Journal of General Virology (2000), 81, 2573±2604.

Printed in Great Britain

...................................................................................................................................................................................................................................................................................

Update on adenovirus and its vectors

W. C. Russell
Biomolecular Sciences Building, School of Biology, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK

Introduction and IVa2 (Fig. 1). The virus genome is a linear, double-
Adenoviruses have been characterized extensively since stranded DNA with a terminal protein (TP) attached
their initial description in the early 1950s (Hillemann & Werner, covalently to the 5« termini (Rekosh et al., 1977), which
1954 ; Rowe et al., 1953) and there is now a panoply of have inverted terminal repeats (ITRs). The virus DNA is
observations on the properties of many of the virus gene intimately associated with the highly basic protein VII and a
products. Nevertheless, there is still a lack of understanding of a small peptide termed mu (Anderson et al., 1989). Another
number of the molecular mechanisms that operate in the protein, V, is packaged with this DNA±protein complex and
infected cell, particularly in respect of how the virus gene appears to provide a structural link to the capsid via protein
products interact with cellular components and of the nature of VI (Matthews & Russell, 1995). The virus also contains a
the responses mounted by the host in response to infection. virus-encoded protease (Pr) (Weber, 1976 ; Webster et al.,
In this regard, it is signi®cant that, although there were 1989), which is necessary for processing of some of the
almost 4000 references to adenoviruses in the 3 years from structural proteins to produce mature infectious virus.
1997 to 1999, most of these have been concerned with the Members of the adenovirus family (Adenoviridae) infect a
results of investigations using adenoviruses as vectors and great variety of post-mitotic cells, even those associated with
relatively few have dealt with the basic virology and highly differentiated tissues such as skeletal muscle, lung, brain
immunology of virus infection. Indeed, it is now accepted and heart. Since they deliver their genome to the nucleus and
that the initial enthusiasm for utilizing adenovirus gene can replicate with high efficiency, they are prime candidates for
vectors in therapy was rather prematurely optimistic and was the expression and delivery of therapeutic genes. They have a
perhaps over-hyped. In its place, there is a realization that wide host-range and are currently divided into three genera with
targetting the vector effectively is not so straightforward and further subdivision into species (also termed subgenera or
that, more importantly, the efficacy of host defences has not subgroups) A to F. Division of human serotypes, based mainly
been fully appreciated and must be adequately addressed. on immunological criteria, has historically been the basis of
classi®cation (Benko% et al., 1999 ; Lukashok & Horwitz,
This paper reviews the current knowledge in the ®eld of
1998 ; Mautner, 1989). Some adenoviruses produce tumours in
adenovirus vectors as well as advances in our understanding
animals and can transform cells in vitro, but no reference to
of the properties of the adenovirus gene products. Particular
these characteristics will be made here.
emphasis has been made on developments over the last two
to three years. There have been a number of reviews
examining different aspects of the vector ®eld, and the Early events in adenovirus infection
reader is referred to these (Benihoud et al., 1999 ; Hitt et al.,
1997 ; Zhang, 1999) for more comprehensive coverage. The The adenovirus infectious cycle can be clearly de®ned
expectation remains that a better understanding of the total into two phases. The ®rst or ` early ' phase covers the entry
spectrum of the virus±cell and virus±host interactions will of the virus into the host cell and the passage of the virus
lead to the design of vectors that provide more efficient genome to the nucleus, followed by the selective
delivery along with minimal deleterious host reactions. transcription and translation of the early genes. These early
events modulate the functions of the cell so as to facilitate
the replication of the virus DNA and the resultant
General properties of adenoviruses transcription and translation of the late genes. This leads to
Adenoviruses have a characteristic morphology (Stewart et the assembly in the nucleus of the structural proteins and the
al., 1993), with an icosahedral capsid consisting of three major maturation of infectious virus. The early phase in a
proteins, hexon (II), penton base (III) and a knobbed ®bre (IV), permissive cell can take about 6±8 h (depending on a number
along with a number of other minor proteins, VI, VIII, IX, IIIa of extraneous factors), while the late phase is normally much
more rapid, yielding virus in another 4±6 h.
Author for correspondence : Willie Russell. Fax The adsorption of virus to target cell receptors involves
-44 1334 462595. e-mail wcr!st-andrews.ac.uk high-affinity binding to cell receptors via the knob portion of

CFHD
0001-7217 # 2000 SGM
W. C. Russell

Fig. 1. Structure of adenovirus. The locations of the capsid and cement components are reasonably well de®ned.
In contrast, the disposition of the core components and the virus DNA is largely conjectural.

the ®bre ; for a review see Chroboczek et al. (1995). The prime transmembrane and cytoplasmic domains (Tomko et al., 1997),
receptor for the human subgroup C adenoviruses was shown to with the extracellular domain being sufficient for attachment
be identical to that for coxsackie B virus (Bergelson et al., (Wang & Bergelson, 1999). A more recent study has indicated
1997) and has therefore been termed the coxsackie}adenovirus that the adenovirus CAR does not completely overlap the
receptor (CAR). This has subsequently been shown to be a coxsackievirus receptor (Tomko et al., 2000). A comprehensive
plasma membrane protein of 46 kDa belonging to the survey of representative members of all the human adenovirus
immunoglobulin superfamily and to contain extracellular, species A to F (Roelvink et al., 1998) suggested that they all

CFHE
bound to CAR with the exception of members of the subgroup Review: Update on adenovirus vectors
B, which appear to recognize a different receptor (Stevenson et
al., 1995). In the same study, it was also noted that adenovirus
serotype 41 (Ad41) (of subgroup F) has two ®bres of different
situations such as the intestinal epithelium (Croyle et al.,
1998 c) while in others, such as hepatocytes, they have a
lengths, and only one of them binds to CAR. Since Ad41 readily
minimal role (Hautala et al., 1998).
infects cells of the gastrointestinal tract, it seems likely that the
other ®bre will adsorb to a different cell receptor, perhaps Interaction of the virus with the plasma membrane can
induce a number of signalling pathways and there is good
displayed on enterocytes. Some cells, such as those of
evidence for the activation of the phosphoinositide-3-OH kinase
haemopoietic origin, appear to be largely refractory to
(PI-3K) pathway, which in turn triggers the Rho family of
productive infection by human adenoviruses 2 and 5 and do not
GTPases and the polymerization and reorganization of actin to
display CAR molecules on their plasma membranes (Mentel et
facilitate endocytosis (Li et al., 1998 ; Rauma et al., 1999). As
al., 1997). This suggests that receptor recognition could be one
early as 20 min post-infection, activation of the Raf}mitogen-
of the key factors involved in cell tropism. In an attempt to
activated protein kinase (MAPK) pathway and consequential
modify cell tropism, ®breless adenoviruses have been
production of IL-8 have been observed (Bruder & Kovesdi,
constructed. Not surprisingly, these particles showed drastically
1997). Since the activation of Raf}MAPK is insensitive to the
reduced infectivities and were extremely unstable. The low level
addition of cycloheximide and is sensitive to prior heating of the
of infectivity that could be detected possibly operated by
virus inoculum at 56 °C, it seems plausible that the initial events
integrin-dependent pathways, which have been demonstrated to
at the cell membrane are triggered by a structural component,
operate in some cell systems (Huang et al., 1996 ; see below).
and this could be via the penton base, since it is heat sensitive
Recent investigations have succeeded in de®ning the receptor-
(Russell et al., 1967). Triggering these pathways may act as an `
binding motif on the three-dimensional structure of the ®bre
early-warning system ' for the induction of defence mechanisms
head (Kirby et al., 1999 ; Santis et al., 1999) as well as on the
induced in the host (see below).
CAR (Kirby et al., 2000). It has also been shown that receptor
recognition can be altered by switching ®bre heads from other As noted above, progress of the virus through the endosomes
subgroups (Miyazawa et al., 1999). Experiments have also been and into the cell cytoplasm is normally mediated by clathrin and
carried out that have substituted or added other receptor-binding the coated pit pathway (Wang et al., 1998). Thereafter, the
motifs (Hidaka et al., 1999) in the ®bre knob ; see below. It is virus-encoded protease appears to assist in the further disruption
of the virus capsid by the proteolysis of the structural protein VI
interesting to note that some adenovirus serotypes seem to have
(Greber et al., 1996), which functions as a linker between the
additional speci®cities of binding, suggesting that the CAR
capsid and the core components (Matthews
receptor may be part of a family of receptors (Segerman et al.,
2000). Indeed, another receptor, the histocompatibility class I & Russell, 1994, 1995). The partially disrupted virus is then transported to
molecule, also a member of the immunoglobulin superfamily, the nuclear membrane and the genome is passaged through the nuclear pore
has been shown (Hong et al., 1997) to be available for the and into the nucleus, where the primary transcription events are initiated. The
subgroup C viruses. Moreover, a recent observation suggests passage through the cytoplasm to the nucleus has been postulated to be
that Ad37, a member of subgroup D, appears to bind to mediated by the association of the virus core (the virus DNA and the
sialoglycoprotein receptors (Arnberg et al., 2000), indicating covalently attached TP, together with the basic proteins VII and V and mu
that receptor speci®cities are wider than was at ®rst thought. peptide) with a cellular protein, p32 (Matthews & Russell, 1998 b). The p32
protein is primarily located in the mitochondria but can also be detected in the
nucleus, and it has been suggested that it is a component of a cellular
After the initial interaction of the virus with the ®bre
transport system that shuttles between the mito-chondria and the nucleus and
receptor, entry of the virus proceeds via clathrin-mediated
that the virus can hijack this system to gain access to the nucleus. This
endocytosis. The critical recognition mechanism for this process
passage to the nucleus is relatively rapid and also involves the participation of
is an RGD motif that is exposed on the penton base (Stewart et
dynein and microtubules (Leopold et al., 2000 ; Suomalainen et al., 1999).
al., 1997) and interacts with cellular av integrins (Wickham et
Virus-like particles can be detected at the nuclear membrane by electron
al., 1993). There appears to be direct binding of the virus penton
microscopy within 1 h of infection (Dales & Chardonnet, 1973) and virus
base to the integrins in the presence of divalent cations (Mathias
DNA and proteins V and VII can be detected within the nucleus between 1
et al., 1998). Integrins normally react with the extracellular
and 2 h (Greber et al., 1997 ; Matthews & Russell, 1998 a). Once inside the
matrix to facilitate adhesion, differen-tiation and other cell±cell
nucleus, the genome is targetted to the nuclear matrix (NM), where the TP
phenomena (Meredith et al., 1996). They form a large family of
forms a tight complex with the cellular CAD pyrimidine synthesis enzyme
heterodimeric receptors and it appears that integrins avb3 and
and possibly other NM components (Angeletti & Engler, 1998 ; Fredman &
avb5 both support adenovirus internalization. It is noteworthy
Engler, 1993). It is interesting that nuclear lamin B, which is a component of
that integrin avb5 is expressed on human bronchial epithelial
the NM, readily binds to p32 (Simos &
cells, a major site of primary adenovirus infection in vivo (Mette
et al., 1993). Integrins may also play an important part in
de®ning tropism in some

CFHF
W. C. Russell

Fig. 2. Transcription of the adenovirus genome. The early transcripts are outlined in green, the late in blue. Arrows indicate
the direction of transcription. The gene locations of the VA RNAs are denoted in brown. MLP, Major late promoter.

Georgatos, 1994) and this may allow for the the nucleus and the activation of NF-jB-responsive genes,
disassociation of p32 from the incoming genome. among the latter being the E3 gene promoter (Deryckere &
Burgert, 1996) (see below). Phosphorylation of I jB by a kinase
Transcription and replication complex, IKK, appears to be crucial for the proteolysis of I jB.
As noted above, adenovirus transcription can be de®ned The protein p53 is a tumour suppressor that regulates the
largely as a two-phase event, early and late, respectively transcription of a variety of genes involved in cell cycle arrest
occurring before and after virus DNA replication (Fig. 2). and apoptosis. In normal cells, p53 is present in small amounts,
Transcription is accompanied by a complex series of splicing but levels increase in response to genotoxic and other stresses.
events, with four early ` cassettes ' of gene transcription The regulation of p53 seems to mainly at the protein level,
termed E1, E2, E3 and E4 (Fig. 2). utilizing the cellular protein mdm2, which binds to p53 and acts
The E1 gene products can be subdivided further into E1A as a ubiquitin ligase, targetting p53 to the proteosome for
and E1B. E1A itself has two major components sharing degradation. Another cellular protein, p19arf, also contributes to
substantial stretches of sequence that are termed 289R (or 13S) this system by binding to mdm2, blocking its ligase activity and
and 243R (or 12S), based on the number of amino acid residues. thereby stabilizing p53 (de Stanchina et al., 1998 ; Honda &
These E1A proteins are primarily concerned with modulating Yasuda, 1999 ; Tao & Levine, 1999 ; Weber et al., 1999). An
cellular metabolism to make the cell more susceptible to virus additional factor in this regulation has also been uncovered by
replication. At the risk of being oversimplistic, it is convenient the ®nding that p53 can be modi®ed by the small ubiquitin-like
to de®ne cellular metabolism as being devoted principally to modi®er (SUMO), leading to activation of p53 (Rodriguez et
promoting accurate cell division while retaining speci®c al., 1999).
cellular functions. In so doing, the cell has devised mechanisms E1A proteins interfere with the processes of cell division and
to defend this process from external interference and to remove with the regulation of NF-jB and p53, and do this by a great
any defective cells. The former operates by invoking the innate variety of strategies involving both direct and indirect
and adaptive immune systems (see below), and some of these interaction with cellular proteins. They can also modulate
pathways appear to be regulated via the transcription factor NF- transcription patterns in favour of virus transcription. A
jB, while the latter is mainly carried out by the induction of summary of the characteristics of E1A is found in Table 1 and
apoptosis via a number of routes, one being the transcription Fig. 3. It should be pointed out that many of the properties
factor p53 (for a review of p53 pathways see Prives & Hall, ascribed to E1A in Table 1 are based on in vitro studies,
1999). whereas the availability of the relevant cellular components in
NF-jB is a nuclear transactivator that is released by vivo will depend on the nature of the infected cell and its
proteolysis of an associated inhibitory factor, IjB, in the metabolic state. Moreover, other virus gene products can
cytoplasm (Hay et al., 1999 b), thus leading to its migration to modulate these cellular interactions signi®cantly. For instance,

CFHG
 Review: Update on adenovirus vectors

Table 1. Properties of E1A proteins

Many of the E1A-interacting proteins listed have been mapped to the E1 protein sequences and the locations of those in bold are shown in
Fig. 3. It should be noted that no attempt has been made to distinguish between the 243 and 289R formats ; they share substantial sequence
and differences are more important in the transformation processes. CDK, Cyclin-dependent kinase.

Property Reference(s)

Bind to p21 and related CDK inhibitors, thereby stimulating cell division and growth Keblusek et al. (1999)
Bind to cyclins A and E±CDK complexes, which regulate passage to cell DNA synthesis Faha et al. (1993)
Bind to the p300}CBP family of transactivators, which play a key role in regulating the Arany et al. (1995), Jones (1995), Dorsman
transcription of many components of the cell cycle. Cellular supply of CBP appears et al. (1997), Lipinski et al. (1999)
to be self-limiting and sequestration by E1A probably displaces other factors, thereby
modulating normal cellular pathways
Binding of E1A also modulates the acetyltransferase activities associated with p300}CBP Reid et al. (1998), Chakravarti et al. (1999),
(pCAF), probably by altering nucleosome structure and thereby transcriptional patterns Hamamori et al. (1999), Perissi et al. (1999)
Binding of E1A to the p300}CBP family inhibits the activity of the transactivator Look et al. (1998), McDonald & Reich (1999),
STAT-1, which is required for induction of responses to interferon (see text) and also Paulson et al. (1999), Putzer et al. (2000)
inhibits the activation of the caspase pathway to apoptosis independent of p53
Bind to the Rb}p130 family of ` pocket proteins', some of which can function as oncogenes, Zantema & van der Eb (2000)
and in so doing both interfere and promote the activation of a wide range of transcripts One
important factor that is released from Rb on E1A binding is E2F ; this is critical for the Brehm et al. (1998)
synthesis of a range of S-phase components as well as the activation of the adenovirus E2
gene cassette. Binding of E1A may also affect the acetyltransferase activities of Rb
E1A interacts via a PLDLS motif at its C-terminal region with a protein (CtBP) which also Meloni et al. (1999)
binds to Rb at the same motif and then functions as a transcriptional co-repressor
E1A activates the promoter of the PCNA gene in HeLa cells. but represses it in rat cells, Kannabiran et al. (1999)
possibly a factor in the transformation process. This effect appears to be mediated via
Rb and involves p53
Interact with a multiprotein complex Sur-2, thereby stimulating the transcription of Boyer et al. (1999 b)
virus genes
Binding to the TATA-box-binding protein (TBP) and TBP-associated protein Mazzarelli et al. (1997)
(TAF) provides a route to transcriptional regulation
E1A proteins can stimulate the induction of apoptosis by a number of routes. An Hale & Braithwaite (1999)
important one is by promoting the synthesis of p53 and maintaining its integrity.
Following binding to pRb, release of the transcription factors E2F and ETF can lead
directly to transcription of p53 and to the transcription of the p19arf gene
The product of the latter can interact with mdm2 and thus prevent its interaction with Lin et al. (1994), Honda & Yasuda (1999), Tao
p53 and consequential proteolysis via the ubiquitin}SUMO pathway & Levine (1999) ; Weber et al. (1999), Rodriguez et al.
(1999)
The stability of p53 can also be maintained by the interaction of E1A with Sug1, a Grand et al. (1999 a)
subunit of the proteosome complex that is required for the proteolysis of p53
In contrast, it has also been shown that transcriptional activation of p53 can be achieved by Somasundaram & El-Deiry (1997),
binding of CBP to a TRAM motif, which, at the same time, prevents the binding of mdm2 O'Connor et al. (1999)
and therefore stabilizes p53. The sequestration of CBP by E1A eliminates this interaction
Another route to activation of apoptosis is by sensitization of cells to stimuli such as Shisler et al. (1996), Shao et al. (1997, 1999)
ionizing radiation, DNA damage, TNF and Fas ligand. This is mediated by
inhibiting the IjB kinases, which are critical for the release of NF- jB to the nucleus,
and requires the binding of E1A to p300}CBP
E1A can also bind to UBC9, a protein involved in the SUMO enzymatic pathway. Desterro et al. (1999), Hateboer et al.
Binding to E1A may interfere with the SUMO modi®cation (and stabilization) of (1996), Rodriguez et al. (1999)
cellular proteins such as p53

the E4 gene products can co-operate with E1A in a variety The E1B 19K gene product is analogous to that from the
of ways (Goodrum & Ornelles, 1999 ; Hall et al., 1998 ; cellular Bcl-2 gene. This gene product is concerned with
Yun et al., 1999). The E1B gene product 19K also seems prolonging cell survival by interacting and ablating members of
to function co-operatively with E1A and p53 in promoting the Bax family (whose transcription can be promoted by p53),
oncogenesis and transformation (Kannabiran et al., 1999), which induce apoptosis and necrosis (Han et al., 1996) (see
mainly by ensuring that the downstream consequences of below). A number of studies have examined the interaction of
cell cycle release do not induce apoptosis. the other E1B gene product, 55K, with p53. This interaction

CFHH
 W. C. Russell

Fig. 3. Binding map of proteins to E1A. The locations have been determined on the basis of in vitro studies using
deletion and mutational analysis. The abbreviations are explained in Table 1. CR1, CR2 and CR3 are constant
regions present in a wide range of adenoviruses. PLDLS and LXCXE etc. are recognition motifs. E2F-1 A. D.
homology refers to sequences that can displace E2F-1 from Rb and p300.

appears to be direct and involves the co-localization of the proapoptotic pathways (see below). A recent review on these
complex to the cytoplasm (Grand et al., 1999 b). Interest in this proteins can be consulted for further information on the
interaction was stimulated by the development of an E1B- molecular mechanisms involved (Wold & Chinnadurai, 2000).
deleted vector that was claimed to act as an oncolytic virus to The gene products derived from the E4 cassette (termed orfs
target tumours with defective p53 genes (Bischoff et al., 1996). 1±6}7) mainly facilitate virus messenger RNA metabolism
Further investigations have shown that, while the interaction (sometimes in association with E1B gene products) (Goodrum
reduces the transcription of p53, it also seems to be necessary & Ornelles, 1999 ; Weigel & Dobbelstein, 2000) and provide
for virus replication (Ridgway et al., 1997) and for transport of functions to promote virus DNA replication and shut-off of host
viral RNAs (Horridge & Leppard, 1998). However, the protein synthesis (Halbert et al., 1985) (see Fig. 4 B). They are also
dependence on intact or wild-type p53 is not essential (Harada associated with resistance to lysis by CTLs (Kaplan et al., 1999).
& Berk, 1999 ; Vollmer et al., 1999) and, more signi®cantly, Recently, some of the E4 products have been shown to bind to
the selectivity for replication in defective p53 cells was not apparent when DNA-activated protein kinase, thereby preventing viral DNA
a wide range of cells was tested (Hay et al., 1999 a). The involvement of concatenation. Since the same kinase is needed for activation of the
an E4 gene product, E4orf6 (see below), in the interaction of E1B 55K p53 gene following DNA damage, it has been suggested that such a
with p53 also in¯uences the stability of p53. In addition, E1B 55k has an characteristic could block one of the pathways to apoptosis (Boyer
effect on late virus mRNA transcription (Harada & Berk, 1999) and et al., 1999 a ; Weiden & Ginsberg, 1994). It is signi®cant that
functions as an important effector of in¯ammation in vivo (Ginsberg et many of the characteristics of both the E1B and E4 gene products
al., 1999). are related to counteracting the activities of those encoded by E1A,
The E2 gene products are subdivided into E2A (DBP) thus E4 orf4 seems to inhibit the E1A activation of the E2F
and E2B (pTP and Pol). These provide the machinery for promoter (Mannervik et al., 1999). E4 orf6 has a direct effect on the
replication of virus DNA (Hay et al., 1995) and the transactivation of p53 (Boivin et al., 1999) by interacting with E1B
ensuing transcription of late genes, and this is mediated 55k when it binds to p53, thereby releasing it to be degraded (Boyer
by interaction with a number of cellular factors. & Ketner, 2000). E4 orf3 also appears to relieve the E1B
The E3 genes, which are dispensable for the replication of inactivation of p53 independently (Konig et al., 1999) and to play a
virus in tissue culture, provide a compendium of proteins that role in relocating sites of replication and transcription in the nucleus
subverts the host defence mechanisms (see below) and their (pods) (Doucas et al., 1996 ; Leppard
properties are summarized in Fig. 4 (A). One of these E3 gene
products has been termed the adenovirus death protein (ADP), & Everett, 1999). Recent investigations have also shown
since it facilitates late cytolysis of the infected cell and thereby that p53 can interfere with the binding of the E1A-
releases progeny virus more efficiently (Tollefson et al., 1996). regulated transcription factor p120E4F, thereby repressing
The E3 gp19K is localized in the ER membrane and binds the the activities of the E4 promoter (Sandy et al., 2000). A
MHC class I heavy chain and prevents transport to the cell review of the E4 gene products can be consulted for
surface, where it would be recognized by CTLs. This gene further details (Leppard, 1997).
product, in addition, delays the expression of MHC I (Bennett et Adenoviruses also transcribe a set of RNAs (see Fig. 2) that
al., 1999). The E3 proteins RIDa&b and 14.7k inhibit are not translated, termed the VA RNAs, and these play a role in
combating cellular defence mechanisms (see below).

CFHI
 Review: Update on adenovirus vectors

Fig. 4. Characteristics of E3 and E4 gene products. Horizontal arrows indicate the relative directions of transcription.

