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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 822-833
Intermittent, Repetitive Corticosteroid-Induced Upregulation of
Platelet Levels After Adenovirus-Mediated Transfer to the Liver of a
Chimeric Glucocorticoid-Responsive Promoter Controlling the
Thrombopoietin cDNA
By
Ko Narumi,
Motoyoshi Suzuki,
Wenru Song,
Malcolm A.S. Moore, and
Ronald G. Crystal
From the Division of Pulmonary and Critical Care Medicine, The New
York Hospital-Cornell Medical Center, New York; and James Ewing
Laboratory of Developmental Hematopoiesis, Memorial Sloan-Kettering
Cancer Center, New York, NY.
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ABSTRACT |
For many in vivo gene therapy clinical applications, it is desirable
to control the expression of the transferred transgene using
pharmacologic agents. To evaluate the feasibility of accomplishing this
using corticosteroids, pharmacologic agents widely used in clinical
medicine, we constructed replication deficient adenoviral (Ad) vectors
containing an expression cassette with a chimeric promoter comprised of
five glucocorticoid response elements (GRE) and the chloramphenicol
acetyltransferase reporter gene (AdGRE.CAT) or the murine
thrombopoietin cDNA (AdGRE.mTPO). In vitro studies showed the vectors
functioned as expected, with marked glucocorticoid-induced upregulation
of the CAT or mTPO transgenes. To evaluate the inducibility of the GRE
promoter in vivo, the AdGRE.CAT vector was administered intravenously
to C57B1/6 mice, and CAT activity was quantified in liver before and
after intraperitoneal administration of dexamethasone. The GRE promoter
activity was dependent on the dexamethasone dose, with a 100-fold
increase in CAT expression with 50 µg dexamethasone, similar to the
levels observed in vivo with the Rous sarcoma virus long terminal
repeat constitutive promoter. After dexamethasone administration,
maximum CAT activity was observed at day 2, with a slow decline to
baseline levels by 2 weeks. Based on these observations, we
hypothesized that a single administration of an Ad vector-mediated transfer of the chimeric GRE inducible promoter driving the mTPO cDNA
would enable repetitive administration of corticosteroids to
repetitively upregulate platelet levels for 1 to 2 weeks. The data show
that this occurs, with dexamethasone administration every 3 weeks
associated with 1-week elevations (at each 3-week interval) of serum
mTPO levels, megakaryocyte numbers in bone marrow, and platelet levels
fourfold to sixfold over baseline. Thus, with the appropriate promoter,
it is possible to use a commonly used pharmacologic agent to upregulate
the expression of a newly transferred gene on demand.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE SUCCESSFUL application of in vivo
gene transfer technology to treat human disease requires the ability to
transfer the gene of interest to a specific organ without significant
toxicity, and to express the transferred gene at the level, and for the duration, required by the clinical problem. One strategy to control the
expression of this newly transferred gene is to transfer the gene in
the context of an expression cassette with a promoter that can be
regulated by exogenous agents.1 Examples of inducible promoters that have been evaluated in the context of in vivo gene transfer include the metallothionein promoter,2,3 the
tetracycline-responsive promoter,4-6 a chimeric promoter
with multiple cyclic adenosine monophosphate (cAMP) response elements
superimposed on a minimal fragment of the 5 -flanking region of the
cystic fibrosis transmembrane conductance regulator (CFTR)
gene,7,8 a chimera of the thymidine kinase promoter and the
thyroid hormone/retinoic acid-response element,9 complement
factor 3 and serum amyloid A3 promoters responsive to inflammatory
stimuli,10 and the EGR1 promoter responsive to
radiation.11
The focus of this study is to evaluate the hypothesis that transfer of
expression cassettes containing a chimeric promoter with multiple
glucocorticoid response elements (GRE)12 driving a
transgene will function in vivo in the liver to upregulate expression of the transgene following administration of corticosteroids. To
evaluate this concept, we have constructed adenovirus (Ad) gene
transfer vectors with multiple GRE12 driving a reporter
gene (chloramphenicol acetyl transferase [CAT])13 or
the murine thrombopoietin cDNA (mTPO, the megakaryocyte growth and maturation factor that stimulates
thrombopoiesis14-17), and used the Ad vectors
to transfer these chimeric cassettes to the liver of C57B1/6 mice. The
data show that the expression cassettes are responsive in vivo to
systemically administered dexamethasone in a dose-dependent fashion,
but with little if any expression without administration of
dexamethasone. As a functional demonstration of this strategy, with the
mTPO cDNA in the expression cassette, intermittent administration of
dexamethasone to the mice is associated with intermittent elevation of
murine TPO levels in serum, numbers of megakaryocyte in bone marrow,
and blood platelet levels.
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MATERIALS AND METHODS |
Construction of replication-deficient adenovirus vectors.
Plasmids used for recombination with the adenovirus type 5 (Ad5)
backbone were prepared by inserting the expression cassette containing
the chimeric promoter described by Mader and White12 using
five GRE from the rat tyrosine aminotransferase gene18 in
tandem with the insertion of adenovirus 2 major late promoter (Ad2MLP)
TATA box/initiation site (referred to as the "GRE promoter") and
a reporter gene (CAT gene13 or the mTPO cDNA [courtesy of D. Eaton, Genentech, South San Francisco, CA]14-17)
into pCMV.SV2+19 after removing the cytomegalovirus (CMV)
early promoter/enhancer from the plasmid (Fig
1). The replication-deficient Ad vectors AdGRE.CAT and AdGRE.mTPO were generated by cotransfecting the plasmids
and the pJM17 Ad5-based backbone20 into the 293 embryonic kidney cell line (HEK 293, CRL1573; American Type Culture Collection [ATCC], Rockville, MD), grown in improved Eagle's minimum essential media (Biofluids, Rockville, MD) containing 10% fetal bovine serum, 2 mmol/L glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin. Two
vectors were used as a positive control, the AdRSV.CAT vector, containing CAT gene controlled by the Rous sarcoma virus long terminal
repeat (RSV-LTR) promoter.21 The AdNull vector containing the CMV promoter22 but no transgene in the expression
cassette was used as a negative control.23 All of the Ad
vectors were purified by cesium chloride density gradient
ultracentrifugation, and the titers of the virus stocks were determined
by plaque-forming assay on 293 cells.24-26
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| Fig 1.
