CONFERENCE PAPER

Gutless adenovirus: last-generation adenovirus

for gene therapy
R Alba
1
, A Bosch
1
and M Chillon
1,2
1
Gene Therapy Laboratory, Department of Biochemistry and Molecular Biology, Center of Animal Biotechnology and Gene Therapy
(CBATEG), Universitat Auto`noma de Barcelona, Bellaterra, Spain; and
2
Institut Catala` de Recerca i Estudis Avancats (ICREA),
Barcelona, Spain
Last-generation adenovirus vectors, also called helper-depen-
dent or gutless adenovirus, are very attractive for gene therapy
because the associated in vivo immune response is highly
reduced compared to rst- and second-generation adenovirus
vectors, while maintaining high transduction efciency and
tropism. Nowadays, gutless adenovirus is administered in
different organs, such as the liver, muscle or the central
nervous system achieving high-level and long-term transgene
expression in rodents and primates. However, as devoid of all
viral coding regions, gutless vectors require viral proteins
supplied in trans by a helper virus. To remove contamination
by a helper virus from the nal preparation, different systems
based on the excision of the helper-packaging signal have
been generated. Among them, Cre-loxP system is mostly
used, although contamination levels still are 0.11% too high
to be used in clinical trials. Recently developed strategies to
avoid/reduce helper contamination were reviewed.
Gene Therapy (2005) 12, S18S27. doi:10.1038/sj.gt.3302612
Keywords: adenovirus; gutless; helper-dependent vectors; in vivo gene therapy
Introduction
Gene therapy for most genetic diseases requires expres-
sion of the therapeutic protein for the whole life of
the patient. In order to be efcient for the treatment
of genetic disorders, a gene therapy vector has to meet
several conditions: (i) safety, which can be better
achieved with a nonintegrative vector, as it avoids the
risk for insertional mutagenesis; (ii) ability to be easily
and inexpensively produced at a large-scale in the
laboratory; (iii) stability in target cells, which is favored
with low-immunogenic vectors; and (iv) high-capacity
allowing the possibility of introducing full-length DNA
sequences for most genes, long full-length cDNAs,
endogenous promoters or additional regulatory
sequences such as enhancers or insulators, which can
provide a tightly regulated expression of the therapeutic
gene, similar to physiologic conditions. Adenoviruses
and especially gutless adenoviruses seem to accomplish
most of these conditions as they are not integrative, they
have a capacity of 36 kb, they can be easily produced at
high titers in the laboratory and can be delivered to a
large mass of cells, and, contrary to the rst-generation
adenovirus in which the expression of wild-type adeno-
viral genes stimulates the immune system, gutless
adenoviruses show long-term stability in many tissues.
As seen in Table 1, adenovirus is one of the rst elections
in clinical trials, being the most used vector in the last
5 years (19992004) (visit www.wiley.co.uk/genmed/
clinical for more information). Nowadays, adenovirus
vectors are applied to treat cancer, monogenic disorders,
vascular diseases and others complications.
Basically, wild-type adenovirus is associated with
mild diseases like conjunctivitis, pharyngitis and, in a
high percentage, with colds or acute respiratory diseases
but not to tumoral pathways or other viral alterations.
1
In
the early 1950s, adenovirus was used for the rst time
as a preventive vaccine for respiratory diseases. These
studies supposed a great advance since they allowed the
knowledge of aspects related to associated immune
responses and adverse secondary effects.
As gene transfer vector, the adenovirus has a high
transfection efciency, both in quiescent and in dividing
cells, it does not integrate; allows easy capsid modication
in order to retarget its tropism to different tissues; and
high titers (up to 10
13
particles/ml) are routinely obtained.
In addition, it is noteworthy that in the last 10 years, new
scale-up adenovirus production systems have been
developed, facilitating its use in human clinical trials.
Well-characterized human serotypes Ad2 and Ad5
from group C are the classic adenoviruses used as
vectors. In order to produce safe and nonreplicative
vectors, rst-generation adenoviral vectors containing
the whole viral genome with the exception of the E1
region were developed.
2
To propagate rst-generation
adenoviruses, several E1-expressing cell lines have been
generated: 293,
3
911,
4
N52.E6
5
and PER.C6.
6
Although E1-deleted vectors cannot replicate in vivo,
residual expression from adenoviral genes triggers a
cytotoxic T lymphocyte (CTL) immune response towards
infected cells,
7
which nally leads to the elimination of
transduced cells and, therefore, to the lost of therapeutic
gene expression.