Other characteristics of these early gene products are comes into play. After the onset of virus DNA replication, the
described below, and a cartoon depicting the effects of some of IVa2 and IX genes are expressed at high levels (see Fig. 2) and
them on a few cellular pathways is provided in Fig. 5. DNA transcription via the MLP is fully functional by speci®c
replication begins from both DNA termini and requires activation. This is accomplished via the IX and IVa2 gene
sequences within the ITRs as origins of replication (Hay et al., products (Lutz & Kedinger, 1996 ; Lutz et al., 1997) and is also
1995). Thereafter, late transcription ensues, with ®ve cassettes in¯uenced by effective competition for the limiting tran-
of transcripts (termed L1 to L5) resulting from a complex series scription factors (Fessler & Young, 1998). The encapsidation
of splicing events. These lead to the production of the virus process is governed by the presence in the virus DNA of a
structural components and the encapsidation and maturation of packaging signal at the conventional left end, which consists of
virus particles in the nucleus. A key player in the control of a series of AT-rich sequences (Hearing et al., 1987). These
transcription is the major late promoter (MLP), which is events are accompanied by major changes in the nuclear
attenuated during transcription of the early genes. However, it infrastructure and the permeabilization of the nuclear mem-
should be noted that there is a low basal level of late brane (Rao et al., 1996 ; Tollefson et al., 1996). This facilitates
transcription occurring early in infection, even before the MLP the egress of the virus into the cytoplasm and is followed by

CFHJ
W. C. Russell

Fig. 5. A cartoon (not to scale) illustrating some of the sites of action of the virus and virus gene products (in red) on a
few of the cellular pathways (in yellow). A virus particle at the receptor site is in green.

the disintegration of the plasma membrane and the release microbial peptides termed defensins (Ganz & Lehrer, 1998) and
of virus from the cell. it has been shown that these compounds can provide signi®cant
protection from adenovirus infection (Gropp et al., 1999).
Defence mechanisms Indeed, an adenovirus vector expressing a defensin has been
Adenoviruses induce only very low levels of morbidity in utilized to supplement innate defences (Bals et al., 1999). Some
general and this characteristic initially seemed to make them tissues, on receiving the appropriate signal (perhaps via the
attractive as gene vectors. However, on closer scrutiny, it Raf}MAPK pathway ; see above), will release multiple chemo-
becomes evident that a well-orchestrated host defence is the kines that, in turn, recruit neutrophils and invoke an in¯am-
key factor in the suppression of virus spread during infection matory response (Charles et al., 1999 ; Muruve et al., 1999).
and, since this could be just as effective against a vector, it Innate defence mechanisms such as recruitment of macro-
becomes important to unravel the complex molecular phages, activation of complement and natural killer (NK) cells
operations involved in mounting the host defences. have been shown to play a signi®cant role in clearing an
Host cells have a range of strategies to combat any adenovirus infection in vivo (Worgall et al., 1997 a, 1999). The
incursion by an intruder ; these can be considered as innate transcription factor NF-jB appears to be a key regulator of the
and adaptive. With respect to the former, it has recently been innate antiviral response (Ferreira et al., 1999), since it can
established that some epithelial cells release 3±4 kDa anti- activate the transcription of cytokines and adhesion molecules,

CFIA
leading to the production of a range of proin¯ammatory Review: Update on adenovirus vectors
cytokines and the orchestration of other signalling pathways.
It has been claimed that adenovirus infection, especially at
high multiplicities, can lead to the activation of NF- jB at Apoptosis
early stages of infection (Clesham et al., 1998 ; Lieber et al.,
As another means of combating virus infection, the cell can
1998). One mechanism for achieving this could be by the
redirect its metabolism to switch on its apoptosis circuits. Cells
binding of E1A to the p65 subunit of NF- jB, although it is
have complex mechanisms for ensuring that their integrity is not
also apparent that this activation can be suppressed by E1B
compromised and they have devised a fall-back strategy to
19K (Pahl et al., 1996 ; Schmitz et al., 1996). Reference has
switch on proapoptotic proteins when speci®c alarm pathways
been made above to the very early induction, possibly by the
are activated (see Fig. 5). Chief among these is the tumour
interaction of the penton base in the virus with cellular
suppressor p53, which regulates the transcription of genes
integrins, of the Raf}MAPK and other pathways. These may
play a role in the activation of NF- jB (Ghoda et al., 1997) as involved in cell cycle arrest and apoptosis. Among the latter are
well as in the early release of chemokines (Kuhnel et al., members of the Bax family (Pearson et al., 2000), which interact
2000 ; Muruve et al., 1999) and interferons, which are with mitochondria and are involved in the induction of caspases,
important components of the innate response to infection. leading to apoptosis. There are other members of this family,
However, the induction of these endogenous genes following such as Bcl-2, which function to inhibit apoptosis and they carry
infection appears to be quite cell dependent, with human this out by binding to Btf, an important trans-criptional
endothelial cells displaying a range of signalling molecules at repressor. Btf promotes cell death (Kasof et al., 1999) by
24 h, a scenario not seen in human dermal ®broblasts or inducing the permeabilization of mitochondrial membranes
alveolar macrophages (Ramalingam et al., 1999). (Imazu et al., 1999) and releasing cytochrome c, thereby
initiating the caspase cascade. Adenoviruses can subvert the
operation of this pathway by utilizing virus gene products from
its E1 cassette ; thus, E1B 19K can inactivate Bax (Han et al.,
Interferons 1996 ; Ohi et al., 1999) and has a similar function to Bcl-2 in
binding to Btf, thus counteracting the proapoptotic response of
The interferons are subdivided into two main classes, type I
the E1A gene product in activating p53. A parallel mechanism
(containing interferons a and b) and type II (interferon c).They
of apoptosis is mediated by TNFa, which is secreted by
are cellular proteins ranging in size from 15 to 35 kDa and are
monocytes and lymphocytes following activation as part of the
released from cells very early after infection by viruses and as a
innate response. This cytokine appears to play a signi®cant part
result of other insults to the cell and display a fair degree of cell
in the elimination of adenovirus vectors (Elkon et al., 1997) and
speci®city. In the case of adenoviruses, induction seems to be
functions by activating cytosolic phospholipase A2 (pL A2),
by interaction with a structural component, since they can be
which permeabilizes cell membranes, releasing arachidonic acid
produced by virus particles in the absence of protein synthesis
(Wolf & Laster, 1999) and initiating the production of
(Reich et al., 1988). The interferons function by binding to cell
prostaglandins and leukotrienes, which also play a role in
receptors, thereby activating the cellular Jak}STAT pathways,
in¯ammation (Krajcsi et al., 1996). This pathway is normally
which lead to STAT complexes being transferred to the nucleus
modulated by Bcl-2 and its virus analogue, E1B 19K (see
and binding to interferon-response elements (ISREs) on the
above), via downregulation of I jB transcription, thus releasing
cellular DNA. The ISREs regulate the transcription of a range of
NF-jB to the nucleus (de Moissac et al., 1999). In this regard, it
gene products, such as a dsRNA-induced protein kinase (PRK)
is intriguing to note that the ability of p53 to induce apoptosis
and a 2«±5« oligoadenylate synthetase as well as a variety of
requires the participation of NF- jB (Ryan et al., 2000), implying
immunomodulators. These form an impressive array of weapons
a degree of co-operative ` cross-talk'. It is important, however, to
to combat the intracellular activities of the invading virus ; for a
note that many of these effects seem to be cell speci®c : thus, in
recent review see Goodbourn et al. (2000). Adenoviruses are
endothelial cells, Bcl-2 serves to protect the cells from both
generally refractive to interferons, since they have provided
apoptosis and proin¯ammatory responses (Badrichani et al.,
themselves with a number of strategies to overcome this assault
1999). Another route of TNFa action is by the direct induction
on their activities. Thus, gene products from E1A downregulate
of caspases (Kimura & Gelmann, 2000). In contrast, in
the STAT activators (Look et al., 1998 ; McDonald & Reich,
oligodendrocytes, apoptosis by TNF a appears to be mediated by
1999 ; Paulson et al., 1999 ; Leonard & Sen, 1996, 1997).
p53 and involves initiating the JNK signalling pathway
(Ladiwala et al., 1999). TNF-induced apoptosis can be ablated
It has also been claimed (Feigenblum et al., 1998) that by E3 gene products (Lukashok et al., 2000) (see Fig. 5). Other
adenoviruses induce an interferon-regulatory factor (IRF) key players in apoptosis are Fas and Fas ligand interactions, and
at later stages of infection that plays a role in these have been shown to be the major mediators of the
cytopathogenicity. In addition, the VA RNAs (Mathews & elimination of adenovirus vectors from the liver (Chirmule et
Shenk, 1991) bind to and inactivate PRK. al., 1999). In this case, the E3 gene products RID a and RIDb
cause Fas to be removed from the cell surface and degraded
(Tollefson et al., 1998). A cellular protein termed

CFIB
W. C. Russell
 FIP-3 (Li et al., 1999 b) has also been implicated in these
proapoptotic events. FIP-3 appears to be a scaffolding
component of the IKK complex (Ye et al., 2000) and blocks the
proliferation of B cells to provide immunoglobulins for the
release of NF-jB by inhibiting the kinase activity of IKK (Fig.
humoral response (see below). Very few attempts have been
5) (Li et al., 1999 b). Adenoviruses modulate these events via made to examine the adenovirus antigens involved in the
the E3 gene product 14.7K, which binds to FIP-3 (Li et al., 1999 b) and initiation of the proliferative response. A study on the lymphoid
effectively restores NF-jB transcription and thereby cell survival. Many of cells from one individual suggested that either the ®bre or IIIa
these apoptotic mechanisms involve the activation of a range of proteases, structural polypeptides could be targets (Souberbielle & Russell,
such as caspases, and it has been demonstrated that inhibition of the related 1995). However, a more general investigation noted that
ICE-like proteases can boost adenovirus yields (Chiou & White, 1998). A proliferative responses to the un-common Ad35 occurred in
recent investigation has also shown that E1A can induce apoptosis by individuals without any serological evidence of previous Ad35
activation of caspase-8 and is independent of the status of p53 (Putzer et al., +
infection (Flomenberg et al., 1995), implying that CD4 T cells
2000). Interferons can also act proapoptotically by inducing caspase-8
recognized a conserved antigen. This suggests that this arm of
(Balachandran et al., 2000), and this can be ampli®ed in infected cells via the
the immune system may play a role in modulating infection with
dsRNA route (Tanaka et al., 1998). In this case, however, the inhibition of
a wide range of serotypes.
interferon induction by E1A suppresses this apoptotic response. From the
above discussion it will be seen that there is a complex interplay of cellular
and virus components seeking to control cell survival and promotion of virus The humoral response
replication and spread. Thus, in utilizing adenoviruses as vectors, it is critical The humoral response is a major component of the defence
to take these factors into account in devising the optimum conditions for strategy of the host and depends on the ability of B cells,
delivery and effective expression of the transgene. elaborating surface immunoglobulins, to recognize a speci®c
epitope on a foreign antigen. This recognition initiates a massive
Cellular immune responses proliferation via T helper cells and thus the release of speci®c
+ immunoglobulins of various classes into plasma to interact
T cells provide an effective defence via both CD8 directly with these antigens. Where these are important in the
+ initial interaction with the host cell, virus infection can be
cytotoxic cells (CTLs) and CD4 helper cells. CTLs
neutralized very efficiently. Given the importance of the ®bre
function by recognizing a virus antigen in a complex with
class I proteins of the MHC on the cell surface. This event and penton base in the recognition of the receptors (see above),
releases perforin, resulting in cell lysis, thereby eliminating it is not surprising that adenovirus-neutralizing antibodies are
the infected cells even at an early stage before any virus is directed against epitopes on these capsid components (Gahery-
assembled and released. The recognition mechanism depends Segard et al., 1997, 1998 ; Willcox & Mautner, 1976).
on a virus antigen being available to complex successfully However, there are also antigens on the hexon that induce
within the ER membrane with an MHC component and then neutralizing antibodies, and these seem to function by
being trans-ported to the plasma membrane. The complex aggregating virus particles and thereby inhibiting adsorption.
formation is a function of the nature of the cell being The efficacy of the humoral response in the case of adenovirus
infected, as well as the MHC status of the host. It also gene therapy is very important and depends on the nature of pre-
appears that different virus gene products can provide the
existing immunity as well as the route and target of infection
target depending on their ability to interact with a particular
MHC. It is signi®cant, however, that there appears to be (Harvey et al., 1999).
some cross-reactivity of human CTLs in recognizing For humans, there are 51 different adenovirus serotypes,
different adenovirus subgroups (Smith et al., 1998). classi®ed on the basis of their speci®c neutralizing abilities,
Adenoviruses can combat this cellular strategy as and protection by humoral antibodies is therefore tightly
described above by utilizing E3 gp19K to retain the MHC restricted to a given serotype. Further subdivision into species or
antigens in the ER and hence disrupt the recognition subgenera A to F has also been made, using a variety of criteria
process (Kvist et al., 1978). E4 gene products have also (Benko% et al., 1999). Type-speci®c antigens have been
been demonstrated to function in the inhibition of T cell described in the ®bre that are associated with the trimeric knob
cytolysis (Kaplan et al., 1999). and the proximal regions of the stem (Watson et al., 1988 ; and
The CD4 helper cells are important in mounting a W. C. Russell, unpublished data). In the case of the hexon, the
proliferative response to infection. This is mediated in a similar type-speci®c epitopes reside, not surprisingly, on the hexon
fashion by recognition of a virus target antigen in association surface, whereas the internal antigens are conserved, being
with class II MHC. These helper T cells can thereby stimulate critical in the formation of the capsid structure, and therefore
have a very much wider ` group ' speci®city. Group-speci®c
hexon anti-bodies have been used extensively as general
adenovirus diagnostic reagents.

Adenoviruses as vectors
Adenoviruses can infect a wide variety of cell types and

CFIC
tissues in both dividing and non-dividing cells. This charac- Review: Update on adenovirus vectors
teristic, together with their relative ease of preparation and
puri®cation, has led to their extensive use as gene vectors.
The virus can incorporate only about 2 kb of foreign DNA Second- and third-generation vectors
without signi®cant affects on its stability or its infectivity, and
The next approach was to construct vectors (using suitable
the introduction of longer sequences therefore requires the
complementing cell lines) with some or all of the E2 genes
removal of some or all of the virus genes. There are a range of
excised (Lusky et al., 1998 ; Moorhead et al., 1999) and hence
techniques for constructing recombinant adenoviruses, and these
with the capacity to replicate virus DNA and to produce RCAs
are described in detail elsewhere (Hitt et al., 1997 ; Tashiro et
removed. Generation of RCAs could also be prevented by
al., 1999 ; Zhang, 1999).
constructing cell lines that do not contain adenovirus sequences
Vectors can be utilized for : (i) cancer therapy to deliver that overlap those in the vector (Fallaux et al., 1998, 1999).
genes that will lead to tumour suppression and elimination ;
Nevertheless, the host immune response was still a major
(ii) gene therapy, i.e. to deliver genes to tissues to augment
impediment to achieving persistent transgene expression and
defective genes ; (iii) supplementary therapy to deliver
was particularly evident when repeated infections were
genes, expression of which will combat disease processes.
attempted. A number of studies con®rmed that the infecting
recombinant virus itself was sufficient to induce the immune
response, perhaps not surprising in view of the early activation
First-generation vectors of signalling cascades noted above and the potent antigenicity of
the capsid components.
In the ®rst generation of vectors, the E1 and}or E3 gene
cassettes were removed, allowing the introduction of up to 6±5 Other, rather more sophisticated vectors (third generation)
kb of foreign DNA, often under the control of a heterologous have been constructed by deleting other virus genes
promoter. In the case of the E1 deletions, care was taken to
(Amal®tano et al., 1998) and the latest of these have all or
ensure the retention of the ITR and the packaging sequences.
nearly all of the virus genes removed. These so-called `
Removal of the E1 region had the additional apparent advantage
gutless ' vectors (Hardy et al., 1997 ; Kumar-Singh &
of impairing the transcription of the E2 genes (which are E1
Chamberlain, 1996 ; Lieber et al., 1999 ; Morsy et al., 1998 ;
Steinwaerder et al., 1999) originally retained only the ITR
dependent) and consequently the replication of virus DNA and
and packaging sequences and required helper virus and
the production of the virus capsid proteins. However, it will be
appropriate com-plementing cells for propagation, followed
evident from the description of the E1 genes given above that
by careful puri-®cation. Nevertheless, there were problems
there is also the disadvantage of the cellular environment being
associated with these techniques, mainly due to
much less conducive to vector transcription. The defective E1
contaminating helper virus and vector instability. A further
viruses could be propagated by infection of 293 cells (Graham
development, which prevented the packaging of the helper
et al., 1977), which provide the E1 gene products in trans.
virus, involved the use of the Cre-lox helper-dependent
Although many of the initial studies in vitro provided much
system (Chen et al., 1996 ; Hartigan-O'Connor et al., 1999 ;
promise, it soon became evident that the expression of the
Ng et al., 1999 ; Parks et al., 1996 ; Tashiro et al., 1999).
transgene in vivo was only transient and was depressed because
of the overwhelming immune response, mounted mainly against Other methods to simplify and improve the construction of
the virus capsid antigens as well as the expressed transgene. One vectors have been described (He et al., 1998 ; Mizuguchi &
of the reasons for this was the observation that many cells Kay, 1998). A more comprehensive review (Hitt et al., 1997)
harboured E1-like proteins that allowed the E2 genes to provides details of most of the different techniques available for
function, albeit at reduced levels. In turn, this facilitated virus construction of vectors. One factor in fabricating these vectors is
DNA replication and the synthesis of the late structural antigens the need to maintain the vector size for efficient DNA packaging
and the production of replication-competent adeno-virus (RCA). (Parks & Graham, 1997). This has been achieved by using `
It also became evident that, at higher m.o.i., the E1 dependence stuffer ' DNA, although the nature of this stuffer segment has
of E2 gene transcription could be ablated. Bearing in mind these been shown to in¯uence transgene expression (Parks et al., 1999
problems, a number of strategies have been adopted in an b). These latest vectors have increased expression dramatically
attempt to minimize the production of RCA (Hehir et al., 1996 ; in vivo (Morral et al., 1999 ; Morsy et al., 1998 ; Ji et al., 1999).
Gao et al., 2000). Furthermore, as described above, removal of However, it has become clear that the retention of some of the
the E1B products also effectively disarmed one of the E4 genes is important in combating the T cell response (Kaplan
mechanisms for combating proapoptotic defences. In the case of et al., 1999 ; Lusky et al., 1999 ; Yew et al., 1999) and more
the E3-deleted vectors, there were similar sequelae as a result of recent vectors have been modi®ed accordingly (Gorziglia et al.,
the elimination of the E3 gene-mediated defences against host 1999).
responses (Poller et al., 1996). An extension of this approach involves the formation of
hybrid vectors with adeno-associated virus (AAV) ITRs, which
facilitate transgene integration (Lieber et al., 1999 ; Recchia et
al., 1999). A similar strategy has been developed recently by
using the long terminal repeats of Maloney leukaemia virus
 CFID
W. C. Russell

(Zheng et al., 2000) and has shown promise both in vitro and in with anti-CD4 reagents (Poller et al., 1996). This latter protocol
vivo in a model system in facilitating transgene persistence. reduced the population of T helper cells, which are needed for
Hybrids with other viruses such as Epstein±Barr virus and the activation of B cells and for the production of neutralizing
retroviruses have also been developed (Caplen et al., 1999 ; Tan antibodies. Another study implicated factors other than the
et al., 1999). Adenoviruses derived from other species (avian, capsid antigens in modulating the humoral response. It was
ovine, bovine, canine) have been investigated as vectors for shown that deletion of the E4 gene cassette diminished Th2 and
human gene therapy, since they do not normally invoke endemic B cell activities and it was postulated that an E4 gene product
humoral immunity (Hofmann et al., 1999 ; Kremer et al., 2000 ; facilitated antigen presentation and the production of IL-6 and
Michou et al., 1999 ; Reddy et al., 1999 ; Zakhartchouk et al., IL-8, which are important in B cell maturation (Armentano et
1998). Animal adenovirus vectors have also been used for al., 1997). Not surprisingly, a number of studies demonstrated
animal vaccination (Hammond et al., 2000 ; Rasmussen et al., that the administration of immunosuppressive agents such as
1999). cyclosporin, cyclophosphamide (Smith et al., 1996), FK506
(Ilan et al., 1997), deoxyspergualin (Kaplan & Smith, 1997) and
Strategies for ensuring effective vectors CTLa4 Ig (Jooss et al., 1998 b) enhanced the persistence of the
transgene product. Induction of tolerance has also been shown
The effectiveness of gene therapy is governed in the main by
to be successful in some cases, leading to signi®cant transgene
the ability of the vector to be delivered to the relevant tissue
persistence (Ilan et al., 1996, 1998 ; Lee et al., 1999 b). Another
and, once there, to express the gene product in appropriate
route to immunosuppression was brought into play by the co-
quantities. This, of course, is exactly what the virus has sought
administration of an adenovirus vector with another that had a
to achieve in the course of evolution, and has been
transgene expressing soluble CD8 or CD8 fused to the
demonstrably successful in that adenoviruses are very preva-lent
extracellular regions of a TNFa receptor (Peng et al., 1999).
without causing excessive morbidity. This seems to have been
This procedure successfully inhibited the action of TNF a and
accomplished by utilizing virus gene products to delay the early
signi®cantly reduced the humoral antibody responses to both
innate and immune host defences, thus ensuring that the primary
adenovirus and the transgene product. Another, more direct
infection produces large amounts of virus. Propagation to other
approach to minimizing antibody neutralization was achieved
host cells can then be accomplished before the infected host's
by covalently linking polyethylene glycol (O'Riordan et al.,
full immunological armoury is deployed successfully. Whether
1999) or a hydrophilic polymer based on N-(2-
adenoviruses are able to adopt other strategies to ensure their
hydroxypropyl)methacrylamide (HPMA) to the capsid
continuance, such as persistence or latency in the presence of an
components of the virus (Fisher et al., 2000). This latter
immune response, has never been adequately demonstrated,
procedure also allowed retargetting of the vector. It should be
although adenovirus sequences can be detected in a proportion
noted, however, that the antibody response to the adminis-
of the lungs from healthy individuals (Eissa et al., 1994 ; Elliott
tration of a virus vector is in¯uenced signi®cantly by the pre-
et al., 1995) as well as patients with pulmonary disease (Keicho
existing antibody status and by the route of administration
et al., 1999) (for reviews see Lukashok et al., 2000 ; Mahr &
(Harvey et al., 1999). Some improvement in transgene
Gooding, 1999). However, the facts that adenovirus immunity
persistence can be achieved by repeated administration with
appears to be long lasting and that antibodies to the common
vectors of different serotypes (Parks et al., 1999 a), although
serotypes 2 and 5 can be detected in almost 90 % of individuals
this has its limitations in view of the T cell cross-reactivities
do suggest that persistence may be a factor in virus survival.
described above. Nevertheless, in spite of this plethora of
This latter characteristic is obviously a desirable feature of an
techniques available to minimize the humoral antibody re-
effective vector in some applications, but to achieve such an
sponse, there is no doubt that the inability to ablate the response
outcome in the tissue of choice will require a much greater
effectively remains a major impediment to exploi-tation of
understanding of the total spectrum of molecular mechanisms
vectors (Benihoud et al., 1999).
that operate in infection and of the resulting immunity.
In terms of T cells, a number of investigations have
indicated that adenovirus gene delivery can elicit a complex
Minimizing immune and apoptotic responses. In view of the + +
panoply of cellular immune responses. CD4 and CD8 T
importance of the immune response in relation to transgene
cells speci®c for the transgene product as well as the vector
persistence, a number of studies have been carried out to can all be elaborated with variations dependent on the route
unravel the role of the different arms of the immunological of administration, the target organ and other factors such as
repertoire. Most of them have been carried out in model the host genotype (van Ginkel et al., 1997) and development
rodent systems, but a few have involved human subjects. status (Kass-Eisler et al., 1994). In addition, for delivery to
Humoral responses can be mounted, as noted above, with the lung, innate immune mechanisms involving the migration
a single immunization (Juillard et al., 1995), but this can be of alveolar macrophages seem to be very important (Worgall
modi®ed to some extent by ensuring the retention of the E3 et al., 1997 b).
gene cassette in the vector as well as by treatment of the host Apoptosis can also play an important part in minimizing

