Review TRENDS in Biotechnology
3 March 2003 117
Latest development in viral vectors for
gene therapy
Kenneth Lundstrom
Regulon Inc./BioXtal., Chemin des Croisettes 22, CH-1066 Epalinges, Switzerland
Gene therapy includes the application of various viral
vectors, which represent most types and families of
viruses, suitable for infection of mammalian host cells.
Both hereditary diseases and acquired illnesses, such as
cancer, can be targeted. Because of the various proper-
ties of each viral vector, the definition of their appli-
cation range depends on factors such as packaging
capacity, host range, cell- or tissue-specific targeting,
replication competency, genome integration and dur-
ation of transgene expression. Recent engineering of
modified viral vectors has contributed to improved
gene delivery efficacy.
Gene therapy as a modern therapeutic has been around for
nearly a decade. Although great hope was founded on
rapid breakthrough, the progress has been slower than
anticipated. The first major gene therapy success was the
retrovirus-based treatment of infants suffering from the X
chromosome-linked severe combined immune deficiency
(SCID-X1) and showed the real potential of long-term
or even permanent cure of hereditary disease [1]. How-
ever, the field suffered some setbacks, such as the
unfortunate fatal case of adenovirus-based treatment of
a non-life-threatening disease, Ornithine Transcarbamy-
lase deficiency [2], and the recent discovery that one of the
SCID-X1-treated patients developed a leukemia-like con-
dition [3]. Nevertheless, it also led to improved control of
study design and monitoring, and has directly impacted
vector development and the engineering of alternative
delivery vehicles [4]. Four factors directly related to gene
therapy vectors have hampered the progress.
† Inefficient gene delivery: although many non-viral
and viral vectors demonstrated high gene delivery
efficacy in cell lines, their potency in vivo has been
disappointingly modest.
† Targeting of expression: related to gene delivery issues,
specific targeting to the cells or tissue of interest is
extremely important to avoid expression of therapeutic
and, specifically in cancer therapy applications, toxic
gene products in healthy tissue.
† Duration of expression: the establishment of long-term
expression has been hampered by poor replication and
stability of episomal vectors and inefficient or in-
appropriate integration of vectors into the host genome.
† Safety: a prerequisite for clinical gene therapy trials
must be to ensure that appropriate safety standards are
Corresponding author: Kenneth Lundstrom (kenneth.lundstrom@mepnet.org).
http://tibtec.trends.com 0167-7799/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0167-7799(02)00042-2
Vol.21 No.
met. In this context, vectors that are replication-
deficient and for which the potential probability of
homologous recombination is reduced to theoretically
acceptable levels need to be engineered. Moreover,
positioned genome integration and reduced cytotoxicity
and immunogenicity are very important.
Overall, there have been major improvements in all
aspects of gene delivery vector development and targeting
of gene expression during the past few years. However,
it rapidly became obvious that there are no universally
applicable ideal viral vector systems available. The
various features of each vector and type of disease to be
treated needs to be defined before decisions are made as to
which vector type should be applied. Several properties of
the vectors need to be addressed before making decisions
on which system to use, and it is therefore appropriate to
make a comparison between the various viral vectors used
in gene therapy today (Table 1). The properties of adeno-
associated virus (AAV), adenovirus, alphavirus, herpes
simplex virus, lentivirus and retrovirus vectors are
presented here and their potential applications described.
Less attention is paid to reviewing results from clinical
trials. An insight into novel technologies developed for
various viral vectors to make them more efficient and
targeted to specific host cells or tissues is presented.
Adeno-associated virus
AAV has established its position as one of the most popular
gene delivery systems. This is mainly because of the long-
term and efficient transgene expression in various cell
types in many tissues such as liver, muscle, retina and the
central nervous system [5]. However, there are some
disadvantages associated with the application of AAV. The
packaging capacity is relatively restricted and the large-
scale production inefficient. Furthermore, the pre-existing
immunity to human AAV vectors is comparable to
adenovirus and the integration into the host genome (if
it occurs at all) is random, which can lead to unexpected
activation or inhibition of endogenous gene expression.