Schematic representation of the expression cassette
including the chimeric GRE promoter with the CAT gene or mTPO cDNA as reporter genes. The chimeric promoter includes five GRE from the rat
tyrosine aminotransferase gene inserted upstream of the adenovirus-2 major late promoter (Ad2MLP).12 The SV40 polyA stop signal
follows the reporter genes.
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In vitro evaluation of AdGRE.CAT.
To evaluate the time-dependent response of the GRE promoter in the
context of an Ad vector to the addition of dexamethasone in vitro, HeLa
cells (105/well) were incubated in Dulbecco's modified
Eagle's medium (GIBCO-BRL, Grand Island, NY) containing 10% fetal
bovine serum, and infected with the AdGRE.CAT or AdRSV.CAT at a
multiplicity of infection (moi) of 25 for 90 minutes. After 24 hours,
the cells were incubated with or without dexamethasone (25 nmol/L;
Sigma, St Louis, MO) for 1 to 48 hours. The promoter function was
assessed by measuring CAT activity27 and standardized by
total cellular protein concentration (BCA protein assay; Pierce
Chemical, Rockford, IL).
To demonstrate that the modulation of the chimeric GRE promoter by
dexamethasone was mediated via the glucocorticoid receptor, HeLa cells
(105/well) were infected with AdGRE.CAT (25 moi) and, after
24 hours, the cells were incubated with various concentrations of
glucocorticoid receptor antagonist, mifepristone (RU486,
10 7 to 105 mol/L; BIOMOL, Plymouth
Meeting, PA), and dexamethasone (25 nmol/L). Parallel studies were
performed with HeLa cells (105/well) infected with
AdGRE.CAT (25 moi) and, 24 hours later, incubated with: (1)
dexamethsone (25 nmol/L), and then after 6 hours with RU486
(105 mol/L) for an additional 18 hours; or (2) RU468
(105 mol/L), and then after 6 hours with dexamethasone
(25 nmol/L) for an additional 18 hours. The promoter function was
assessed by measuring CAT activity and standardized by total protein
concentration.
Experimental animals.
C57B1/6 mice (6 to 10 weeks of age) were from Charles River
Laboratories (Wilmington, MA). The age, sex, and body weight (>20 g)
of mice were matched for each experiment. Each experiment included at
least three animals for each data point.
In vivo evaluation of AdGRE.CAT.
To quantify the dose response of the AdGRE.CAT vector to dexamethasone
in vivo, AdGRE.CAT (108, 5 × 108,
109 plaque-forming units [pfu]) was administered
intravenously to C57B1/6 mice in 100 µL phosphate-buffered saline
(PBS), pH 7.4. To activate the GRE promoter, the animals were treated
(24 hours after Ad vector administration) with a single dose of 50 µg
dexamethasone intraperitoneally. Based on the knowledge that
intravenous administration of Ad vectors is associated with more than
90% of the vector genome transferred to the liver,28,29 we
focused the analysis on liver. Two days after vector administration,
the liver was removed, and homogenized and centrifuged to remove
debris. CAT activity was measured as described by Neumann et
al,27 and values were reported after standardization to
total protein concentration.
To evaluate the dose response of dexamethasone to AdGRE.CAT-mediated
gene expression, C57B1/6 mice were treated with various doses of
dexamethasone (1 to 500 µg) intraperitoneally 24 hours after
intravenous administration of AdGRE.CAT (5 × 108 pfu).
Two days after vector administration, CAT activity in the liver was
quantified and standardized to total protein concentration.
To examine the time-dependent response of the GRE promoter to
dexamethasone in the liver, the AdGRE.CAT vector
(5 × 108 pfu) or AdRSV.CAT (as a positive control
vector, 5 × 108 pfu) was administered intravenously to
C57B1/6, mice and 24 hours later 50 µg dexamethasone was administered
intraperitoneally. CAT activity in the liver, standardized to total
protein concentration, was evaluated before and 1 to 14 days after Ad
vector administration.
To quantify the time response of the GRE promoter to repeated
administration of dexamethasone, the AdGRE.CAT vector
(5 × 108 pfu) was administered intravenously to C57B1/6
mice and dexamethasone was administered intraperitoneally (50 µg/d)
either 1 day or up to 6 days after Ad vector administration. CAT
activity in the liver, referenced to total protein concentration, was
evaluated before and 1 to 14 days after Ad vector administration.
In vitro evaluation of AdGRE.mTPO.
To evaluate the upregulation of the mTPO cDNA in the GRE.mTPO cassette
transferred to HeLa cells by the AdGRE.mTPO vector, HeLa cells were
infected (90 minutes, 37°C) in a serum-free medium with
AdGRE.mTPO, at moi of 5 or 25. After 24 hours, 25 nmol/L dexamethasone
was added to the culture medium to stimulate the GRE promoter. To
access the upregulation of mTPO at the mRNA level, total RNA (10 µg)
was isolated using guanidine isothiocyanate phenol-chloroform
extraction,30 separated on a 1% agarose gel containing 2.2 mol/L formaldehyde, transferred to a nylon membrane (NYTRAN; Schleicher
& Schuell, Keene, NH), and hybridized with a 32P-labeled
mTPO cDNA probe and a glyceroaldehyde-3-phosphate dehydrogenase (GAPDH)
cDNA probe31 (as an internal control) prepared by random priming and evaluated by autoradiography.32 To access the
upregulation of mTPO at the protein level, Western blotting was
performed by infecting HeLa cells with AdGRE.mTPO (25 moi), AdNull (25 moi), or AdCMV.mTPO (5 moi). After 24 hours, the cells were incubated with dexamethasone (25 nmol/L) or without dexamethasone for an additional 24 hours. Cells were lysed in 100 mmol/L Tris-HCl, pH 7.5, 75 mmol/L NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS),
20 mmol/L EDTA, 0.1% phenylmethylsulfonyl fluoride (Sigma), and 1 µg/mL aprotinin (Sigma). Total protein (50 µg per lane) was
separated on 10% SDS-polyacrylamide gels, transferred to
nitrocellulose membrane (BIORAD, Hercules, CA), and incubated with a
goat anti-mTPO antibody (R&D, Minneapolis, MN) at a 1:1,000 dilution.