CFIE
 Review: Update on adenovirus vectors

transgene expression, and this can be combated to a signi®cant into the C-terminal domain of the ®bre protein. This
extent by using vectors that express Bcl-2 both with and without procedure facilitated binding to other cells without altering
NF-jB inhibitors (Bilbao et al., 1999 a, b ; de Moissac et al., the en-dogenous binding, but this technique showed that
1999 ; Lieber et al., 1998), the expression of the inhibitors speci®c delivery could be obtained in cells where the normal
encouraging greater transgene persistence in mouse livers. The CAR was not expressed (Hidaka et al., 1999). A similar
role of the E3 14.7K protein in attenuating in¯ammation was strategy using a variety of ligands proved promising in a
shown neatly by constructing transgenic mice in which this gene model system for mouse gliomas (Staba et al., 2000).
was expressed selectively by using a human SP-C promoter. One problem in attempting to produce vectors with novel
There was a signi®cant difference in lung in¯am-mation and receptors is the need for simultaneous development of culture
prolonged transgene expression when an E1}E3-deleted vector cells that would allow good propagation of the vector. A neat
was administered (Harrod et al., 1998). A systematic approach to dealing with this has been demonstrated by
investigation of the target proteins for CTLs and their incorporating six histidine residues (a His tag) into the H1 loop
histocompatibility restriction was undertaken in a murine model of the ®bre knob (Krasnykh et al., 1998 ; Michael et al., 1995)
of liver gene therapy and revealed that the levels of CTL and then using the modi®ed virus to infect human glioma cells
responses to adenovirus antigens and to the transgene product successfully (which lack normal receptors), which themselves
were varied and very dependent on the MHC haplotype of the had been modi®ed to display a single-chain antibody against the
host. A range of adenovirus antigens were examined in this His tag (Douglas et al., 1999).
system (pTP, Pol, DBP, hexon, penton and ®bre) and the A similar method, using a peptide from in¯uenza virus
structural proteins, especially hexon, appeared to be the major haemagglutinin inserted into either the ®bre or the penton
targets (Jooss et al., 1998 a). Another survey examined both base, was used successfully to infect cells expressing the
apoptosis and antibody formation in different strains of mice as single-chain antibody ligand (Einfeld et al., 1999). This
a result of infection with an adenovirus vector and also approach could, in principle, be developed to construct
concluded that there were differing responses depending on the vectors that have lost their native tropisms through mutation
mouse strain (Schowalter et al., 1999). An interesting study with of the receptor-binding site on the ®bre (Bewley et al.,
nude mice, where the immune system was ablated, also 1999 ; Kirby et al., 1999 ; Santis et al., 1999) and the RGD
concluded that the persistence of a transgene in mouse lung motif on the penton base (Chiu et al., 1999 ; Mathias et al.,
depended on the nature of the vector backbone and on the host 1998) and therefore have the capacity to infect cells with
background (Kaplan et al., 1997). These results suggest that the other speci®cities. It has also been shown that delivery can
efficacy of therapy with adenovirus vectors will exhibit be inhibited by protective extracellular matrices (van
considerable heterogeneity in human populations. Deutekom et al., 1999) and that there are also anatomical
barriers to overcome (Fechner et al., 1999).
Factors that affect delivery of transgenes. The primary cellular A survey of a range of human adenovirus serotypes has
receptors for adenoviruses appear to be distributed so widely in demonstrated that some of them exhibit different and wider host
cells that effective and speci®c delivery to target cells would tropisms, indicating that factors other than CAR must also
normally be precluded. On the other hand, a number of tissues operate. Thus, a chimeric type 2 adenovirus with a type 17 ®bre
and cells express very little, if any, of these receptors (Leon et can enhance gene transfer to airway epithelia (Zabner et al.,
al., 1998). Thus, the apical surfaces of ciliated airway epithelia, 1999), in contrast with type 2 on its own. Similar use could be
so important in dealing with treatment of cystic ®brosis, do not made of the properties of adenoviruses of subgenus H ; thus,
appear to have CAR available (Walters et al., 1999 ; Zabner et Ad41 binds selectively to differentiated gut enterocytes (Croyle
al., 1997), and the same is true of some primary tumours (Li et et al., 1998 b). Viruses of subgroup D infect primary central
al., 1999 c ; Miller et al., 1998). To permit targetted gene nervous system cells more efficiently than do subgroup C
delivery, therefore, novel strategies need to be developed and a (Chillon et al., 1999). One other strategy to ablate the binding
number of vectors have been constructed in an attempt to do properties of the ®bre is to use ®breless virus. In this case,
this. Some of these have bispeci®c conjugates that can ablate infectivity is reduced drastically but entry to cells can still be
the normal receptor binding and introduce novel tropisms, e.g. achieved at low levels via the RGD motif in the penton base
by using growth factor receptor (Miller et al., 1998), CD3 (Legrand et al., 1999 ; Von Seggern et al., 1999). There is
(Wickham et al., 1997), ®broblast growth factor (Printz et al., additional evidence that virus uptake can be mediated via the
2000), heparin (Wickham et al., 1996) or gastric releasing penton base alone and that the interaction with integrins can lead
peptide (Hong et al., 1999 a). In this way, in¯ammatory vascular to a different route to the nucleus (Hong et al., 1999 b).
endothelial cells exhibiting E selectin can be targetted by Other, more non-speci®c ways of bypassing normal
complexing an anti-selectin E MAb with an anti-FLAG MAb receptor-mediated entry are by transfection with the aid of
and then attaching this dual antibody to a vector expressing the cationic lipids and polymers and by using calcium phosphate
FLAG epitope (Harari et al., 1999). Another, more direct (Alton et al., 1999 ; Campain et al., 1998 ; Croyle et al., 1998
approach has been to incorporate binding motifs a ; Dodds et al., 1999 ; Fasbender et al., 1997, 1998 ; Lee et al.,

CFIF
W. C. Russell
 1999 a ; Qiu et al., 1998). Although not strictly a vectorial
procedure, the ability of adenovirus to enter cells efficiently has
been exploited by condensing a plasmid with polyethyl-enimine
and then complexing with psoralen-inactivated adeno-virus
1999). Another strategy, involving the incorporation into
the transgene of tissue ` silencer ' elements in conjunction
(Baker et al., 1997 ; Bischof et al., 1999 ; Edgell et al., 1998). A
with ubiquitous promoters to drive tissue-speci®c
variation, with a simpler technique involving the ability of the
expression in neuronal cells, has been implemented
virus mu peptide to package and deliver DNA to the nucleus
successfully (Millecamps et al., 1999).
with the aid of liposomes, has also been developed recently
From the above discussion, it will be evident that, by
(Murray et al., 2000). A peptide derived from adeno-virus ®bre
modi®cation of receptor-binding characteristics coupled with a
has recently been shown to target to the nucleolus and may
judicious use of promoters}enhancers, much more effective
provide a vehicle for gene delivery (Zhang et al., 1999). A
delivery and expression of a transgene can be achieved.
combinatorial approach, using adenovirus transduct-ion and
plasmid transfection as well as lipofection, can also lead to
enhancement of expression (Dunphy et al., 1999). Application of adenovirus vectors
The ability of adenovirus vectors to deliver and express
Ensuring expression of the transgene. Assuming that there is effective genes at high yields, especially in vitro, has been amply
delivery of the transgene to the host cell, the next step in successful demonstrated over the last 15 years and has been well
expression depends greatly on the efficiency of promoter and documented. However, the supremacy of the immune response
enhancer elements. While the backbone E1A promoter may be in vivo has been a limiting factor in the practical development of
sufficient in some cases, heterologous promoters have often been vectors. Thus, it is critical to control or to suppress the immune
inserted to provide better ex-pression. These promoters may be response to the vector and the transgene where persistent
speci®c to tissues or cells such as prostate (Rodriguez et al., 1997 ; expression is needed in the case of supplementing the activity of
Yu et al., 1999), muscle (Acsadi et al., 1998 ; Amal®tano et al., a faulty gene. In contrast, cancer therapy may bene®t from the
1999), lung (Harrod et al., 1998), liver (Sandig et al., 1996) or induction of a vigorous immune response. With a better
oligodendrocyte (Horwitz et al., 1997). More ubiquitous promoters, understanding of the molecular and immuno-logical factors that
such as those derived from the immediate-early human operate in vivo, great strides have been made over the last few
cytomegalovirus (HCMV) promoter, Rous sarcoma virus (RSV) or years in the construction of more effective vectors, and some of
the phosphoglycerate kinase gene (Millecamps et al., 1999), have these will be described here.
also been inserted. The HCMV promoter has been utilized
extensively, since it is relatively powerful and requires the insertion Gene therapy in the treatment of cancer
of only a small number of bases (380 bp). An interesting A variety of techniques have been adopted to suppress or
comparison (Sallenave et al., 1998) was made of the effectiveness eliminate tumour cells, the approach in each case depending
of a series of adenovirus vectors with three different promoters, the largely on the type and location of the tumour. Most of the
adeno-virus MLP, HCMV and MCMV (mouse cytomegalovirus) vectors have been developed by using in vitro models of the
promoters. In vitro studies using human and rat alveolar and tumours and then by testing in the appropriate animal,
pulmonary cells showed the MCMV promoter to be very much sometimes using tumour-transplantation methodologies. A
more effective than MLP, with the HCMV promoter being number of these approaches have now moved on to clinical
intermediate. A similar result was obtained in vivo using rat lung. It trials. The therapies in use can be divided into three groups :
was also noted that, although the HCMV promoter was much better (i) tumour suppressors, (ii) oncolytic and sensitizing drug
than the endogenous E1A promoter in inducing persistent therapy and (iii) vaccines.
expression, this was also in¯uenced by the nature of the vector
backbone (Armentano et al., 1997). Careful optimization of the (i) Tumour suppressors. Mutations in the p53 gene that lead to
CMV promoter}enhancer sequences can also lead to signi®cant loss of function have been implicated in the development of a
improvements in expression (Massie et al., 1998). The RSV wide variety of human tumours (Wills et al., 1994). To remedy
promoter seems to function well in liver (Gorziglia et al., 1999) and this defect and to induce apoptosis in the tumour cells, a number
has been used in a vector in phase I clinical trials on mesothelioma of vectors incorporating wild-type p53 have been constructed.
patients (Sterman et al., 1998) and effectively in ovine vectors Initial studies, using appropriate tumour cell lines and then
(Hofmann et al., 1999). The incorporation of endogenous promoters animal model systems, demonstrated the efficacy of these
and enhancers into the vector construct can also improve the approaches with anaplastic thyroid cancer (Blagosklonny et al.,
transgene expression and persistence dramatically, as demonstrated 1998), human malignant gliomas (Cirielli et al., 1999 ; Li et al.,
for apolipo-protein A (De Geest et al., 2000). A similar result was 1999 a) and breast cancer (Putzer et al., 1998). In some cases,
demonstrated for endogenous liver promoters (Pastore et al., combination with an immunomodulatory gene such as IL-2
(Putzer et al., 1998) or with a cytotoxic drug such as adriamycin
(Blagosklonny et al., 1998) proved more effective. Clinical
trials testing the efficacies of these vectors in the treatment of
lung, head, neck and liver cancers are under way. However, one
CFIG of the problems inherent in this approach is the
has been explored in a mouse model (Anderson et al., 1998). More recent
investigations have illustrated dramatically the importance of the
ARF±mdm2±p53 interactions in regulating p53 expression, and the discovery
desirability of efficient targetting of the vector, and direct
of a range of mutations in ARF and in related transcription factors such as
inoculation via the hepatic artery for treatment of liver cancer
Twist (Maestro et al., 1999) in a variety of tumours (Eischen et al., 1999 ;
Sanchez-Cespedes et al., 1999 ; Taniguchi et al., 1999) have Review: Update on adenovirus vectors
suggested that vectors expressing other components of the p53
pathway might be equally productive. Other approaches to
since administration of ganciclovir will be followed by its
induce apoptosis have been explored, and these have involved
phosphorylation by HSV-tk to yield a chain terminator for DNA
inhibiting cyclin-dependent kinases, which are critical to the cell
synthesis. This technique has been used in situ for head and neck
cycle. Indeed, one of these, p16, has been shown to be defective
tumours (Goebel et al., 1998) and phase 1 trials have been
in many human tumour cell lines, and vectors expressing p21,
completed for malignant mesothelioma (Sterman et al., 1998)
p15 and p16 have shown promise in model tumour systems
and for prostate carcinoma (Herman et al., 1999). A vector with
(Tsao et al., 1999). The use of vectors expressing proapoptotic
both E1 and E4 deleted has been constructed recently for
proteins such as Fas ligand and caspase-8 has been limited by
delivery of the HSV-tk gene (Lanuti et al., 1999), apparently
the difficulty of production of the vectors. However, the
with better results. Cytosine deaminase (CD) has also been used
construction of complementing cell lines expressing adenovirus
in colon carcinoma as a suicide gene in an adenovirus vector,
E3 14.7k or the poxvirus serpin gene CrmA has recently
with co-administration of 5-¯uorocytosine (Hirschowitz et al.,
provided a route for good production and exploration of their
1995). A fusion gene of HSV-tk and CD has also been inserted
properties (Bruder et al., 2000). Another intriguing strategy to
into vectors to treat prostate carcinoma (Blackburn et al., 1999).
encourage apoptosis has been the use of ribozymes such as anti-
A trimodal therapy involving a double suicide vector of HSV-tk
H-ras for bladder cancer (Irie et al., 1999), anti-Bcl-2 for
and CD combined with radiotherapy has provided very effective
prostate cancer (Dorai et al., 1999) and anti-HER2 for breast
tumour reduction in a cervical carcinoma xenograft model
cancer (Suzuki et al., 2000).
(Rogulski et al., 2000). Another interesting approach has been to
co-administer with the CD vector another containing the gene
(ii) Oncolytic and sensitizing drug therapy. Direct application of
for uracil phosphoribosyl-transferase. In a rat tumour model, this
wild-type adenovirus to tumours was attempted soon after their
increased the sensitivity of the system very signi®cantly
discovery in the 1950s, but only local effects were
(Adachi et al., 2000). A so-called ` bystander effect ' appears to
demonstrated, and it was not until 1996 that it was claimed that
amplify the cytotoxicity with these therapies (Zhang & DeGroot,
an adenovirus that had a mutation in E1B 55k would replicate
2000).
selectively in p53-defective tumour cells (Bischoff et al., 1996)
and therefore could function as an oncolytic virus. This led to Vectors containing suicide genes have been complemented
the commercial development of the mutant (Onyx with vectors delivering a variety of cytokines in an attempt to
boost the in situ cytotoxicity (Cao et al., 1998).
15) and, although a number of publications have shown
In all these systems, it was evident that the efficacy of
convincingly that the original premises did not hold up (Hay
treatment could be enhanced considerably if targetting could
et al., 1999 a ; Ridgway et al., 1997 ; Vollmer et al., 1999), it
be made more speci®c, and tissue-speci®c promoters have
is still claimed that the virus is effective by intravenous
been incorporated into vectors to facilitate this (Hart, 1996).
administration in treatment of some tumours (Heise et al.,
Applications to breast cancer (Manome et al., 1994), liver
1999). Combination with standard chemotherapy also looks
cancer (Kaneko et al., 1995) and melanoma (Siders et al.,
promising (Heise et al., 1997), and phase 3 clinical trials for
1998 a) have been described. However, although speci®city
head and neck tumours using Onyx are under way.
was obtained in vivo, results were quite often disappointing
Moreover, a recent study using the E1B-deleted virus in
because of relatively low promoter activity. In an attempt to
parallel with an adenovirus vector expressing IL-2 has shown
improve this, an ingenious approach has been developed
complete regression of a p53-de®cient pancreatic tumour in
using the Cre-lox system and a tumour-speci®c antigen in a
a mouse model (Motoi et al., 2000).
model system, with promising results (Kijima et al., 1999).
Another strategy that has been developed for the selective
elimination of tumour cells is to deliver a prodrug enzyme via a Reference should also be made to the importance of
vector into target cells and then to administer a non-toxic drug retaining the E1A genes in vectors in these cases because of
that can be converted into a cytotoxic agent in situ (Crystal, their ability to promote p53 transcription and to enhance the
1999). Herpes simplex virus thymidine kinase (HSV-tk) has sensitivity of tumour cells to cytotoxic agents (Brader et al.,
been used extensively as a so-called suicide gene, 1997 ; Cook et al., 1999 ; Wildner et al., 1999) and to
radiation therapies (Martin-Duque et al., 1999).

(iii) Vaccines. Strategies to invoke anti-tumour cell immunity

have been explored using vectors by introducing a variety of
immunomodulatory genes and}or tumour-speci®c antigens.
Many cytokines can be effective in this way : thus, IL-2 can
induce CTLs, enhance NK cell activity and promote tumour-
in®ltrating lymphocytes, and high doses of recombinant IL-2
and IL-2-expressing vector have been successful in reducing
tumour load in animal models. Nevertheless, problems of
toxicity have become apparent (Toloza et al., 1996) and later

CFIH
W. C. Russell
developments concentrated on the more-directed delivery of
other cytokines such as IL-12 (Bramson et al., 1996 ; Gambotto
et al., 1999 ; Mazzolini et al., 2000 ; Siders et al., 1998 b),
disease caused by mutations in the CFTR gene that result in
sometimes in combination with IL-2 (Addison et al., 1998) and
poor chloride ion conductance and increased sodium ion uptake.
tumour antigen (Hirschowitz & Crystal, 1999). Intratumoural
Since the defect is manifested primarily in the lung,
injection of vectors expressing IL-2 or IL-12 in combination
adenoviruses, with their apparent propensity to infect this organ,
with a vector expressing lymphotactin have been successful in a
would appear to be the vector of choice to deliver the
murine breast cancer model system (Emtage et al., 1999). The
therapeutic gene. However, a multiplicity of investigations have
recognition that many tumours exhibit tumour-speci®c antigens
demonstrated that there are many barriers to successful
encouraged the use of vectors expressing these antigens as a
transgene expression. Among these is the inability of the
means of boosting anti-tumour immunity and led to a trial on
standard Ad2}5 vectors to infect the well-differentiated airway
metastatic melanomas using adenovirus vectors expressing
epithelial cells and alveolar macrophages, mainly due to the lack
either MART 1 or gp100 melanoma antigens (Rosenberg et al.,
of CARs (Kaner et al., 1999 ; Pickles et al., 1998 ; Walters et
1998). The results demonstrated that high doses of the vector
al., 1999). Attempts have been made to improve uptake by
could be administered safely, but that the immune response to
using cationic lipids and calcium phosphate co-precipitates
the vector ablated any longer-term anti-tumour response. In the
(Alton et al., 1999 ; Fasbender et al., 1997, 1998 ; Lee et al.,
case of a model system of colorectal cancer (Li et al., 1997),
1999 a). A more promising approach has been the construction
expression of a tumour antigen via an adenovirus vector resulted
of a chimeric adenovirus vector with serotype 17 ®bre, which
in signi®cant tumour regression and induction of immunity to
displays an increased binding to airway epithelial cells (Zabner
further tumour challenge. Another promising approach to
et al., 1999). Another barrier to successful transgene expression
boosting anti-tumour immunity was modelled on the ability of
was the speci®c pulmonary-associated T helper cell response
dendritic cells to present antigens effectively in vivo. It has been
(van Ginkel et al., 1997), and later studies have shown the
proposed that one useful strategy would be to isolate dendritic
importance of retaining E4 genes in the vector to counteract this
cells from a patient and then to modify them by infection with
assault (Armentano et al., 1997 ; Chirmule et al., 1998 ; Lusky
an adenovirus vector expressing the appropriate tumour antigen,
et al., 1999 ; Yew et al., 1999). A further, natural in-hibitor is
followed by readministration to the patient in combination with
derived from the ability of airway epithelia to release
standard therapies (Crystal, 1999). Studies with human dendritic
antimicrobial peptides with anti-adenovirus properties (Gropp et
cells have shown that dendritic cells modi®ed with adenovirus
al., 1999), and a study of bronchoalveolar lavage ¯uid has
vectors are not perturbed in terms of their maturation and
indicated the presence of adenovirus inhibitors in addition to
function (Rea et al., 1999 ; Zhong et al., 1999) and signi®cant
neutralizing antibodies (Bastian & Bewig, 1999). Many of these
reduction of lung metastases was achieved with murine
factors probably account for the results from human trials with
dendritic cells (Wan et al., 1999). A murine dendritic cell model
earlier vectors in which, although efficacy of transfer was
of melanoma also showed a signi®cant boost to anti-melanoma
demonstrated, the overall efficiency was low and expression of
immunity using an adenovirus vector (Tuting et al., 1999).
the transgene was of relatively short duration (Zuckerman et al.,
Considerable improvement in the efficiency of targetting of the
1999). It remains to be seen whether the later vectors can
vector to dendritic cells can be obtained by using a bispeci®c
improve the efficiency and persistence of the transgene.
antibody to redirect the virus to CD40 receptors on the cells
(Tillman et al., 1999). This technique also facilitated the Considerable effort using adenovirus vectors has been
maturation of the cells and thereby enhanced their immuno- devoted to trying to repair the gene defects responsible for
stimulatory characteristics. Vectors expressing CD40 ligand muscular dystrophy. In a mouse model system, vectors encoding
have also been introduced directly into tumour cells and dystrophin can be delivered to muscle ®bres with some
facilitate antigen presentation (Crystal, 1999). Other strategies improvement of function, but this is only transient, because of a
to do this involved the vector expression of granulocyte± potent immune response against the vector and transgene (Yang
macrophage colony-stimulating factors (Ozawa et al., 1999) and et al., 1998 ; Yuasa et al., 1998). Attempts to circumvent this by
increasing the tolerogenicity of the dendritic cells with a vector using improved vectors (Kumar-Singh & Chamberlain, 1996)
encoding TGF b1 (Lee et al., 1999 b). A recent study has shown and utrophin (a homologue of dystrophin) (Gilbert et al., 1999)
that dendritic cells can be infected with a vector independent of showed better transgene persistence. One of the barriers to
their CAR status and that the cells can elicit the appropriate effective gene transfer relates to the lack of receptors for the
CTL responses (Linette et al., 2000). vectors being used, and a vector with a polylysine-modi®ed
®bre improved uptake into muscle cells signi®cantly (Bouri et
al., 1999). It has been shown that the immune response in the
muscle ®bre cells appears to be mediated via dendritic cells,
Gene therapy for genetic diseases and it has been suggested that AAV vectors may be better
Cystic ®brosis is a relatively common, inherited, recessive vehicles for delivery of the transgene (Jooss et al., 1998 c). A
canine model system has also demonstrated the signi®cance of
the immune response, since treatment with cyclosporin after
adenovirus delivery of

CFII
dystrophin gives a more sustained improvement of muscle Review: Update on adenovirus vectors
function (Howell et al., 1998).
A number of vectors have been constructed to alleviate
gene defects in other tissues, and similar problems in was made by the observation that vector expression of an
terms of delivery have been encountered. inhibitor of NF-jB inhibited the production of TNFa in
macrophages (Foxwell et al., 1998), and later investigations
Supplementary therapy showed that inhibition of NF-jB was accompanied by the
inhibition of proin¯ammatory cytokines, but not the main
With an increasingly ageing population, therapy for human
in¯ammatory mediators like IL-10 (Bondeson et al., 1999 a,
neurodegenerative diseases such as Parkinson's offers a major
b). These results therefore pinpointed the important role of
challenge. Adenoviruses, with their ability to infect post-mitotic
NF-jB in in¯ammation and as a therapeutic target.
cells coupled with a potentially high transduction efficiency and
Nevertheless, it should be pointed out that TNF a production
low pathogenicity in the immunologically privileged site of the
did not appear to be NF- jB-dependent in some other cells,
central nervous system, should provide effective vectors for
such as monocytes (Hayes et al., 1999).
neuronal gene therapy. Two major strategies have been
examined for delivering therapeutic genes. One involves direct Other applications
intracerebral injection of the vector and the other uses ex vivo
gene therapy, where cells can be modi®ed in vitro by vector Adenoviruses have been useful vectors for the production of
infection and then transplanted into the relevant areas of the a number of proteins for more-detailed molecular analysis, and
brain (Barkats et al., 1998). Neuro-progenitor cells (Fisher, the reader is referred to the very comprehensive review by Hitt
1997) and human astrocytes (Ridet et al., 1999) can apparently et al. (1997). Adenovirus vaccines have been tested thoroughly
provide autologous cellular vehicles for ex vivo modi®cation for safety, as a result of problems with adenovirus respiratory
and expansion. A tetracycline-regulated adenovirus vector disease in the American military (Chanock et al., 1966), and this
expressing tyrosine hydroxylase, a rate-limiting enzyme in the has facilitated the development of recombinant adenovirus
synthesis of dopamine, has shown considerable promise in vaccines for human immunode®ciency virus (Bruce et al.,
1999) and rabies virus (Matthews et al., 1999 ; Yarosh et al.,
model systems using the ex vivo technique (Corti et al., 1999 a,
1996). A comprehensive review of earlier vaccine develop-
b). In an attempt to utilize adenovirus vectors to alleviate
ments is available (Graham & Prevec, 1992).
Huntington's disease, a con-struct expressing brain-derived
neurotrophic factor was found to give promising results in a rat This review has only touched on a relatively small
model system (Bemelmans et al., 1999). Reference has also number of the applications of vector technology and is
been made above to the use of neuronal silencer elements in certainly not comprehensive, but is illustrative of the
adenovirus constructs in restricting expression to neuronal cells,
considerable progress that has been made ; Table 2
provides further examples of applications.
with the expectation that such a strategy would avoid the side-
effects due to ectopic expression of transgenes (Millecamps et Safety considerations
al., 1999).
The protocols for developing a virus vector for clinical use
Over the last few years, there has been considerable progress
invariably employ a series of steps that begin with a study of
in our understanding of arthritic diseases and the role that
infection of appropriate cells in vitro, followed by application to
cytokines play in invoking the in¯ammatory processes that
an animal model system of some kind. This latter step can bring
occur in joints and synovial ¯uids. A major discovery has been
to light some of the problems inherent in delivery to the site of
the role that TNFa plays in the induction of rheumatoid arthritis,
interest and in the expression and persistence (where that is
amply demonstrated by the success of clinical trials with TNF
required) of the transgene. Very often, the efficiency of delivery
antibody (Maini et al., 1999) and TNF receptor (Franklin, 1999).
has been found to be poor and the expression of the transgene to
Adenovirus vectors have been extremely useful in unravelling
be only transient, although some of these effects may be a
the signi®cance of some of the cytokines and their pathways in
function of a different distribution of receptors in the model
this disease process. Thus, direct delivery of TNF a receptor and
systems. With a greater understanding of the nature of the
cytokine IL-1 using adenovirus vectors has shown synergistic
responses in vivo, many of these problems can be alleviated to
bene®ts, both direct and distal, in a rat model system
some extent, as described above, by constructing vectors that
(Ghivizzani et al., 1998). Similarly, TNFa receptor showed
improve targetting and minimize immune responses to both the
signi®cant synergy with an Epstein±Barr virus homologue of
vector and the transgene (Miller & Whelan, 1997). Nevertheless,
IL-10 in an animal model (Kim et al., 2000 ; Lechman et al.,
in the course of in vivo studies, it has become apparent that there
1999). Use of a vector expressing IL-4 has demonstrated that
can be acute injury and in¯ammation of infected tissues quite
this cytokine can provide considerable protection for cartilage
independent of the possibility of helper virus replication. This
from in¯ammatory processes (Lubberts et al., 1999). In contrast,
was particularly apparent in the liver (Lieber et al., 1997), where
vector delivery of IL-12 accelerated the disease process (Parks
it was shown that a vector can induce production of chemokines
et al., 1998). A signi®cant advance
within 1 h, even with psoralen-inactivated virus (Muruve et al.,
1999). This was followed by an in¯ux of neutrophils into mouse
liver and, with

CFIJ
W. C. Russell

Table 2. Some recent vector applications

APC, Adenomatous polyposis coli ; GM±CSF, granulocyte±macrophage colony-stimulating
factor ; PKC, protein kinase C.