Different AAV serotypes have shown remarkably different
expression patterns because of differences in cell entry and
intracellular activities. For instance, AAV1 is suitable for
expression in skeletal muscle and retina, whereas AAV5
transduces neuronal and lung cells efficiently [6]. By
contrast, AAV2 is characterized for its long-term, albeit
poor, expression levels and for this reason, the discovery of
novel rhesus monkey serotypes AAV7 and AAV8 might be
118 Review TRENDS in Biotechnology
Table 1. Viral vectors used for gene delivery
Vector Packaging capacity Host range
AAV Low , 4 kb Broad, infects both non-dividing and
dividing cells
Adenovirus Medium , 7.5 kb Broad low transduction of neurons
Alphaviruses Medium , 7.5 kb Broad, neuron and glial cell-specific
strains
Herpes simplex virus High . 30 kb Broad, neurons, stem cells, muscle cells
Lentivirus Medium 8 kb Broad, dividing and non-dividing cells
Retrovirus Medium 8 kb Restricted, dividing cells only
of interest for human gene therapy applications [7].
In particular, AAV8 demonstrated high levels of up to
100-fold higher factor IX expression in liver cells than for
any other AAV serotype tested. Moreover, the expression
levels were not compromised by preimmunization with
other AAV serotypes.
Application of the dimerizer-inducible transcriptional
regulatory system for AAV has allowed pharmacological
regulation of heterologous gene expression in vivo [8].
Using this procedure, erythropoietin (Epo) expression was
regulated by administration of rapamycin. The Epo level
was negligible in the subretinal space of nude mice but was
detected at maximal level three days after systemic
administration of rapamycin. The Epo levels returned to
baseline levels three weeks after withdrawal of the drug.
The rapamycin-inducible system gave similar results in
primates [8].
An interesting indirect observation indicated that
AAV vectors could mediate sensitization of human tumor
cells towards chemotherapeutic drugs. In this way,
infection of AAV type 2 increased cisplatin-induced frag-
mentation in HeLa and A549 cells [9]. This phenomenon
was serotype-specific because no sensitization was
observed after AAV-5 infection.
Adenovirus
The key features that have made adenoviruses such
popular gene therapy vectors have been the ability to
generate high-titer virus stocks and the high-level
heterologous gene expression. Although early versions of
adenoviruses showed toxic side effects and strong immune
responses, newer second- and third-generation vectors
with many of the viral genes deleted, have demonstrated
significant improvements [10]. The restricted tropism of
adenovirus has also limited their range of application.
Although there are indications that adenovirus can infect
CD34þ cells, the transduction rates in many hematopoeitic
cells have been fairly modest. Screening of various
adenovirus serotypes showed that serotypes 35 and 11
demonstrated the best interaction of virus with CD34þ
cells, which triggered the construction of a novel chimeric
Ad5/35 vector with the Ad35 fiber incorporated into the
Ad5 capsid [11]. The chimeric Ad5/35 vectors showed a
superior transduction efficiency of hematopoietic cells. In
another approach, adenovirus vectors were retargeted to
renal carcinoma cells (RCC) in an attempt to develop a
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Vol.21 No.3 March 2003
Clinical Features
trials
þ Slow expression onset, genome integration
(^ ), long-term expression, inefficient large-
scale virus production
þ Transient expression, strong immunogenicity
þ Transient, but extreme, expression levels; low
immunogenicity
2 Latent infection, long-term expression, low
toxicity (mutants)
2 Genome integration, long-term expression,
safety concerns low titers, production
inefficient
þ Genome integration, long-term expression
treatment for metastatic renal cell carcinoma, which is one
of the most treatment-resistant human diseases [12].
Although RCCs seemed to be devoid of Coxsackie-
adenovirus receptor (CAR) (i.e. adenovirus receptors)
they showed abundant expression of integrins (avb3 and
avb5), which are putative receptors for Ad3. Generation of
chimeric fibers composed of Ad5 shaft and Ad3 knob
resulted in efficient retargeting of RCCs. Alternatively,
this was also achieved by the introduction of the RGD (in
single-letter aminoacid code) motif into the H1 loop of the
Ad fiber knob domain.
