells
between the left end of the Ad genome with the
transgene contained in a shuttle plasmid and the right
end of the Ad genome circularized as a plasmid with
head-to-head ITRs and a bacterial origin of replication
and selection marker in the E1 region [14]. Because of
the low efficiency of this method, the vector genome is
now usually obtained by direct cloning, recombination
or transposition techniques in E. coli, then released from
the plasmid or cosmid backbone, and transfected into the
producer cell line for rescue (comprehensively reviewed
in [15]). First-generation vectors can have an additional
deletion in E3 which is dispensable for viral replication in
cell culture, yielding a total transgene capacity of these
vectors of 8 kb. Major advantages of these vectors include
that they can be produced in high titers of up to 1012
to 1013 vector particles per ml, and that they transduce
quiescent as well as proliferating cells.
Disadvantages of these vectors became apparent
from many experiments in animals and from clinical
studies in humans. In spite of the E1 deletion, viral
genes are expressed at low levels in cells transduced
with first-generation vectors, causing direct toxicity and
immunogenicity of the viral gene products. A cytotoxic T
lymphocyte (CTL)-mediated immune response often leads
to the clearance of vector-transduced cells and to short
duration of transgene expression [16,17]. In addition,
a higher transgene capacity would often be desirable.
The construction of second-generation Ad vectors
characterized by additional deletions or inactivation
(e.g., temperature-sensitive mutants) of E2 [18] and/or
Copyright 2004 John Wiley & Sons, Ltd.

E4 functions [1921], which are complemented by

more complex producer cell lines, improved transgene
persistence and decreased the inflammatory response in
some studies. However, the effects of these modifications
were discussed controversially, and the second-generation
vectors did not ultimately solve the immunogenicity or the
capacity problem. In parallel, more radically gutted Ad
vectors were developed.

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

regulated manner and amounts as required are not

available today, making these vectors more difficult to
produce. In an early version of this vector system,
an E1-deleted helper virus was engineered to carry
a partially defective packaging signal [28] containing
only three of the seven AT-rich elements representing
the Ad5 packaging signal [29] with impaired packaging
function, so that the recombinant vector DNA with the
wild-type packaging signal was preferentially packaged
into capsids during production. However, the helper virus
was difficult to produce, and undesired amplification of
the mutated packaging sequence could prevent vector
production. More efficient systems currently in use for
HC-Ad vector production are based on a recombinasemediated excision of the packaging signal in the
helper virus, which is flanked by loxP recognition
sites [30,31] or by frt recognition sites [32]. The
recombinant vector carrying the transgene is produced
in 293 cells constitutively expressing bacteriophage
PI Cre recombinase [33] or yeast Flp recombinase
[32], respectively. After coinfection of these cells with
the helper virus, its packaging signal is excised by
recombination, and the helper-virus DNA replicates and
provides all the structural proteins, but is excluded from
the capsids while the recombinant vector DNA is efficiently
packaged. After several rounds of amplification, the HCAd vector can be purified from remaining contaminating
helper-virus particles with unexcised packaging signals by
CsCl density gradient centrifugation due to differences in
the densities of the particles (Figure 3).
Helper-virus contamination of HC-Ad vectors prepared
with the recombinase-based systems is usually below 1%.
However, homologous recombination between vector and
helper-virus DNA at the left terminus within the packaging

HC-Ad Vector

Helper Virus
xP xP
lo lo

signal can occur during DNA replication, resulting in a

helper virus with only one loxP/frt recognition site left
which will not be selected against in the producer cell
line. This prevents vector production and leads to higher
helper-virus contamination. Helper-virus versions with
reduced sequence homology to the vectors left terminus
currently being developed and vector versions with a
loxP/frt site positioned at the left end of the packaging
signal can address this problem.