Alkaline phosphatase-conjugated swine anti-goat secondary antibody
(Boehringer Mannheim, Indianapolis, IN) was used at 1:1,000 dilution
and developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl
phosphate (BIORAD).
In vivo evaluation of AdGRE.mTPO.
Preliminary evaluation of the in vivo response of the inducible GRE
promoter to dexamethasone was performed by administering the AdGRE.mTPO
(5 × 108) vector intravenously to C57B1/6 mice and
dexamethasone (50 µg/d) intraperitoneally on 1 to 3 successive days.
As a negative control, AdNull (109 pfu) was administered
intravenously to C57B1/6 mice with or without dexamethasone (50 µg/d;
at 1 to 3 days after Ad vector administration). Platelet levels were
determined in blood samples drawn from the tail vein with a capillary
pipette (Unopette; Fisher Scientific, Springfield, MA) using a Neubauer
hemocytometer (Fisher Scientific) 4 to 22 days after Ad vector
administration.
Repeated in vivo upregulation of the GRE promoter in the AdGRE.mTPO
by dexamethasone.
To examine the ability of repeated administration of dexamethasone to
repetitively upregulate the GRE promoter after one-time administration
of the AdGRE.mTPO vector, the AdGRE.mTPO vector (5 × 108 pfu) was administered intravenously to C57B1/6
mice and dexamethasone (50 µg/d) was administered intraperitoneally
on 3 consecutive days starting at days 1, 22, and 43. The animals were
then assessed for the amount of Ad genome DNA in liver, anti-Ad vector
cytotoxic T-lymphocytes (CTL) and anti-Ad neutralizing antibodies, mTPO concentration in serum, megakaryocyte number in bone marrow, and the
level of blood platelets before and at various times 1 to 64 days after
vector administration.
To quantify the amount of AdGRE.mTPO Ad genome DNA in the liver over
time, total genomic DNA was extracted from the liver by homogenizing in
3 mL lysis buffer (20 mmol/L Tris-HCl, pH 7.5, 600 mmol/L NaCl, 1%
SDS, 10 mmol/L EDTA, 200 µg/mL proteinase K [Boehringer-Mannheim]).
DNA was digested with HindIII, subjected to agarose gel
electrophoresis, transferred to a nylon membrane (NYTRAN), and
hybridized with a 32P-labeled Ad5 E4 cDNA probe, as
described by Worgall et al.29 The relative amount of Ad
genome was quantified by phosphorimager (Molecular Dynamics, Sunnyvale,
CA).
To evaluate for the presence of CTL against AdGRE.mTPO, splenocytes
were isolated from animals 43 days after administration of AdGRE.mTPO,
mixed with irradiated syngeneic C57SV cells infected with AdGRE.mTPO
(cell density 6 × 106 cells/mL), and cultured for 5 days.33 The 51Cr assay was performed as
described previously.34 Briefly, the in vitro stimulated
splenocytes were incubated 4 hours with 51Cr-labeled
syngeneic C57SV target cells (104, uninfected or infected
with AdGRE.mTPO, or AdNull) at an effector/target ratio of 6, 20, and 60. The percent specific cytotoxicity was calculated as:
% Specific Lysis = (Mean dpm of Test 51Cr
Release  Mean dpm of Spontaneous 51Cr Release) × 100/(Mean dpm of Maximum Isotope Release Mean dpm of Spontaneous
51Cr Release).
To quantify the humoral immune response directed toward intravenously
administered AdGRE.mTPO vector after dexamethasone administration, serum samples were obtained from animals before and 22 to 64 days after
Ad administration. Serum anti-Ad neutralizing antibodies were measured
by evaluating the ability of the serum to prevent infection of A549
cells (human lung carcinoma cell line, CCL 185; ATCC) by wild-type
adenovirus type 5 (Ad5) as previously described.35 The A549
cells were seeded in 96-well plates (Falcon 3072; Becton Dickinson,
Lincoln Park, NJ) at a density of 3 × 104 cells/well.
Serum samples were diluted serially and added in twofold serial
dilution to a 96-well plate. Ad5 (1 moi) was added, and the plates were
incubated for 1 hour at 37°C. The mixture was then added to the A549
cells, and the cells were incubated until the serum free-control wells
exhibited greater than 95% cytopathic effect (typically 5 to 8 days).
The neutralizing antibody titer (per 4.5 µL serum) was calculated at
the product of the reciprocal of the initial dilution times the
reciprocal of the dilution in the last well showing greater than 95%
cytopathic effect.
The mTPO concentration in serum samples was determined in a
double-sandwich enzyme-linked immunosorbent assay (ELISA; mouse TPO
immunoassay kit, R&D) following the protocol provided by the manufacturer. To evaluate the number of megakaryocytes in the bone
marrow, femurs were removed from mice, fixed in 4% paraformaldehyde in
PBS, and stained with hematoxylin and eosin. The number of megakaryocytes in bone marrow was evaluated as number of cells/high power field. Platelet number was evaluated by counting as described above.
Statistical analysis.
The results are expressed as mean ± standard error of the mean.