The efficacy of adenovirus delivery might be severely
hampered because most people have been exposed to
natural adenoviruses infections, even when using replica-
tion-deficient vectors. In a novel approach, the surfaces of
viral particles were coated with a multivalent copolymer
based on poly-[N-(2-hydroxypropyl) methacrylamide]
(pHPMA) [13]. To improve targeting, fibroblast growth
factor (FGF) and vascular endothelial growth factor
(VEGF) were incorporated in the polymer, which resulted
in targeting of bFGF receptor-positive A549 cells. Target-
ing of endothelial human umbilical endothelial cells with
polymer-VEGF coated adenovirus was also highly efficient
[13]. The in vivo targeting of polymer-bFGF ADLacZ virus
in nude mice bearing intraperitoneal xenografts of human
SUIT2 cells was highly efficient. Separation of human
tumor cells from murine cells by FACS (fluorescence acti-
vated cell sorting) demonstrated that all b-galactosidase
expression was present in the tumor cells. In addition, the
polymer-coated adenovirus particles were able to shield
against antibody recognition. This should further improve
the possibilities of readministration of adenovirus to
achieve improved therapeutic efficacy. Polymer-coating
will also permit the incorporation of tumor-specific surface
antigens and biological effectors to improve tissue pene-
tration and broaden the tropism. In another approach, the
PEGylation of the adenovirus capsid protein prolonged
transgene expression after systemic delivery of E1-deleted
adenovirus, and allowed partial readministration with
native virus [14].
To enable chromosomal integration of adenovirus
vectors to expand the duration of expression in vivo,
hybrid Ad-AAV vectors were designed [15]. The vector
engineered contained the first generation adenovirus
vector and the inverted terminal repeat (ITR) sequences
of AAV flanking a reporter gene. The hybrid vectors could
 Review TRENDS in Biotechnology
be efficiently produced as by-products of adenovirus-AAV
amplification. The Ad-AAV vector stably transduced cell
lines at comparable efficacy to AAV and the chrosomal
integration appeared to be random.
Alphaviruses
Replication-deficient vectors have been engineered for
several alphavirus species such as Semliki Forest virus
(SFV), Sindbis virus and Venezuelan equine encephalitis
virus (VEE) [16]. The recombinant particles from these
vectors are generated by co-transfection of alphavirus
replicon and helper vector RNA, which therefore upon
infection of host cells leads to one round of RNA
replication, but no further production of virus progeny.
Alphavirus vectors have mainly been used for recombinant
protein expression in cell lines, in expression studies in
neurons and for vaccine production [17]. However, the
rapid high-titer production, broad host range and extreme
transgene expression capacity have made these vectors
attractive for gene therapy applications (Fig. 1). Intratu-
moral injections with SFV vectors expressing reporter
genes and therapeutic genes have demonstrated efficacy in
animal models. SFV-based expression of interleukin-12
(IL-12) in a B16 melanoma mouse model resulted in
significant tumor regression [18], as did consecutive intra-
tumoral administration of SFV-GFP (GFP, green fluor-
escent protein) particles into a human lung carcinoma
mouse model [19]. In a recent approach, dendritic cells
isolated from bone marrow were transduced with SFV-IL-12
to treat mice with brain tumors and resulted in prolonged
survival of animals with established tumors [20]. In
another study, Sindbis particles expressing the human
papillomavirus E7 antigen linked to the Herpes simplex
virus VP22 protein were generated in a new packaging cell
line [21]. The VP22 protein facilitates the spread of the
linked antigen to neighbouring cells, which in this case
resulted in a significantly increased number of E7-specific
CD8þ T cell precursors and improved antitumor response
in C57BL/6 mice.