Replicative, oncolytic Ad vectors

Conditionally, replication-competent adenoviruses (no
transgene) and Ad vectors (with therapeutic transgene,
e.g., thymidine kinase in E3) are intended to replicate and
spread exclusively in tumor cells [34,35]. Many tumors
have loss-of-function mutations in the cell cycle regulatory
proteins p53 and/or pRb. One type of conditionally
replicative (CR-) Ad vectors is characterized by a
deletion in the gene encoding the 55 kDa E1B protein
which normally binds to and inactivates p53, thereby
activating the cell cycle. These CR-Ad vectors should
productively infect and lyse p53-negative tumor cells, but
not normal, untransformed, p53-positive cells. With the
same rationale, CR-Ad vectors with a mutation in the
pRb-binding domain of the E1A gene products (26 kDa
E1A, 32 kDa E1A) have been designed for selective
replication in pRb-negative tumor cells. In another type of
CR-Ad vectors, tumor selectivity and oncolytic potential
independent of the p53/pRb status has been achieved to
some extent by putting the wild-type E1A gene under
control of a tumor-specific promoter such as the prostatespecific antigen (PSA) promoter or the -fetoprotein
(AFP) promoter [34,35]. CR-Ad vectors are currently
under evaluation in clinical trials for various carcinomas;
however, efficacy in these trials has been limited so far
[35,36].

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.

Safety, toxicity and immunogenicity

problems
When the classical HEK293 cells are being used
for vector production, recombination between the left
terminus of first-generation Ad vector or helper-virus DNA
and partially overlapping E1 sequences in the cellular
genome may result in the generation of E1-positive,
replication-competent adenovirus (RCA), a serious safety
risk. As a consequence, vector production under GMP
standards for drug development will likely require the use
of more recent cell lines which have been transformed
with well-defined, non-overlapping E1 DNA fragments
excluding recombination, e.g., PER.C6 [12] or N52.E6
[13].
In addition to the toxicity of viral proteins expressed
from first-generation vectors (27 days after application)
and the cellular immune response against vector-encoded,
J Gene Med 2004; 6: S164S171.

S168

C. Volpers and S. Kochanek

MHC-presented proteins (>7 days), Ad vector particles

cause an acute, immediate toxicity (first hours) following
systemic application, which likely involves Kupffer cells
in the liver [37,38]. After intravenous injection Ad
particles are efficiently taken up by macrophages, in
particular Kupffer cells, resulting in a strong activation
and rapid chemokine expression of these cells and
limiting transduction of hepatocytes. The transient
removal of Kupffer cells prior to Ad vector injection by
bisphosphonate drugs like clodronate has been shown to
reduce vector-mediated toxicity, to increase vector uptake
into hepatocytes, and even to reduce humoral immune
responses against transgenic proteins [39,40].
Type-specific, neutralizing antibodies raised against Ad
vector particles can prevent successful reapplication of
the vector. Apart from transient immunosuppression at
the time of gene transfer, approaches to overcome this
hurdle include the sequential use of vectors based on
different serotypes (e.g., Ad2 and Ad5) [41,42], and the
use of vectors based on animal adenoviruses (e.g., canine
or ovine Ad) [43] for readministration.

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

or chemically engineered structural modification of the

capsid and the use of bispecific adaptor molecules
directing vector attachment to alternative cell surface
receptors. These approaches to expanding or shifting
the Ad vector tropism are reviewed in Table 3. The
detailed structural and functional understanding of the
fiber protein composed of an N-terminal tail for penton
base binding, a rigid shaft, and a globular knob domain
responsible for fiber trimerization and CAR interaction
[62,63], has allowed for identification of the most suitable
regions for incorporation of peptide ligands (HI-loop and
C-terminus of the knob domain). At the same time, it
has helped in developing strategies to abolish the natural
Ad vector tropism, like the construction of knobless Ad
vectors [61] and vectors with CAR interaction-ablating
point mutations in the knob [60]. These modifications
aim at reducing background reactivity in CAR-expressing
non-target cells and at increasing the specificity of the
vectors for application in vivo.
In addition to transductional targeting strategies,
the use of tissue-specific promoters (transcriptional
targeting) has been shown to contribute in confining
transgene expression to certain tissues. For example,
the von Willebrand factor promoter and the vascular
endothelial growth factor receptor type-1 (flt-1) promoter
have been used for endothelial-specific expression control
in the context of Ad vectors [64,65].

Strategies for long-term expression in

proliferating cells
Ad vectors transduce quiescent as well as proliferating
cells. With the development of HC-Ad vectors, longterm expression in many quiescent cell types has become

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

Example (target receptor or cells)

Ref.