Statistical comparisons were made using the unpaired two-tailed Student's t-test, unless otherwise noted.
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RESULTS |
In vitro evaluation of the AdGRE.CAT vector.
Consistent with the previous observations that the transient or stable
transfection of the plasmid containing the GRE promoter with a CAT
reporter gene provides a low level of basal level of gene expression in
HeLa cells in vitro that can be upregulated with
dexamethasone,12 transfer of the GRE promoter CAT
expression cassette into HeLa cells using the AdGRE.CAT adenovirus
vector demonstrated a low basal level of CAT reporter gene expression that was markedly upregulated induced by the addition of dexamethasone (Fig 2). Transfer to HeLa cells of the
highly active RSV promoter-CAT expression cassette with the AdRSV.CAT
vector resulted in high levels of CAT expression, but with little
increase with the addition of dexamethasone (+dexamethasone v
no dexamethasone, P > .05 for 1, 6, and 24 hours after
dexamethasone addition, threefold increase at 48 hours,
P < .03; Fig 2A). In marked contrast, dexamethasone was
required to achieve high levels of CAT activity following transfer of
the GRE.CAT expression cassette at all time points more than 1 hour
after dexamethasone addition (1 hour, P > .1; 6 hours,
17-fold increase, P < .0003; 24 hours, 75-fold increase, P < .0007; 48 hours, 85-fold increase,
P < .002; Fig 2B). In contrast to the increase with the
addition of dexamethasone, the expression of CAT was minimally
increased from 1 to 48 hours without dexamethasone (fivefold,
P < .02). Importantly, the level of CAT expression achieved
at 48 hours with the AdGRE.CAT vector plus dexamethasone was similar to
that achieved with AdRSV.CAT and dexamethasone (P > .5).
Thus, the AdGRE.CAT vector expressed at low levels of the reporter
gene, with little "leak" of expression over 48 hours, but with
marked (85-fold) upregulation at 48 hours with dexamethasone, achieving
the same levels as a highly active, constitutive viral promoter.
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| Fig 2.
AdGRE.CAT-mediated in vitro time course of CAT activity
in HeLa cells upregulated by dexamethasone (Dex). (A) HeLa cells
infected with AdRSV.CAT (25 moi), a control vector driven by the
RSV-LTR constitutive promoter. (B) HeLa cells infected with AdGRE.CAT (25 moi). For both panels, after 24 hours, cells were incubated with or
without dexamethasone (25 nmol/L) for an additional 1 to 48 hours, and
CAT activity was quantified relative to total protein concentration.
Shown are data for AdRSV.CAT with dexamethasone ( ) or without
dexamethasone ( ); and for AdGRE.CAT infection with dexamethasone
( ) or without dexamethasone ( ). The data are presented as mean ± SEM of three independent experiments.
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To show that the upregulation of this GRE promoter was mediated by the
glucocorticoid receptor, we examined the inhibitory effect of the
glucocorticoid receptor antagonist RU486 on AdGRE.CAT-mediated gene
expression in vitro. The addition of RU486 led to a dose-dependent inhibition of dexamethasone-stimulated CAT activity in HeLa cells (P < .002, all doses of RU486 compared with no RU486 but
with dexamethasone, Fig 3A). When RU486 was
added to HeLa cells in vitro after 6 hours of incubation with
dexamethasone, there was some inhibition of CAT activity (compared with
dexamethasone with added RU486 simultaneously, panel A,
P < .0004), but the inhibitory effect of RU486 was far more
if added 6 hours before dexamethasone (compared with dexamethasone
added first, P < .0001; compared with dexamethasone added
simultaneously, P > .2) (Fig 3B). These data are
consistent with the hypothesis that activation of GRE promoter of AdGRE.CAT vector by dexamethasone occurs via the
glucocorticoid receptor.
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| Fig 3.
Effect of the glucocorticoid receptor antagonist RU486 on
GRE promoter activity after AdGRE.CAT infection of HeLa cells in vitro.
The cells were incubated with the AdGRE.CAT vector (25 moi) for 24 hours to transfer the GRE.CAT expression cassette, followed by the
addition of dexamethasone (Dex) and RU486 as indicated. (A) Effect of
dexamethasone and RU486 added together. Twenty-four hours after
infection with AdGRE.CAT, dexamethasone (25 nmol/L) and various
concentrations of RU486 (107 to 105
mol/L) were added together, and the incubation continued for 24 hours.
Shown are data for CAT activity with dexamethasone () or without
dexamethasone (). (B) Effect of dexamethasone or RU486 added in
series. Twenty-four hours after vector administration, the cells were
pre-incubated for 6 hours with dexamethasone, then with RU486 for an
additional 18 hours. Parallel cultures were evaluated in reverse order
(RU486 for 6 hours and then dexamethasone for an additional 18 hours).
Shown is CAT activity at the end of the incubation. The results are
expressed as the mean ± SEM of three experiments.
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In vivo evaluation of AdGRE.CAT.
Based on the knowledge that Ad-mediated gene transfer to the liver can
be efficiently achieved by the intravenous route of vector
administration,28,29 the responsiveness of the GRE promoter to dexamethasone was evaluated after administration of the Ad CAT-expressing vectors via the intravenous route to C57B1/6 mice. Consistent with the data obtained from in vitro Ad transfection of HeLa
cells, CAT activity of the liver following intravenous administration
of AdGRE.CAT was upregulated by dexamethasone (50 µg) with vector
doses of 5 × 108 pfu (41-fold, P < .000006,
comparison to no dexamethasone treatment) and 109 pfu
(fivefold, P < .000005, comparison to no dexamethasone at 109 pfu), but not at 108 pfu
(P > .3; Fig 4A). Since
109 pfu Ad administration without dexamethasone provides a
higher level of basal level of gene expression compared with 5 × 108 pfu, but no additional advantage in the presence of
dexamethasone, 5 × 108 pfu of the Ad vector was used in
subsequent experiments.