Correspondence: Dr M Chillon, Gene Therapy Laboratory, Department of
Biochemistry and Molecular Biology, Center of Animal Biotechnology and
Gene Therapy (CBATEG), Edici H, Universitat Auto`noma de Barcelona,
Bellaterra 08193, Spain
Gene Therapy (2005) 12, S18S27
& 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00
www.nature.com/gt
To avoid this problem, second-generation adenoviral
vectors combining deletion of different early regions
(E17E3 and E2/E4) were generated (Figure 1). Deletion
of these regions permits to accommodate up to 14 kb and
hence increase the vector cloning capacity.
8,9
However,
second-generation Ad vectors still do not avoid in vivo-
associated immunogenicity and toxicity due to residual
gene expression from remaining viral genes.
Third-generation vectors, called gutless or gutted
Ad, devoid of all coding viral regions were recently
generated. They are also called helper-dependent adeno-
viruses because of the need of a helper adenovirus that
carries all coding regions, and high-capacity adenoviruses
because they can accommodate up to 36 kb of DNA.
Briey, the gutless adenovirus only keeps the 5
0
and 3
0
inverted terminal repeats (ITRs) and the packaging
signal (C) from the wild-type adenovirus.
Vector capsids package efciently only 75105% of the
whole adenovirus genome.
1014
As therapeutic expres-
sion cassettes usually do not reach up to 36 kb, there is a
need to use stuffer DNA in order to complete the genome
size for encapsidation. Initially, it was believed that
stuffer DNAs unique role was the participation in the
packaging of the vector genome. Thus, the rst DNA
stuffer used was from lambda phage, yeast, bacterial and
non-human DNA.
14
However, rst injections of gutless
vectors carrying lambda DNA elicited CTL immune
response because peptides from stuffer were presented
in the cell membrane, and it became evident that DNA
stuffer played an important role in the stabilization of
viral DNA into the cell.
15,16
The rst choice was then
DNA from mammalian and human introns since they
seemed to favor maintenance of the gutless genome into
the cell for long periods of time.
15
For example, intronic
sequences from the HPRT gene containing matrix
attachment regions (MAR) elements have been used as
stuffer DNA showing increased DNA stability and no
induction of a CTL response. Sequences from other
human loci have also been used with similar or better
results.
17
However, not all intronic sequences are appropriate
DNA stuffer and, thus, proper candidates should avoid
the following sequences (follow next characteristics)
(i) coding regions, (ii) repetitive sequences (like alu
sequences), (iii) hot spots for recombination, (iv) regions
that can interfere with the expression of the transgene,
and (v) toxic or immunogenic regions. On the contrary,
MAR that seem to stabilize the adenovirus genome into
the nucleus and permit long periods of gene expression
are recommended.
17
Production of gutless adenovirus vectors
Since gutless vectors are devoid of all viral genes,
proteins needed for its genome replication, packaging
and capsid formation must be supplied in trans. This
is achieved by coinfection of the gutless with a helper
adenovirus. However, since both helper and gutless
vectors have the same viral capsid, separation must be
addressed before purication. Thus far, strategies have
been based on reducing the packaging efciency of the
helper genome compared to the gutless genome, either
by mutating its packaging signal,
1820
by the different
Ad5 genome
First
generation
Second
generation
Helper
Dependent Ad

0 36000 10000 20000 30000

MLP
E1 E3
E4 E2A E2B
L1 L2 L3 L4 L5
ITR ITR
Transgene
Transgene
Transgene
E1
E1 E2 E3
E3
E4
Figure 1 Map of adenovirus serotype 5 genome and different generations of adenoviral vectors. Early transcripts are represented by E1E4 regions and late
transcripts are represented by L1L5 regions. MLP: major late promoter; C: packaging signal.
Table 1 Vectors used in gene therapy clinical trials
Total number of
protocols (2004)
Protocol number
increase (19992004)
Retrovirus 263 83
Adenovirus 258 172
Lipofection 85 10
Adeno-associated virus 25 21
Other viruses 148 100
Others 183 148
Data from www.wiley.co.uk/genmed/clinical.
Gutless adenovirus
R Alba et al
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Gene Therapy
size of its genome
13
(genomes bigger or smaller than
the optimal do not package efciently), or by specic
elimination of its packaging signal during viral produc-
tion.