Genetic incorporation of peptide ligand into: Fiber HI-loop

Fiber C-terminus
Hexon
Penton base
Protein IX
Genetic incorporation of fiber protein from different serotype
(Pseudotyping or fiber swap)

RGD peptide (integrins v 3 , v 5 )

Poly-lysine (heparan sulfate containing receptors)
RGD peptide (integrins v 3 , v 5 )
LDV peptide (integrin 4 1 )
Poly-lysine (heparan sulfate containing receptors)
Ad16 fiber (endothelial cells, smooth muscle cells)
Ad17 fiber (airway epithelial cells)
Ad35 fiber (hematopoietic stem cells)
biotinylated SCF bound via avidin (hematopoietic cells)
sss.17 peptide coupled (airway epithelial cells)
FGF incorporated (tumor cells)
anti-FLAG mab/anti-integrin v mab {Ad vector with FLAG tag}
anti-knob scFv/anti-EGFR scFv
anti-knob scFv/EGF
sCAR/EGF
anti-EGFR mab {Ad vector with Z-domain}

[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]

Point mutations in the region aa 485493 (DG loop)

Removal of fiber shaft repeats 822 and the knob domain

[60]
[61]

Chemical modification of the capsid: Biotinylation

PEGylation
Polymer coating
Use of adaptor molecule: chemically crosslinked antibodies
recombinant fusion protein
monoclonal antibody
Ablation of the natural tropism for CAR
Point mutations in the fiber knob domain
Removal of the fiber knob domain

Copyright 2004 John Wiley & Sons, Ltd.

J Gene Med 2004; 6: S164S171.

S169

Adenoviral Vectors

feasible. Since the adenoviral genome remains episomal

after infection, however, all Ad vector types are rapidly
eliminated from proliferating cells, like regenerating liver
cells, cutaneous basal stem cells, hematopoietic tissue
or tumor cells. To address this problem, several groups
have started to incorporate elements from other virus
systems into Ad vectors which allow for either a controlled
episomal replication or a chromosomal integration of the
transgene. In one approach, cre recombinase is expressed
from the Ad vector in order to excise a loxP-flanked DNA
fragment in cis or trans which carries an Epstein-Barr virus
EBNA-1 gene cassette and an ori P element in addition
to the transgene. Upon excision and circularization,
the transgene-carrying circular episome replicates in
synchrony with the host cell [66,67]. Another strategy
involves the construction of an adenovirus/retrovirus
chimeric vector [68,69]. Proliferating target cells are
converted into transient retroviral producer cells by
transduction with two Ad vectors which deliver retroviral
packaging functions (gag/pol/env) and LTR-flanked
retroviral vector sequences (with transgene), respectively.
Progeny retroviral vector particles that are released
from the transient producer cells can infect neighboring
cells, leading to the chromosomal integration of the
transgene sequence they carry. Similarly, adenovirus/AAV
chimera have been designed making use of the AAV rep
protein-mediated stable integration [70,71]. Finally, a
eukaryotic transposase (Sleeping Beauty) has recently
been employed to stably integrate transgene sequences
from HC-Ad vectors into the target cell genome [72].

Ad vectors in clinical trials

Ten years ago, in 1993, the first patient was enrolled
in a clinical trial using Ad vectors for gene therapy. To
date, more than 170 clinical studies with Ad vectors
have been performed in humans, mostly phase I and
II trials for cancer treatment, but also for vascular
disorders and monogenic diseases, especially cystic
fibrosis [73,74]. Several oncolytic CR-Ad vectors are
in phase III trials [75,76]. The first clinical study for
evaluation of HC-Ad vectors (in hemophilia patients)
has recently been initiated [73,77]. About 27% of all
human gene transfer protocols so far, and most of the
human studies with viral vectors initiated during the
past 3 years, have involved Ad vectors for gene delivery
[73,78], reflecting the high expectations towards this
vector system. However, therapeutic efficacy of Ad vectors
in these trials has been very limited, low efficiency of
gene delivery into the target cells and short duration
of transgene expression likely being the predominant
causes.
Ad vectors have usually been well tolerated in
most of these clinical studies, with light fever, chills,
shivering and local pain as the most common side effects
[36,74]. In September 1999, an 18-year-old male patient
(Jesse Gelsinger) with partial deficiency of ornithine
Copyright 2004 John Wiley & Sons, Ltd.