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| Fig 4.
In vivo upregulation of CAT activity in the liver by
dexamethasone (Dex) after intravenous administration of the AdGRE.CAT vector to C57B1/6 mice. (A) Dexamethasone-induced expression of CAT as
a function of vector dose. The AdGRE.CAT vector (108,
5 × 108 or 109 pfu) was administered
intravenously to C57B1/6 mice and 24 hours later, dexamethasone (50 µg) was administered intraperitoneally. (B) Expression of CAT as a
function of dexamethasone dose. The AdGRE.CAT vector
(5 × 108 pfu) was administered intravenously to C57B1/6
mice, and 24 hours later various doses of dexamethasone (1, 5, 10, 50, 100, 500 µg) were administered intraperitoneally. Two days later, CAT
activity was quantified in the liver. Shown are data for AdGRE.CAT
infection with dexamethasone () or without dexamethasone (). The
data are presented as mean ± SEM of three experiments.
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Evaluation of the dose dependency of the GRE promoter-mediated CAT
reporter activity with various amounts of dexamethasone (0 to 500 µg)
in vivo showed that the CAT activity in the liver of C57B1/6 mice after
AdGRE.CAT (5 × 108 pfu) administration increased with
doses of 1 to 50 µg dexamethasone, and reached a plateau with 50 to
500 µg dexamethasone, with the plateau 93-fold greater than no
dexamethasone (P < .02, all comparisons to no
dexamethasone; Fig 4B). Based on these observations, 50 µg
dexamethasone was used to induce the GRE promoter in vivo in subsequent
experiments.
Based on the dependency on the dose of Ad-vector (Fig 4A) and
dependency on dexamethasone (Fig 4B), evaluation of the time dependency
of the 5 × 108 pfu AdGRE.CAT vector-mediated CAT activity
in the liver of C57B1/6 mice with a single administration of
dexamethasone (50 µg) demonstrated that the maximum CAT activity was
observed at 2 to 4 days after Ad vector administration
(P < .000002, both comparisons to no dexamethasone
treatment at the same day), with a decrease to baseline levels at 2 weeks (Fig 5A). Repetitive daily
administration of dexamethasone (50 µg daily for 6 days after Ad
vector administration) induced a similar increase in CAT activity at 2 to 4 days, but it was sustained for 7 days (P < .001, days
2 to 7 + dexamethasone compared with no dexamethasone), compared to
the decrease after 4 days with a single dexamethasone administration
(Fig 5B). The maximum value achieved with multiple administrations of
dexamethasone was similar to that of the peak value achieved with the
AdRSV.CAT positive control vector-mediated CAT activity
(P > .1, compare Fig 5B with Fig 5C).
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| Fig 5.
In vivo time-dependent expression of CAT in response to
dexamethasone (Dex) after administration of the AdGRE.CAT vector. CAT
activity in the liver was evaluated before and 1 to 14 days after
intravenous administration of the vector to C57B1/6 mice. (A)
Time-dependent expression of CAT following administration of the
AdGRE.CAT vector and dexamethasone. AdGRE.CAT (5 × 108
pfu) was administered intravenously and dexamethasone and (50 µg) was
administered intraperitoneally at day 1. Shown are data for CAT
expression with dexamethasone () or without dexamethasone (). (B)
Effect of multiple daily administration of dexamethasone on CAT
expression after administration of the AdGRE.CAT vector. The vector
administration was identical to (B), but dexamethasone (50 µg/d) was
administered intraperitoneally on 6 consecutive days. (C)
Time-dependent expression of CAT following intravenous administration
of AdGRE.CAT (5 × 108 pfu; ) or AdRSV.CAT
(5 × 108 pfu; ); no dexamethasone was administered.
Shown are data for AdGRE.CAT after 6 days' administration of
dexamethasone () or without dexamethasone (). The data are
presented as mean ± SE of three independent experiments.
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In vitro evaluation of the AdGRE.mTPO vector.
Based on the success of CAT reporter gene upregulation by using GRE
promoter in vitro and in vivo, we applied this inducible promoter Ad
vector strategy to one clinically applicable model in which an Ad
vector was used to transfer the GRE promoter controlling the murine TPO
cDNA. Evaluation of the AdGRE.mTPO vector in vitro confirmed that this
vector expressed the mTPO cDNA in vitro as expected (Fig
6). In this regard, a 1.5-kb mRNA
transcript of the murine thrombopoietin cDNA was visible in HeLa cells
infected with AdGRE.mTPO (5 or 25 moi) incubated with 25 nmol/L
dexamethasone (Fig 6A, lanes 3 and 4) but no mTPO mRNA was detected
without the addition of dexamethasone (lanes 1 and 2). Cells infected with the positive control vector, AdCMV.mTPO, demonstrated expression of the same size mTPO transcripts compared with AdGRE.mTPO with dexamethasone (not shown). The mRNA level for GAPDH, used as internal control, did not change with the addition of dexamethasone in cells
infected with the AdGRE.mTPO vector.
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| Fig 6.
Dexamethasone (Dex) stimulated expression of mouse TPO
(mTPO) mRNA transcripts and mTPO protein in HeLa cells after infection with the AdGRE.mTPO vector. (A) Northern analyses of total RNA (10 µg/lane) isolated from HeLa cells infected with Ad vectors. Cells
were infected with AdGRE.mTPO (5 or 25 moi), an Ad vector containing a
chimeric corticosteroid inducible promoter controlling the expression
of the mTPO cDNA (lanes 1 through 4). After 24 hours, the cells were
incubated with dexamethasone (25 nmol/L; lanes 3 and 4) or without
dexamethasone (lanes 1 and 2) for an additional 24 hours. The RNA was
hybridized with a mTPO cDNA probe (top) or a GAPDH (bottom) probe. Lane
1, AdGRE.CAT (5 moi) alone; lane 2, AdGRE.CAT (25 moi) alone; lane 3, AdGRE.CAT (5 moi) + Dex; lane 4, AdGRE.CAT (25 moi) + Dex. (B)
Western blot analysis of total protein (50 µg/lane) isolated from
HeLa cells infected with Ad vectors. Cells were infected with either
AdNull (25 moi, lanes 1 and 2), or AdGRE.mTPO (25 moi, lanes 3 and
4). After 24 hours, the cells were incubated with
dexamethasone (25 nmol/L) (lanes 2 and 4) or without dexamethasone
(lanes 1 and 3) for an additional 24 hours. Anti-mTPO serum was used to
detect the 35-kD mTPO protein.