21
Initial strategies consisted in the use of helper-
dependent adenoviruses carrying a defective packaging
signal.
13
These vectors had extensive regions of deleted
viral genome, but they still have some coding regions.
Cotransfection of a wild-type adenovirus as a helper
with this gutless permitted to propagate both adeno-
viruses. After several passages, the gutless adenovirus
was puried by CsCl
2
density gradient. However, high
levels of helper virus and multiple recombinations
between both vectors were detected. This, together with
important complications in large-scale production and
purication, led to the development of new strategies.
An important advance was the specic removal of the
packaging signal by Cre recombinase. Parks and colla-
borators
21
developed a production system where the
helper adenovirus had a packaging signal anked by two
loxP sites, and amplication was performed in Cre
recombinase-expressing cell lines (293Cre). When the
helper adenovirus entered the cell, its packaging signal
was excised preventing the inclusion of its genome into
the viral particle, but retaining all coding regions for the
viral proteins needed to produce the gutless vectors
(Figure 2). Using this system, average helper contamina-
tion ranged over 0.110% compared to gutless vectors.
These levels of helper contamination seem to be due
to the limited efciency of packaging signal excision
associated to low recombinase activity or with low
endogenous levels of recombinases. This is caused either
by adenovirus-mediated host cell shut off or by
cytotoxicity of high levels of Cre recombinase.
22,23
Although the nal level of helper contamination is low,
further reduction is desirable to minimize any potential
toxicity associated with the helper virus, especially when
high doses are required.
24
Other recombinases such as FLP have also been used
to remove the packaging signal. FLP is a yeast site-
specic recombinase, which catalyzes recombination
between frt sites.
25
Philip Ng and collaborators
26
created
new FLP-expressing cell lines (293FLP and 293CreFLP)
and a helper adenovirus with a packaging signal anked
by frt sites. Cre and FLP seem to have equivalent
efciency in the production of gutless adenovirus with a
nal helper contamination of 0.11%. A recent and
elegant improvement in the Cre-loxP system has been
the reversion of the packaging signal of the helper virus
in order to avoid generation of a replication competent
adenovirus (RCA) by recombination with the gutless
vector, which has reduced the helper contamination to
levels down to 0.020.1%.
18
However, Cre and FLP are
bidirectional recombinases that permit excision of the
packaging signal and also its re-entrance, favoring
contamination by the helper adenovirus. Improving
HELPER Ad GUTLESS Ad GENOME
CRE/293 CELL LINE
COTRANSFECTION
HUMAN STUFFER DNA

+
RECOMBINASE
CRE
loxP
Sequences

ADENOVIRUS GENOME
ITR ITR

+
VIRAL
PROTEINS
O.1-10%
HELPER ADENOVIRUS
CONTAMINATION
GUTLESS ADENOVIRUS
ITR
ITR
PLASMID
OR VIRUS
Figure 2 Generation of gutless adenovirus using the Cre/loxP system. Gutless and helper genomes are cotransfected in permissive 293 Cre-expressing cells,
where both genomes are amplied and viral proteins produced. Then, packaging signal of the helpers genome is excised by Cre recombinase, preventing its
packaging into the viral capsid, while gutless genome is still packageable. Efciency of the excision process allows 9099.9% purity of the gutless vector.
Gutless adenovirus
R Alba et al
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Gene Therapy
the excision efciency of Cre and FLP in cell lines
also improves the opposite reaction. Thus, the use of
unidirectional recombinases, such as FC31, is an
attractive alternative to reduce contamination levels with
the helper adenovirus. FC31 is a unidirectional recombi-
nase, which recognizes homologous sequences called
attB/attP and shows an excision efciency similar to that
of Cre in 293 cells.
27
When FC31 excises attB/attP-
anked sequences, it creates new sequences called attR/
attL preventing the opposite reaction. Thus, the helper
adenovirus carrying an attB/attP-anked packaging
signal opens the door to a new gutless production
system (authors unpublished results).
Another approach to reduce helper Ad contamination
is based on size-restricted protein IX-deleted helper. pIX
is a hexon-associated protein essential for packaging full-
length viral genomes. A 293 pIX-expressing cell line
permitting the growth of a full-length pIX-defective
helper was generated.