transcarbamylase (OTC) died after administration of a

very high dose (3.8 1013 total particles) of a secondgeneration Ad vector carrying a functional OTC gene [79],
the only serious adverse event of that kind in a clinical
trial employing Ad vectors so far. His death most likely
resulted as a direct consequence of the gene therapy
from a systemic, Ad vector-induced shock syndrome,
due to a cytokine cascade that led to disseminated
intravascular coagulation, acute pulmonary complications
and multiorgan failure [80]. Since then, the responsible
approval authorities in the US have been reevaluating Ad
vector dosage, safety and toxicity in clinical trials, and
stringent guidelines have been issued for studies to be
continued [74,80].

Future aspects and applications

In the future, predominant clinical applications for firstand second-generation Ad vectors will be vaccination
against infectious diseases as well as tumor therapy
and vaccination, conditions in which short-term gene
expression is sufficient for a therapeutic effect. Here, the
immunogenicity associated with the vectors might even
be helpful. In contrast, HC-Ad vectors will be applied
in conditions that require long-term gene expression
(e.g., monogenic disorders), expression of more than
one gene or large genes, and low vector doses. In vivo
applications of HC-Ad vectors have included gene transfer
into liver (e.g., secreted gene products like factor VIII, 1antitrypsin) [27,81], muscle (e.g., dystrophin) [82,83],
eye (e.g., protective proteins like pigment-epithelial
derived growth factor, PEDF) [84,85], and the CNS
[26], and encouraging results from animal studies will
provide the basis for more clinical trials employing HCAd vectors in the future. Further development of HC-Ad
vector technology will have to focus on the optimization
of stuffer DNAs which might support long-term expression
[86], the adaptation of targeting strategies proved
successful with first-generation vectors [87], and the
design of an efficient production system avoiding any
helper-virus contamination, such as recently attempted
by constructing a baculoviral vector to provide the
helper functions [88]. With regard to oncolytic CRAd vectors for viral tumor therapy, further efforts will
have to be directed towards increasing the therapeutic
window, which will be crucial both for the local
treatment of a primary tumor and even more for the
systemic treatment of a disseminated disease. Finally,
for all types of Ad vectors, the further development
of chromatographic purification procedures previously
applied to first-generation Ad vectors by several groups
[89,90] as an alternative to the classic CsCl equilibrium
centrifugation will streamline vector production from
small-scale amounts for high-throughput functional
genomics studies to large-scale amounts needed for
clinical trials and applications.
J Gene Med 2004; 6: S164S171.