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The 35-kD mouse thrombopoietin protein was visible in HeLa
cells infected with AdGRE.mTPO (25 moi) plus 25 nmol/L dexamethasone (Fig 6B, lane 4), but no mTPO protein was detected without the administration of dexamethasone (Fig 6B, lane 3). In the context that
the mTPO cDNA codes for the full length of the TPO coding sequences,
and the fully glycosylated secreted mTPO is a 70-kD protein,36,37 it is likely that the 35-kD band represents
intracellular, nonglycosylated mTPO. Cells infected with the positive
control vectorAdC-MV.mTPO demonstrated the same size mTPO protein
compared with AdGRE.mTPO (not shown). Infection with the AdNull vector did not yield mTPO bands (without or with dexamethasone, Fig 6B, lanes
1 and 2).
In vivo evaluation of the AdGRE.mTPO.
Based on the in vitro observation that levels of mTPO mRNA transcripts
derived from the AdGRE.mTPO vector could be upregulated by
dexamethasone, we hypothesized that an increased expression of mTPO by
AdGRE.mTPO with dexamethasone administration would induce an increase
in the platelet count in vivo. As expected, AdGRE.mTPO
(5 × 108 pfu) administration together with
dexamethasone (50 µg per day for 1 to 3 days after vector
administration) was associated with the upregulation of the platelet
levels (Fig 7A). The peak platelet level
was observed at day 8 (P < .05, all dexamethasone
strategies of administration compared with no dexamethasone), but the
platelet levels remained elevated at 15 days (P < .002, all
dexamethasone strategies of administration compared with no
dexamethasone), decreasing to baseline by day 22 (P > .4
for dexamethasone at days 0 and 0 and 1 compared with no
dexamethasone), except where dexamethasone was administered for 3 days
(P < .04 compared with no dexamethasone). Administration of
AdNull (5 × 108 pfu) as a negative control vector, with
or without dexamethasone, administered for 3 consecutive days showed no
upregulation of platelet number compared with the normal range of
platelet levels (P > .06, all comparisons; Fig 7B).
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| Fig 7.
In vivo upregulation of platelet number by dexamethasone
(Dex) after intravenous administration of the of AdGRE.mTPO vector. The
AdGRE.mTPO vector (5 × 108 pfu) was administered
intravenously. (A) Dose-dependent increase in platelet number in
C57B1/6 mice after dexamethasone administration for 1 to 3 successive
days (50 µg/d) intraperitoneally. Platelet number was evaluated
before and 4 to 22 days after vector administration. Shown are the
platelet levels without dexamethasone (), and with dexamethasone
administered for 1 day (), 2 days (), and 3 days (). (B)
Platelet levels after administration of the AdNull control vector and
dexamethasone. As a negative control, the AdNull vector (5 × 108 pfu) was injected intravenously to C57B1/6
mice with dexamethasone administered on 3 successive days (50 µg/d;
intraperitoneal; ) or without dexamethasone (). The platelet
level was counted before and 4 to 22 days after vector administration.
The data are presented as mean ± SE of three independent
experiments.
|
|
Intermittent upregulation of platelet levels in vivo.
To evaluate the hypothesis that intermittent administration of
dexamethasone will result in repeated upregulation of expression of the
GRE.mTPO expression cassette transferred to the liver by the AdGRE.mTPO
vector, the AdGRE.mTPO vector was administered one time intravenously
to C57B1/6 mice and dexamethasone was administered intermittently.
After administration of the AdGRE.mTPO vector (5 × 108), dexamethasone was administered
intraperitoneally (50 µg/dose) on 3 consecutive days starting at days
1, 22, and 43. Quantification of the amount of vector DNA in the liver
showed a decrease in the first 4 days after vector administration, but
thereafter the rate of decrease slowed dramatically, and the Ad genome
could be easily detected in the liver 64 days after vector
administration (Fig 8). The amount of Ad
genome in the liver with or without dexamethasone was similar at all
time points (P > .05) except for day 64 where the group
receiving dexamethasone had 2.9-fold more AdGRE.mTPO genome in the
liver compared with the untreated group (P < .002).
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| Fig 8.
Amount of adenovirus genome DNA in the liver after
intravenous administration of the AdGRE.mTPO vector. Following
administration of AdGRE.mTPO (5 × 108 pfu),
dexamethasone (Dex) was administered intraperitoneally (50 µg/dose)
on 3 consecutive days starting at days 1, 22, and 43. Following 10 minutes and 1, 4, 22, and 64 days after vector administration, the liver was removed, and the amount of vector genome
determined by Southern analysis and quantified by phosphorimager. Data
are presented as relative percent of data at 10 minutes on day 0 (defined as 100%). Shown are data for adenovirus genome with
dexamethasone () and without dexamethasone (). The data are
presented as mean ± SE of three independent experiments.
|
|
Mice receiving dexamethasone and mice not receiving dexamethasone both
developed CTL directed against the Ad vector (Fig 9A and
B). The splenocytes recovered at day 43 (before the timing of the third cycle of dexamethasone administration)
exhibited similar levels of destruction of target cells either infected with AdNull or infected with the AdGRE.mTPO vector, independent of
dexamethasone treatment. These data are consistent with the concept
that the CTL were directed toward the Ad vector-derived antigens, not
the mTPO transgene, and that dexamethasone therapy did not prevent the
development of anti-vector CTL.