28
The use of gutless vectors with
genomes smaller than 36 kb gives a selective advantage
in the pIX system. Plaque-forming unit assay showed
a reduction by 1000 times of helper contamination
compared to the classical Cre-loxP system. However,
southern blot analysis revealed helper contamination
levels of 0.2% (similar to Cre-loxP), meaning that the
majority of helper adenoviruses was packaged into the
capsid. Combining both Cre and pIX size-restricted
systems allowed to reduce the number of helper
adenovirus particles, but defective helper virions still
remain in the nal preparation.
To address the helper contamination issue, other
strategies are based on using nonadenovirus vectors as
helpers, like herpes simplex virus-1
29
and baculovirus.
30
However, low production efciency
29
and 2% of RCA
generation
30
make their use not feasible in large-scale
production.
As important as the design of a helper-free gutless
adenovirus system is its production in large or industrial
scale. Large-scale production of gutless adenovirus is
complex and less efcient since it requires successive
time-consuming gutless:helper adenovirus coinfections.
Unfortunately, this process increases the risk of reorga-
nizations in the vector genome by homologous recombi-
nation with viral E1 sequences present in permissive 293
cells, which can nally lead to the generation of RCAs. To
avoid this, several laboratories have developed permis-
sive cells, such as N52.E6
5
and PERC6,
6
that do not
contain viral sequences prone to recombine. However, to
achieve large quantities of high quality helper-free
gutless vectors needed for clinical assays, it is funda-
mental to develop and improve the methodology in the
amplication and purication steps as well as in vector
quality. Thus, development of several cell lines able to
grow in suspension, like human 293S,
31
PER.C6,
32
and
293Cre suspension cell lines
18
facilitates large-scale
amplication in bioreactors, although purication meth-
ods like non-ultracentrifuge-based methods and elimina-
tion of helper contamination still need to be improved.
Gutless adenovirus and immune response
Systemic delivery of rst-generation adenoviral vectors
is known to induce a strong hosts immune response,
resulting in the rapid elimination of vector-transduced
cells and the generation of neutralizing antibodies
against the transgene products and the adenovirus
capsid. Both nonspecic innate and adaptive immune
responses are involved when rst- and second-genera-
tion adenoviral vectors are administered (see Schagen
et al
33
for an extensive review). Thus, the innate immune
response is rapidly developed after virus entry by
induction of inammatory gene expression and further
recruitment of macrophages, neutrophil and natural
killer cells, leading to an 8090% of rst-generation
vector removal from the liver in 24 h.
34
Basically, innate
immunity is triggered by the adenovirus particle, is
Ad-dose dependent and does not require viral gene
expression.
3537
In a second step, adaptive cellular and humoral
immune responses are developed about 47 days after
delivery. At this time, a second peak of cytokine and
chemokine gene expression and inammation occurs
leading to lymphocytic inltrates and to the induction
of adenovirus-specic CTL.
35
Initially, cellular immune
response is activated when antigen-presenting cells
(APCs) uptake adenovirus particles, process the particles
into small oligopeptides and present them through the
major histocompatibility complex (MHC) class-I mole-
cules at the cell surface. Further binding of CD8+ T cells
to the MHC class-I/peptide complex induces formation
of Ad-or transgene-product-specic CTLs. Therefore, the
novo synthesis does not seem to be required to initiate the
process.
33
However, for late inammation, the expression
of viral genes still encoded within Ad vectors plays
a signicant role. In immunocompetent hosts, this
response limits the duration of transgene expression
and results in adenovirus vector clearance within a few
weeks of administration.
7,34
On the other hand, adaptive humoral immune
response is initiated by the binding of adenovirus
particles to the surface immunoglobulin of B cells.
33
After internalization and virus processing, the adeno-
virus-derived epitopes are presented at the surface of the
B cell by MHC-II molecules. Exposure of these cells to
cytokines from activated CD4
+
-Th2 helper cells will
result in differentiated plasma cells secreting antibodies
towards the adenoviral capsid.
38
High titers of antibodies
against capsid proteins, either pre-existing because of
previous exposure to natural virus or generated as a
result of vector administration, may inhibit subsequent
dosing with the same vector.
Different strategies to circumvent innate and adaptive
immune responses have been developed. However, most
of them present secondary complications and/or their
use in human patients is questionable. These strategies
include macrophage depletion,
39,40
use of immunosup-
pressive agents (cyclosporin A, cyclophosphamide,
dexamethasone, FK506, Interleukin-12 and deoxyper-
gualin),
4146
use of antibodies to deplete CTLs,
47,48
blockade of costimulatory interactions between APCs,
T and B cells,
4952
intrathymic administration of adeno-
virus,
53
oral tolerization,
54
use of vectors derived from
non-crossreacting serotypes,
55,56
use of adenoviruses
from other species
57,58
and coating vectors with inert
chemicals like polyethylene glycol (PEG).