S170

References
1. Shenk T. Adenoviridae: the viruses and their replication. In
Virology, Fields BN, Knipe DM, Howley PM (eds). LippincottRaven Publishers: Philadelphia, 1996; 21112148.
2. Horwitz MS. Adenoviruses. In Virology, Fields BN, Knipe DM,
Howley PM (eds). Lippincott-Raven Publishers: Philadelphia,
1996; 21492171.
3. Doerfler W, Boehm P (eds). The Molecular Repertoire of
Adenoviruses. Springer: Berlin, 1995.
4. Stewart PL. Adenovirus structure. In Adenoviral Vectors for Gene
Therapy, Curiel DT, Douglas JT (eds). Academic Press: San
Diego, 2002; 118.
5. Bergelson JM, Cunningham JA, Droguett G, et al. Isolation of a
common receptor for coxsackie B viruses and adenoviruses 2
and 5. Science 1997; 275: 13201323.
6. Tomko RP, Xu R, Philipson L. HCAR and MCAR: the human
and mouse cellular receptors for subgroup C adenoviruses and
group B coxsackieviruses. Proc Natl Acad Sci U S A 1997; 94:
33523356.
7. Roelvink PW, Lizonova A, Lee JGM, et al. The coxsackievirusadenovirus receptor protein can function as a cellular attachment
protein for adenovirus serotypes from subgroups A, C, D, E, and
F. J Virol 1998; 72: 79097915.
8. Wickham TJ, Mathias P, Cheresh DA, et al. Integrins v 3
and v 5 promote adenovirus internalization but not virus
attachment. Cell 1993; 73: 309319.
9. Li E, Brown SL, Stupack DG, et al. Integrin alpha(v)beta1 is an
adenovirus coreceptor. J Virol 2001; 75: 54055409.
10. Greber UF, Willetts M, Webster P, et al. Stepwise dismantling of
adenovirus 2 during entry into cells. Cell 1993; 75: 477486.
11. Graham FL, Smiley J, Russel WC, et al. Characteristics of a
human cell line transformed by DNA from human adenovirus
type 5. J Gen Virol 1977; 36: 5974.
12. Fallaux FJ, Bout A, van der Velde I, et al. New helper cells
and matched early region 1-deleted adenovirus vectors prevent
generation of replication-competent adenoviruses. Hum Gene
Ther 1998; 9: 19091917.
13. Schiedner G, Hertel S, Kochanek S. Efficient transformation of
primary human amniocytes by E1 functions of Ad5: generation
of new cell lines for adenoviral vector production. Hum Gene
Ther 2000; 11: 21052116.
14. Hitt M, Bett AJ, Addison CL, et al. Techniques for human
adenovirus vector construction and characterization. Methods
Mol Genet 1995; 7: 1330.
15. Danthinne X, Imperiale MJ. Production of first generation
adenovirus vectors: a review. Gene Ther 2000; 7: 17071714.
16. Yang Y, Nunes FA, Berencsi K, et al. Cellular immunity to viral
antigens limits E1-deleted adenoviruses for gene therapy. Proc
Natl Acad Sci U S A 1994; 91: 44074411.
17. Yang Y, Li Q, Ertl HCJ, et al. Cellular and humoral immune
responses to viral antigens create barriers to lung-directed
gene therapy with recombinant adenoviruses. J Virol 1995;
69: 20042015.
18. Engelhardt JF, Ye X, Doranz B, et al. Ablation of E2A in
recombinant adenoviruses improves transgene persistence and
decreases inflammatory response in mouse liver. Proc Natl Acad
Sci U S A 1994; 91: 61966200.
19. Armentano D, Sookdeo CC, Hehir KM, et al. Characterization of
an adenovirus gene transfer vector containing an E4 deletion.
Hum Gene Ther 1995; 6: 13431353.
20. Gao GP, Yang Y, Wilson JM. Biology of adenovirus vectors with
E1 and E4 deletions for liver-directed gene therapy. J Virol 1996;
70: 89348943.
21. Wang Q, Greenburg G, Bunch D, et al. Persistent transgene
expression in mouse liver following in vivo gene transfer with
a delta E1/delta E4 adenovirus vector. Gene Ther 1997; 4:
393400.
22. Kochanek S, Schiedner G, Volpers C. High-capacity gutless
adenoviral vectors. Curr Opin Mol Ther 2001; 3: 454463.
23. Kochanek S. High-capacity adenoviral vectors for gene transfer
and somatic gene therapy. Hum Gene Ther 1999; 10:
24512459.
24. Morral N, Parks RJ, Zhou H, et al. High doses of a helperdependent adenoviral vector yield supraphysiological levels of
alpha1 -antitrypsin with negligible toxicity. Hum Gene Ther 1998;
9: 27092716.
Copyright 2004 John Wiley & Sons, Ltd.