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| Fig 9.
Evaluation of the cellular and humoral immune response
against AdGRE.mTPO vector with or without dexamethasone (Dex)
administration in vivo. After administration of the AdGRE.mTPO vector
(5 × 108), dexamethasone was administered
intraperitoneally (50 µg/dose) on 3 consecutive days starting at days
1, 22, and 43. (A and B) CTL at day 43 after administration of
AdGRE.mTPO with and without dexamethasone. Splenocytes were evaluated
for their ability to lyse syngeneic cells infected with AdGRE.mTPO or
AdNull. Data are presented as percent lysis of target cells mixed at
various ratios with splenocytes relative to the total amount of
51Cr that could be released by lysing 100% of the cells.
Shown are data for uninfected target cells ("alone," ), target
cells infected with AdNull (), and target cells infected with
AdGRE.mTPO (). (A) Mice receiving AdGRE.mTPO vector alone (no Dex).
(B) Mice receiving AdGRE.mTPO vector plus dexamethasone administration (+Dex). (C) Serum concentration (titer/4.5 µL) of neutralizing antibody directed against Ad vectors before and 22 to 64 days following
AdGRE.mTPO administration (5 × 108 pfu) with or without
dexamethasone. The dashed line indicates the limit of sensitivity of
the assay (titer < 10). Shown are data for serum anti-Ad
neutralizing antibodies titer with dexamethasone () and without
dexamethasone (). The data are presented as individual time points
for each animal.
|
|
To evaluate the anti-Ad humoral immune response following intravenously
administered AdGRE.mTPO vector without or with dexamethasone administration, the sera recovered before and at days 22, 43, and 64 were evaluated for the presence of anti-Ad neutralizing antibodies (Fig
9C). Anti-Ad neutralizing antibodies were present in both groups at day
22 to 64. There were no significant differences among animals receiving
AdGRE.mTPO vector receiving dexamethasone and animals receiving
AdGRE.mTPO vector alone (P > .3, Mann-Whitney test).
Consistent with the presence of the vector genome in the liver (Fig 8),
and independent of the anti-Ad vector CTL and the anti-Ad vector
neutralizing immunity (Fig 9), there were intermittent increases in
mTPO serum levels after administration of the AdGREm.TPO vector (Fig
10A). In this regard, there was a peak of
mTPO level in serum at day 2 (13-fold, P < .00005), day 24 (9-fold, P < .000005), and day 45 (4-fold,
P < .0000005) (all compared with no dexamethasone treatment
on the same day). For each cycle of increase of mTPO levels, there was
a rapid decrease to baseline levels by 1 week after each dexamethasone
administration. Without dexamethasone, the serum mTPO levels remained
similar or only slightly elevated (<twofold) compared with the
background range (2.9 to 6.6 ng/mL; days 2 to 24, P > .07;
days 26 to 50, P < .02; day 64, P > .9; all
comparisons to 0 time).
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| Fig 10.
Intermittent upregulation of mTPO levels in
serum, megakaryocyte number in bone marrow, and blood platelet levels
in C57B1/6 mice by intermittent administration of dexamethasone (Dex)
following one-time intravenous administration of the AdGRE.mTPO vector. After administration of AdGRE.mTPO (5 × 108 pfu),
dexamethasone was administered intraperitoneally (50 µg/dose) on 3 consecutive days starting at days 1, 22, and 43. (A) The mTPO
concentration in serum evaluated before and 4 to 64 days after vector
administration. Shown are data for mTPO level in serum with
dexamethasone () and without dexamethasone (). (B) The number of
megakaryocytes in bone marrow before and 4 to 64 days following vector
administration. Shown are data for megakaryocytes with dexamethasone
() and without dexamethasone (). (C) Platelet levels before and 4 to 64 days after vector administration. Shown are data for platelet
levels with dexamethasone () and without dexamethasone (). The
data are presented as mean ± SE of three independent experiments.
|
|
In parallel with the intermittent elevation of serum mTPO levels, the
numbers of megakaryocytes in bone marrow showed intermittent elevations
of megakaryocyte numbers of day 4 (8-fold, P < .00002), day
26 (15-fold, P < .0005), and day 47 (11-fold,
P < .00005); all comparisons to no dexamethasone treatment
at the same day; Fig 10B. All peak numbers of megakaryocytes were at 4 days after the first day of dexamethasone administration for each
cycle, with a rapid decrease to baseline levels within 4 days after the last administration of dexamethasone (for each cycle). Similar to the
mTPO in serum, without dexamethasone, the megakaryocyte number in
marrow remained the same as baseline (P > .1, all
comparisons to 0 time). Importantly, there were no pathologic changes
in the bone marrow, such as myelofibrosis, after 64 days (and three
cycles of stimulation with dexamethasone, not shown).