Site/disease Transgene(s) Reference

Tumours
Prostate p16 Steiner et al. (2000)
PKC Fujii et al. (2000)
Cytolytic virus Rodriguez et al. (1997)
Fas ligand Hedlund et al. (1999)
Colon GM±CSF}IL-2 Diaz et al. (1998)
CD suicide gene Topf et al. (1998)
APC Shih et al. (2000)
Lymphotactin-suicide Ju et al. (2000)
Cervix p21 Tsao et al. (1999)
Papillomavirus p21 He et al. (2000)
Ovary Stomatostatin receptor Rogers et al. (1999)
Cytolytic virus}IL-6 Rancourt et al. (1999)
Endometrium p53}p21 Ramondetta et al. (2000)
Gliomas Caspase-3}Fas ligand Shinoura et al. (2000)
Carcinoembryonic antigen-producing cancer HSV-tk Kijima et al. (1999)
Other conditions
Autoimmune diabetes Adenovirus E3 von Herrath et al. (1997)
Glycogen storage disease II Glucosidase Amal®tano et al. (1999)
CNS conditions (via astrocytes) Ridet et al. (1999)
Factor VIII de®ciency Factor VIII Balagu et al. (2000)
OTC de®ciency OTC Batshaw et al. (1999)
Liver graft Bcl-2 Bilbao et al. (1999 a)
Leishmania infection IL-12 Gabaglia et al. (1999)
Tay±Sachs disease Hexosaminidases Guidotti et al. (1999)
Motor neurone disease Neurotrophic factor Haase et al. (1999)

high virus inputs, to hepatic necrosis and apoptosis. It seems previously that host responses to vector were very
evident that the innate system, possibly via NF- jB mediation, is dependent on host genotype, and this unfortunate incident
a major player that must be taken into account even with so- emphasizes the need to understand the basic factors that
called gutless vectors, and consideration could therefore be control host± vector responses.
given, in appropriate cases, to concomitant introduction of a It should be noted that there have been a number of
vector that expresses inhibitors of this pathway. This problem reports of in¯ammatory responses in animals, not only
has been highlighted by the recent tragic death of a patient following vector administration to the liver. Thus, airway
undergoing adenovirus vector therapy for a defect in the neurogenic in¯ammation was apparent in a rat cystic
ornithine transcarbamylase (OTC) pathway. The preliminary ®brosis model system, but this could be alleviated by
enquiries appear to point to a massive activation of innate pharmacological methods (Piedimonte et al., 1997).
immunity followed by systemic in¯ammation (Marshall, 1999). A signi®cant improvement was seen by utilizing gutless
It may be signi®cant that a large dose of virus was given in an vectors in a rat model system for leptin delivery, where liver
effort to get enough functioning OTC genes into the liver. toxicity, in¯ammation and cellular in®ltration were reduced
Apparently, this dramatic reaction had not been noted before, signi®cantly compared with an E1-deleted vector (Morsy et al.,
and it was suggested that this response was an unusual one 1998). However, the problems and considerable costs of scaling
related to this particular individual's genetic repertoire. How- up, puri®cation and production of these vectors are not
ever, it has now also come to light that a substantial number of insigni®cant. The need for careful evaluation of long-term
adverse effects had not been reported adequately in some in¯ammatory responses in human therapy has been underlined
clinical trials. These events highlight the necessity to carry out further by the report of chronic brain in¯ammation following the
trials using monitoring procedures that have the con®dence of successful inhibition of a glioma using cytotoxic gene therapy
the public as well as all of the parties that are directly involved. (Dewey et al., 1999). In this report, persistence and expression
As indicated above, use of animal model systems had indicated of the HSV-tk gene could be found in the brain

CFJA
 Review: Update on adenovirus vectors

some 3 months later and co-incident with the presence of a

variety of in¯ammatory markers. In contrast, a study examining References
the toxicity associated with suicide gene therapy in prostate Acsadi, G., O'Hagan, D., Lochmuller, H., Prescott, S., Larochelle, N.,
cancer found that toxicity was not a major factor, even with Nalbantoglu, J., Jani, A. & Karpati, G. (1998). Interferons impair early
multiple applications (Shalev et al., 2000). The importance of transgene expression by adenovirus-mediated gene transfer in muscle
cells. Journal of Molecular Medicine 76, 442±450.
the route of vector delivery has been demonstrated by the
Adachi, Y., Tamiya, T., Ichikawa, T., Terada, K., Ono, Y., Matsumoto,
®nding that vascular in¯ammation occurred in a rabbit vascular
K., Furuta, T., Hamada, H. & Ohmoto, T. (2000). Experimental gene
model when delivery was by the intraluminal route, but was therapy for brain tumors using adenovirus-mediated transfer of cytosine
greatly reduced when the adventitial route was used (Schneider deaminase gene and uracil phosphoribosyltransferase gene with 5-
et al., 1999). Another, perhaps surprising ®nding in a porcine ¯uorocytosine. Human Gene Therapy 11, 77±89.
model was that direct myocardial administration of a vector Addison, C. L., Bramson, J. L., Hitt, M. M., Muller, W. J., Gauldie, J. &
proved successful without any apparent in¯ammatory responses, Graham, F. L. (1998). Intratumoral coinjection of adenoviral vectors
although the experiment was continued for only 28 days and expressing IL-2 and IL-12 results in enhanced frequency of regression
therefore longer-term chronic toxicity was not assessed (Patel et of injected and untreated distal tumors. Gene Therapy 5, 1400±1409.
al., 1999). A study in sheep, in contrast, led to the conclusion Alton, E. W., Stern, M., Farley, R., Jaffe, A., Chadwick, S. L., Phillips, J.,
Davies, J., Smith, S. N., Browning, J., Davies, M. G., Hodson, M. E.,
that administration of adenovirus vectors in utero was
Durham, S. R., Li, D., Jeffery, P. K., Scallan, M., Balfour, R., Eastman, S. J.,
accompanied by in¯ammatory and ®brotic responses (Iwamoto Cheng, S. H., Smith, A. E., Meeker, D. & Geddes, D. M. (1999).
et al., 1999). Cationic lipid-mediated CFTR gene transfer to the lungs and nose of
patients with cystic ®brosis : a double-blind placebo-controlled
trial. Lancet 353, 947±954.
Amal®tano, A., Hauser, M. A., Hu, H., Serra, D., Begy, C. R. &
Future developments Chamberlain, J. S. (1998). Production and characterization of
Adenovirus vectors have been in vogue for over 15 years and improved adenovirus vectors with the E1, E2b, and E3 genes
deleted. Journal of Virology 72, 926±933.
the early enthusiasm in their therapeutic application has now
Amal®tano, A., McVie-Wylie, A. J., Hu, H., Dawson, T. L., Raben, N.,
been tempered by the realization that there is, as yet, no
Plotz, P. & Chen, Y. T. (1999). Systemic correction of the muscle
evidence of signi®cant clinical success. As noted above, the disorder glycogen storage disease type II after hepatic targeting of a
protective responses of the host had been underestimated and it modi®ed adenovirus vector encoding human acid-alpha-glucosidase.
is only in the last few years has there been some understanding Proceedings of the National Academy of Sciences, USA 96, 8861±8866.
of the complexities of virus expression and the host responses. Anderson, C. W., Young, M. E. & Flint, S. J. (1989). Characterization of
In this connection, it should be noted that most of the clinical the adenovirus 2 virion protein, mu. Virology 172, 506±512.
trials presently under way have utilized the earlier ®rst- and Anderson, S. C., Johnson, D. E., Harris, M. P., Engler, H., Hancock,
second-generation vectors. This rather nega-tive scenario has W., Huang, W. M., Wills, K. N., Gregory, R. J., Sutjipto, S., Wen, S. F.,
been compounded by the news of the death of the patient Lofgren, S., Shepard, H. M. & Maneval, D. C. (1998). p53 gene therapy
in a rat model of hepatocellular carcinoma : intra-arterial delivery of a
undergoing vector therapy. Nevertheless, great progress has
recombinant adenovirus. Clinical Cancer Research 4, 1649±1659.
been made and lessons should have been learned. Principal
Angeletti, P. C. & Engler, J. A. (1998). Adenovirus preterminal protein
among these is the necessity to explore the many factors
binds to the CAD enzyme at active sites of viral DNA replication on the
involved in vector administration in depth before venturing into nuclear matrix. Journal of Virology 72, 2896±2904.
clinical application. Thus, as well as trying to understand the Arany, Z., Newsome, D., Oldread, E., Livingston, D. M. & Eckner,
molecular nature of both the innate and active immune R. (1995). A family of transcriptional adaptor proteins targeted by
responses in the context of genetic background, the target organ, the E1A oncoprotein. Nature 374, 81±84.
the route of administration etc., the possibility of Armentano, D., Zabner, J., Sacks, C., Sookdeo, C. C., Smith, M. P., St
pharmacological and vectorial supplements will need to be George, J. A., Wadsworth, S. C., Smith, A. E. & Gregory, R. J. (1997).
examined. This, of course, implies that much longer de- Effect of the E4 region on the persistence of transgene expression
velopment protocols will be required and much greater patience from adenovirus vectors. Journal of Virology 71, 2408±2416.
will need to be exercised by the institutions and companies that Arnberg, N., Edlund, K., Kidd, A. H. & Wadell, G. (2000). Adenovirus
invest in this technology. Nevertheless, adenovirus vectors offer type 37 uses sialic acid as a cellular receptor. Journal of Virology 74, 42±48.
great promise and should not be abandoned in the light of these Badrichani, A. Z., Stroka, D. M., Bilbao, G., Curiel, D. T., Bach, F.
H. & Ferran, C. (1999). Bcl-2 and Bcl-XL serve an anti-
early mishaps in clinical trials.
in¯ammatory function in endothelial cells through inhibition of NF-
kappaB. Journal of Clinical Investigation 103, 543±553.
Baker, A., Saltik, M., Lehrmann, H., Killisch, I., Mautner, V., Lamm, G.,
The author is indebted to his colleagues, who supplied advice on
Christofori, G. & Cotten, M. (1997). Polyethylenimine (PEI) is a simple,
both the text and the compilation of the ®gures : viz. Rick Randall,
inexpensive and effective reagent for condensing and linking plasmid
Ron Hay, Martin Ryan, Bernie Precious and Alex Houston. Thanks
DNA to adenovirus for gene delivery. Gene Therapy 4, 773±782.
also to David Matthews, who supplied the basis of Fig. 1, and Phil
Gallimore and Andy Turnell for Fig. 3. Balachandran, S., Roberts, P. C., Kipperman, T., Bhalla, K. N.,
Compans, R. W., Archer, D. R. & Barber, G. N. (2000). Alpha}beta

CFJB
W. C. Russell
 interferons potentiate virus-induced apoptosis through activation of
the FADD}caspase-8 death signaling pathway. Journal of Virology
74, 1513±1523.
Balagu, C., Zhou, J., Dai, Y., Alemany, R., Josephs, S. F., Blackburn, R. V., Galoforo, S. S., Corry, P. M. & Lee, Y. J. (1999).
Andreason, G., Hariharan, M., Sethi, E., Prokopenko, E., Jan, H., Adenoviral transduction of a cytosine deaminase}thymidine kinase
Lou, Y. C., Hubert-Leslie, D., Ruiz, L. & Zhang, W. W. (2000). fusion gene into prostate carcinoma cells enhances prodrug and
Sustained high-level expression of full-length human factor VIII radiation sensitivity. International Journal of Cancer 82, 293±297.
and restoration of clotting activity in hemophilic mice using a Blagosklonny, M. V., Giannakakou, P., Wojtowicz, M., Romanova,
minimal adenovirus vector. Blood 95, 820±828. L. Y., Ain, K. B., Bates, S. E. & Fojo, T. (1998). Effects of p53-expressing
Bals, R., Weiner, D. J., Moscioni, A. D., Meegalla, R. L. & Wilson, J. M. adenovirus on the chemosensitivity and differentiation of anaplastic thyroid
(1999). Augmentation of innate host defense by expression of a cathelicidin cancer cells. Journal of Endocrine Metabolism 83, 2516±2522.
antimicrobial peptide. Infection and Immunity 67, 6084±6089. Boivin, D., Morrison, M. R., Marcellus, R. C., Querido, E. &
Barkats, M., Bilang-Bleuel, A., Buc-Caron, M. H., Castel-Barthe, M. N., Branton, P. E. (1999). Analysis of synthesis, stability,
Corti, O., Finiels, F., Horellou, P., Revah, F., Sabate, O. & Mallet, J. phosphorylation, and interacting polypeptides of the 34-kilodalton
(1998). Adenovirus in the brain : recent advances of gene therapy for product of open reading frame 6 and of the early region 4 protein of
neurodegenerative diseases. Progress in Neurobiology 55, 333±341. human adenovirus type 5. Journal of Virology 73, 1245±1253.
Bastian, A. & Bewig, B. (1999). Inhibition of adenovirus-mediated gene Bondeson, J., Browne, K. A., Brennan, F. M., Foxwell, B. M. &
transfer by bronchoalveolar lavage ¯uid. Gene Therapy 6, 637±642. Feldmann, M. (1999 a). Selective regulation of cytokine induction
by adenoviral gene transfer of IjBa into human macrophages :
Batshaw, M. L., Wilson, J. M., Raper, S., Yudkoff, M. & Robinson,
lipopoly-saccharide-induced, but not zymosan-induced,
M. B. (1999). Recombinant adenovirus gene transfer in adults with
proin¯ammatory cyto-kines are inhibited, but IL-10 is nuclear
partial ornithine transcarbamylase de®ciency (OTCD). Human factor-jB independent. Journal of Immunology 162, 2939±2945.
Gene Therapy 10, 2419±2437.
Bondeson, J., Foxwell, B., Brennan, F. & Feldmann, M. (1999 b).
Bemelmans, A. P., Horellou, P., Pradier, L., Brunet, I., Colin, P. &
Mallet, J. (1999). Brain-derived neurotrophic factor-mediated De®ning therapeutic targets by using adenovirus : blocking NF- jB
protection of striatal neurons in an excitotoxic rat model of inhibits both in¯ammatory and destructive mechanisms in rheumatoid
synovium but spares anti-in¯ammatory mediators. Proceedings of the
Huntington's disease, as demonstrated by adenoviral gene transfer.
National Academy of Sciences, USA 96, 5668±5673.
Human Gene Therapy 10, 2987±2997.
Bouri, K., Feero, W. G., Myerburg, M. M., Wickham, T. J.,
Benihoud, K., Yeh, P. & Perricaudet, M. (1999). Adenovirus vectors for
Kovesdi, I., Hoffman, E. P. & Clemens, P. R. (1999). Polylysine
gene delivery. Current Opinions in Biotechnology 10, 440±447.
modi®cation of adenoviral ®ber protein enhances muscle cell
Benko,% M., Harrach, B. & Russell, W. C. (1999). Adenoviridae. In Virus transduction. Human Gene Therapy 10, 1633±1640.
Taxonomy. Seventh Report of the International Committee on Taxonomy of
Boyer, J. L. & Ketner, G. (2000). Genetic analysis of a potential
Viruses, pp. 227±238. Edited by M. H. V. van Regenmortel, C. M. Fauquet
zinc-binding domain of the adenovirus E4 34k protein. Journal of
& D. H. L. Bishop. San Diego : Academic Press.
Biological Chemistry 275, 14969±14978.
Bennett, E. M., Bennink, J. R., Yewdell, J. W. & Brodsky, F. M. (1999).
Boyer, J., Rohleder, K. & Ketner, G. (1999 a). Adenovirus E4 34k and E4
Cutting edge : adenovirus E19 has two mechanisms for affecting 11k inhibit double strand break repair and are physically associated with the
class I MHC expression. Journal of Immunology 162, 5049±5052.
cellular DNA-dependent protein kinase. Virology 263, 307±312.
Bergelson, J. M., Cunningham, J. A., Droguett, G., Kurt-Jones, E. A.,
Boyer, T. G., Martin, M. E., Lees, E., Ricciardi, R. P. & Berk, A. J.
Krithivas, A., Hong, J. S., Horwitz, M. S., Crowell, R. L. & Finberg, R.
(1999 b). Mammalian Srb}mediator complex is targeted by
W. (1997). Isolation of a common receptor for coxsackie B viruses and
adenovirus E1A protein. Nature 399, 276±279.
adenoviruses 2 and 5. Science 275, 1320±1323.
Brader, K. R., Wolf, J. K., Hung, M. C., Yu, D., Crispens, M. A., van Golen,
Bewley, M. C., Springer, K., Zhang, Y. B., Freimuth, P. & Flanagan,
K. L. & Price, J. E. (1997). Adenovirus E1A expression enhances the
J. M. (1999). Structural analysis of the mechanism of adenovirus
sensitivity of an ovarian cancer cell line to multiple cytotoxic agents through
binding to its human cellular receptor, CAR. Science 286, 1579±1583.
an apoptotic mechanism. Clinical Cancer Research 3, 2017±2024.
Bilbao, G., Contreras, J. L., Gomez-Navarro, J., Eckhoff, D. E.,
Bramson, J. L., Hitt, M., Addison, C. L., Muller, W. J., Gauldie, J.
Mikheeva, G., Krasnykh, V., Hynes, T., Thomas, F. T., Thomas, J.
& Graham, F. L. (1996). Direct intratumoral injection of an
M. & Curiel, D. T. (1999 a). Genetic modi®cation of liver grafts
adenovirus expressing interleukin-12 induces regression and long-
with an adenoviral vector encoding the Bcl-2 gene improves organ
lasting immunity that is associated with highly localized expression
preservation. Transplantation 67, 775±783.
of interleukin-12. Human Gene Therapy 7, 1995±2002.
Bilbao, G., Contreras, J. L., Zhang, H. G., Pike, M. J., Overturf, K.,
Brehm, A., Miska, E. A., McCance, D. J., Reid, J. L., Bannister, A.
Mikheeva, G., Krasnykh, V. & Curiel, D. T. (1999 b). Adenovirus-
J. & Kouzarides, T. (1998). Retinoblastoma protein recruits histone
mediated gene expression in vivo is enhanced by the antiapoptotic
deacetylase to repress transcription. Nature 391, 597±601.
bcl-2 gene. Journal of Virology 73, 6992±7000.
Bruce, C. B., Akrigg, A., Sharpe, S. A., Hanke, T., Wilkinson, G.
Bischof, J., Vietor, I., Cotten, M. & Huber, L. A. (1999). Transient
W. G. & Cranage, M. P. (1999). Replication-de®cient recombinant
transfection of mammary epithelial cells with a
adenoviruses expressing the human immunode®ciency virus Env
PEI}DNA}adenovirus system. Biological Chemistry 380, 269±273.
antigen can induce both humoral and CTL immune responses in
Bischoff, J. R., Kirn, D. H., Williams, A., Heise, C., Horn, S., Muna, M., mice. Journal of General Virology 80, 2621±2628.
Ng, L., Nye, J. A., Sampson-Johannes, A., Fattaey, A. & McCormick,
F. (1996). An adenovirus mutant that replicates selectively in p53- Bruder, J. T. & Kovesdi, I. (1997). Adenovirus infection stimulates
de®cient human tumor cells. Science 274, 373±376.
the Raf}MAPK signaling pathway and induces interleukin-8
expression. Journal of Virology 71, 398±404.
Bruder, J. T., Appiah, A., Kirkman, W. M., III, Chen, P., Tian, J.,
Reddy, D., Brough, D. E., Lizonova, A. & Kovesdi, I. (2000). Improved
CFJC
Campain, J. A., Matassa, A. A., Felgner, P. L., Barnhart, K. M., Curiel, D. T. &
Harrison, G. S. (1998). Lipid- and adenoviral-mediated gene transfer into AIDS-
Kaposi's sarcoma cell lines. Cancer Gene Therapy 5, 131±143.
production of adenovirus vectors expressing apoptotic transgenes.
Cao, X., Ju, D. W., Tao, Q., Wang, J., Wan, T., Wang, B. M., Zhang, W.
Human Gene Therapy 11, 139±149.
& Hamada, H. (1998). Adenovirus-mediated GM-CSF gene and Review: Update on adenovirus vectors
cytosine deaminase gene transfer followed by 5-¯uorocytosine
adminis-tration elicit more potent antitumor response in tumor-
bearing mice. Gene Therapy 5, 1130±1136. Cook, J. L., Routes, B. A., Walker, T. A., Colvin, K. L. & Routes, J.
Caplen, N. J., Higginbotham, J. N., Scheel, J. R., Vahanian, N., Yoshida, Y.,
M. (1999). E1A oncogene induction of cellular susceptibility to
Hamada, H., Blaese, R. M. & Ramsey, W. J. (1999). Adeno-retroviral
killing by cytolytic lymphocytes through target cell sensitization to
apoptotic injury. Experimental Cell Research 251, 414±423.
chimeric viruses as in vivo transducing agents. Gene Therapy 6, 454±459.
Corti, O., Sabate, O., Horellou, P., Colin, P., Dumas, S., Buchet,
Chakravarti, D., Ogryzko, V., Kao, H. Y., Nash, A., Chen, H., Nakatani,
D., Buc-Caron, M. H. & Mallet, J. (1999 a). A single adenovirus
Y. & Evans, R. M. (1999). A viral mechanism for inhibition of p300 and
vector mediates doxycycline-controlled expression of tyrosine
PCAF acetyltransferase activity. Cell 96, 393±403.
hydroxylase in brain grafts of human neural progenitors. Nature
Chanock, R. M., Ludwig, W., Heubner, R. J., Cate, T. R. & Chu, L. Biotechnology 17, 349±354.
W. (1966). Immunization by selective infection with type 4
Corti, O., Sanchez-Capelo, A., Colin, P., Hanoun, N., Hamon, M. &
adenovirus grown in human diploid tissue cultures. I. Safety and Mallet, J. (1999 b). Long-term doxycycline-controlled expression of
lack of oncogenicity and tests for potency in volunteers. Journal of human tyrosine hydroxylase after direct adenovirus-mediated gene
the American Medical Association 195, 445±452. transfer to a rat model of Parkinson's disease. Proceedings of the
Charles, P. C., Chen, X., Horwitz, M. S. & Brosnan, C. F. (1999). National Academy of Sciences, USA 96, 12120±12125.
Differential chemokine induction by the mouse adenovirus type-1 in Croyle, M. A., Roessler, B. J., Hsu, C. P., Sun, R. & Amidon, G. L.
the central nervous system of susceptible and resistant strains of (1998 a). Beta cyclodextrins enhance adenoviral-mediated gene
mice. Journal of Neurovirology 5, 55±64. delivery to the intestine. Pharmacy Research 15, 1348±1355.
Chen, L., Anton, M. & Graham, F. L. (1996). Production and Croyle, M. A., Stone, M., Amidon, G. L. & Roessler, B. J. (1998 b).
characterization of human 293 cell lines expressing the site-speci®c In vitro and in vivo assessment of adenovirus 41 as a vector for gene
recombinase Cre. Somatic Cell Molecular Genetics 22, 477±488. delivery to the intestine. Gene Therapy 5, 645±654.
Chillon, M., Bosch, A., Zabner, J., Law, L., Armentano, D., Welsh, Croyle, M. A., Walter, E., Janich, S., Roessler, B. J. & Amidon, G. L.
M. J. & Davidson, B. L. (1999). Group D adenoviruses infect (1998 c). Role of integrin expression in adenovirus-mediated gene
primary central nervous system cells more efficiently than those delivery to the intestinal epithelium. Human Gene Therapy 9, 561±573.
from group C. Journal of Virology 73, 2537±2540.
Crystal, R. G. (1999). In vivo and ex vivo gene therapy strategies to
Chiou, S. K. & White, E. (1998). Inhibition of ICE-like proteases treat tumors using adenovirus gene transfer vectors. Cancer
inhibits apoptosis and increases virus production during adenovirus Chemotherapy and Pharmacology 43, S90±S99.
infection. Virology 244, 108±118.
Dales, S. & Chardonnet, Y. (1973). Early events in the interaction
Chirmule, N., Hughes, J. V., Gao, G. P., Raper, S. E. & Wilson, J. of adenoviruses with HeLa cells. IV. Association with microtubules
M. (1998). Role of E4 in eliciting CD4 T-cell and B-cell responses and the nuclear pore complex during vectorial movement of the
to adenovirus vectors delivered to murine and nonhuman primate inoculum. Virology 56, 465±483.
lungs. Journal of Virology 72, 6138±6145.
De Geest, B., Van Linthout, S., Lox, M., Collen, D. & Holvoet, P.
Chirmule, N., Moscioni, A. D., Qian, Y., Qian, R., Chen, Y. & (2000). Sustained expression of human apolipoprotein A-I after
Wilson, J. M. (1999). Fas±Fas ligand interactions play a major role adenoviral gene transfer in C57BL}6 mice : role of apolipoprotein
in effector functions of cytotoxic T lymphocytes after adenovirus A-I promoter, apolipoprotein A-I introns, and human apolipoprotein
vector-mediated gene transfer. Human Gene Therapy 10, 259±269. E enhancer. Human Gene Therapy 11, 101±112.
Chiu, C. Y., Mathias, P., Nemerow, G. R. & Stewart, P. L. (1999). de Moissac, D., Zheng, H. & Kirshenbaum, L. A. (1999). Linkage
Structure of adenovirus complexed with its internalization receptor, of the BH4 domain of Bcl-2 and the nuclear factor kappaB signaling
avb5 integrin. Journal of Virology 73, 6759±6768. pathway for suppression of apoptosis. Journal of Biological
Chroboczek, J., Ruigrok, R. W. H. & Cusack, S. (1995). Chemistry 274, 29505±29509.
Adenovirus ®ber. In The Molecular Repertoire of Adenoviruses, pp. Deryckere, F. & Burgert, H. G. (1996). Tumor necrosis factor alpha
163±200. Edited by W. Doer¯er & P. Bohm. Berlin : Springer. induces the adenovirus early 3 promoter by activation of NF-
Cirielli, C., Inyaku, K., Capogrossi, M. C., Yuan, X. & Williams, J. A. kappaB. Journal of Biological Chemistry 271, 30249±30255.
(1999). Adenovirus-mediated wild-type p53 expression induces apop- de Stanchina, E., McCurrach, M. E., Zindy, F., Shieh, S. Y.,
tosis and suppresses tumorigenesis of experimental intracranial human Ferbeyre, G., Samuelson, A. V., Prives, C., Roussel, M. F., Sherr,
malignant glioma. Journal of Neuroncology 43, 99±108. C. J. & Lowe, S. W. (1998). E1A signaling to p53 involves the p19
Clesham, G. J., Adam, P. J., Proudfoot, D., Flynn, P. D., Efstathiou, S. (ARF) tumor suppressor. Genes & Development 12, 2434±2442.
& Weissberg, P. L. (1998). High adenoviral loads stimulate NF Desterro, J. M., Rodriguez, M. S., Kemp, G. D. & Hay, R. T. (1999).
kappaB-dependent gene expression in human vascular smooth Identi®cation of the enzyme required for activation of the small ubiquitin-
muscle cells. Gene Therapy 5, 174±180. like protein SUMO-1. Journal of Biological Chemistry 274, 10618±10624.
Dewey, R. A., Morrissey, G., Cowsill, C. M., Stone, D., Bolognani, F.,
Dodd, N. J., Southgate, T. D., Klatzmann, D., Lassmann, H., Castro,
M. G. & Lowenstein, P. R. (1999). Chronic brain in¯ammation and
persistent herpes simplex virus 1 thymidine kinase expression in
survivors of syngeneic glioma treated by adenovirus-mediated gene
therapy : implications for clinical trials. Nature Medicine 5, 1256±1263.
Diaz, R. M., Todryk, S., Chong, H., Hart, I. R., Sikora, K., Dorudi, S. & Vile,
R. G. (1998). Rapid adenoviral transduction of freshly resected tumour
explants with therapeutically useful genes provides a rationale for genetic
immunotherapy for colorectal cancer. Gene Therapy 5, 869±879.