C. Volpers and S. Kochanek

25. Morsy MA, Gu M, Motzel S, et al. An adenoviral vector deleted
for all viral coding sequences results in enhanced safety and
extended expression of a leptin transgene. Proc Natl Acad Sci
U S A 1998; 95: 78667871.
26. Thomas CE, Schiedner G, Kochanek S, et al. Peripheral
infection with adenovirus causes unexpected long-term brain
inflammation in animals injected intracranially with firstgeneration, but not with high-capacity, adenovirus vectors:
towards realistic long-term neurological gene therapy for chronic
diseases. Proc Natl Acad Sci U S A 2000; 97: 74827487.
27. Schiedner G, Morral N, Parks RJ, et al. Genomic DNA transfer
with a high-capacity adenovirus vector results in improved
in vivo gene expression and decreased toxicity. Nat Genet 1998;
18: 180183.
28. Kochanek S, Clemens PR, Mitani K, et al. A new adenoviral
vector: replacement of all viral coding sequences with 28 kb of
DNA independently expressing both full-length dystrophin and
-galactosidase. Proc Natl Acad Sci U S A 1996; 93: 57315736.
29. Graeble M, Hearing P. Adenovirus type 5 packaging domain is
composed of a repeated element that is functionally redundant.
J Virol 1990; 64: 20472056.
30. Parks RJ, Chen L, Anton M, et al. A helper-dependent
adenovirus vector system: removal of helper virus by Cremediated excision of the viral packaging signal. Proc Natl Acad
Sci U S A 1996; 93: 13 56513 570.
31. Hardy S, Kitamura M, Harris-Stansil T, et al. Construction of
adenovirus vectors through Cre-lox recombination. J Virol 1997;
71: 18421849.
32. Umana P, Gerdes CA, Stone D, et al. Efficient FLPe recombinase
enables scalable production of helper-dependent adenoviral
vectors with negligible helper-virus contamination. Nat
Biotechnol 2001; 19: 582585.
33. Chen L, Anton M, Graham FL. Production and characterization
of human 293 cell lines expressing the site-specific recombinase
Cre. Somat Cell Mol Genet 1996; 22: 477488.
34. Alemany R, Balague C, Curiel DT. Replicative adenoviruses for
cancer therapy. Nat Biotechnol 2000; 18: 723727.
35. Kirn D. Replication-selective oncolytic adenovirus E1-region
mutants: virotherapy for cancer. In Adenoviral Vectors for Gene
Therapy, Curiel DT, Douglas JT (eds). Academic Press: San
Diego, 2002; 329348.
36. Vorburger SA, Hunt KK. Adenoviral gene therapy. Oncologist
2002; 7: 4659.
37. Lieber A, He CY, Meuse L, et al. The role of Kupffer cell
activation and viral gene expression in early liver toxicity after
infusion of recombinant adenovirus vectors. J Virol 1997; 71:
87988807.
38. Muruve DA, Barnes MJ, Stillman IE, et al. Adenoviral gene
therapy leads to rapid induction of multiple chemokines and
acute neutrophil-dependent hepatic injury in vivo. Hum Gene
Ther 1999; 10: 965976.
39. Alemany R, Suzuki K, Curiel DT. Blood clearance rates of
adenovirus type 5 in mice. J Gen Virol 2000; 81: 26052609.
40. Wolff G, Worgall S, van Rooijen N, et al. Enhancement of in vivo
adenovirus-mediated gene transfer and expression by prior
depletion of tissue macrophages in the target organ. J Virol
1997; 71: 624629.
41. Parks RJ, Evelegh CM, Graham FL. Use of helper-dependent
adenoviral vectors of alternative serotypes permits repeat vector
administration. Gene Ther 1999; 6: 15651573.
42. Morral N, ONeal W, Rice K, et al. Administration of helperdependent adenoviral vectors and sequential delivery of
different vector serotype for long-term liver-directed gene
transfer in baboons. Proc Natl Acad Sci U S A 1999; 96:
12 81612 821.
43. Moffatt S, Hays J, Hogen-Esch H, et al. Circumvention of vectorspecific neutralizing antibody response by alternating use of
human and non-human adenoviruses: implications in gene
therapy. Virology 2000; 272: 159167.
44. Dmitriev I, Krasnykh V, Miller CR, et al. An adenovirus vector
with genetically modified fibers demonstrates expanded
tropism via utilization of a coxsackievirus and adenovirus
receptor-independent cell entry mechanism. J Virol 1998; 72:
97069713.
45. Wickham TJ, Roelvink PW, Brough DE, et al. Adenovirus
targeted to heparan-containing receptors increases its gene
delivery efficiency to multiple cell types. Nat Biotechnol 1996;
14: 15701573.
J Gene Med 2004; 6: S164S171.