Although the transient nature of expression from
alphavirus will prevent any long-term presence and
spread of virus, the broad host range and the strong
preference of neuron-specific transgene expression could
severely limit gene therapy applications. Preliminary
attempts for cell or tissue-specific targeting of Sindbis
particles indicated that the introduction of IgG-binding
domains of protein A into the E2 envelope protein resulted
in a 105-fold reduction in the infection rate of normal host
cells and allowed infection of cells treated with a
monoclonal antibody against a surface protein [22]. In
another approach, SFV particles have been encapsulated
into liposomes, which allowed systemic delivery to cancer
tissue in mouse models with implanted human tumors
[23]. Because of the differences in normal and tumor
vasculature composition, encapsulated SFV-LacZ par-
ticles were specifically targeted to tumors, leading to
high b-galactosidase expression in implanted human
tumors after intraperitoneal injection of mice. Systemic
delivery of high concentrations of encapsulated SFV
particles did not cause toxicity in normal tissue. Recently,
a phase I clinical trial on advanced melanoma and kidney
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Vol.21 No.3 March 2003 119
Adenovirus AAV
(Tumors, hematopoietic
cells)
(liver, muscle,
retina)
Polymer-
coated
Hematopoietic
adenovirus
(tumors)
cells
Alphavirus
(tumors)
Retrovirus Liposome-
(tumors, stem,
hematopoietic
Stem
cells
encapsulated
alphavirus
cells) (systemic
delivery)
Lentivirus
(CNS, liver,
Herpes simplex virus
muscle)
(CNS, PNS, muscle,
hematopoietic, stem cells)
TRENDS in Biotechnology
Fig. 1. Clinical applications of viral vectors. Viral vectors are directly injected into
specific tissues or administered systemically. Ex vivo applications are also
possible.
carcinoma using encapsulated SFV particles expressing
the p40 and p35 subunits of IL-12 showed no liposome or
SFV-related toxicity, but transient increase of IL-12 levels
in the serum of patients. Because of the encapsulation
procedure, multiple readministration of SFV particles was
possible.
Herpes simplex virus
The typical characteristics of Herpes simplex virus (HSV)
are the large genome size (152 kb), neurotropism and the
latent phase of the life cycle, which means that the viral
genome remains episomally in the cell nucleus for the
lifetime of the host organism. Although full genome HSV is
cytotoxic to neurons, multiple gene deletions can be made
to still generate viable HSV vectors. For instance, the
deletion of four immediate early (IE) genes ICP0, ICP4,
ICP22 and ICP47 resulted in an entirely nontoxic vector,
which persists for extended time in host cells [24]. Because
of the latency, HSV vectors are highly attractive for
application areas where life-long sustaining of transgene
expression is desirable. However, in the case of cancer gene
therapy or other approaches in which only high-level
transient expression is required, other vectors might be
more appropriate.
Neurological diseases, characterized by chronic path-
ologies requiring long-term expression of therapeutic
genes, are suitable targets for HSV vectors. HSV vectors
containing latency active promoters LAP1 and LAP2 have
been engineered for long-term expression (up to 300 days)
in the peripheral nervous system (PNS) [25]. HSV-based
expression of nerve growth factor (NGF) protected
primary dorsal root ganglion neurons from an oxidative
insult with H2O2. In animal models NGF expression also
protected sensory neurons from toxic insult, which implied
a prophylactic application in treatment of chemotherapy-
induced peripheral neuropathy. In another application,
chronic pain was targeted by prepro-enkephalin expres-
sion from a conditionally replicating HSV vector after
inoculation into the footpads of mice [26]. The study
showed that the recombinantly expressed protein was
120 Review TRENDS in Biotechnology
similarly processed and released as native prepro-enkeph-
alin. HSV vectors have also found applications in the CNS.
However, because wild-type HSV causes encephalitis, it
was essential to develop nontoxic replication-defective
vectors with deletions in the IE genes (ICP4, ICP22,
ICP27) [27].
Other possible targets for HSV are malignant gliomas,
skeletal muscle and stem cells. For instance, HSV1716, a
selectively competent mutant, showed persistence in
cultured human glioma cells from tumor tissue of a patient
treated 2.5 years earlier [28]. The wild-type HSV17 lysed
cells within three days, whereas HSV1716 persisted in the
glioma cells, which indicated a potential of tumor killing
over extended periods of time. Replication-defective HSV
vectors are attractive for targeting gliomas because of
their high infectivity and requirement of low multiplicity.