CFJD
W. C. Russell Dodds, E., Piper, T. A., Murphy, S. J. & Dickson, G.
(1999). Cationic lipids and polymers are able to enhance
 adenoviral infection of cultured mouse myotubes. Journal of
Neurochemistry 72, 2105±2112.
Dorai, T., Perlman, H., Walsh, K., Shabsigh, A., Goluboff, E. T., Olsson, C.
Fallaux, F. J., van der Eb, A. J. & Hoeben, R. C. (1999). Who's afraid of
A. & Buttyan, R. (1999). A recombinant defective adenoviral agent
expressing anti-bcl-2 ribozyme promotes apoptosis of bcl-2-expressing replication-competent adenoviruses? Gene Therapy 6, 709±712.
human prostate cancer cells. International Journal of Cancer 82, 846±852. Fasbender, A., Zabner, J., Chillon, M., Moninger, T. O., Puga, A.
Dorsman, J. C., Teunisse, A. F. A. S., Zantema, A. & van der Eb, A. J.
P., Davidson, B. L. & Welsh, M. J. (1997). Complexes of
(1997). The adenovirus 12 E1A proteins can bind directly to proteins of
adenovirus with polycationic polymers and cationic lipids increase
the p300 transcription co-activator family, including the CREB-binding the efficiency of gene transfer in vitro and in vivo. Journal of
protein CBP and p300. Journal of General Virology 78, 423±426. Biological Chemistry 272, 6479±6489.
Doucas, V., Ishov, A. M., Romo, A., Juguilon, H., Weitzman, M. Fasbender, A., Lee, J. H., Walters, R. W., Moninger, T. O., Zabner, J.
D., Evans, R. M. & Maul, G. G. (1996). Adenovirus replication is & Welsh, M. J. (1998). Incorporation of adenovirus in calcium
coupled with the dynamic properties of the PML nuclear structure. phosphate precipitates enhances gene transfer to airway epithelia in
Genes & Development 10, 196±207. vitro and in vivo. Journal of Clinical Investigation 102, 184±193.
Douglas, J. T., Miller, C. R., Kim, M., Dmitriev, I., Mikheeva, G., Fechner, H., Haack, A., Wang, H., Wang, X., Eizema, K., Pauschinger, M.,
Schoemaker, R., Veghel, R., Houtsmuller, A., Schultheiss, H. P., Lamers,
Krasnykh, V. & Curiel, D. T. (1999). A system for the propagation
of adenoviral vectors with genetically modi®ed receptor J. & Poller, W. (1999). Expression of coxsackie adenovirus receptor and av-
speci®cities. Nature Biotechnology 17, 470±475. integrin does not correlate with adenovector targeting in vivo indicating
anatomical vector barriers. Gene Therapy 6, 1520±1535.
Dunphy, E. J., Redman, R. A., Herweijer, H. & Cripe, T. P. (1999).
Reciprocal enhancement of gene transfer by combinatorial Feigenblum, D., Walker, R. & Schneider, R. J. (1998). Adenovirus
adenovirus transduction and plasmid DNA transfection in vitro and induction of an interferon-regulatory factor during entry into the
in vivo. Human Gene Therapy 10, 2407±2417. late phase of infection. Journal of Virology 72, 9257±9266.
Edgell, C. J., Curiel, D. T., Hu, P. C. & Marr, H. S. (1998). Efficient Ferreira, V., Sidenius, N., Tarantino, N., Hubert, P., Chatenoud,
gene transfer to human endothelial cells using DNA complexed to L., Blasi, F. & Korner, M. (1999). In vivo inhibition of NF-kappa B
adenovirus particles. Biotechniques 25, 264±268, 270±272. in T-lineage cells leads to a dramatic decrease in cell proliferation
Einfeld, D. A., Brough, D. E., Roelvink, P. W., Kovesdi, I. & and cytokine production and to increased cell apoptosis in response
Wickham, T. J. (1999). Construction of a pseudoreceptor that to mitogenic stimuli, but not to abnormal thymopoiesis. Journal of
mediates trans-duction by adenoviruses expressing a ligand in ®ber Immunology 162, 6442±6450.
or penton base. Journal of Virology 73, 9130±9136. Fessler, S. P. & Young, C. S. (1998). Control of adenovirus early
Eischen, C. M., Weber, J. D., Roussel, M. F., Sherr, C. J. & gene expression during the late phase of infection. Journal of
Cleveland, J. L. (1999). Disruption of the ARF±Mdm2±p53 tumor Virology 72, 4049±4056.
suppressor pathway in Myc-induced lymphomagenesis. Genes & Fisher, L. J. (1997). Neural precursor cells : applications for the
Development 13, 2658±2669. study and repair of the central nervous system. Neurobiology and
Eissa, N. T., Chu, C. S., Danel, C. & Crystal, R. G. (1994). Evaluation Disease 4, 1±22.
of the respiratory epithelium of normals and individuals with cystic Fisher, K., Stallwood, Y., Ulbrich, K., Mautner, V. & Seymour, L.
®brosis for the presence of adenovirus E1a sequences relevant to the (2000). Protection and retargeting of adenovirus using a
use of E1a® adenovirus vectors for gene therapy for the respiratory multifunctional hydrophilic polymer. Molecular Therapy 1, S57.
manifestations of cystic ®brosis. Human Gene Therapy 5, 1105±1114. Flomenberg, P., Piaskowski, V., Truitt, R. L. & Casper, J. T. (1995).
Elkon, K. B., Liu, C. C., Gall, J. G., Trevejo, J., Marino, M. W., Characterization of human proliferative T cell responses to adenoviruses.
Abrahamsen, K. A., Song, X., Zhou, J. L., Old, J. L., Crystal, R. G. & Journal of Infectious Diseases 171, 1090±1096.
Falck-Pederson, E. (1997). Tumor necrosis factor alpha plays a central
Foxwell, B., Browne, K., Bondeson, J., Clarke, C., de Martin, R.,
role in immune-mediated clearance of adenoviral vectors. Proceedings
Brennan, F. & Feldmann, M. (1998). Efficient adenoviral infection
of the National Academy of Sciences, USA 94, 9814±9819.
with IjB a reveals that macrophage tumor necrosis factor a
Elliott, W. M., Hayashi, S. & Hogg, J. C. (1995). Immunodetection production in rheumatoid arthritis is NF-jB dependent. Proceedings
of adenoviral E1A proteins in human lung tissue. American Journal of the National Academy of Sciences, USA 95, 8211±8215.
of Respiratory Cell Molecular Biology 12, 642±648.
Franklin, C. M. (1999). Clinical experience with soluble TNF p75
Emtage, P. C., Wan, Y., Hitt, M., Graham, F. L., Muller, W. J., Zlotnik, A.
receptor in rheumatoid arthritis. Seminars on Arthritis and
& Gauldie, J. (1999). Adenoviral vectors expressing lymphotactin Rheumatism 29, 172±181.
and interleukin 2 or lymphotactin and interleukin 12 synergize to facilitate tumor
regression in murine breast cancer models. Human Gene Therapy 10, 697±709. Fredman, J. N. & Engler, J. A. (1993). Adenovirus precursor to
terminal protein interacts with the nuclear matrix in vivo and in
Faha, B., Harlow, E. & Lees, E. (1993). The adenovirus E1A- vitro. Journal of Virology 67, 3384±3395.
associated kinase consists of cyclin E±p33cdk2 and cyclin
Fujii, T., Garcia-Bermejo, M. L., Bernabo, J. L., Caamano, J., Ohba,
A±p33cdk2. Journal of Virology 67, 2456±2465.
M., Kuroki, T., Li, L., Yuspa, S. H. & Kazanietz, M. G. (2000).
Fallaux, F. J., Bout, A., van der Velde, I., van der Wollenberg, D. J., Hehir,
Involvement of protein kinase C d (PKCd) in phorbol ester-induced
K. M., Keegan, J., Augur, C., Cramer, S. J., van Ormondt, H., van der Eb,
apoptosis in LNCaP prostate cancer cells. Lack of proteolytic cleavage
A. J., Valerio, D. & Hoeben, R. C. (1998). New helper cells and matched
of PKCd. Journal of Biological Chemistry 275, 7574±7582.
early region 1-deleted adenovirus vectors prevent generation of replication-
competent adenoviruses. Human Gene Therapy 9, 1909±1917. Gabaglia, C. R., Pedersen, B., Hitt, M., Burdin, N., Sercarz, E. E.,
Graham, F. L., Gauldie, J. & Braciak, T. A. (1999). A single intra-
muscular injection with an adenovirus-expressing IL-12 protects
BALB}c mice against Leishmania major infection, while treatment
CFJE with an IL-4-expressing vector increases disease susceptibility in
B10.D2 mice. Journal of Immunology 162, 753±760.
adenoviruses : routes of immunization modulate virus-induced Ig subclass shifts.
European Journal of Immunology 27, 653±659.
Gahery-Segard, H., Farace, F., Godfrin, D., Gaston, J., Lengagne, R., Tursz, T.,
Gahery-Segard, H., Juillard, V., Gaston, J., Lengagne, R.,
Boulanger, P. & Guillet, J. G. (1998). Immune response to recombinant capsid
Pavirani, A., Boulanger, P. & Guillet, J. G. (1997). Humoral
proteins of adenovirus in humans : anti®ber and anti-penton base antibodies have
immune response to the capsid components of recombinant
a synergistic effect on neutralizing activity. Journal of Virology 72, Review: Update on adenovirus vectors
2388±2397.
Gambotto, A., Tuting, T., McVey, D. L., Kovesdi, I., Tahara, H., Lotze,
M. T. & Robbins, P. D. (1999). Induction of antitumor immunity by
Grand, R. J., Parkhill, J., Szestak, T., Rookes, S. M., Roberts, S. &
direct intratumoral injection of a recombinant adenovirus vector Gallimore, P. H. (1999 b). De®nition of a major p53 binding site on
expressing interleukin-12. Cancer Gene Therapy 6, 45±53. Ad2E1B58K protein and a possible nuclear localization signal on
the Ad12E1B54K protein. Oncogene 18, 955±965.
Ganz, T. & Lehrer, R. I. (1998). Antimicrobial peptides of vertebrates.
Current Opinions in Immunology 10, 41±44. Greber, U. F., Webster, P., Weber, J. & Helenius, A. (1996). The
role of the adenovirus protease on virus entry into cells. EMBO
Gao, G. P., Engdahl, R. K. & Wilson, J. M. (2000). A cell line for high-yield Journal 15, 1766±1777.
production of E1-deleted adenovirus vectors without the emergence of
replication-competent virus. Human Gene Therapy 11, 213±219. Greber, U. F., Suomalainen, M., Stidwill, R. P., Boucke, K.,
Ebersold, M. W. & Helenius, A. (1997). The role of the nuclear pore
Ghivizzani, S. C., Lechman, E. R., Kang, R., Tio, C., Kolls, J., Evans,
complex in adenovirus DNA entry. EMBO Journal 16, 5998±6007.
C. H. & Robbins, P. D. (1998). Direct adenovirus-mediated gene transfer of
interleukin 1 and tumor necrosis factor alpha soluble receptors to rabbit Gropp, R., Frye, M., Wagner, T. O. & Bargon, J. (1999). Epithelial
knees with experimental arthritis has local and distal anti-arthritic effects. defensins impair adenoviral infection : implication for adenovirus-
Proceedings of the National Academy of Sciences, USA 95, 4613±4618. mediated gene therapy. Gene Therapy 10, 957±964.
Ghoda, L., Lin, X. & Greene, W. C. (1997). The 90-kDa ribosomal Guidotti, J. E., Mignon, A., Haase, G., Caillaud, C., McDonell, N.,
S6 kinase (pp90rsk) phosphorylates the N-terminal regulatory Kahn, A. & Poenaru, L. (1999). Adenoviral gene therapy of the
domain of IjBa and stimulates its degradation in vitro. Journal of Tay±Sachs disease in hexosaminidase A-de®cient knock-out mice.
Biological Chemistry 272, 21281±21288. Human Molecular Genetics 8, 831±838.
Gilbert, R., Nalbantoglu, J., Petrof, B. J., Ebihara, S., Guibinga, G. H., Haase, G., Pettmann, B., Bordet, T., Villa, P., Vigne, E.,
Tinsley, J. M., Kamen, A., Massie, B., Davies, K. E. & Karpati, G. (1999). Schmalbruch, H. & Kahn, A. (1999). Therapeutic bene®t of ciliary
Adenovirus-mediated utrophin gene transfer mitigates the dystrophic neurotrophic factor in progressive motor neuronopathy depends on
phenotype of mdx mouse muscles. Human Gene Therapy 10, 1299±1310. the route of delivery. Annals of Neurology 45, 296±304.
Ginsberg, H. S., Moldawer, L. L. & Prince, G. A. (1999). Role of Halbert, D. N., Cutt, J. R. & Shenk, T. (1985). Adenovirus early region 4
the type 5 adenovirus gene encoding the early region 1B 55-kDa encodes functions required for efficient DNA replication, late gene
protein in pulmonary pathogenesis. Proceedings of the National expression, and host cell shutoff. Journal of Virology 56, 250±257.
Academy of Sciences, USA 96, 10409±10411. Hale, T. K. & Braithwaite, A. W. (1999). The adenovirus oncoprotein E1a
Goebel, E. A., Davidson, B. L., Graham, S. M. & Kern, J. A. (1998). stimulates binding of transcription factor ETF to transcriptionally activate
Tumor reduction in vivo after adenoviral mediated gene transfer of the p53 gene. Journal of Biological Chemistry 274, 23777±23786.
the herpes simplex virus thymidine kinase gene and ganciclovir Hall, A. R., Dix, B. R., O'Carroll, S. J. & Braithwaite, A. W. (1998).
treatment in human head and neck squamous cell carcinoma. p53-dependent cell death}apoptosis is required for a productive
Otolaryngology and Head and Neck Surgery 119, 331±336. adenovirus infection. Nature Medicine 4, 1068±1072.
Goodbourn, S., Didcock, L. & Randall, R. E. (2000). Interferons : Hamamori, Y., Sartorelli, V., Ogryzko, V., Puri, P. L., Wu, H. Y.,
cell signalling, immune modulation, antiviral responses and virus Wang, J. Y., Nakatani, Y. & Kedes, L. (1999). Regulation of histone
counter-measures. Journal of General Virology 81, 2341±2365. acetyltransferases p300 and PCAF by the bHLH protein twist and
Goodrum, F. D. & Ornelles, D. A. (1999). Roles for the E4 orf6, adenoviral oncoprotein E1A. Cell 96, 405±413.
orf3, and E1B 55-kilodalton proteins in cell cycle-independent Hammond, J. M., McCoy, R. J., Jansen, E. S., Morrissy, C. J.,
adenovirus replication. Journal of Virology 73, 7474±7488. Hodgson, A. L. & Johnson, M. A. (2000). Vaccination with a single
Gorziglia, M. I., Lapcevich, C., Roy, S., Kang, Q., Kadan, M., Wu, dose of a recombinant porcine adenovirus expressing the classical
V., Pechan, P. & Kaleko, M. (1999). Generation of an adenovirus swine fever virus gp55 (E2) gene protects pigs against classical
vector lacking E1, E2a, E3, and all of E4 except open reading frame swine fever. Vaccine 18, 1040±1050.
3. Journal of Virology 73, 6048±6055. Han, J., Sabbatini, P., Perez, D., Rao, L., Modha, D. & White, E. (1996).
Graham, F. L. & Prevec, L. (1992). Adenovirus-based expression The E1B 19K protein blocks apoptosis by interacting with and
vectors and recombinant vaccines. Biotechnology 20, 363±390. inhibiting the p53-inducible and death-promoting Bax protein.
Graham, F. L., Smiley, J., Russell, W. C. & Nairn, R. (1977). Genes & Development 10, 461±477.
Charac-teristics of a human cell line transformed by DNA from Harada, J. N. & Berk, A. J. (1999). p53-Independent and -dependent
human adenovirus type 5. Journal of General Virology 36, 59±72. requirements for E1B-55K in adenovirus type 5 replication. Journal
Grand, R. J., Turnell, A. S., Mason, G. G., Wang, W., Milner, A. E., of Virology 73, 5333±5344.
Mymryk, J. S., Rookes, S. M., Rivett, A. J. & Gallimore, P. H. (1999 a). Harari, O. A., Wickham, T. J., Stocker, C. J., Kovesdi, I., Segal, D.
Adenovirus early region 1A protein binds to mammalian SUG1 ± a M., Huehns, T. Y., Sarraf, C. & Haskard, D. O. (1999). Targeting an
regulatory component of the proteasome. Oncogene 18, 449±458. adenoviral gene vector to cytokine-activated vascular endothelium
via E-selectin. Gene Therapy 6, 801±807.
Hardy, S., Kitamura, M., Harris-Stansil, T., Dai, Y. & Phipps, M. L.
(1997). Construction of adenovirus vectors through Cre-lox recom-
bination. Journal of Virology 71, 1842±1849.
Harrod, K. S., Hermiston, T. W., Trapnell, B. C., Wold, W. S. & Whitsett, J.
A. (1998). Lung-speci®c expression of adenovirus E3-14.7K in transgenic
mice attenuates adenoviral vector-mediated lung in¯ammation and enhances
transgene expression. Human Gene Therapy 9, 1885±1898.
Hart, I. R. (1996). Transcriptionally targeted gene therapy. Current
Topics in Microbiology and Immunology 213, 19±25.