Adenoviral Vectors
46. Vigne E, Mahfouz I, Dedieu JF, et al. RGD inclusion in the hexon
monomer provides adenovirus type 5-based vectors with a
fiber knob-independent pathway for infection. J Virol 1999;
73: 51565161.
47. Wickham TJ, Carrion ME, Kovesdi I. Targeting of adenovirus
penton base to new receptors through replacement of its RGD
motif with other receptor-specific peptide motifs. Gene Ther
1995; 2: 750756.
48. Dmitriev IP, Kashentseva EA, Curiel DT. Engineering of
adenovirus vectors containing heterologous peptide sequences
in the C terminus of capsid protein IX. J Virol 2002; 76:
68936899.
49. Havenga MJE, Lemckert AAC, Grimbergen JM, et al. Improved
adenovirus vectors for infection of cardiovascular tissues. J Virol
2001; 75: 33353342.
50. Zabner J, Chillon M, Grunst T, et al. A chimeric type 2
adenovirus vector with a type 17 fiber enhances gene transfer
to human airway epithelia. J Virol 1999; 73: 86898695.
51. Shayakhmetov DM, Papayannopoulou T, Stamatoyannopoulos G, et al. Efficient gene transfer into human CD34+ cells by a
retargeted adenovirus vector. J Virol 2000; 74: 25672583.
52. Smith JS, Keller JR, Lohrey NC, et al. Redirected infection of
directly biotinylated recombinant adenovirus vectors through
cell surface receptors and antigens. Proc Natl Acad Sci U S A
1999; 96: 88558860.
53. Romanczuk H, Galer CE, Zabner J, et al. Modification of an
adenoviral vector with biologically selected peptides: a novel
strategy for gene delivery to cells of choice. Hum Gene Ther
1999; 10: 26152626.
54. Fisher KD, Stallwood Y, Green NK, et al. Polymer-coated
adenovirus permits efficient retargeting and evades neutralizing
antibodies. Gene Ther 2001; 8: 341348.
55. Wickham TJ, Segal D, Roelvink PW, et al. Targeted adenovirus
gene transfer to endothelial and smooth muscle cells by using
bispecific antibodies. J Virol 1996; 70: 68316838.
56. Haisma HJ, Grill J, Curiel DT, et al. Targeting of adenoviral
vectors through a bispecific single-chain antibody. Cancer Gene
Ther 2000; 7: 901904.
57. Watkins SJ, Mesyanzhinov VV, Kurochkina LP, et al. The
adenobody approach to viral targeting: specific and enhanced
adenoviral gene delivery. Gene Ther 1997; 4: 10041012.
58. Dmitriev I, Kashentseva E, Rogers BE, et al. Ectodomain of
coxsackievirus and adenovirus receptor genetically fused to
epidermal growth factor mediates adenovirus targeting to
epidermal growth factor receptor-positive cells. J Virol 2000;
74: 68756884.
59. Volpers C, Thirion C, Biermann V, et al. Antibody-mediated
targeting of an adenovirus vector modified to contain a synthetic
immunoglobulin G-binding domain in the capsid. J Virol 2003;
77: 20932104.
60. Kirby I, Davison E, Beavil AJ, et al. Mutations in the DG loop of
adenovirus type 5 fiber knob protein abolish high-affinity binding
to its cellular receptor CAR. J Virol 1999; 73: 95089514.
61. Magnusson MK, Hong SS, Boulanger P, et al. Genetic retargeting
of adenovirus: novel strategy employing deknobbing of the
fiber. J Virol 2001; 75: 72807289.
62. Xia D, Henry LJ, Gerard RD, et al. Crystal structure of the
receptor-binding domain of adenovirus type 5 fiber protein
at 1.7 A resolution. Structure 1994; 2: 12591270.
63. Kirby I, Davison E, Beavil AJ, et al. Identification of contact
residues and definition of the CAR-binding site of adenovirus
type 5 fiber protein. J Virol 2000; 74: 28042813.
64. Nicklin SA, Reynolds PN, Brosnan MJ, et al. Analysis of cellspecific promoters for viral gene therapy targeted at the vascular
endothelium. Hypertension 2001; 38: 6570.
65. Reynolds PN, Nicklin SA, Kaliberova L, et al. Combined
transductional and transcriptional targeting improves the
specificity of transgene expression in vivo. Nat Biotechnol 2001;
19: 838842.
66. Leblois H, Roche C, Di Falco N, et al. Stable transduction of
actively dividing cells via a novel adenoviral/episomal vector.
Mol Ther 2000; 1: 314322.

Copyright 2004 John Wiley & Sons, Ltd.