5961
Diverse in vivo studies in mice suggested that, in the
absence of an immune response, rst-generation adeno-
viral vector DNA is maintained as a stable episome in the
host cell.
41,62,63
Last-generation helper-dependent or
Gutless adenovirus
R Alba et al
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Gene Therapy
gutless adenovirus vectors display reduced long-term
toxicity and prolonged transgene expression compared
to rst-generation vectors after administration to per-
ipheral organs of immunologically na ve animals.
56,6468
Lack of coding viral genes may account for reduced
adaptive cellular immune response after systemic deliv-
ery of gutless vectors. Initially, gutless vectors are
capable of transducing dendritic cells and stimulating
Ad-specic T-cell responses, independent of viral gene
transcription.
69
However, the expression of viral genes is
required for T cells to exert their effector functions in the
liver,
70
which possibly explains the vector persistence
and improved transgene expression following transduc-
tion with gutless vectors compared to results with rst-
generation Ad vectors.
As expected, systemic delivery of gutless vectors still
induces adaptive humoral response against the vector
capsid as for rst-generation Ad vectors. Indeed, the
development of Ad-specic antibodies does not con-
tribute to the elimination of Ad-transduced cells and
therefore does not affect the persistence of transgene
expression. However, Ad-specic antibodies will bind
the readministered Ad vector and thereby prevent cell
entry and promote opsonization by macrophages.
Humoral response can also be developed towards
circulating antigen induced by gutless adenoviral trans-
fer.
70
Vectors that mediate transgene expression in APCs
trigger antibody formation because they increase the
probability of neoantigen presentation by APCs,
71
and,
hence, careful selection of tissue-specic promoters may
signicantly improve adenovirus-associated toxicity
proles and diminish or abolish APC transduction and
transgene expression.
72
In addition, systemic adminis-
tration of gutless vectors in a clinical setting might be
inefcient because of the presence of circulating neu-
tralizing antibodies against the same or crossreactive
serotype as a consequence of a natural infection or as a
result of previous vector administration. A large propor-
tion of the human population has signicant levels of
antibodies against adenoviruses;
73
thus, the capacity to
overcome a pre-existing immunity is a fundamental
problem. Different successful strategies to circumvent
pre-existing immunity have been applied, such as the
use of alternative gutless serotypes
15
and the use of a
non-human gutless adenovirus.
58
Thus, administration
of gutless CAV-2 vectors allows the stable, high-level
expression in neurons throughout the rat central nervous
system (CNS).
20
It is noteworthy that even in the
presence of an active peripheral immunization with an
adenovirus that completely eliminates expression from
rst-generation vectors within 60 days, gutless vectors
are able to maintain a long-term transgene expression
in the brain
74,75
due to reduced inammation both in
intensity and in duration in the brains of the preimmu-
nized animals.
As innate immune responses are dependent on the
viral capsid or particle, innate responses stimulated by
gutless vectors are similar to those stimulated by rst-
generation adenovirus vectors. Thus, dose-dependent
acute inammation was reported by Brunetti-Pierri and
colleagues
76
in non-human primates following the
administration of high-dose gutless vectors. However,
innate response, as these recently reported, may be
reduced by PEG modication, probably due to lower
vector uptake by Kupffer cells in vivo.
77,78
Surprisingly, gutless vectors have also been proved to
be very efcient in vaccination, due to their longer
duration of expression, their lower antiviral reactivity
and their higher levels of transgene protein in dendritic
cells compared to the same amount of rst-generation
Ad vectors.
79
In vivo administration of gutless adenovirus
Gutless adenoviruses have been administered in vivo to
different tissues in rodents, dogs and also to non-human
primates. Owing to the high efciency of adenoviruses in
targeting hepatocytes, most of the toxicity studies were
carried out in the liver, following intravenous adminis-
tration of the vector.
68
Gutless vectors have been shown
to express non-immunogenic transgenes during the
whole life of the mouse, while expression driven
by rst-generation adenovirus lasted at most for 3
months.
8082
Therapeutic genes carried by gutless vectors have
been administered to the liver of mouse models for
different diseases, such as hemophilia A and B,
83,84
obesity,
66
familial hypercholesterolemia,
81,8587
ornithine
transcarbamylase deciency,
88
diabetes
89
and chronic
viral hepatitis.