CFJF
W. C. Russell Hartigan-O'Connor, D., Amal®tano, A. & Chamberlain,
J. S. (1999).
 Improved production of gutted adenovirus in cells expressing
adenovirus preterminal protein and DNA polymerase. Journal of
Virology 73, 7835±7841.
Harvey, B. G., Hackett, N. R., El-Sawy, T., Rosengart, T. K., Intravenous administration of ONYX-015, a selectively replicating
Hirschowitz, E. A., Lieberman, M. D., Lesser, M. L. & Crystal, R. G. adenovirus, induces antitumoral efficacy. Cancer Research 59, 2623±2628.
(1999). Variability of human systemic humoral immune responses Herman, J. R., Adler, H. L., Aguilar-Cordova, E., Rojas-Martinez, A.,
to adenovirus gene transfer vectors administered to different organs. Woo, S., Timme, T. L., Wheeler, T. M., Thompson, T. C. & Scardino, P.
Journal of Virology 73, 6729±6742. T. (1999). In situ gene therapy for adenocarcinoma of the prostate : a
phase I clinical trial. Human Gene Therapy 10, 1239±1249.
Hateboer, G., Hijmans, E. M., Nooij, J. B., Schlenker, S., Jentsch,
S. & Bernards, R. (1996). mUBC9, a novel adenovirus E1A- Hidaka, C., Milano, E., Leopold, P. L., Bergelson, J. M., Hackett, N. R.,
interacting protein that complements a yeast cell cycle defect. Finberg, R. W., Wickham, T. J., Kovesdi, I., Roelvink, P. & Crystal, R. G.
Journal of Biological Chemistry 271, 25906±25911. (1999). CAR-dependent and CAR-independent pathways of
adenovirus vector-mediated gene transfer and expression in human
Hautala, T., Grunst, T., Fabrega, A., Freimuth, P. & Welsh, M. J.
®broblasts. Journal of Clinical Investigation 103, 579±587.
(1998). An interaction between penton base and alpha v integrins
plays a minimal role in adenovirus-mediated gene transfer to Hillemann, M. R. & Werner, J. R. (1954). Recovery of a new agent
hepatocytes in vitro and in vivo. Gene Therapy 5, 1259±1264. from patients with acute respiratory illness. Proceedings of the
Society for Experimental Biology and Medicine 85, 183±188.
Hay, R. T., Freeman, A., Leith, I., Monaghan, A. & Webster, A. (1995).
Hirschowitz, E. A. & Crystal, R. G. (1999). Adenovirus-mediated
Molecular interactions during adenovirus DNA replication. In The
Molecular Repertoire of Adenoviruses, pp. 31±48. Edited by W. expression of interleukin-12 induces natural killer cell activity and
Doer¯er & P. Bohm. Berlin : Springer. complements adenovirus-directed gp75 treatment of melanoma lung
metastases. American Journal of Respiratory Cell and Molecular
Hay, J. G., Shapiro, N., Sauthoff, H., Heitner, S., Phupakdi, W. & Biology 20, 935±941.
Rom, W. N. (1999 a). Targeting the replication of adenoviral gene
Hirschowitz, E. A., Ohwada, A., Pascal, W. R., Russi, T. J. & Crystal,
therapy vectors to lung cancer cells : the importance of the
R. G. (1995). In vivo adenovirus-mediated gene transfer of the
adenoviral E1b-55kD gene. Human Gene Therapy 10, 579±590.
Escherichia coli cytosine deaminase gene to human colon
Hay, R. T., Vuillard, L., Desterro, J. M. & Rodriguez, M. S. (1999 b). carcinoma-derived tumors induces chemosensitivity to 5-
Control of NF-jB transcriptional activation by signal induced ¯uorocytosine. Human Gene Therapy 6, 1055±1063.
proteolysis of IjB a. Philosophical Transactions of the Royal Hitt, M. M., Addison, C. L. & Graham, F. L. (1997). Human
Society Series B 354, 1601±1609. adenovirus vectors for gene transfer into mammalian cells.
Hayes, A. L., Smith, C., Foxwell, B. M. & Brennan, F. M. (1999). CD45- Advances in Pharmacology 40, 137±206.
induced tumor necrosis factor alpha production in monocytes is Hofmann, C., Loser, P., Cichon, G., Arnold, W., Both, G. W. &
phosphatidylinositol 3-kinase-dependent and nuclear factor-kappaB- Strauss, M. (1999). Ovine adenovirus vectors overcome preexisting
independent. Journal of Biological Chemistry 274, 33455±33461. humoral immunity against human adenoviruses in vivo. Journal of
He, T. C., Zhou, S., da Costa, L. T., Yu, J., Kinzler, K. W. & Vogelstein, Virology 73, 6930±6936.
B. (1998). A simpli®ed system for generating recombinant adenoviruses.
Honda, R. & Yasuda, H. (1999). Association of p19 (ARF) with
Proceedings of the National Academy of Sciences, USA 95, 2509±2514. Mdm2 inhibits ubiquitin ligase activity of Mdm2 for tumor
He, Z., Wlazlo, A. P., Kowalczyk, D. W., Cheng, J., Xiang, Z. Q., suppressor p53. EMBO Journal 18, 22±27.
Giles-Davis, W. & Ertl, H. C. (2000). Viral recombinant vaccines to Hong, S. S., Karayan, L., Tournier, J., Curiel, D. T. & Boulanger,
the E6 and E7 antigens of HPV-16. Virology 270, 146±161. P. A. (1997). Adenovirus type 5 ®ber knob binds to MHC class I a2
Hearing, P., Samulski, R. J., Wishart, W. L. & Shenk, T. (1987). domain at the surface of human epithelial and B lymphoblastoid
Identi®cation of a repeated sequence element required for efficient cells. EMBO Journal 16, 2294±2306.
encapsidation of the adenovirus type 5 chromosome. Journal of Hong, S. S., Galaup, A., Peytavi, R., Chazal, N. & Boulanger, P.
Virology 61, 2555±2558. (1999 a). Enhancement of adenovirus-mediated gene delivery by
Hedlund, T. E., Meech, S. J., Srikanth, S., Kraft, A. S., Miller, G. use of an oligopeptide with dual binding speci®city. Human Gene
J., Schaack, J. B. & Duke, R. C. (1999). Adenovirus-mediated Therapy 10, 2577±2586.
expression of Fas ligand induces apoptosis of human prostate Hong, S. S., Gay, B., Karayan, L., Dabauvalle, M. C. & Boulanger,
cancer cells. Cell Death and Differentiation 6, 175±182. P. (1999 b). Cellular uptake and nuclear delivery of recombinant
Hehir, K. M., Armentano, D., Cardoza, L. M., Choquette, T. L., adenovirus penton base. Virology 262, 163±177.
Berthelette, P. B., White, G. A., Couture, L. A., Everton, M. B., Horridge, J. J. & Leppard, K. N. (1998). RNA-binding activity of
Keegan, J., Martin, J. M., Pratt, D. A., Smith, M. P., Smith, A. E. & the E1B 55-kilodalton protein from human adenovirus type 5.
Wadsworth, S. C. (1996). Molecular characterization of replication- Journal of Virology 72, 9374±9379.
competent variants of adenovirus vectors and genome modi®cations to Horwitz, M. S., Evans, C. F., McGavern, D. B., Rodriguez, M. &
prevent their occurrence. Journal of Virology 70, 8459±8467. Oldstone, M. B. (1997). Primary demyelination in transgenic mice
Heise, C., Sampson-Johannes, A., Williams, A., McCormick, F., expressing interferon-gamma. Nature Medicine 3, 1037±1041.
Von Hoff, D. D. & Kirn, D. H. (1997). ONYX-015, an E1B gene- Howell, J. M., Lochmuller, H., O'Hara, A., Fletcher, S., Kakulas, B. A.,
attenuated adenovirus, causes tumor-speci®c cytolysis and Massie, B., Nalbantoglu, J. & Karpati, G. (1998). High-level dystrophin
antitumoral efficacy that can be augmented by standard expression after adenovirus-mediated dystrophin minigene transfer to
chemotherapeutic agents. Nature Medicine 3, 639±645. skeletal muscle of dystrophic dogs : prolongation of expression with
Heise, C. C., Williams, A. M., Xue, S., Propst, M. & Kirn, D. H. (1999). immunosuppression. Human Gene Therapy 9, 629±634.
Huang, S., Kamata, T., Takada, Y., Ruggeri, Z. M. & Nemerow, G.
R. (1996). Adenovirus interaction with distinct integrins mediates
CFJG separate events in cell entry and gene delivery to hematopoietic
cells. Journal of Virology 70, 4502±4508.
into the host thymus permits long-term gene therapy in Gunn rats. Journal of Clinical
Investigation 98, 2640±2647.
Ilan, Y., Jona, V. K., Sengupta, K., Davidson, A., Horwitz, M. S., Roy-
Ilan, Y., Attavar, P., Takahashi, M., Davidson, A., Horwitz, M. S.,
Chowdhury, N. & Roy-Chowdhury, J. (1997). Transient immuno-suppression
Guida, J., Roy Chowdhury, N. & Roy Chowdhury, J. (1996). Induction
with FK506 permits long-term expression of therapeutic genes introduced into the
of central tolerance by intrathymic inoculation of adenoviral antigens
liver using recombinant adenoviruses in the rat. Hepatology 26, 949±956.
Ilan, Y., Sauter, B., Roy Chowdhury, N., Reddy, B. V. N., Thummala, Review: Update on adenovirus vectors
N. R., Droguett, G., Davidson, A., Ott, M., Horwitz, M. S. & Roy
Chowdhury, J. (1998). Oral tolerization to adenoviral proteins permits
repeated adenovirus-mediated gene therapy in rats with pre-existing genetic program of human alveolar macrophages by adenovirus
immunity to adenoviruses. Hepatology 27, 1368±1376. vectors in vitro is feasible but inefficient, limited in part by the low
Imazu, T., Shimizu, S., Tagami, S., Matsushima, M., Nakamura, level of expression of the coxsackie}adenovirus receptor. American
Y., Miki, T., Okuyama, A. & Tsujimoto, Y. (1999). Bcl-2}E1B 19 Journal of Respiratory Cell and Molecular Biology 20, 361±370.
kDa-interacting protein 3-like protein (Bnip3L) interacts with bcl- Kannabiran, C., Morris, G. F. & Mathews, M. B. (1999). Dual action
2}Bcl-xL and induces apoptosis by altering mitochondrial of the adenovirus E1A 243R oncoprotein on the human proliferating
membrane permeability. Oncogene 18, 4523±4529. cell nuclear antigen promoter : repression of transcriptional
Irie, A., Anderegg, B., Kashani-Sabet, M., Ohkawa, T., Suzuki, T., Halks- activation by p53. Oncogene 18, 7825±7833.
Miller, M., Curiel, D. T. & Scanlon, K. J. (1999). Therapeutic efficacy of an Kaplan, J. M. & Smith, A. E. (1997). Transient immunosuppression
adenovirus-mediated anti-H-ras ribozyme in experimental bladder cancer. with deoxyspergualin improves longevity of transgene expression
Antisense & Nucleic Acid Drug Development 9, 341±349. and ability to readminister adenoviral vector to the mouse lung.
Iwamoto, H. S., Trapnell, B. C., McConnell, C. J., Daugherty, C. & Human Gene Therapy 8, 1095±1104.
Whitsett, J. A. (1999). Pulmonary in¯ammation associated with re- Kaplan, J. M., Armentano, D., Sparer, T. E., Wynn, S. G.,
peated, prenatal exposure to an E1, E3-deleted adenoviral vector in Peterson, P. A., Wadsworth, S. C., Couture, K. K., Pennington, S.
sheep. Gene Therapy 6, 98±106. E., St George, J. A., Gooding, L. R. & Smith, A. E. (1997).
Ji, L., Bouvet, M., Price, R. E., Roth, J. A. & Fang, B. (1999). Characterization of factors involved in modulating persistence of
Reduced toxicity, attenuated immunogenicity and efficient transgene expression from recombinant adenovirus in the mouse
mediation of human p53 gene expression in vivo by an adenovirus lung. Human Gene Therapy 8, 45±56.
vector with deleted E1±E3 and inactivated E4 by GAL4±TATA Kaplan, J. M., Armentano, D., Scaria, A., Woodworth, L. A., Pennington, S.
replacement. Gene Therapy 6, 393±442. E., Wadsworth, S. C., Smith, A. E. & Gregory, R. J. (1999). Novel role for
Jones, N. (1995). Transcriptional modulation by the adenovirus E1A E4 region genes in protection of adenovirus vectors from lysis by cytotoxic
gene. Current Topics in Microbiology and Immunology 199, 59±80. T lymphocytes. Journal of Virology 73, 4489±4492.
Jooss, K., Ertl, H. C. & Wilson, J. M. (1998 a). Cytotoxic T- Kasof, G. M., Goyal, L. & White, E. (1999). Btf, a novel death-
lymphocyte target proteins and their major histocompatibility promoting transcriptional repressor that interacts with Bcl-2-related
complex class I restriction in response to adenovirus vectors proteins. Molecular and Cell Biology 19, 4390±4404.
delivered to mouse liver. Journal of Virology 72, 2945±2954.
Kass-Eisler, A., Falck-Pedersen, E., Elfenbein, D. H., Alvira, M.,
Jooss, K., Turka, L. A. & Wilson, J. M. (1998 b). Blunting of Buttrick, P. M. & Leinwand, L. A. (1994). The impact of developmental
immune responses to adenoviral vectors in mouse liver and lung stage, route of administration and the immune system on adenovirus-
with CTLA4Ig. Gene Therapy 5, 309±319. mediated gene transfer. Gene Therapy 1, 395±402.
Jooss, K., Yang, Y., Fisher, K. J. & Wilson, J. M. (1998 c). Transduction of Keblusek, P., Dorsman, J. C., Teunisse, A. F. A. S., Teunissen, H.,
dendritic cells by DNA viral vectors directs the immune response to van der Eb, A. J. & Zantema, A. (1999). The adenoviral E1A
transgene products in muscle ®bers. Journal of Virology 72, 4212±4223. oncoproteins interfere with the growth-inhibiting effect of the cdk-
Ju, D. W., Tao, Q., Cheng, D. S., Zhang, W., Zhang, M., Hamada, H. & Cao, CIP /WAF
inhibitor p21 " ". Journal of General Virology 80, 381±390.
X. (2000). Adenovirus-mediated lymphotactin gene transfer improves
Keicho, N., Higashimoto, Y., Bondy, G. P., Elliott, W. M., Hogg, J.
therapeutic efficacy of cytosine deaminase suicide gene therapy in C. & Hayashi, S. (1999). Endotoxin-speci®c NF-kappaB activation
established murine colon carcinoma. Gene Therapy 7, 329±338. in pul-monary epithelial cells harboring adenovirus E1A. American
Juillard, V., Villefroy, P., Godfrin, D., Pavirani, A., Venet, A. & Guillet, Journal of Physiology 277, L523±L532.
J. G. (1995). Long-term humoral and cellular immunity induced by a
Kijima, T., Osaki, T., Nishino, K., Kumagai, T., Funakoshi, T., Goto, H.,
single immunization with replication-defective adenovirus recombinant
Tachibana, I., Tanio, Y. & Kishimoto, T. (1999). Application of the
vector. European Journal of Immunology 25, 3467±3473.
Cre recombinase}loxP system further enhances antitumor effects in
Kaneko, S., Hallenbeck, P., Kotani, T., Nakabayashi, H., McGarrity, G., cell type-speci®c gene therapy against carcinoembryonic antigen-
Tamaoki, T., Anderson, W. F. & Chiang, Y. L. (1995). Adenovirus- producing cancer. Cancer Research 59, 4906±4911.
mediated gene therapy of hepatocellular carcinoma using cancer-
Kikuchi, T. & Crystal, R. G. (1999). Anti-tumor immunity induced
speci®c gene expression. Cancer Research 55, 5283±5287.
by in vivo adenovirus vector-mediated expression of CD40 ligand
Kaner, R. J., Worgall, S., Leopold, P. L., Stolze, E., Milano, E., Hidaka, in tumor cells. Human Gene Therapy 10, 1375±1387.
C., Ramalingam, R., Hackett, N. R., Singh, R., Bergelson, J., Finberg,
Kim, K. N., Watanabe, S., Ma, Y., Thornton, S., Giannini, E. H. & Hirsch, R.
R., Falck-Pedersen, E. & Crystal, R. G. (1999). Modi®cation of the
(2000). Viral IL-10 and soluble TNF receptor act synergistically to inhibit
collagen-induced arthritis following adenovirus-mediated gene transfer.
Journal of Immunology 164, 1576±1581.
Kimura, K. & Gelmann, E. P. (2000). Tumor necrosis factor-alpha
and fas activate complementary fas-associated death domain-
dependent pathways that enhance apoptosis induced by gamma-
irradiation. Journal of Biological Chemistry 275, 8610±8617.
Kirby, I., Davison, E., Beavil, A. J., Soh, C. P., Wickham, T. J., Roelvink, P.
W., Kovesdi, I., Sutton, B. J. & Santis, G. (1999). Mutations in the DG loop
of adenovirus type 5 ®ber knob protein abolish high-affinity binding to its
cellular receptor CAR. Journal of Virology 73, 9508±9514.
Kirby, I., Davison, E., Beavil, A. J., Soh, C. P., Wickham, T. J., Roelvink, P.
W., Kovesdi, I., Sutton, B. J. & Santis, G. (2000). Identi®cation of

CFJH
W. C. Russell
 contact residues and de®nition of the CAR-binding site of adenovirus
type 5 ®ber protein. Journal of Virology 74, 2804±2813.
Konig, C., Roth, J. & Dobbelstein, M. (1999). Adenovirus type 5
E4orf3 protein relieves p53 inhibition by E1B-55-kilodalton Leonard, G. T. & Sen, G. C. (1997). Restoration of interferon
protein. Journal of Virology 73, 2253±2262. responses of adenovirus E1A-expressing HT1080 cell lines by
overexpression of p48 protein. Journal of Virology 71, 5095±5101.
Krajcsi, P., Dimitrov, T., Hermiston, T. W., Tollefson, A. E., Ranheim,
T. S., Vande Pol, S. B., Stephenson, A. H. & Wold, W. S. (1996). Leopold, P. L., Kreitzer, G., Miyazawa, N., Rempel, S., P®ster, K.
The adenovirus E3-14.7K protein and the E3-10.4K}14.5K complex K., Rodriguez-Boulan, E. & Crystal, R. G. (2000). Dynein- and
of proteins, which independently inhibit tumor necrosis factor microtubule-mediated translocation of adenovirus serotype 5 occurs
(TNF)-induced apoptosis, also independently inhibit TNF-induced after endosomal lysis. Human Gene Therapy 11, 151±165.
release of arachidonic acid. Journal of Virology 70, 4904±4913. Leppard, K. N. (1997). E4 gene function in adenovirus, adenovirus
Krasnykh, V., Dmitriev, I., Mikheeva, G., Miller, C. R., Belousova, vector and adeno-associated virus infections. Journal of General
N. & Curiel, D. T. (1998). Characterization of an adenovirus vector Virology 78, 2131±2138.
containing a heterologous peptide epitope in the HI loop of the Leppard, K. N. & Everett, R. D. (1999). The adenovirus type 5 E1b
®ber knob. Journal of Virology 72, 1844±1852. 55K and E4 Orf3 proteins associate in infected cells and affect
Kremer, E. J., Boutin, S., Chillon, M. & Danos, O. (2000). Canine ND10 components. Journal of General Virology 80, 997±1008.
adenovirus vectors : an alternative for adenovirus-mediated gene Li, W., Berencsi, K., Basak, S., Somasundaram, R., Ricciardi, R. P.,
transfer. Journal of Virology 74, 505±512. Gonczol, E., Zaloudik, J., Linnenbach, A., Maruyama, H., Miniou, P. &
Kuhnel, F., Zender, L., Paul, Y., Tietze, M. K., Trautwein, C., Manns, M. Herlyn, D. (1997). Human colorectal cancer (CRC) antigen CO17-
& Kubicka, S. (2000). NFjB mediates apoptosis through 1A}GA733 encoded by adenovirus inhibits growth of established CRC
transcriptional activation of Fas (CD95) in adenoviral hepatitis. Journal of cells in mice. Journal of Immunology 159, 763±769.
Biological Chemistry 275, 6421±6427. Li, E., Stupack, D., Bokoch, G. M. & Nemerow, G. R. (1998).
Kumar-Singh, R. & Chamberlain, J. S. (1996). Encapsidated adenovirus Adenovirus endocytosis requires actin cytoskeleton reorganization
minichromosomes allow delivery and expression of a 14 kb dystrophin mediated by Rho family GTPases. Journal of Virology 72, 8806±8812.
cDNA to muscle cells. Human Molecular Genetics 5, 913±921. Li, H., Alonso-Vanegas, M., Colicos, M. A., Jung, S. S.,
Kvist, S., Ostberg, L., Persson, H., Philipson, L. & Peterson, P. A. Lochmuller, H., Sadikot, A. F., Snipes, G. J., Seth, P., Karpati, G.
(1978). Molecular association between transplantation antigens and cell & Nalbantoglu, J. (1999 a). Intracerebral adenovirus-mediated p53
surface antigen in adenovirus-transformed cell line. Proceedings of the tumor suppressor gene therapy for experimental human glioma.
National Academy of Sciences, USA 75, 5674±5678. Clinical Cancer Research 5, 637±642.
Ladiwala, U., Li, H., Antel, J. P. & Nalbantoglu, J. (1999). p53 induction by Li, Y., Kang, J., Friedman, J., Tarassishin, L., Ye, J., Kovalenko, A.,
tumor necrosis factor-alpha and involvement of p53 in cell death of human Wallach, D. & Horwitz, M. S. (1999 b). Identi®cation of a cell protein
oligodendrocytes. Journal of Neurochemistry 73, 605±611. (FIP-3) as a modulator of NF- jB activity and as a target of an
Lanuti, M., Gao, G. P., Force, S. D., Chang, M. Y., El Kouri, C., adenovirus inhibitor of tumor necrosis factor a-induced apoptosis.
Amin, K. M., Hughes, J. V., Wilson, J. M., Kaiser, L. R. & Albelda, Proceedings of the National Academy of Sciences, USA 96, 1042±1047.
S. M. (1999). Evaluation of an E1E4-deleted adenovirus expressing Li, Y., Pong, R. C., Bergelson, J. M., Hall, M. C., Sagalowsky, A. I.,
the herpes simplex thymidine kinase suicide gene in cancer gene Tseng, C. P., Wang, Z. & Hsieh, J. T. (1999 c). Loss of adenoviral
therapy. Human Gene Therapy 10, 463±475. receptor expression in human bladder cancer cells : a potential impact
Lechman, E. R., Jaffurs, D., Ghivizzani, S. C., Gambotto, A., Kovesdi, on the efficacy of gene therapy. Cancer Research 59, 325±330.
I., Mi, Z., Evans, C. H. & Robbins, P. D. (1999). Direct adenoviral gene
Lieber, A., He, C. Y., Meuse, L., Schowalter, D., Kirillova, I., Winther, B.
transfer of viral IL-10 to rabbit knees with experimental arthritis
& Kay, M. A. (1997). The role of Kupffer cell activation and viral
ameliorates disease in both injected and contralateral control knees.
gene expression in early liver toxicity after infusion of recombinant
Journal of Immunology 163, 2202±2208.
adenovirus vectors. Journal of Virology 71, 8798±8807.
Lee, J. H., Zabner, J. & Welsh, M. J. (1999 a). Delivery of an
Lieber, A., He, C. Y., Meuse, L., Himeda, C., Wilson, C. & Kay, M.
adenovirus vector in a calcium phosphate coprecipitate enhances
A. (1998). Inhibition of NF-jB activation in combination with bcl-2
the therapeutic index of gene transfer to airway epithelia. Human
Gene Therapy 10, 603±613. expression allows for persistence of ®rst-generation adenovirus
vectors in the mouse liver. Journal of Virology 72, 9267±9277.
Lee, W. C., Wan, Y. H., Li, W., Fu, F., Sime, P. J., Gauldie, J., Thomson, A.
W., Fung, J. J., Lu, L. & Qian, S. (1999 b). Enhancement of dendritic cell Lieber, A., Steinwaerder, D. S., Carlson, C. A. & Kay, M. A. (1999).
tolerogenicity by genetic modi®cation using adenoviral vectors encoding Integrating adenovirus±adeno-associated virus hybrid vectors
cDNA for TGF beta 1. Transplantation Proceedings 31, 1195. devoid of all viral genes. Journal of Virology 73, 9314±9324.
Legrand, V., Spehner, D., Schlesinger, Y., Settelen, N., Pavirani, Lin, J., Chen, J., Elenbaas, B. & Levine, A. J. (1994). Several
A. & Mehtali, M. (1999). Fiberless recombinant adenoviruses : virus hydrophobic amino acids in the p53 amino-terminal domain are
matu-ration and infectivity in the absence of ®ber. Journal of required for transcriptional activation, binding to mdm-2 and the
Virology 73, 907±919. adenovirus 5 E1B 55-kD protein. Genes & Development 8, 1235±1246.
Leon, R. P., Hedlund, T., Meech, S. J., Li, S., Schaack, J., Hunger, Linette, G. P., Shankara, S., Longerich, S., Yang, S., Doll, R., Nicolette,
S. P., Duke, R. C. & DeGregori, J. (1998). Adenoviral-mediated C., Preffer, F. I., Roberts, B. L. & Haluska, F. G. (2000). In vitro
gene transfer in lymphocytes. Proceedings of the National priming with adenovirus}gp100 antigen-transduced dendritic cells
Academy of Sciences, USA 95, 13159±13164. reveals the epitope speci®city of HLA-A*0201-restricted CD8- T cells
in patients with melanoma. Journal of Immunology 164, 3402±3412.
Leonard, G. T. & Sen, G. C. (1996). Effects of adenovirus E1A
protein on interferon-signaling. Virology 224, 25±33. Lipinski, K. S., Fax, P., Wilker, B., Hennemann, H., Brockmann, D. &
Esche, H. (1999). Differences in the interactions of oncogenic adenovirus 12
early region 1A and nononcogenic adenovirus 2 early region 1A with the
cellular coactivators p300 and CBP. Virology 255, 94±105.
CFJI
Lubberts, E., Joosten, L. A., van den Bersselaar, L., Helsen, M. M., Bakker, A. C.,
van Meurs, J. B., Graham, F. L., Richards, C. D. & van den Berg, W. B. (1999).
Adenoviral vector-mediated overexpression of IL-4 in the knee joint of mice with
Look, D. C., Roswit, W. T., Frick, A. G., Gris-Alevy, Y., Dickhaus,
collagen-induced arthritis prevents cartilage destruction. Journal of Immunology 163,
D. M., Walter, M. J. & Holtzman, M. J. (1998). Direct suppression of
Stat1 function during adenoviral infection. Immunity 9, 871±880. 4546±4556.
Lukashok, S. A. & Horwitz, M. S. (1998). New perspectives in adenoviruses. Review: Update on adenovirus vectors
Current Clinical Topics in Infectious Diseases 18, 286±305.
Lukashok, S. A., Tarassishin, L., Li, Y. & Horwitz, M. S. (2000). An
adenovirus inhibitor of tumor necrosis factor alpha-induced and human malignant tumors. International Journal of Oncology
apoptosis complexes with dynein and a small GTPase. Journal of 15, 1163±1168.
Virology 74, 4705±4709. Massie, B., Couture, F., Lamoureux, L., Mosser, D. D., Guilbault, C.,
Jolicoeur, P., Belanger, F. & Langelier, Y. (1998). Inducible over-expression
Lusky, M., Christ, M., Rittner, K., Dieterle, A., Dreyer, D., Mourot, B.,
of a toxic protein by an adenovirus vector with a tetracycline-regulatable
Schultz, H., Stoeckel, F., Pavirani, A. & Mehtali, M. (1998). In vitro
and in vivo biology of recombinant adenovirus vectors with E1, expression cassette. Journal of Virology 72, 2289±2296.
E1}E2A, or E1}E4 deleted. Journal of Virology 72, 2022±2032. Mathews, M. B. & Shenk, T. (1991). Adenovirus-associated RNA
Lusky, M., Grave, L., Dieterle, A., Dreyer, D., Christ, M., Ziller, C., and translational control. Journal of Virology 65, 5657±5662.
Furstenberger, P., Kintz, J., Ali Hadji, D., Pavirani, A. & Mehtali, M. Mathias, P., Galleno, M. & Nemerow, G. R. (1998). Interactions of
(1999). Regulation of adenovirus-mediated transgene expression by soluble recombinant integrin av b5 with human adenoviruses.
the viral E4 gene products : requirement for E4 ORF3. Journal of Journal of Virology 72, 8669±8675.
Virology 73, 8308±8319. Matthews, D. A. & Russell, W. C. (1994). Adenovirus
Lutz, P. & Kedinger, C. (1996). Properties of the adenovirus IVa2 protein±protein interactions : hexon and protein VI. Journal of
gene product, an effector of late-phase-dependent activation of the General Virology 75, 3365±3374.
major late promoter. Journal of Virology 70, 1396±1405. Matthews, D. A. & Russell, W. C. (1995). Adenovirus
Lutz, P., Rosa-Calatrava, M. & Kedinger, C. (1997). The product of protein±protein interactions : molecular parameters governing the
the adenovirus intermediate gene IX is a transcriptional activator. binding of protein VI to hexon and the activation of the adenovirus
Journal of Virology 71, 5102±5109. 23K protease. Journal of General Virology 76, 1959±1969.
McDonald, C. & Reich, N. C. (1999). Cooperation of the Matthews, D. A. & Russell, W. C. (1998 a). Adenovirus core protein
transcriptional coactivators CBP and p300 with Stat6. Journal of V is delivered by the invading virus to the nucleus of the infected
Interferon and Cytokine Research 19, 711±722. cell and later in infection is associated with nucleoli. Journal of
Maestro, R., Dei Tos, A. P., Hamamori, Y., Krasnokutsky, S., Sartorelli, V., General Virology 79, 1671±1675.
Kedes, L., Doglioni, C., Beach, D. H. & Hannon, G. J. (1999). Twist Matthews, D. A. & Russell, W. C. (1998 b). Adenovirus core protein V
is a potential oncogene that inhibits apoptosis. Genes & interacts with p32 ± a protein which is associated with both the mitochondria
Development 13, 2207±2217. and the nucleus. Journal of General Virology 79, 1677±1685.
Mahr, J. A. & Gooding, L. R. (1999). Immune evasion by adenoviruses. Matthews, D. A., Cummings, D., Evelegh, C., Graham, F. L. &
Immunological Reviews 168, 121±130. Prevec, L. (1999). Development and use of a 293 cell line
Maini, R., St Clair, E. W., Breedveld, F., Furst, D., Kalden, J., expressing lac repressor for the rescue of recombinant adenoviruses
Weisman, M., Smolen, J., Emery, P., Harriman, G., Feldmann, M. expressing high levels of rabies virus glycoprotein. Journal of
& Lipsky, P. (1999). In¯iximab (chimeric anti-tumour necrosis General Virology 80, 345±353.
factor alpha mono-clonal antibody) versus placebo in rheumatoid Mautner, V. (1989). Adenoviridae. In Andrewes ' Viruses of Vertebrates, 5th
arthritis patients receiving concomitant methotrexate : a randomised edn, pp. 249±282. Edited by J. S. Porter®eld. London : Ballie're Tindall.
phase III trial. ATTRACT Study Group. Lancet 354, 1932±1939.
Mazzarelli, J. M., Mengus, G., Davidson, I. & Ricciardi, R. P. (1997).
Mannervik, M., Fan, S., Strom, A. C., Helin, K. & Akusjarvi, G. (1999). The transactivation domain of adenovirus E1A interacts with the C
Adenovirus E4 open reading frame 4-induced dephosphorylation terminus of human TAFII135. Journal of Virology 71, 7978±7983.
inhibits E1A activation of the E2 promoter and E2F-1-mediated Mazzolini, G., Qian, C., Narvaiza, I., Barajas, M., Borras-Cuesta,
transactivation independently of the retinoblastoma tumor
F., Xie, X., Duarte, M., Melero, I. & Prieto, J. (2000). Adenoviral
suppressor protein. Virology 256, 313±321.
gene transfer of interleukin 12 into tumors synergizes with adoptive
Manome, Y., Abe, M., Hagen, M. F., Fine, H. A. & Kufe, D. W. (1994). T cell therapy both at the induction and effector level. Human Gene
Enhancer sequences of the DF3 gene regulate expression of the herpes Therapy 11, 113±125.
simplex virus thymidine kinase gene and confer sensitivity of human
breast cancer cells to ganciclovir. Cancer Research 54, 5408±5413. Meloni, A. R., Smith, E. J. & Nevins, J. R. (1999). A mechanism for
Rb}p130-mediated transcription repression involving recruitment of
Marshall, E. (1999). Gene therapy death prompts review of the CtBP corepressor. Proceedings of the National Academy of
adenovirus vector. Science 286, 2244±2245. Sciences, USA 96, 9574±9579.
Martin-Duque, P., Sanchez-Prieto, R., Romero, J., Martinez-Lamparero, A.,
Mentel, R., Dopping, G., Wegner, U., Seidel, W., Liebermann, H. &
Cebrian-Sagarriga, S., Guinea-Viniegra, J., Dominguez, C., Lleonart, M.,
Dohner, L. (1997). Adenovirus-receptor interaction with human
Cano, A., Quintanilla, M. & Ramon, Y. C. S. (1999).
lymphocytes. Journal of Medical Virology 51, 252±257.
In vivo radiosensitizing effect of the adenovirus E1A gene in murine
Meredith, J. E., Jr, Winitz, S., Lewis, J. M., Hess, S., Ren, X. D.,
Renshaw, M. W. & Schwartz, M. A. (1996). The regulation of
growth and intracellular signaling by integrins. Endocrinology
Reviews 17, 207±220.
Mette, S. A., Pilewski, J., Buck, C. A. & Albelda, S. M. (1993).
Distribution of integrin cell adhesion receptors on normal bronchial
epithelial cells and lung cancer cells in vitro and in vivo. American Journal
of Research in Respiratory Cell and Molecular Biology 8, 562±572.
Michael, S. I., Hong, J. S., Curiel, D. T. & Engler, J. A. (1995).
Addition of a short peptide ligand to the adenovirus ®ber protein.
Gene Therapy 2, 660±668.