S171
67. Tan BT, Wu L, Berk AJ. An adenovirus-Epstein-Barr virus hybrid
vector that stably transforms cultured cells with high efficiency.
J Virol 1999; 73: 75827589.
68. Feng M, Jackson WH, Goldman CK, et al. Stable in vivo gene
transduction via a novel adenoviral/retroviral chimeric vector.
Nat Biotechnol 1997; 15: 866870.
69. Reynolds PN, Feng M, Curiel DT. Chimeric viral vectors the
best of both worlds? Mol Med Today 1999; 5: 2531.
70. Fisher KJ, Kelley WM, Burda JF, et al. A novel adenovirusadeno-associated virus hybrid vector that displays efficient
rescue and delivery of the AAV genome. Hum Gene Ther 1996;
7: 20792087.
71. Recchia A, Parks RJ, Lamartina S, et al. Site-specific integration
mediated by a hybrid adenovirus/adeno-associated virus vector.
Proc Natl Acad Sci U S A 1999; 96: 26152620.
72. Yant SR, Ehrhardt A, Mikkelsen JG, et al. Transposition from a
gutless adeno-transposon vector stabilizes transgene expression
in vivo. Nat Biotechnol 2002; 20: 9991005.
73. Journal of Gene Medicine website. Available: http://www.
wiley.co.uk/wileychi/genmed.
74. NIH website. Available: http://www4.od.nih.gov/oba/rac/
clinicaltrial.htm.
75. Cohen EE, Rudin CM. ONYX-015. Onyx Pharmaceuticals. Curr
Opin Investig Drugs 2001; 2: 17701775.
76. Wilson DR. Viral-mediated gene transfer for cancer treatment.
Curr Pharm Biotechnol 2002; 3: 151164.
77. High KA. Gene transfer as an approach to treating hemophilia.
Circ Res 2001; 88: 137144.
78. Ferber D. Gene therapy: safer and virus-free? Science 2001; 294:
16381642.
79. Raper SE, Yudkoff M, Chirmule N, et al. A pilot study of in vivo
liver-directed gene transfer with an adenoviral vector in partial
ornithine transcarbamylase deficiency. Hum Gene Ther 2002;
13: 163175.
80. NIH Report. Assessment of adenoviral vector safety and toxicity:
Report of the National Institutes of Health Recombinant DNA
Advisory Committee. Hum Gene Ther 2002; 13: 313.
81. Balague C, Zhou J, Dai Y, et al. Sustained high-level expression
of full-length human factor VIII and restoration of clotting
activity in hemophilic mice using a minimal adenovirus vector.
Blood 2000; 95: 820828.
82. Clemens PR, Kochanek S, Sunada Y, et al. In vivo muscle gene
transfer of full-length dystrophin with an adenoviral vector that
lacks all viral genes. Gene Ther 1996; 3: 965972.
83. Haecker SE, Stedman HH, Balice-Gordon RJ, et al. In vivo
expression of full-length human dystrophin from adenoviral
vectors deleted of all viral genes. Hum Gene Ther 1996; 7:
19071914.
84. Kreppel F, Luther TT, Semkova I, et al. Long-term transgene
expression in the RPE after gene transfer with a highcapacity adenoviral vector. Invest Ophthalmol Vis Sci 2002; 43:
19651970.
85. Kumar-Singh R, Farber DB. Encapsidated adenovirus minichromosome-mediated delivery of genes to the retina: application to
the rescue of photoreceptor degeneration. Hum Mol Genet 1998;
7: 18931900.
86. Parks RJ, Bramson JL, Wan Y, et al. Effects of stuffer DNA on
transgene expression from helper-dependent adenovirus vectors.
J Virol 1999; 73: 80278034.
87. Biermann V, Volpers C, Hussmann S, et al. Targeting of highcapacity adenoviral vectors. Hum Gene Ther 2001; 12:
17571769.
88. Cheshenko N, Krougliak N, Eisensmith RC, et al. A novel system
for the production of fully deleted adenovirus vectors that does
not require helper adenovirus. Gene Ther 2001; 8: 846854.
89. Huyghe BG, Liu X, Sutjipto S, et al. Purification of a type
5 recombinant adenovirus encoding human p53 by column
chromatography. Hum Gene Ther 1995; 6: 14031416.
90. Green AP, Huang JJ, Scott MO, et al. A new scalable method for
the purification of recombinant adenovirus vectors. Hum Gene
Ther 2002; 13: 19211934.

J Gene Med 2004; 6: S164S171.