Furthermore, gliomas are generally highly localized. The
use of the HSV thymidine kinase (TK) gene has proven
highly efficient as a form of therapy. HSV-TK as such does
not kill cells but converts the prodrug ganciclovir (GCV) to
its active form. In addition to HSV-TK transduced cells,
neighbouring cells are also killed after GCV adminis-
tration because of the so-called by-stander effect [29]. The
advantage of using muscular delivery of replication-
defective HSV for hereditary diseases such as various
forms of muscular dystrophy is the capacity of the HSV
vectors to accommodate, for instance, the large dystrophin
gene. Again, the use of triple IE mutant (ICP4, ICP22,
ICP27) HSV-dystrophin vectors showed substantially
reduced cytotoxicity and expression of dystrophin in
dystrophin-null myotubes. However, in vivo dystrophin
staining was observed in dystrophin-null muscle, although
mainly in areas close to the injection site [30]. Interest-
ingly, stem cells were shown to be susceptible to HSV
infections. For instance, monkey CD34þ cells transduced
with IE mutant HSV vectors, which were transplanted
into monkeys with skin autographs, showed reporter gene
expression for more than three weeks [31]. This suggested
that potentially therapeutic gene products can be intro-
duced into stem cells with the aid of HSV vectors.
Retroviruses
The pioneering work on gene delivery in vivo was
performed with retrovirus vectors, typically using murine
leukemia virus (MLV). Advantageous features of retro-
viruses are their ability to integrate into the host genome
and therefore sustain heterologous gene expression for
extended time periods. The inability of retrovirus vectors
to infect non-dividing cells has, however, restricted their
potential applications. To overcome limitations of host cell
tropism, retrovirus vectors have been pseudotyped with
envelope proteins from other viruses such as the G
glycoprotein from vesicular stomatitis virus (VSV) [32].
VSV G pseudotyped retroviruses are less labile and can be
concentrated to high titers and also show a much broader
host range than the wild-type retrovirus. Vector develop-
ment has been intense for retroviruses; for instance,
self-inactivating and self-activating vectors have been
engineered [33]. This can be achieved by the introduction
of directly repeated sequences, which leads to high-
frequency deletions during reverse transcription. Targeting
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Vol.21 No.3 March 2003
of retroviruses has also been accomplished by modifying
the envelope structure, as in the case of pseudotyping,
displaying recognition sequences on the surface, or apply-
ing tissue-specific promoters. Tumor cells were specifically
targeted by displaying an scFv antibody to the carcino-
embryonic antigen (CEA) on retrovirus particles [34].
Substantial tumor regression, up to 70%, was observed
after subcutaneous administration of recombinant retro-
virus to SCID mice with MKN-45 tumors.
Retrovirus vectors were subjected to the first clinical
trial on human gene therapy to correct adenosine
deaminase (ADA) deficiency [35]. White-blood cells iso-
lated from patients were infected ex vivo with an MLV-
based vector expressing ADA and a neomycin marker
gene. After selection with G418, neomycin-resistant cells
were isolated and re-introduced into patients. The treat-
ment improved the physical condition of the patients and
the ADA-containing provirus was stable in the blood for
several years. Retrovirus vectors have also demonstrated
some promising results in cancer therapy and bone
marrow transplantation. The introduction of retrovirus
particles expressing HSV-TK and administration of GCV
suggested that the treatment of graft-versus-host disease
was efficient [36]. The demonstration of the full correction
of the SCID-X1 phenotype in infants is a further indication
of the efficacy of retrovirus vectors [1].
Lentivirus
Although lentiviruses belong to retroviruses, their special
features have made it appropriate to describe them
separately. Many of the lentivirus vectors used in gene
therapy are based on the human immunodeficiency virus
(HIV) [37]. Typically, HIV vectors can accommodate fairly
large gene inserts and can provide long-term expression
through chromosomal integration. In contrast to conven-
tional retrovirus particles, lentivirus vectors can also
infect efficiently non-dividing cells and can therefore be
applied for expression in neuronal cells. Intron-containing
constructs have been successfully introduced into recent
versions of lentivirus vectors [38].
Recently, a series of improved lentivirus vectors were
developed for transduction of hepatocytes in vivo [39].
Various promoters, such as the human cytomegalovirus
(CMV), the human phosphoglycerate kinase (PGK) and
the mouse albumin promoter, were introduced into the
HIV-1-based vector. These vectors showed enhanced
nuclear translocation in hepatocytes and improved trans-
gene expression. Ex vivo reporter gene delivery was highly
efficient for rat hepatocytes. Moreover, intravenous
administration into SCID mice resulted in 30% transduc-
tion rate of parenchymal and nonparenchymal liver cells.