CFJJ
W. C. Russell Michou, A. I., Lehrmann, H., Saltik, M. & Cotten, M.
(1999). Mutational analysis of the avian adenovirus
 CELO, which provides a basis for gene delivery vectors. Journal of
Virology 73, 1399±1410.
Millecamps, S., Kiefer, H., Navarro, V., Geoffroy, M. C., Robert, J.
J., Finiels, F., Mallet, J. & Barkats, M. (1999). Neuron-restrictive binding protein B5 inhibits apoptosis induced by Nip3 by forming a
silencer elements mediate neuron speci®city of adenoviral gene heterodimer through the C-terminal hydrophobic region. Cell Death
expression. Nature Biotechnology 17, 865±869. and Differentiation 6, 314±325.
O'Riordan, C. R., Lachapelle, A., Delgado, C., Parkes, V., Wadsworth,
Miller, N. & Whelan, J. (1997). Progress in transcriptionally targeted and
S. C., Smith, A. E. & Francis, G. E. (1999). PEGylation of adenovirus
regulatable vectors for genetic therapy. Human Gene Therapy 8, 803±815.
with retention of infectivity and protection from neutralizing antibody
Miller, C. R., Buchsbaum, D. J., Reynolds, P. N., Douglas, J. T.,
in vitro and in vivo. Human Gene Therapy 10, 1349±1358.
Gillespie, G. Y., Mayo, M. S., Raben, D. & Curiel, D. T. (1998).
Ozawa, H., Ding, W., Torii, H., Hosoi, J., Seiffert, K., Campton, K.,
Differential susceptibility of primary and established human glioma
cells to adenovirus infection : targeting via the epidermal growth Hackett, N. R., Topf, N., Crystal, R. G. & Granstein, R. D. (1999).
factor receptor achieves ®ber receptor-independent gene transfer. Granulocyte±macrophage colony-stimulating factor gene transfer to
Cancer Research 58, 5738±5748. dendritic cells or epidermal cells augments their antigen-presenting
function including induction of anti-tumor immunity. Journal of
Miyazawa, N., Leopold, P. L., Hackett, N. R., Ferris, B., Worgall, S.,
Investi-gative Dermatology 113, 999±1005.
Falck-Pedersen, E. & Crystal, R. G. (1999). Fiber swap between
adenovirus subgroups B and C alters intracellular trafficking of Pahl, H. L., Sester, M., Burgert, H. G. & Baeuerle, P. A. (1996).
adenovirus gene transfer vectors. Journal of Virology 73, 6056±6065. Activation of transcription factor NF- jB by the adenovirus E3}19K
protein requires its ER retention. Journal of Cell Biology 132, 511±522.
Mizuguchi, H. & Kay, M. A. (1998). Efficient construction of a
recombinant adenovirus vector by an improved in vitro ligation Parks, R. J. & Graham, F. L. (1997). A helper-dependent system for
method. Human Gene Therapy 9, 2577±2583. adenovirus vector production helps de®ne a lower limit for efficient
Moorhead, J. W., Clayton, G. H., Smith, R. L. & Schaack, J. DNA packaging. Journal of Virology 71, 3293±3298.
(1999). A replication-incompetent adenovirus vector with the Parks, R. J., Chen, L., Anton, M., Sankar, U., Rudnicki, M. A. & Graham, F.
preterminal protein gene deleted efficiently transduces mouse ears. L. (1996). A helper-dependent adenovirus vector system : removal of helper
Journal of Virology 73, 1046±1053. virus by Cre-mediated excision of the viral packaging signal. Proceedings
Morral, N., O'Neal, W., Rice, K., Leland, M., Kaplan, J., Piedra, P. A., of the National Academy of Sciences, USA 93, 13565±13570.
Zhou, H., Parks, R. J., Velji, R., Aguilar-Cordova, E., Wadsworth, S., Parks, E., Strieter, R. M., Lukacs, N. W., Gauldie, J., Hitt, M.,
Graham, F. L., Kochanek, S., Carey, K. D. & Beaudet, A. L. (1999). Graham, F. L. & Kunkel, S. L. (1998). Transient gene transfer of IL-
Administration of helper-dependent adenoviral vectors and 12 regulates chemokine expression and disease severity in
sequential delivery of different vector serotype for long-term liver- experimental arthritis. Journal of Immunology 160, 4615±4619.
directed gene transfer in baboons. Proceedings of the National Parks, R., Evelegh, C. & Graham, F. (1999 a). Use of helper-
Academy of Sciences, USA 96, 12816±12821. dependent adenoviral vectors of alternative serotypes permits repeat
Morsy, M. A., Gu, M., Motzel, S., Zhao, J., Lin, J., Su, Q., Allen, H., vector administration. Gene Therapy 6, 1565±1573.
Franlin, L., Parks, R. J., Graham, F. L., Kochanek, S., Bett, A. J. &
Parks, R. J., Bramson, J. L., Wan, Y., Addison, C. L. & Graham, F. L.
Caskey, C. T. (1998). An adenoviral vector deleted for all viral
(1999 b). Effects of stuffer DNA on transgene expression from helper-
coding sequences results in enhanced safety and extended
dependent adenovirus vectors. Journal of Virology 73, 8027±8034.
expression of a leptin transgene. Proceedings of the National
Academy of Sciences, USA 95, 7866±7871. Pastore, L., Morral, N., Zhou, H., Garcia, R., Parks, R. J., Kochanek,
S., Graham, F. L., Lee, B. & Beaudet, A. L. (1999). Use of a liver-
Motoi, F., Sunamura, M., Ding, L., Duda, D. G., Yoshida, Y., Zhang,
speci®c promoter reduces immune response to the transgene in
W., Matsuno, S. & Hamada, H. (2000). Effective gene therapy for
adenoviral vectors. Human Gene Therapy 10, 1773±1781.
pancreatic cancer by cytokines mediated by restricted replication-
competent adenovirus. Human Gene Therapy 11, 223±235. Patel, S. R., Lee, L. Y., Mack, C. A., Polce, D. R., El-Sawy, T., Hackett, N.
R., Ilercil, A., Jones, E. C., Hahn, R. T., Isom, O. W., Rosengart, T. K.
Murray, K. D., Etheridge, C. J., Shah, S. I., Matthews, D. A., Russell,
& Crystal, R. G. (1999). Safety of direct myocardial administration
W. C., Miller, A. D. & Gurling, H. M. D. (2000). Synthetic viral vector
complexes : enhanced cationic liposome-mediated transfection using
of an adenovirus vector encoding vascular endothelial growth factor
121. Human Gene Therapy 10, 1331±1348.
the viral DNA-binding protein Mu. Gene Therapy (in press).
Paulson, M., Pisharody, S., Pan, L., Guadagno, S., Mui, A. L. & Levy,
Muruve, D. A., Barnes, M. J., Stillman, I. E. & Libermann, T. A. (1999).
D. E. (1999). Stat protein transactivation domains recruit p300}CBP
Adenoviral gene therapy leads to rapid induction of multiple
chemokines and acute neutrophil-dependent hepatic injury in vivo. through widely divergent sequences. Journal of Biological
Human Gene Therapy 10, 965±976. Chemistry 274, 25343±25349.
Ng, P., Parks, R. J., Cummings, D. T., Evelegh, C. M., Sankar, U. & Pearson, A. S., Spitz, F. R., Swisher, S. G., Kataoka, M., Sarkiss,
M. G., Meyn, R. E., McDonnell, T. J., Cristiano, R. J. & Roth, J. A.
Graham, F. L. (1999). A high-efficiency Cre}loxP-based system for
(2000). Up-regulation of the proapoptotic mediators Bax and Bak
construction of adenoviral vectors. Human Gene Therapy 10, 2667±2672.
after adenovirus-mediated p53 gene transfer in lung cancer cells.
O'Connor, M. J., Zimmermann, H., Nielsen, S., Bernard, H. U. & Clinical Cancer Research 6, 887±890.
Kouzarides, T. (1999). Characterization of an E1A±CBP interaction
Peng, Y., Trevejo, J., Zhou, J., Marino, M. W., Crystal, R. G., Falck-
de®nes a novel transcriptional adapter motif (TRAM) in
CBP}p300. Journal of Virology 73, 3574±3581. Pedersen, E. & Elkon, K. B. (1999). Inhibition of tumor necrosis factor alpha
by an adenovirus-encoded soluble fusion protein extends transgene
Ohi, N., Tokunaga, A., Tsunoda, H., Nakano, K., Haraguchi, K., Oda,
expression in the liver and lung. Journal of Virology 73, 5098±5109.
K., Motoyama, N. & Nakajima, T. (1999). A novel adenovirus E1B19K-
Perissi, V., Dasen, J. S., Kurokawa, R., Wang, Z., Korzus, E., Rose, D.
W., Glass, C. K. & Rosenfeld, M. G. (1999). Factor-speci®c modu-
lation of CREB-binding protein acetyltransferase activity. Proceedings
CGAA of the National Academy of Sciences, USA 96, 3652±3657.
well-differentiated airway epithelium is responsible for inefficient gene transfer.
Journal of Virology 72, 6014±6023.
Piedimonte, G., Pickles, R. J., Lehmann, J. R., McCarty, D., Costa, D. L.
Pickles, R. J., McCarty, D., Matsui, H., Hart, P. J., Randell, S. H. &
& Boucher, R. C. (1997). Replication-de®cient adenoviral vector for gene transfer
Boucher, R. C. (1998). Limited entry of adenovirus vectors into
potentiates airway neurogenic in¯ammation. American Journal of Research in Respiratory
Cell and Molecular Biology 16, 250±258.
Poller, W., Schneider-Rasp, S., Liebert, U., Merklein, F., Thalheimer, Review: Update on adenovirus vectors
P., Haack, A., Schwaab, R., Schmitt, C. & Brackmann, H. H. (1996).
Stabilization of transgene expression by incorporation of E3 region
genes into an adenoviral factor IX vector and by transient anti-CD4 Recchia, A., Parks, R. J., Lamartina, S., Toniatti, C., Pieroni, L.,
Palombo, F., Ciliberto, G., Graham, F. L., Cortese, R., La Monica,
treatment of the host. Gene Therapy 3, 521±530.
N. & Colloca, S. (1999). Site-speci®c integration mediated by a
Printz, M. A., Gonzalez, A. M., Cunningham, M., Gu, D. L., Ong, hybrid adenovirus}adeno-associated virus vector. Proceedings of
M., Pierce, G. F. & Aukerman, S. L. (2000). Fibroblast growth
the National Academy of Sciences, USA 96, 2615±2620.
factor 2-retargeted adenoviral vectors exhibit a modi®ed
biolocalization pattern and display reduced toxicity relative to Reddy, P. S., Idamakanti, N., Chen, Y., Whale, T., Babiuk, L. A.,
native adenoviral vectors. Human Gene Therapy 11, 191±204. Mehtali, M. & Tikoo, S. K. (1999). Replication-defective bovine
adenovirus type 3 as an expression vector. Journal of Virology 73,
Prives, C. & Hall, P. A. (1999). The p53 pathway. Journal of Pathology
9137±9144.
187, 112±126.
Reich, N., Pine, R., Levy, D. & Darnell, J. E., Jr (1988). Transcription of
Putzer, B. M., Bramson, J. L., Addison, C. L., Hitt, M., Siegel, P.
interferon-stimulated genes is induced by adenovirus particles but is
M., Muller, W. J. & Graham, F. L. (1998). Combination therapy with
suppressed by E1A gene products. Journal of Virology 62, 114±119.
interleukin-2 and wild-type p53 expressed by adenoviral vectors
potentiates tumor regression in a murine model of breast cancer. Reid, J. L., Bannister, A. J., Zegerman, P., Martinez-Balbas, M. A. &
Human Gene Therapy 9, 707±718. Kouzarides, T. (1998). E1A directly binds and regulates the P}CAF
acetyltransferase. EMBO Journal 17, 4469±4477.
Putzer, B. M., Stiewe, T., Parssanedjad, K., Rega, S. & Esche, H.
(2000). E1A is sufficient by itself to induce apoptosis independent Rekosh, D. M., Russell, W. C., Bellet, A. J. & Robinson, A. J. (1977).
of p53 and other adenoviral gene products. Cell Death and Identi®cation of a protein linked to the ends of adenovirus DNA.
Differentiation 7, 177±188. Cell 11, 283±295.
Qiu, C., De Young, M. B., Finn, A. & Dichek, D. A. (1998). Cationic Ridet, J. L., Corti, O., Pencalet, P., Hanoun, N., Hamon, M.,
liposomes enhance adenovirus entry via a pathway independent of the Philippon, J. & Mallet, J. (1999). Toward autologous ex vivo gene
®ber receptor and av-integrins. Human Gene Therapy 9, 507±520. therapy for the central nervous system with human adult astrocytes.
Ramalingam, R., Ra®i, S., Worgall, S., Hackett, N. R. & Crystal, R. G. Human Gene Therapy 10, 271±280.
(1999). Induction of endogenous genes following infection of Ridgway, P. J., Hall, A. R., Myers, C. J. & Braithwaite, A. W. (1997).
− +
human endothelial cells with an E1 E4 adenovirus gene transfer p53}E1b58kDa complex regulates adenovirus replication. Virology
vector. Journal of Virology 73, 10183±10190. 237, 404±413.
Ramondetta, L., Mills, G. B., Burke, T. W. & Wolf, J. K. (2000). Rodriguez, R., Schuur, E. R., Lim, H. Y., Henderson, G. A.,
Adenovirus-mediated expression of p53 or p21 in a papillary serous Simons, J. W. & Henderson, D. R. (1997). Prostate attenuated
endometrial carcinoma cell line (SPEC-2) results in both growth replication com-petent adenovirus (ARCA) CN706 : a selective
inhibition and apoptotic cell death : potential application of gene cytotoxic for prostate-speci®c antigen-positive prostate cancer cells.
therapy to endometrial cancer. Clinical Cancer Research 6, 278±284. Cancer Research 57, 2559±2563.
Rancourt, C., Piche, A., Gomez-Navarro, J., Wang, M., Alvarez, R. Rodriguez, M. S., Desterro, J. M., Lain, S., Midgley, C. A., Lane, D. P.
D., Siegal, G. P., Fuller, G. M., Jones, S. A. & Curiel, D. T. (1999). & Hay, R. T. (1999). SUMO-1 modi®cation activates the transcriptional response
Interleukin-6 modulated conditionally replicative adenovirus as an of p53. EMBO Journal 18, 6455±6461.
antitumor}cytotoxic agent for cancer therapy. Clinical Cancer Roelvink, P. W., Lizonova, A., Lee, J. G., Li, Y., Bergelson, J. M., Finberg,
Research 5, 43±50. R. W., Brough, D. E., Kovesdi, I. & Wickham, T. J. (1998). The
Rao, L., Perez, D. & White, E. (1996). Lamin proteolysis facilitates nuclear coxsackievirus-adenovirus receptor protein can function as a
events during apoptosis. Journal of Cell Biology 135, 1441±1455. cellular attachment protein for adenovirus serotypes from subgroups
Rasmussen, U. B., Benchaibi, M., Meyer, V., Schlesinger, Y. & A, C, D, E, and F. Journal of Virology 72, 7909±7915.
Schughart, K. (1999). Novel human gene transfer vectors : Rogers, B. E., McLean, S. F., Kirkman, R. L., Della Manna, D.,
evaluation of wild-type and recombinant animal adenoviruses in Bright, S. J., Olsen, C. C., Myracle, A. D., Mayo, M. S., Curiel, D.
human-derived cells. Human Gene Therapy 10, 2587±2599. T. & Buchsbaum, D. J. (1999). In vivo localization of ["""In]-
Rauma, T., Tuukkanen, J., Bergelson, J. M., Denning, G. & DTPA-d-Phe1-octreotide to human ovarian tumor xenografts
Hautala, T. (1999). rab5 GTPase regulates adenovirus endocytosis. induced to express the somatostatin receptor subtype 2 using an
Journal of Virology 73, 9664±9668. adenoviral vector. Clinical Cancer Research 5, 383±393.
Rea, D., Schagen, F. H., Hoeben, R. C., Mehtali, M., Havenga, M. J., Rogulski, K. R., Wing, M. S., Paielli, D. L., Gilbert, J. D., Kim, J. H.
Toes, R. E., Melief, C. J. & Offringa, R. (1999). Adenoviruses activate & Freytag, S. O. (2000). Double suicide gene therapy augments the
human dendritic cells without polarization toward a T-helper type 1- antitumor activity of a replication-competent lytic adenovirus
inducing subset. Journal of Virology 73, 10245±10253. through enhanced cytotoxicity and radiosensitization. Human Gene
Therapy 11, 67±76.
Rosenberg, S. A., Zhai, Y., Yang, J. C., Schwartzentruber, D. J.,
Hwu, P., Marincola, F. M., Topalian, S. L., Restifo, N. P., Seipp, C.
A., Einhorn, J. H., Roberts, B. & White, D. E. (1998). Immunizing
patients with metastatic melanoma using recombinant adenoviruses
encoding MART-1 or gp100 melanoma antigens. Journal of the
National Cancer Institute 90, 1894±1900.
Rowe, W. P., Huebner, R. J., Gilmore, L. K., Parrot, R. H. & Ward,
T. G. (1953). Isolation of a cytopathic agent from human adenoids
undergoing spontaneous degradation in tissue culture. Proceedings
of the Society for Experimental Biology and Medicine 84, 570±573.