90,91
Therapeutic levels of most proteins
have been documented for a long-term duration. In some
cases the antibody response against a nonendogenous
transgene led to a signicant decrease in the circulating
levels of the therapeutic protein. However, when the
transgene expression was driven by a liver-specic
promoter, the immune response was lower, efcacy of
the therapeutic protein increased and secondary effects
derived from systemic circulation of the therapeutic
protein were undetectable.
90,92
The encouraging results
obtained in rodents had promoted the preclinical studies
in larger animal models. This is the case for hemophilia
A and B dogs,
72,83
which resulted in minimal acute liver
toxicity and transient expression of the transgene
allowing for partial or complete correction of hemo-
philia. As in rodents, the use of liver-specic promoters
avoided the development of neutralizing antibodies
against the therapeutic protein, although the expression
was lower than with a viral ubiquitous promoter.
72
Long-term expression of gutless-driven transgene
was also documented in baboons for more than 1 year;
however, a decrease in the levels of the protein was
observed over time.
56
Toxicity in non-human primates
was assessed using different doses of gutless adenovirus
and the results obtained showed that administration of
high doses of adenovirus (10
13
viral particles/kg)
induced a strong activation of the innate inammatory
response that caused the death of the animal.
76
In order to circumvent the decrease in transgene
expression overtime, readministration with gutless vec-
tors has been attempted in mice using different adeno-
virus serotypes. While readministration with different
serotypes is possible using rst-generation adeno-
virus,
55,56,9395
the level of transgene expression is lower
after the second administration, probably due to cross-
reacting CTLs between the different serotype proteins.
96
However, as gutless vectors do not contain viral genes,
readministration of a different serotype leads to the same
levels of the therapeutic protein as in na ve animals
without showing liver toxicity.
15,81
Liver toxicity was also
Gutless adenovirus
R Alba et al
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Gene Therapy
absent after repeated administration of the same ser-
otype of gutless;
67
however, it resulted in a drop of 30-
to 100-fold in transgene expression compared to na ve
animals.
15
Efciency of regulated promoters was also evaluated
using gutless vectors as they offer the possibility of
incorporating all the genetic components of an inducible
system plus other additional sequences such as insula-
tors or silencers that have been shown to avoid leakiness
of the uninduced promoter.
97,98
When these promoters
were driven by rst-generation adenoviruses, only one
induction was possible; a signicant hepatotoxicity was
developed when the animals were treated with the
inducer, followed by a rapid loss of vector DNA.
99
This
was not the case with gutless vectors, where expression
was reinduced three and four times over a period of 2
months. Similar results were obtained with different
inducible systems such as tetracycline, tamoxifen or
mifepristone.
9799
Stability of gutless vectors was evaluated during
hepatocyte cell cycle. Surprisingly, after two-third partial
hepatectomy, gutless vector genomes were reduced by
50%, while rst-generation Ad vector copies were
reduced by 71%. More importantly, episomal plasmid
DNA-injected mice showed a reduction of 99% in DNA
copy number. The decrease in vector copy number/cell
correlated with transgene expression. Several hypotheses
have been proposed to explain the persistence of
episomal gutless vectors in dividing hepatocytes, like
centromeric function of the Ad genome or nuclear
retention activity of the Ad terminal protein, but
conclusive results have not been obtained to date.
100
Adenoviruses target the liver very efciently; how-
ever, they are less effective in other cell types lacking the
CAR or adenovirus receptor. Retargeting of gutless
vectors has been attempted by the incorporation of
polylysine or RGD in the HI loop of the adenoviral ber
protein, which can be obtained by adding ber-modied
helper viruses in the last amplication step of the gutless
vector production. Polylysine has been shown to
effectively transduce mature muscle cells in vitro and
in vivo,
101
while RGD increased the efciency of
transducing ovarian carcinoma cell lines, primary
vascular smooth muscle cells and primary human
endothelial cells.
102
Duchenne muscular dystrophy (DMD), an X-linked
lethal disorder that affects 1 in 3500 males, is caused by
genetic mutations in the dystrophin gene. The cDNA for
full-length dystrophin is approximately of 14 kb, far
above the size of most gene transfer viral vectors. Thus,
the highcapacity of gutless adenoviruses has opened the
possibility to treat DMD animal models
103
not only with
one full-length dystrophin cDNA but also with two
copies of the therapeutic gene.