Finally, the platelet levels intermittently increased concomitantly
with the intermittent administration of dexamethasone (Fig 10C). Three
peaks of platelet levels were observed, at day 8 (4-fold,
P < .0001), day 29 (6-fold, P < .00001), and
day 50 (4-fold, P < .00001); all compared with no
dexamethasone treatment at the same day. All of the peaks were at day 8 after first dexamethasone administration for each cycle, with a slow
decrease to baseline levels within 2 weeks after each administration of
dexamethasone. Without dexamethasone administration, the platelet count
remained within the normal range. Interestingly, the second peak of
platelets was higher than the first peak. One explanation for this
phenomenon is that thrombopoietin has an effect on primitive
hematopoietic cells, such as stem cells.38
| |
DISCUSSION |
Now that gene transfer has proven to be feasible in experimental
animals and humans,39 efforts have focused on a myriad of
challenges that must be overcome before gene transfer can be used
therapeutically. For many of these applications one challenge is to be
able to control the expression of the newly transferred gene. The
present study shows this can be achieved by using an adenovirus vector
to transfer to the liver of C57B1/6 mice an expression cassette
containing a chimeric promoter comprised of multiple glucocorticoid
response elements driving the CAT reporter gene or the murine
thrombopoietin cDNA, a gene coding for a secreted thrombopoietic
hormone that activates bone marrow megakayocyte, resulting in elevation
of blood platelet levels.14-17 The ability to control the
GRE expression cassette in vivo was dependent on the dose of the vector
and on the amount of dexamethasone administered, with maximum levels of
transgene expression similar to that achieved with the active
constitutive RSV viral promoter. Strikingly, administration of the
AdGRE.mTPO vector followed by dexamethasone every 3 weeks was
associated with intermittent elevations of serum mTPO levels, megakaryocyte numbers in bone marrow and blood platelet levels fourfold
to sixfold over baseline, ie, intermittent regulation of the
transferred gene with intermittent changes in platelet-related phenotype. In the context that corticosteroids are widely used in
clinical medicine, and can be used safely when administered intermittently, this strategy may be useful for a variety of clinical applications.
Advantages of using a glucocorticoid responsive promoter.
There are a variety of advantages to using a glucocorticoid response
promoter to intermittently control a newly transferred gene. First, the
glucocorticoid receptor is expressed in a variety of cell types, and
the structure and function of the ligand and receptor, as well as the
signal transduction, and transcriptional response elements for
corticosteroids are well understood.40,41 Second, ligand
activation of corticosteroid receptor-dependent transcription is
specific and dose dependent.42-47 In this context, the
chimeric promoter was responsive to the dose and chronicity of
dexamethasone administration. Third, corticosteroids can be administered by a variety of routes, and the safety profile of corticosteroid in humans is well defined.48 Finally, the
expression cassette of the chimeric GRE promoter is small (total 1 kb
for the promoter and the polyA stop signal), and thus is adaptable to a
variety of vector systems.
One interesting characteristic observed of the chimeric GRE promoter is
that there was very little "leak" of transgene expression without
the administration of glucocorticoids, at least for applications involving gene transfer to the liver. In this regard, even with the
very sensitive CAT reporter gene, little CAT activity was observed in
the liver at AdGRE.CAT vector doses of  5 × 108 pfu
without added dexamethasone. However, an impressive, dose-dependent upregulation of CAT was observed with dexamethasone, reaching levels
100-fold above baseline. Further, the upregulation of expression could
be sustained by repetitive daily administration of dexamethasone, maintaining increased CAT levels in liver for 1 week after six daily
doses of dexamethasone compared to a 1-day peak level with single dose.
These properties compare favorably with other controllable promoters
that have been evaluated in in vivo, ex vivo/in vivo, and transgenic
experimental animal models.2-11,49-63 The dose of dexamethasone used in this study (175 mg) are equivalent to doses of 1 to 1.5 g/d of methylprednisolone in humans. Although such doses are
used in short-term human therapies, additional studies will have to be
performed to determine the lowest dose that will turn on the GRE
promoter in vivo before this strategy is applied to humans.
Application to controlling platelet levels.
There is increasing evidence that gene transfer vectors can be used
effectively in vivo to express secreted hematopoietic hormones such as
erythropoietin, thrombopoietin, granulocyte-monocyte colony-stimulating
factor, and granulocyte colony-stimulating factor.63-70 For
all of these hematologic mediators, overexpression and/or
inappropriate persistence of expression could be associated with
adverse effects from the relevant transgene both from inappropriate levels of the blood element, abnormalities in bone marrow, or nonhematologic adverse effects.68,69,71 Although the data in the present study are not necessarily applicable to all hematologic hormones, it shows the ability to upregulate a potent
hematologic-mediator, thrombopoietin, intermittently for a 1-week
period over 2 months. In the context of the potential for adverse
effects such as stroke from excess platelet levels, such a strategy may
be useful for gene transfer applications using transfer of the
thrombopoietin cDNA for the available upregulation of platelet levels.
Use of the mTPO cDNA as a reporter gene.
The mTPO cDNA is a useful "reporter" for gene transfer studies.
First, the phenotype for successful expression is clear (platelet levels), and easily carried out, requiring only a hemocytometer. Second, because measurement of platelet levels requires only 20 µL of
blood, the phenotype can be assessed in the same mouse over months,
using repetitive blood sampling from the tail vein with a capillary
pipette. Third, because the mTPO cDNA is of murine origin, it serves to
code for an autologous "reporter" that is not recognized by the
murine immune system. Finally, like  1-antitrypsin,72,73 the combination of the mTPO cDNA and Ad vectors can persist for considerable periods in strains such as C57B1/6, despite the generation of that anti-Ad cytotoxic T cells, consistent with the emerging concept
that anti-Ad cellular immunity does not limit Ad vector expression in
all applications.34,68,74-76
| |
FOOTNOTES |
Submitted November 17, 1997;
accepted April 7, 1998.
Supported in part by the National Institutes of Health/National Heart,
Lung and Blood Institute Grants No. P01 HL51746 and P01 HL59312; the
Cystic Fibrosis Foundation (Bethesda, MD); Will Rogers Memorial Fund
(White Plains, NY); and GenVec, Inc (Rockville, MD). M.A.S.M. is
supported by the Gar Reichman Fund of the Cancer Research Institute
(New York, NY).
Address reprint requests to Ronald G. Crystal, MD, Division of
Pulmonary and Critical Care Medicine, The New York Hospital-Cornell Medical Center, 520 E 70th St, ST505, New York, NY 10021; e-mail: nmohamed{at}mail.med.cornell.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Ben-Gary Harvey and Satish Deshmane for helpful advice and
assistance in carrying out these studies; and N. Mohamed for help in
preparation of the manuscript.
| |
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Abstract
Introduction
Abstract