The systemic delivery also demonstrated efficient expres-
sion in the spleen and bone marrow. Interestingly, targeted
expression to the liver could be accomplished by the use of
the albumin promoter. Therapeutic levels of human factor
IX, stable for one year, was achieved after a single
injection. The limitations of using lentivirus vectors in
clinical trials are today mainly because of the lack of
sufficient methods for production of high-titer virus stocks
and the safety concerns related to their origin from HIV,
despite the engineering of packaging cell lines and
 Review TRENDS in Biotechnology
deletions of genes required for viral replication. One
approach to address safety issues has been to develop
lentivirus vectors incapable of replication in human cells.
It has been demonstrated that equine infectious anemia
virus (EIAV) possesses comparable transduction efficiency
in primate and human cells to HIV, and might therefore be
an attractive candidate for gene therapy applications [40].
Conclusions and future development
Although major improvements in all areas of vector
development have been achieved, further work on tech-
nology issues is necessary. Today, several viral vectors can
generate long-term expression in vivo. An important issue
is to establish physiological levels of expression and
therefore ways of regulating the expression. For instance,
the use of transcriptional regulation by drugs as described
for AAV vectors is an interesting approach [8]. Recent
development of inducible promoters, which are turned on
or off depending on cellular concentrations of the target
gene expressed, are also useful. Attention should also be
paid to the choice of therapeutic genes delivered by the
viral vectors. So far, genes encoding both toxic and
immunostimulatory molecules have been applied in cancer
therapy, whereas replacement of defect genes with healthy
counterparts has been the strategy for various hereditary
diseases.
An interesting approach that could lead to major
advancement in gene therapy has been the development
of RNA interference (RNAi) technology, which was
recently demonstrated to also induce stable gene silencing
in mammalian cells [41]. The low efficiency of in vivo gene
delivery has restricted the use of RNAi [42]. However, viral
vectors could play an important role here, and it was
recently demonstrated that the gene-silencing effect could
be obtained in mice through adenovirus delivery vectors
[43]. RNAi could provide, for instance, new means of
preventing tumor cell proliferation and growth. Promising
results start to appear for targeting, mainly through the
use of tissue-specific promoters. Another approach to
achieve cell or tissue-specific targeting, which seems
highly successful, is the coating of viruses with polymers
or liposome structures as has been described for adeno-
viruses [13] and alphaviruses [23]. Coating of viral par-
ticles also addresses the issue of possible reduction of
immune responses by administered vectors, which should
also improve the readministration capacity. One of the
most important issues surrounding gene therapy appli-
cations is the need to ensure the highest possible safety
levels. Much work has been done in this area, which has
resulted in self-inactivating or self-destroying vectors
upon addition of drugs. Deletion of unnecessary viral
genes from the vector constructs has significantly reduced
cytotoxicity and immunogenicity and also prevented the
generation of replication-competent particles and spread
of virus infection.
Use of alternative species or serotypes has improved
the possibilities of readministration. However, the
recent observation of a leukemia-like condition in one
SCID-X1-treated patient raises the question of the risks of
random integration into the host genome [3]. In this case,
linear amplification-mediated PCR analysis suggested
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Vol.21 No.3 March 2003 121
that the T cells of the patient had initially approximately
50 different retrovirus insertion sites, but eventually a
single one in the LMO-2 gene was present in all T cells.
Mutations in the LMO-2 gene have been associated with
childhood cancers, but in this leukemia patient two of his
relatives also developed cancers at an early age and might
therefore indicate an inherited susceptibility. To further
complicate the matter, the form of cancer in the current
patient has never been seen before. However, the patient is
doing well today (Fischer, A), but this incident should act
as a wake-up call to pay attention to diagnostic analysis of
genome integration, but also to developed methods for
targeted insertion. Today, several countries, such as the
USA and United Kingdom have decided that the benefits
are higher than the risks to authorize the continuation of
retrovirus-based therapy. Although we have to be pre-
pared to encounter setbacks, the long-term cure of SCID-
X1 (upto 3.7 years today) and promising preliminary
results from the treatment of hemophilia patients with
AAV vectors and cancer gene therapy with various viral
vectors will hopefully make gene therapy, the medicine of
the future, a reality in a reasonably short time.
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