104,105
In these studies,
neonate skeletal muscles of mdx mice injected with
gutless adenovirus expressed dystrophin for the dura-
tion of the experiment, up to 1 year. At this point, 52%
of the muscle bers showed transgene expression and
functional correction of muscle contractility, and
improved histopathology was reported, correlating with
the level of dystrophin expression. However, a four-fold
decline in the vector DNA copy number was observed
from 10 days to 1 year after vector injection, and the
treated mice developed a signicant humoral response.
No transduced muscle ber loss was reported, indicating
that the decline in DNA copies could be due to instability
of the vector DNA into the host cell.
104
However, loss of
DNA copy number/cell has no consequences in dystro-
phin expression or muscle function in experiments
performed in mice because dystrophin is stable for 26
weeks. When skeletal muscle bers were transduced
before birth (in utero at embryonic day 16), expression of
the reporter protein was stable at least for 5 months
despite the development of antibodies against the
transgene and the adenoviral capsid.
106
However, in this
case, the authors reported no loss of DNA copy number.
Lower levels of toxicity compared to rst-generation
eneration Ad that resulted in higher survival rates of
animals transduced in utero were also documented.
Mdx muscle goes through cycles of necrosis followed
by regeneration, which could account for the higher
efciency of infection of muscle bers from young and
old mdx mice by adenovirus vectors compared to control
animals as reported by the group of JS Chamberlain.
107
On the contrary, the high turnover of muscle cells in the
mdx mice can decrease the stability of nonintegrative
vectors like adenovirus. In 1-year old mdx mice,
transduction of 2530% of skeletal muscle cross-sectional
area with gutless vectors carrying the dystrophin cDNA
under the regulation of a muscle-specic promoter lead
to 40% correction of their high susceptibility of contrac-
tion-induced injury 1 month after treatment.
107
However,
longer time points were not evaluated, but mild immune
cell inltration was reported using mouse dystrophin
cDNA.
Administration of recombinant protein to the CNS
requires the breakdown of the bloodbrain barrier, a
procedure that should not be used for repetitive read-
mnistrations. Thus, permanent transduction of neurons
offers the possibility of stable treatment of diseases
affecting the CNS. Gene transfer to the CNS has been
explored using different vector systems. Despite the
immunoprotection of the CNS, results with rst-genera-
tion adenovirus show a decrease in the expression of the
transgene 2 months after transduction, correlating with
the disappearance of the adenoviral DNA.
108
Acute loss
of transduced cells and chronic inammation could
account for this decline, mainly at high doses.
109
On the
contrary, gutless adenovirus expression can be detected
in the CNS 1 year after striatal injections.
20,110
Moreover
and contrary to what was reported for other tissues,
preimmunization with the same serotype of adenovirus
does not alter gutless stability in the CNS, and does not
signicantly reduce the initial infection of the CNS by
gutless vectors, although a transient acute brain inam-
mation is shown.
75
Gutless vectors have also been used
to successfully express therapeutic genes in the retina
either by transplanting cells previously transduced with
the virus or by direct injection of the vector.
110,111
Other tissues like lung, cardiac muscle, vascular tissue
or dendritic cells have also been targeted with gutless
adenoviruses with similar results as for the muscle or the
liver.
112115
Future considerations
This is a fascinating moment for gutless adenovirus
vectors: their use permits long-term expression in vivo
with reduced and transient cellular immune response in
Gutless adenovirus
R Alba et al
S23
Gene Therapy
animal models for human diseases and, moreover, the
increasing number of groups working in this eld favors
technological advances to escape preimmune response
by developing non-human gutless adenovirus
20
or to
increase stability by generating integrative gutless
vectors,
116,117
or vectors with replication capacity.
118
However, their use in clinical assays is questionable
since helper contamination levels are still high, and
large-scale production in bioreactors is not yet fully
developed. Therefore, future studies will be needed in
these areas to nally bring gutless adenovirus to the
clinic.
Acknowledgements
We would like to acknowledge Dr Merce` Monfar for
critically reading the manuscript. Our work is supported
by MCYT-SAF2003-03256, Marato TV3-2002-031632 and
Instituto de Salud Carlos III (C03/08). AB has a contract
from the Ramon y Cajal Program (Ministerio Educacio n
y Ciencia, Spain), and RA is a recipient of an FI-
Generalitat fellowship.
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