CGAB
W. C. Russell
 Russell, W. C., Valentine, R. C. & Pereira, H. G. (1967). The effect
of heat on the anatomy of the adenovirus. Journal of General
Virology 1, 509±522.
Shisler, J., Duerksen-Hughes, P., Hermiston, T. M., Wold, W. S. &
Ryan, K. M., Ernst, M. K., Rice, N. R. & Vousden, K. H. (2000).
Gooding, L. R. (1996). Induction of susceptibility to tumor necrosis
Role of NF-kappaB in p53-mediated programmed cell death.
Nature 404, 892±897. factor by E1A is dependent on binding to either p300 or p105-Rb
and induction of DNA synthesis. Journal of Virology 70, 68±77.
Sallenave, J. M., Xing, Z., Simpson, A. J., Graham, F. L. & Gauldie, J.
(1998). Adenovirus-mediated expression of an elastase-speci®c inhibitor Siders, W. M., Halloran, P. J. & Fenton, R. G. (1998 a). Melanoma-
(ela®n) : a comparison of different promoters. Gene Therapy 5, 352±360.
speci®c cytotoxicity induced by a tyrosinase
promoter±enhancer}herpes simplex virus thymidine kinase
Sanchez-Cespedes, M., Reed, A. L., Buta, M., Wu, L., Westra, W. H., adenovirus. Cancer Gene Therapy 5, 281±291.
Herman, J. G., Yang, S. C., Jen, J. & Sidransky, D. (1999). Inactivation
Siders, W. M., Wright, P. W., Hixon, J. A., Alvord, W. G., Back, T.
of the INK4A}ARF locus frequently coexists with TP53 mutations in
C., Wiltrout, R. H. & Fenton, R. G. (1998 b). T cell- and NK cell-
non-small cell lung cancer. Oncogene 18, 5843±5849.
independent inhibition of hepatic metastases by systemic
Sandig, V., Loser, P., Lieber, A., Kay, M. A. & Strauss, M. (1996). HBV- administration of an IL-12-expressing recombinant adenovirus.
derived promoters direct liver-speci®c expression of an adenovirally Journal of Immunology 160, 5465±5474.
transduced LDL receptor gene. Gene Therapy 3, 1002±1009.
Simos, G. & Georgatos, S. D. (1994). The lamin B receptor-associated
Sandy, P., Gostissa, M., Fogal, V., Cecco, L. D., Szalay, K., Rooney, protein p34 shares sequence homology and antigenic determinants with the
R. J., Schneider, C. & Del Sal, G. (2000). p53 is involved in the splicing factor 2-associated protein p32. FEBS Letters 346, 225±228.
p120E4F-mediated growth arrest. Oncogene 19, 188±199.
Smith, T. A., White, B. D., Gardner, J. M., Kaleko, M. &
Santis, G., Legrand, V., Hong, S. S., Davison, E., Kirby, I., Imler, J.-L., McClelland, A. (1996). Transient immunosuppression permits
Finberg, R. W., Bergelson, J. M., Mehtali, M. & Boulanger, P. (1999). successful repetitive intravenous administration of an adenovirus
Molecular determinants of adenovirus serotype 5 ®bre binding to its vector. Gene Therapy 3, 496±502.
cellular receptor CAR. Journal of General Virology 80, 1519±1527.
Smith, C. A., Woodruff, L. S., Rooney, C. & Kitchingman, G. R. (1998).
Schmitz, M. L., Indorf, A., Limbourg, F. P., Stadtler, H., Traenckner,
Extensive cross-reactivity of adenovirus-speci®c cytotoxic T cells.
E. B. & Baeuerle, P. A. (1996). The dual effect of adenovirus type 5 Human Gene Therapy 9, 1419±1427.
E1A 13S protein on NF-jB activation is antagonized by E1B 19K. Molecular Cell
Biology 16, 4052±4063. Somasundaram, K. & El-Deiry, W. S. (1997). Inhibition of p53-
mediated transactivation and cell cycle arrest by E1A through its
Schneider, D. B., Sassani, A. B., Vassalli, G., Driscoll, R. M. & Dichek, p300}CBP-interacting region. Oncogene 14, 1047±1057.
D. A. (1999). Adventitial delivery minimizes the proin¯ammatory
Souberbielle, B. E. & Russell, W. C. (1995). Human T cell
effects of adenoviral vectors. Journal of Vascular Surgery 29, 543±550.
proliferative response to polypeptides from adenovirus type 2.
Schowalter, D. B., Himeda, C. L., Winther, B. L., Wilson, C. B. & Journal of Infectious Diseases 172, 1421±1422.
Kay, M. A. (1999). Implication of interfering antibody formation
Staba, M. J., Wickham, T. J., Kovesdi, I. & Hallahan, D. E. (2000).
and apop-tosis as two different mechanisms leading to variable
duration of adenovirus-mediated transgene expression in immune- Modi®cations of the ®ber in adenovirus vectors increase tropism
competent mice. Journal of Virology 73, 4755±4766. for malignant glioma models. Cancer Gene Therapy 7, 13±19.
Steiner, M. S., Zhang, Y., Farooq, F., Lerner, J., Wang, Y. & Lu, Y.
Segerman, A., Mei, Y. F. & Wadell, G. (2000). Adenovirus types
(2000). Adenoviral vector containing wild-type p16 suppresses
11p and 35p show high binding efficiencies for committed
hematopoietic cell lines and are infective to these cell lines. Journal prostate cancer growth and prolongs survival by inducing cell
of Virology 74, 1457±1467. senescence. Cancer Gene Therapy 7, 360±372.
Shalev, M., Kadmon, D., Teh, B. S., Butler, E. B., Aguilar- Steinwaerder, D. S., Carlson, C. A. & Lieber, A. (1999). Generation
Cordova, E., Thompson, T. C., Herman, J. R., Adler, H. L., of adenovirus vectors devoid of all viral genes by recombination
Scardino, P. T. & Miles, B. J. (2000). Suicide gene therapy toxicity between inverted repeats. Journal of Virology 73, 9303±9313.
after multiple and repeat injections in patients with localized Sterman, D. H., Treat, J., Litzky, L. A., Amin, K. M., Coonrod, L.,
prostate cancer. Journal of Urology 163, 1747±1750. Molnar-Kimber, K., Recio, A., Knox, L., Wilson, J. M., Albelda, S.
M. & Kaiser, L. R. (1998). Adenovirus-mediated herpes simplex
Shao, R., Karunagaran, D., Zhou, B. P., Li, K., Lo, S. S., Deng, J.,
Chiao, P. & Hung, M. C. (1997). Inhibition of nuclear factor-jB activity
virus thymidine kinase}ganciclovir gene therapy in patients with
is involved in E1A-mediated sensitization of radiation-induced
localized malignancy : results of a phase I clinical trial in malignant
apoptosis. Journal of Biological Chemistry 272, 32739±32742.
mesothelioma. Human Gene Therapy 9, 1083±1092.
Stevenson, S. C., Rollence, M., White, B., Weaver, L. &
Shao, R., Hu, M. C., Zhou, B. P., Lin, S. Y., Chiao, P. J., von
McClelland, A. (1995). Human adenovirus serotypes 3 and 5 bind to
Lindern, R. H., Spohn, B. & Hung, M. C. (1999). E1A sensitizes
cells to tumor necrosis factor-induced apoptosis through inhibition two different cellular receptors via the ®ber head domain. Journal
of IjB kinases and nuclear factor jB activities. Journal of Biological of Virology 69, 2850±2857.
Chemistry 274, 21495±21498. Stewart, P. L., Fuller, S. D. & Burnett, R. M. (1993). Difference imaging
Shih, I. M., Yu, J., He, T. C., Vogelstein, B. & Kinzler, K. W. (2000). of adenovirus : bridging the resolution gap between X-ray crystal-
The beta-catenin binding domain of adenomatous polyposis coli is lography and electron microscopy. EMBO Journal 12, 2589±2599.
sufficient for tumor suppression. Cancer Research 60, 1671±1676. Stewart, P. L., Chiu, C. Y., Huang, S., Muir, T., Zhao, Y., Chait, B.,
Mathias, P. & Nemerow, G. R. (1997). Cryo-EM visualization of an
Shinoura, N., Muramatsu, Y., Yoshida, Y., Asai, A., Kirino, T. &
Hamada, H. (2000). Adenovirus-mediated transfer of caspase-3 exposed RGD epitope on adenovirus that escapes antibody
with Fas ligand induces drastic apoptosis in U-373MG glioma cells. neutralization. EMBO Journal 16, 1189±1198.
Experimental Cell Research 256, 423±433. Suomalainen, M., Nakano, M. Y., Keller, S., Boucke, K., Stidwill, R. P.
& Greber, U. F. (1999). Microtubule-dependent plus- and minus
end-directed motilities are competing processes for nuclear
targeting of adenovirus. Journal of Cell Biology 144, 657±672.
CGAC
(2000). Adenovirus-mediated ribozyme targeting of HER-2}neu inhibits in vivo growth of
breast cancer cells. Gene Therapy 7, 241±248.

Suzuki, T., Anderegg, B., Ohkawa, T., Irie, A., Engebraaten, O., Halks- Tan, B. T., Wu, L. & Berk, A. J. (1999). An adenovirus±Epstein±Barr virus hybrid
Miller, M., Holm, P. S., Curiel, D. T., Kashani-Sabet, M. & Scanlon, K. J. vector that stably transforms cultured cells with high efficiency. Journal of Virology
73, 7582±7589.
Tanaka, N., Sato, M., Lamphier, M. S., Nozawa, H., Oda, E., Review: Update on adenovirus vectors
Noguchi, S., Schreiber, R. D., Tsujimoto, Y. & Taniguchi, T.
(1998). Type I interferons are essential mediators of apoptotic death
in virally infected cells. Genes and Cells 3, 29±37. van Deutekom, J. C., Cao, B., Pruchnic, R., Wickham, T. J., Kovesdi, I.
Taniguchi, T., Chikatsu, N., Takahashi, S., Fujita, A., Uchimaru, K., & Huard, J. (1999). Extended tropism of an adenoviral vector does not circumvent
Asano, S., Fujita, T. & Motokura, T. (1999). Expression of p16INK4A the maturation-dependent transducibility of mouse skeletal muscle. Journal of
and p14ARF in hematological malignancies. Leukemia 13, 1760±1769.
Genetic Medicine 1, 393±399.
van Ginkel, F. W., McGhee, J. R., Liu, C., Simecka, J. W., Yamamoto,
Tao, W. & Levine, A. J. (1999). P19 (ARF) stabilizes p53 by
M., Frizzell, R. A., Sorscher, E. J., Kiyono, H. & Pascual, D. W. (1997).
blocking nucleo-cytoplasmic shuttling of Mdm2. Proceedings of the
National Academy of Sciences, USA 96, 6937±6941. Adenoviral gene delivery elicits distinct pulmonary-associated T
helper cell responses to the vector and to its transgene. Journal of
Tashiro, F., Niwa, H. & Miyazaki, J. (1999). Constructing adenoviral Immunology 159, 685±693.
vectors by using the circular form of the adenoviral genome cloned
Vollmer, C. M., Ribas, A., Butter®eld, L. H., Dissette, V. B., Andrews,
in a cosmid and the Cre-loxP recombination system. Human Gene
K. J., Eilber, F. C., Montejo, L. D., Chen, A. Y., Hu, B., Glaspy, J. A.,
Therapy 10, 1845±1852.
McBride, W. H. & Economou, J. S. (1999). p53 selective and non-
Tillman, B. W., de Gruijl, T. D., Luykx-de Bakker, S. A., Scheper, R. J., selective replication of an E1B-deleted adenovirus in hepatocellular
Pinedo, H. M., Curiel, T. J., Gerritsen, W. R. & Curiel, D. T. (1999). carcinoma. Cancer Research 59, 4369±4374.
Maturation of dendritic cells accompanies high-efficiency gene
von Herrath, M. G., Efrat, S., Oldstone, M. B. & Horwitz, M. S. (1997).
transfer by a CD40-targeted adenoviral vector. Journal of
Immunology 162, 6378±6383. Expression of adenoviral E3 transgenes in beta cells prevents
autoimmune diabetes. Proceedings of the National Academy of
Tollefson, A. E., Ryerse, J. S., Scaria, A., Hermiston, T. W. & Wold, Sciences, USA 94, 9808±9813.
W. S. (1996). The E3-11±6-kDa adenovirus death protein (ADP) is
Von Seggern, D. J., Chiu, C. Y., Fleck, S. K., Stewart, P. L. &
re-quired for efficient cell death : characterization of cells infected
Nemerow, G. R. (1999). A helper-independent adenovirus vector
with adp mutants. Virology 220, 152±162.
with E1, E3, and ®ber deleted : structure and infectivity of ®berless
Tollefson, A. E., Hermiston, T. W., Lichtenstein, D. L., Colle, C. F., particles. Journal of Virology 73, 1601±1608.
Tripp, R. A., Dimitrov, T., Toth, K., Wells, C. E., Doherty, P. C. & Wold,
Walters, R. W., Grunst, T., Bergelson, J. M., Finberg, R. W., Welsh,
W. S. (1998). Forced degradation of Fas inhibits apoptosis in
M. J. & Zabner, J. (1999). Basolateral localization of ®ber receptors limits
adenovirus-infected cells. Nature 392, 726±730.
adenovirus infection from the apical surface of airway epithelia. Journal of
Toloza, E. M., Hunt, K., Swisher, S., McBride, W., Lau, R., Pang, S., Biological Chemistry 274, 10219±10226.
Rhoades, K., Drake, T., Belldegrun, A., Glaspy, J. & Economou, J. S.
Wan, Y., Emtage, P., Foley, R., Carter, R. & Gauldie, J. (1999).
(1996). In vivo cancer gene therapy with a recombinant interleukin- Murine dendritic cells transduced with an adenoviral vector
2 adenovirus vector. Cancer Gene Therapy 3, 11±17. expressing a de®ned tumor antigen can overcome anti-adenovirus
Tomko, R. P., Xu, R. & Philipson, L. (1997). HCAR and MCAR : neutralizing immunity and induce effective tumor regression.
the human and mouse cellular receptors for subgroup C International Journal of Oncology 14, 771±776.
adenoviruses and group B coxsackieviruses. Proceedings of the
Wang, X. & Bergelson, J. M. (1999). Coxsackievirus and
National Academy of Sciences, USA 94, 3352±3356.
adenovirus receptor cytoplasmic and transmembrane domains are
Tomko, R. P., Johansson, C. B., Totrov, M., Abagyan, R., Frisen, J. & not essential for coxsackievirus and adenovirus infection. Journal of
Philipson, L. (2000). Expression of the adenovirus receptor and its Virology 73, 2559±2562.
interaction with the ®ber knob. Experimental Cell Research 255, 47±55. Wang, K., Huang, S., Kapoor-Munshi, A. & Nemerow, G. (1998).
Topf, N., Worgall, S., Hackett, N. R. & Crystal, R. G. (1998). Regional Adenovirus internalization and infection require dynamin. Journal
`pro-drug ' gene therapy : intravenous administration of an adenoviral vector of Virology 72, 3455±3458.
expressing the E. coli cytosine deaminase gene and systemic administration Watson, G., Burdon, M. G. & Russell, W. C. (1988). An antigenic
of 5-¯uorocytosine suppresses growth of hepatic met-astasis of colon analysis of the adenovirus type 2 ®bre polypeptide. Journal of
carcinoma. Gene Therapy 5, 507±513. General Virology 69, 525±535.
Tsao, Y. P., Huang, S. J., Chang, J. L., Hsieh, J. T., Pong, R. C. & Chen,
Weber, J. (1976). Genetic analysis of adenovirus type 2. III. Temperature
WAF /SDII/CIP
S. L. (1999). Adenovirus-mediated p21 " " gene sensitivity of processing viral proteins. Journal of Virology 17, 462±471.
transfer induces apoptosis of human cervical cancer cell lines. Weber, J. D., Taylor, L. J., Roussel, M. F., Sherr, C. J. & Bar-Sagi,
Journal of Virology 73, 4983±4990.
D. (1999). Nucleolar Arf sequesters Mdm2 and activates p53.
Tuting, T., Steitz, J., Bruck, J., Gambotto, A., Steinbrink, K., Nature Cell Biology 1, 20±26.
DeLeo, A. B., Robbins, P., Knop, J. & Enk, A. H. (1999). Dendritic
Webster, A., Russell, S., Talbot, P., Russell, W. C. & Kemp, G. D.
cell-based genetic immunization in mice with a recombinant
(1989). Characterization of the adenovirus proteinase : substrate
adenovirus encoding murine TRP2 induces effective anti-melanoma
immunity. Journal of Genetic Medicine 1, 400±406. speci®city. Journal of General Virology 70, 3225±3234.
Weiden, M. D. & Ginsberg, H. S. (1994). Deletion of the E4 region of
the genome produces adenovirus DNA concatemers. Proceedings of the
National Academy of Sciences, USA 91, 153±157.
Weigel, S. & Dobbelstein, M. (2000). The nuclear export signal
within the E4orf6 protein of adenovirus type 5 supports virus
replication and cytoplasmic accumulation of viral mRNA. Journal
of Virology 74, 764±772.
Wickham, T. J., Mathias, P., Cheresh, D. A. & Nemerow, G. R. (1993).
Integrins av b3 and av b5 promote adenovirus internalization but not
virus attachment. Cell 73, 309±319.

CGAD
W. C. Russell Wickham, T. J., Roelvink, P. W., Brough, D. E. &
Kovesdi, I. (1996).
 Adenovirus targeted to heparan-containing receptors increases its
gene delivery efficiency to multiple cell types. Nature
Biotechnology 14, 1570±1573.
Wickham, T. J., Lee, G. M., Titus, J. A., Sconocchia, G., Bakacs, T., Yu, D. C., Chen, Y., Seng, M., Dilley, J. & Henderson, D. R. (1999).
Kovesdi, I. & Segal, D. M. (1997). Targeted adenovirus-mediated gene The addition of adenovirus type 5 region E3 enables calydon virus
delivery to T cells via CD3. Journal of Virology 71, 7663±7669. 787 to eliminate distant prostate tumor xenografts. Cancer
Research 59, 4200±4203.
Wildner, O., Morris, J. C., Vahanian, N. N., Ford, H., Jr, Ramsey, W. J.
& Blaese, R. M. (1999). Adenoviral vectors capable of replication Yuasa, K., Miyagoe, Y., Yamamoto, K., Nabeshima, Y., Dickson,
improve the efficacy of HSVtk}GCV suicide gene therapy of cancer. Gene G. & Takeda, S. (1998). Effective restoration of dystrophin-
Therapy 6, 57±62. associated proteins in vivo by adenovirus-mediated transfer of
truncated dystrophin cDNAs. FEBS Letters 425, 329±336.
Willcox, N. & Mautner, V. (1976). Antigenic determinants of
adenovirus capsids. I. Measurement of antibody cross-reactivity. Yun, J., Chae, H. D., Choy, H. E., Chung, J., Yoo, H. S., Han, M. H.
Journal of Im-munology 116, 19±24. & Shin, D. Y. (1999). p53 negatively regulates cdc2 transcription via
the CCAAT-binding NF-Y transcription factor. Journal of
Wills, K. N., Maneval, D. C., Menzel, P., Harris, M. P., Sutjipto, S.,
Biological Chemistry 274, 29677±29682.
Vaillancourt, M.-T., Huang, W.-M., Johnson, D. E., Anderson, S. C.,
Wen, S. F., Bookstein, R., Shepard, H. M. & Gregory, R. J. (1994). Zabner, J., Freimuth, P., Puga, A., Fabrega, A. & Welsh, M. J. (1997).
Development and characterization of recombinant adenoviruses Lack of high affinity ®ber receptor activity explains the resistance
encoding human p53 for gene therapy of cancer. Human Gene of ciliated airway epithelia to adenovirus infection. Journal of
Therapy 5, 1079±1088. Clinical Investigation 100, 1144±1149.
Wold, W. M. & Chinnadurai, G. (2000). Adenovirus proteins that Zabner, J., Chillon, M., Grunst, T., Moninger, T. O., Davidson, B.
regulate apoptosis. In DNA Virus Replication, pp. 200±232. Edited L., Gregory, R. & Armentano, D. (1999). A chimeric type 2
by A. J. Cann. Oxford : Oxford University Press. adenovirus vector with a type 17 ®ber enhances gene transfer to
human airway epithelia. Journal of Virology 73, 8689±8695.
Wolf, L. A. & Laster, S. M. (1999). Characterization of arachidonic acid-
induced apoptosis. Cell Biochemistry and Biophysics 30, 353±368. Zakhartchouk, A. N., Reddy, P. S., Baxi, M., Baca-Estrada, M. E.,
Mehtali, M., Babiuk, L. A. & Tikoo, S. K. (1998). Construction and
Worgall, S., Wolff, G., Falck-Pedersen, E. & Crystal, R. G. (1997 a).
characterization of E3-deleted bovine adenovirus type 3 expressing
Innate immune mechanisms dominate elimination of adenoviral vectors full-length and truncated form of bovine herpesvirus type 1
following in vivo administration. Human Gene Therapy 8, 37±44. glycoprotein gD. Virology 250, 220±229.
Worgall, S., Leopold, P. L., Wolff, G., Ferris, B., Van Roijen, N. &
Zantema, A. & van der Eb, A. (2000). Control of transcription by
Crystal, R. G. (1997 b). Role of alveolar macrophages in rapid
adenovirus-E1A proteins. In DNA Virus Replication, pp. 173±199.
elimination of adenovirus vectors administered to the epithelial surface
Edited by A. J. Cann. Oxford : Oxford University Press.
of the respiratory tract. Human Gene Therapy 8, 1675±1684.
Zhang, W.-W. (1999). Development and application of adenoviral
Worgall, S., Singh, R., Leopold, P. L., Kaner, R. J., Hackett, N. R.,
vectors for gene therapy of cancer. Cancer Gene Therapy 6, 113±138.
Topf, N., Moore, M. A. & Crystal, R. G. (1999). Selective expansion
of alveolar macrophages in vivo by adenovirus-mediated transfer of Zhang, R. & DeGroot, L. J. (2000). Gene therapy of established
the murine granulocyte-macrophage colony-stimulating factor medullary thyroid carcinoma with herpes simplex viral thymidine
cDNA. Blood 93, 655±666. kinase in a rat tumor model : relationship of bystander effect and
antitumor efficacy. Thyroid 10, 313±319.
Yang, L., Lochmuller, H., Luo, J., Massie, B., Nalbantoglu, J.,
Karpati, G. & Petrof, B. J. (1998). Adenovirus-mediated dystrophin Zhang, F., Andreassen, P., Fender, P., Geissler, E., Hernandez, J.
minigene transfer improves muscle strength in adult dystrophic F. & Chroboczek, J. (1999). A transfecting peptide derived from
(MDX) mice. Gene Therapy 5, 369±379. adenovirus ®ber protein. Gene Therapy 6, 171±181.
Yarosh, O. K., Wandeler, A. I., Graham, F. L., Campbell, J. B. & Zheng, C., Baum, B. J., Iadarola, M. J. & O'Connell, B. C. (2000).
Prevec, L. (1996). Human adenovirus type 5 vectors expressing Genomic integration and gene expression by a modi®ed adenoviral
rabies glycoprotein. Vaccine 14, 1257±1264. vector. Nature Biotechnology 18, 176±180.
Ye, J., Xie, X., Tarassishin, L. & Horwitz, M. S. (2000). Regulation Zhong, L., Granelli-Piperno, A., Choi, Y. & Steinman, R. M. (1999).
of the NF-jB activation pathway by isolated domains of FIP3}IKKc, Recombinant adenovirus is an efficient and non-perturbing genetic vector
a component of the IjB-a kinase complex. Journal of Biological for human dendritic cells. European Journal of Immunology 29, 964±972.
Chemistry 275, 9882±9889. Zuckerman, J. B., Robinson, C. B., McCoy, K. S., Shell, R., Sferra, T. J.,
Yew, N. S., Marshall, J., Przybylska, M., Wysokenski, D. M., Ziegler, Chirmule, N., Magosin, S. A., Propert, K. J., Brown-Parr, E. C., Hughes, J.
R. J., Rafter, P. W., Li, C., Armentano, D. & Cheng, S. H. (1999). V., Tazelaar, J., Baker, C., Goldman, M. J. & Wilson, J. M. (1999). A phase I
Increased duration of transgene expression in the lung with plasmid study of adenovirus-mediated transfer of the human cystic ®brosis
DNA vectors harboring adenovirus E4 open reading frame 3. transmembrane conductance regulator gene to a lung segment of individuals
Human Gene Therapy 10, 1833±1843. with cystic ®brosis. Human Gene Therapy 10, 2973±2985.

Published ahead of print (11 August 2000) in JGV Direct as

DOI 10.1099/vir.0.17217-0

CGAE